Dow Splash 311956 2012-07-231

THEME [KBBE.2012.3.
4-02] [Biotechnology for novel biopolymers]

Grant agreement for: Collaborative project
*
Annex I - "Description of Work"

Project acronym: SPLASH Project full title: " Sustainable PoLymers from Algae Sugars and Hydrocarbons " Grant agreement no: 311956 Version date:
Table of Contents
Part A
A.1 Project summary ...................................................................................................................................... 3 A.2 List of beneficiaries ..................................................................................................................................4 A.3 Overall budget breakdown for the project ............................................................................................... 5
Workplan Tables
WT1 List of work packages ............................................................................................................................1 WT2 List of deliverables .................................................................................................................................2 WT3 Work package descriptions ................................................................................................................... 9 Work package 1......................................................................................................................................9 Work package 2....................................................................................................................................13 Work package 3....................................................................................................................................20 Work package 4....................................................................................................................................26 Work package 5....................................................................................................................................31 Work package 6....................................................................................................................................33 Work package 7....................................................................................................................................37 WT4 List of milestones .................................................................................................................................42 WT5 Tentative schedule of project reviews ................................................................................................. 44 WT6 Project effort by beneficiaries and work package ................................................................................45 WT7 Project effort by activity type per beneficiary ...................................................................................... 46 WT8 Project efforts and costs ......................................................................................................................48
Project summary
Project Number
1
A1:
311956
Project Acronym
SPLASH
One form per project General information Project title

3
Sustainable PoLymers from Algae Sugars and Hydrocarbons

4
Starting date
The first day of the month after the signature by the Commission
5
Duration in months Call (part) identifier
48 FP7-KBBE-2012-6-singlestage KBBE.2012.3.4-02: Biotechnology for novel biopolymers green algae, polymer, biopolymer, green naphtha, hydrocarbon, carbohydrate, Botryococcus braunii, Chlamydonomas reinhardtii, base chemicals, polyolefins, polyester, metabolic engineering Abstract
9
Activity code(s) most 7 relevant to your topic
Free keywords
The 4-year SPLASH project will develop a new biobased industrial platform using microalgae as a renewable raw material for the sustainable production and recovery of hydrocarbons and (exo)polysaccharides from the species Botryococcus braunii and further conversion to renewable polymers. The project comprises 20 partners of which 40% SME and several large corporates plus universities and research institutes. Two bioproduction platforms will be explored: (1) green alga Botryococcus braunii on its own and (2) the green microalga Chlamydomonas reinhardtii, to which the unique hydrocarbon and polysaccharides producing genes from Botryococcus will be transferred. SPLASH will deliver knowledge, tools and technologies needed for the establishment of a new industry sector: Industrial Biotechnology with algae and/or algal genes for the manufacture of polyesters and polyolefins. The building blocks for these polymers will be derived from the sugars (polyesters) and hydrocarbons (polyolefins) exuded by the algae: adipic acid from galactose, 2,5-furandicarboxylic acid from glucose, rhamnose and fucose, 1,4-pentanediol from rhamnose and fucose, ethylene from green naphtha, propylene from green naphtha. The conversion of ethylene and propylene to polyolefins is common technology, and will not be included in the project. The sugar-derived building blocks will be converted to new condensation polymers, including poly(ethylene 2,5-furandioate) (PEF) and poly(1,4-pentylene adipate-co-2,5-furandioate). End-use applications include food packaging materials and fibres for yarns, ropes and nets. The project encompasses (1) development of Botryococcus as an industrial production platform, (2) Systems biology analysis, (3) Development of procedures for production, in situ extraction and isolation, (4) product development.
311956 SPLASH - Part A - - Page 3 of 6
List of Beneficiaries
Project Number
1
A2:
311956
Project Acronym
SPLASH
List of Beneficiaries No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Name STICHTING DIENST LANDBOUWKUNDIG ONDERZOEK CENTRE FOR RESEARCH AND TECHNOLOGY HELLAS ORGANIC WASTE SYSTEMS NV PAQUES BV NIELS-HENRIK NORSKER VALUE FOR TECHNOLOGY BVBA AVANTIUM CHEMICALS BV LIFEGLIMMER GMBH PURSUIT DYNAMICS PLC NOVA-INSTITUT FUR POLITISCHE UND OKOLOGISCHE INNOVATION GMBH FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE PNO CONSULTANTS BV UNIVERSIDAD DE HUELVA WAGENINGEN UNIVERSITEIT UNIVERSITAET BIELEFELD WESTFAELISCHE WILHELMS-UNIVERSITAET MUENSTER EGE UNIVERSITESI LANKHORST EURONETE PORTUGAL SA Rhodia Operations Short name DLO CERTH OWS PAQ BIT VFT AVT LG PDX NOVA FRAUNHOFER UCAM PNO UHU WU UNIBI UMUE EGE LEP RHO 311956 SPLASH - Part A - - Page 4 of 6 Country Netherlands Greece Belgium Netherlands Denmark Belgium Netherlands Germany United Kingdom Germany Germany United Kingdom Netherlands Spain Netherlands Germany Germany Turkey Portugal France Project entry 10 month 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Project exit month 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48
Budget Breakdown
Project Number
1
A3:
311956
Project Acronym
SPLASH
One Form per Project Participant number in this 11 project 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total Estimated eligible costs (whole duration of the project) Participant short name DLO CERTH OWS PAQ BIT VFT AVT LG PDX NOVA FRAUNHOFER UCAM PNO UHU WU UNIBI UMUE EGE LEP RHO Fund. 12 % 75.0 75.0 75.0 50.0 75.0 75.0 75.0 75.0 75.0 75.0 75.0 75.0 50.0 75.0 75.0 75.0 75.0 75.0 50.0 50.0 Ind. costs
13
RTD / Innovation (A) 2,071,595.00 199,405.00 173,407.00 560,697.00 533,120.00 138,420.80 581,035.20 1,057,598.40 993,321.60 0.00 144,002.00 618,704.00 0.00 426,763.20 1,054,957.00 617,851.20 595,428.80 106,480.00 19,107.00 145,328.40 10,037,221.60
Demonstration (B) 28,337.00 0.00 0.00 314,706.00 0.00 11,200.00 288,292.80 0.00 30,733.20 0.00 32,556.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3,894.00 231,096.00 940,815.00
Management (C) 373,307.00 0.00 0.00 5,000.00 5,000.00 0.00 5,000.00 10,000.00 10,000.00 5,000.00 0.00 5,000.00 0.00 0.00 10,000.00 5,000.00 5,000.00 0.00 0.00 0.00 438,307.00
Other (D) 17,984.00 0.00 20,567.00 28,701.00 0.00 33,600.00 27,734.40 62,889.60 38,538.00 374,400.00 0.00 0.00 83,229.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Total A+B+C+D 2,491,223.00 199,405.00 193,974.00 909,104.00 538,120.00 183,220.80 902,062.40 1,130,488.00 1,072,592.80 379,400.00 176,558.00 623,704.00 83,229.00 426,763.20 1,064,957.00 622,851.20 600,428.80 106,480.00 23,001.00 376,424.40
Total receipts 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Requested EU contribution 1,959,154.00 149,553.00 150,622.00 471,402.00 404,840.00 143,015.00 612,657.00 866,088.00 808,895.00 379,400.00 124,279.00 469,028.00 83,229.00 320,072.00 801,217.00 468,338.00 451,571.00 79,860.00 11,500.00 188,212.00 8,942,932.00
A S A A T T T T F T A T A T A T T T A F
687,643.00 12,103,986.60
Budget Breakdown
Note that the budget mentioned in this table is the total budget requested by the Beneficiary and associated Third Parties.
A3:
* The following funding schemes are distinguished Collaborative Project (if a distinction is made in the call please state which type of Collaborative project is referred to: (i) Small of medium-scale focused research project, (ii) Large-scale integrating project, (iii) Project targeted to special groups such as SMEs and other smaller actors), Network of Excellence, Coordination Action, Support Action. 1. Project number The project number has been assigned by the Commission as the unique identifier for your project, and it cannot be changed. The project number should appear on each page of the grant agreement preparation documents to prevent errors during its handling. 2. Project acronym Use the project acronym as indicated in the submitted proposal. It cannot be changed, unless agreed during the negotiations. The same acronym should appear on each page of the grant agreement preparation documents to prevent errors during its handling. 3. Project title Use the title (preferably no longer than 200 characters) as indicated in the submitted proposal. Minor corrections are possible if agreed during the preparation of the grant agreement. 4. Starting date Unless a specific (fixed) starting date is duly justified and agreed upon during the preparation of the Grant Agreement, the project will start on the first day of the month following the entry info force of the Grant Agreement (NB : entry into force = signature by the Commission). Please note that if a fixed starting date is used, you will be required to provide a detailed justification on a separate note. 5. Duration Insert the duration of the project in full months. 6. Call (part) identifier The Call (part) identifier is the reference number given in the call or part of the call you were addressing, as indicated in the publication of the call in the Official Journal of the European Union. You have to use the identifier given by the Commission in the letter inviting to prepare the grant agreement. 7. Activity code Select the activity code from the drop-down menu. 8. Free keywords Use the free keywords from your original proposal; changes and additions are possible. 9. Abstract 10. The month at which the participant joined the consortium, month 1 marking the start date of the project, and all other start dates being relative to this start date. 11. The number allocated by the Consortium to the participant for this project. 12. Include the funding % for RTD/Innovation either 50% or 75% 13. Indirect cost model A: Actual Costs S: Actual Costs Simplified Method T: Transitional Flat rate F :Flat Rate
Workplan Tables
Project number
311956
Project title
SPLASHSustainable PoLymers from Algae Sugars and Hydrocarbons

Call (part) identifier
FP7-KBBE-2012-6-singlestage
Funding scheme
Collaborative project
List of work packages

Project Number
1
WT1
311956
Project Acronym
SPLASH
LIST OF WORK PACKAGES (WP) WP Number

53
WP Title Project Management From systems biology to strain engineering Process design: production & downstream processing Product development and testing Process demonstration at pilot scale Process integration: Sustainability assessment and market analysis Dissemination, exploitation and intellectual property management
Type of 54 activity MGT RTD RTD RTD DEM RTD OTHER
Lead beneficiary 55 number 1 12 1 7 9 6 10 Total
Person56 months 27.30 392.80 360.10 98.90 47.20 77.40 61.40 1,065.10
Start month
57
End month
58
WP 1 WP 2 WP 3 WP 4 WP 5 WP 6 WP 7
1 1 1 6 24 1 1
48 48 48 48 48 48 48
311956 SPLASH - Workplan table - - Page 1 of 49
List of Deliverables
Project Number
1
WT2:
311956
Project Acronym
SPLASH
List of Deliverables - to be submitted for review to EC Deliverable Number

61
Deliverable Title Organisation kick-off meeting Installation of advisory boards Periodic report Periodic report Final report Risk Register Audit certificates and bank guarantees Mid term internal review 6- month management report 6- month management report 6- month management report 6-month management report 6-month management report 6-month management report 6-month management report 6-month management report General Assembly Meeting
Estimated WP Lead benefiindicative number ciary number person53 months 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Nature
62
Dissemination level
63
Delivery date
64
D1.1 D1.2 D1.3 D1.4 D1.5 D1.6 D1.7 D1.8 D1.9
0.50 O 1.00 R 2.00 R 2.00 R 4.80 O 2.00 O 1.00 O 2.00 R 0.50 R
CO CO CO CO CO CO CO CO CO
1 6 12 30 48 48 48 30 6
D1.10
0.50 R
CO
12
D1.11
0.50 R
CO
18
D1.12
0.50 R
CO
24
D1.13
0.50 R
CO
30
D1.14
0.50 R
CO
36
D1.15
0.50 R
CO
42
D1.16
0.50 R
CO
48
D1.17
1.00 O
CO
12
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title General Assembly Meeting General Assembly Meeting General Assembly Meeting General Assembly Meeting Plan for the use and dissemination of foreground Plan for the use and dissemination of foreground Annotated genome and transcriptome of B. braunii Measurements of transcripts, proteome and metabolite profiles in B. braunii obtained under different Systems model on regulatory mechanisms for growth and hydrocarbon production Update Systems model on regulatory mechanisms for growth and hydrocarbon production Update Systems model on regulatory
Estimated WP Lead benefiindicative number ciary number person53 months 1 1
Nature
62
Dissemination level
63
D1.18
1.00 O
CO
22
D1.19
1.00 O
CO
30
D1.20
1.00 O
CO
40
D1.21
2.00 O
CO
48
D1.22
1.00 O
CO
30
D1.23
1.00 O
CO
48
D2.1
49.00 O
CO
18
D2.2
16
81.20 R
CO
30
D2.3
76.00 O
CO
24
D2.4
19.00 R
CO
30
D2.5
13.60 R
CO
48
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title mechanisms for growth and hydrocarbon production Mutagenised library of B. braunii and characterization of mutants Verification of a selected number of candidate genes in C. reinhardtii Establishment of molecular tools for metabolic engineering in C. reinhardtii Optimized growth and production of hydrocarbons and polysaccharides Optimised outdoor process Products for WP4 as needed Process for continuous product recovery of hydrocarbons and polysaccharides from Botryococcus Concept for industrial scale biomass production and product recovery Biosafety Analytics of lipid fraction produced by B. braunii
Estimated WP Lead benefiindicative number ciary number person53 months
Nature
62
Dissemination level
63
D2.6
10.00 R
CO
30
D2.7
12
67.00 R
CO
30
D2.8
12
77.00 R
CO
48
D3.1
15
80.00 R
CO
30
D3.2 D3.3
3 3
14 5
116.30 R 36.00 O
CO CO
48 48
D3.4
102.20 R
CO
48
D3.5
19.60 O
CO
48
D3.6 D4.1
3 4
1 11
6.00 R 8.00 R
CO CO
30 30
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title Cracking process to convert hydrocarbons to green naphtha and further to ethylene and propylene Hydrolysis of algae polysaccharides into monomeric sugars Separation of monomeric sugar mixture into individual sugar fractions Conversion of galactose to adipic acid Conversion of fucose and rhamnose to 1,4-pentanediol Conversion of glucose to 2,5-FDCA Conversion of adipic acid and 1,4-pentanediol into polyesters and polyamides Conversion of 2,5-FDCA to PEF films Report on suitability of polyesters for the production of fibers Validation of experimental data resulting from process optimisation
Nature
62
Dissemination level
63
D4.2
11
4.20 R
CO
36
D4.3
5.00 R
CO
24
D4.4
7.00 R
CO
30
D4.5
17.30 R
CO
30
D4.6
12.00 R
CO
30
D4.7
25.00 R
CO
36
D4.8
20
9.00 R
CO
36
D4.9
10.00 R
CO
48
D4.10
19
1.40 R
CO
48
D5.1
17.90 D
CO
44
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title Conversion of hydrocarbons and sugars into biopolymers
Nature
62
Dissemination level
63
D5.2
21.80 D
CO
42
D5.3
Technology, scale up and 5 product commercialization Conceptual design and integrated processes Update conceptual design and integrated processes Update conceptual design and integrated processes Technoeconomic model and sensitivity analysis Update technoeconomic model and sensitivity analysis Report on cradle-to-factorygate LCA Update report on cradle-to-factorygate LCA Update report on cradle-to-factorygate LCA Report on economic assessment and market analysis Report on standardisation Report on social aspects 6
7.50 D
CO
48
D6.1
13.60 R
CO
12
D6.2
3.90 R
CO
30
D6.3
2.90 R
CO
48
D6.4
28.90 R
CO
30
D6.5
3.90 R
CO
48
D6.6
4.90 R
CO
24
D6.7
5.00 R
CO
30
D6.8
5.00 R
CO
48
D6.9
4.30 R
CO
42
D6.10 D6.11
6 6
6 6
2.00 R 3.00 R
CO CO
48 42
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title Communication and dissemination plan Scientific state of the art and IP positions Update on scientific state of the art and IP positions Update on scientific state of the art and IP positions Public website and newsletter, first press release on the project scopes Project leaflet on several languages Update project leaflet on several languages Project brochure Update project brochure Concept for an International conference on industrial microalgae biotechnology Exploitation business plan Update exploitation business plan Pre-market stimulating activities Concept for summer school
Nature
62
Dissemination level
63
D7.1
5.50 O
CO
D7.2
10
4.30 R
CO
12
D7.3
10
2.60 O
CO
30
D7.4
10
1.10 O
CO
48
D7.5
10
8.00 O
PU
D7.6
10
3.50 O
PU
12
D7.7 D7.8 D7.9
7 7 7
10 10 10
2.40 O 3.50 O 1.50 O
PU PU PU
30 30 48
D7.10
10
5.00 O
PU
12
D7.11 D7.12
7 7
10 10
9.00 O 7.00 O
CO CO
30 48
D7.13 D7.14
7 7
10 10
3.00 O 3.00 O
PU PU
42 24
Deliverable Number
61
WT2:
Delivery date
64
Deliverable Title Report on compliance of the SPLASH concept with stakeholders requirements
Nature
62
Dissemination level
63
D7.15
10
2.00 R
CO
48
Total
1,065.10
Work package description

Project Number
1
WT3:
311956
Project Acronym
SPLASH
One form per Work Package Work package number Work package title Start month End month Lead beneficiary number
55 53
WP1
Type of activity 1 48 1
54
MGT
Project Management
Objectives Integral Management of the Project, enabling and stimulating efficient exchange of information and collaboration. Specific objectives Ensuring proper implementation of Work Plan and achievement of SPLASH objectives. Efficient organisation and day-to-day management of the project Preparing the Consortium Agreement Monitoring and ensuring on-time delivery of scientific and financial reports Establishing effective communication with the Commission Ensuring proper interaction between collaborating partners Organisation of project meetings Stimulate / facilitate dissemination of results A special relation will be formed with WP7 (Dissemination & Exploitation). The Dissemination and Exploitation Officer (DEO) is leader of WP6, but with additional responsibilities described below in WP1. Description of work and role of partners Description of work The Overall project Coordinator will fulfil all Tasks as required by the EC and as described in more detail in Chapter 2. Implementation and Paragraph 2.1. PNO will support the coordinators project office by carrying out periodic checks on the produced financial and administrative reports (task 1.3) before they are sent to the Commission. The management tasks can be summarized as follows: Task 1.1 Primary contact Point with the EC and the Scientific Officer and stream-lining of nformation from and to Partners Task 1.2 Scientific project management; compile progress reports, monitoring progress and strategy Task 1.3 Financial and administrative project management including reporting and monitoring Task 1.4 Coordination of legal issues and risk management Task 1.5 Organization of project meetings, including a bilateral meeting with the WP7 (Dissemination & Exploitation) leader, organization of Plenary Kick Off and half-yearly Progress Meetings of the Project Management Team (PMT) and the yearly meetings of the General Assembly (GA) Task 1.6 Coordination activities: communication (i.e. organization of Agendas, Minutes and Action Lists), knowledge management and gender issues Task 1.7 Enhancement of implementation / dissemination; will be done in tight cooperation with WP6 (DEO) and WP7 and includes dissemination, demonstration, training and education Task 1.8

Coordination of the Involvement of the two advisory boards; Scientific Advisory Board (SAB) and the Dissemination and Exploitation Advisory Board (DEIPAB). Task 1.9 Setting up a dedicated Website environment. Person-Months per Participant Participant number
10
WT3:
Participant short name 1 DLO
11
Person-months per participant 27.30 Total 27.30
List of deliverables Deliverable Number

61
Deliverable Title Organisation kick-off meeting Installation of advisory boards Periodic report Periodic report Final report Risk Register Audit certificates and bank guarantees Mid term internal review 6- month management report 6- month management report 6- month management report 6-month management report 6-month management report 6-month management report 6-month management report 6-month management report General Assembly Meeting General Assembly Meeting General Assembly Meeting General Assembly Meeting General Assembly Meeting Plan for the use and dissemination of foreground
Lead beneficiary number 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Estimated indicative personmonths
Nature
62
Dissemination 63 level CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO
Delivery date
64
D1.1 D1.2 D1.3 D1.4 D1.5 D1.6 D1.7 D1.8 D1.9 D1.10 D1.11 D1.12 D1.13 D1.14 D1.15 D1.16 D1.17 D1.18 D1.19 D1.20 D1.21 D1.22
0.50 O 1.00 R 2.00 R 2.00 R 4.80 O 2.00 O 1.00 O 2.00 R 0.50 R 0.50 R 0.50 R 0.50 R 0.50 R 0.50 R 0.50 R 0.50 R 1.00 O 1.00 O 1.00 O 1.00 O 2.00 O 1.00 O
1 6 12 30 48 48 48 30 6 12 18 24 30 36 42 48 12 22 30 40 48 30

61
WT3:
Deliverable Title Plan for the use and dissemination of foreground
Lead beneficiary number 1 Total
Nature
62
Dissemination 63 level CO
Delivery date
64
D1.23
1.00 O 27.30
48
Description of deliverables D1.1) Organisation kick-off meeting: First Meeting with the entire consoritum,both scientific and financial responsibles of each partner will be invited to join [month 1] D1.2) Installation of advisory boards: Set up of Advisory Boards, choice of members, invitations and specific description of tasls [month 6] D1.3) Periodic report: [month 12] D1.4) Periodic report: [month 30] D1.5) Final report: [month 48] D1.6) Risk Register: Risk Register: Development and maintenance of project Risk Register. Risk Register to be updated at months 6, 12, 18, 24, 30, 36, 42 & 48. [month 48] D1.7) Audit certificates and bank guarantees: Audit Certificates and Bank Guarantees: Provision of Audit Certificates and Bank Guarantees as required. [month 48] D1.8) Mid term internal review: Internal Mid-Term review will be organised with Scientific Advisory Board (SAB) in order to get its input [month 30] D1.9) 6- month management report: [month 6] D1.10) 6- month management report: [month 12] D1.11) 6- month management report: [month 18] D1.12) 6-month management report: [month 24] D1.13) 6-month management report: [month 30] D1.14) 6-month management report: [month 36] D1.15) 6-month management report: [month 42] D1.16) 6-month management report: [month 48] D1.17) General Assembly Meeting: [month 12] D1.18) General Assembly Meeting: [month 22] D1.19) General Assembly Meeting: [month 30] D1.20) General Assembly Meeting: [month 40] D1.21) General Assembly Meeting: [month 48] D1.22) Plan for the use and dissemination of foreground: [month 30] D1.23) Plan for the use and dissemination of foreground: [month 48]

Schedule of relevant Milestones Milestone 59 number MS1 MS2 Lead beneficiary number 1 1 Delivery date from 60 Annex I
WT3:
Milestone name Kick-off Meeting Robust project management
Comments 1 Report
48
Contractual deliverables met on time and in full

Project Number
1
WT3:
311956
Project Acronym
SPLASH
55 53
WP2
54
RTD
From systems biology to strain engineering
Objectives The main objective of this WP is to use genomics and systems biology to develop metabolic engineering strategies for hydrocarbon and (exo)polysaccharide production in green algae, thus providing leads for cultivation concepts, improved growth and specific product enhancement that will be exploited in the other WPs. Specific objectives Use a natural hydrocarbon producing alga, B. braunii as a gene source for hydrocarbon biosynthetic genes, as well as those for cell wall carbohydrates Use omics technology together with systems biology modelling to identify the most likely candidate genes to generate strains with altered or enhanced levels of hydrocarbon and/or exopolysaccharide products for downstream processing Use C. reinhardtii strains to verify B. braunii candidate gene products Optimise metabolic engineering strategies for production of lines of C. reinhardtii overproducing one or more of the selected products Optimise metabolic engineering strategies for C. reinhardtii and other algae to facilitate overproduction one or more of the selected products Develop a random mutagenesis and selection platform to improve growth and production characteristics of B. braunii. Description of work and role of partners There will be three strands to WP2: 1. We will sequence the genome of B. braunii race A, and by combination of transcriptomics, proteomics and metabolomics, together with metabolic modelling, construct metabolic maps for the production of hydrocarbons and cell wall polysaccharides. Tasks 2.1 2.3 2. We will verify the identity and function of candidate genes by expression in C. reinhardtii, a unicellular green alga like B. braunii. This will lead to the development of expression platforms for production of the desired products. Optimization of these production lines will be by cross-referencing to the metabolic modelling results from B. braunii. Tasks 2.4 2.5 3. At the same time, we will explore the potential to use B. braunii itself for production of the desired compounds by taking a random mutagenesis approach, and by expressing one or more of the candidate genes in other more industrially-relevant algae, such as Nannochloropsis oculata. Part of Task 2.5 Task 2.1 Reconstruction of the genome sequence, annotation and comparative genomics of B braunii (PRI, UMUE) Genome sequencing is of paramount importance for the understanding of algal biology, and to facilitate exploitation of the biotechnological potential of microalgae. Moreover, the availability of algal genome sequences allows genome-wide comparisons to identify genes of interest, and their genetic context, and it is the prerequisite for the onset of systems biology approaches, including transcriptomics, proteomics and metabolomics. Sequencing of the complete genome of B. braunii will be performed using the latest next generation sequencing technology (454GSflx, Illumina/Solexa). For annotation of genome and EST sequences an automated custom designed annotation pipeline (Cyrille 2) will be used. Subsequently, assigned (putative) functions will be categorized taking into account (putative) metabolic and cellular processes involved in hydrocarbon and polysaccharide biosynthesis and abiotic stress response, using functional categories from NCBI and TAIR.

Furthermore, we will use genomics data from Task 2.2 to enhance functional annotation and identify enzymes to uncharacterized metabolic reactions. Ab initio predicted gene models will be produced using tools such as AUGUSTUS and GenScan, and RNA Seq data will be matched against the genome sequence with BLAT, SIM4, Geneseqer and Genome Threader for open reading frame (ORF) validation, allowing determination of B. braunii gene topology at high resolution. For inclusion of peptide information derived from mass spectrometry we will take advantage of automated proteogenomic annotation pipeline employing GPF and AUGUSTUS. We will use functional annotation using BLASTX based similarity searches to find putative gene functions, and gene sequences that might be implicated or associated with hydrocarbon biosynthesis, such as homologues to the recently identified alkane biosynthesis genes in cyanobacteria , as well as likely candidate glycosyl transferases involved in cell wall polysaccharide biosynthesis. To identify the gene regulatory elements we will align clustered RNA Seq data to genomic sequences, and predicted gene models will be used to identify the putative start site. Upstream sequences will then be extracted and analysed for cis-regulatory motifs using homology based searches and sequence motif identification tools like MEME . Furthermore, we will screen these sequences against the promoter databases such as Transfac. We will then attempt to correlate cis-regulatory transcription-factor-binding motifs with the corresponding gene-expression patterns from Subtask 2.2.1 that are obtained under different environmental conditions. Task 2.2 Analysis of metabolic pathway regulation in B. braunii (PRI, UNIBI, LG, UMUE) To gain insight in the expression of key genes involved in the hydrocarbon and polysaccharides biosynthesis and excretion, and to facilitate assignment of gene function in Task 2.1, we will investigate changes in transcripts, proteins, and metabolite levels in B. braunii grown under different conditions. Accordingly, microalgal cultures will be grown in fully controlled photobioreactor systems as described in WP3. We will expose the cultures to a range of abiotic stresses known to influence hydrocarbon and polysaccharide biosynthesis: for instance low nitrogen leads to a reduction in hydrocarbon biosynthesis, whereas CO2 supplementation enhances levels . Samples will be collected at carefully chosen time points in both exponential growth phase and in static phase, and subsequently analysed for their transcriptomic, proteomic and metabolomic profiles as described below. The bioreactors allow full control of light intensity and temperature (mixing, optical density, dilution rate, pH, nitrogen and carbon dioxide supply and are equipped with on-line gas analysis to monitor carbon dioxide and oxygen exchange. This ensures stable and reproducible cultivation conditions. Subtask 2.2.1 Gene expression in B. braunii (PRI, UNIBI) We will perform complete transcriptome analysis using next generation deep sequencing by Illumina RNA-Seq. Based on frequency coverage, information about the gene-expression levels for each of the mRNAs can be deduced. In addition, RNA-Seq reads can be used to construct single-base resolution gene-models at a genomic scale. We will use the transcriptomics facility of CeBiTec (Center for Biotechnology at Bielefeld University) with up-to-date microarray processing and analysis equipment (Tecan, Agilent). This - and the connection with the exceptional bioinformatics hardware and software platform for storage, computation and presentation of genome and transcriptome data - represent unique features of the CebiTec. Subtask 2.2.2 Proteomics of B. braunii (LG, UMUE) Quantitative proteomic profiles will enable changes in enzyme composition between samples to be detected down to ratios of 2-fold differences. It also provides information such as posttranslational modification that is not revealed from transcriptomics. Furthermore, subcellular fractionation, like isolation of the outer cell wall or oil droplets, will provide dedicated information on local enzyme composition. We will use a high resolution HPLC-FTMS system as recently established for the green alga C. reinhardtii . This state-of-the-art technology enables quantitative comparison of several thousands of peptides between a large number of samples. In addition, we will employ metabolic labelling with isotopically labelled and unlabelled nitrogen (15N/14N). Identification of the peptides is performed by matching the MS/MS (or MSe) signals with the genome sequence information, and vice versa, the peptide signals will vice versa provide further proof for the building of gene models in Task 2.1. To facilitate verification of gene models and exon and intron boundaries we will map the amino acid sequences to the genome using PepSplice and GPF in conjunction with AUGUSTUS. Subtask 2.2.3 Metabolic profiling of B. braunii (UNIBI, LG) Metabolite profiling will enable assessment of the consequences of alteration of gene expression under different growth conditions, and also in the metabolically engineered algal and yeast strains constructed in later tasks. We will use NMR, GC-MS, LC-ESI -TOF-MS/MS techniques and two-dimensional GCxGC-TOF-MS to enhance the number of identified metabolites. For the analysis of metabolites incompatible with GC-MS we will use a coupling
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of liquid chromatography and mass spectrometry (LC-MS) as a complementary method . The information on quantity and quality can then be used in WPs 3, 4, 5 and 6 for product valorization purposes. Task 2.3 Data analysis, reverse engineering and systems models of hydrocarbon and polysaccharide biosynthesis in B. braunii (LG, PRI, WU) The overall objective of this task is to link differences in metabolome and proteome profiles to differences in genetic background, transcripts, growth conditions and biomass production, and thereby provide a large scale view of the cellular biochemical processes underlying hydrocarbon and polysaccharide biosynthesis. Such an integrated approach provides the means to reveal relationships between genes, transcripts, enzymes and metabolites that may not be revealed by classical approaches. We will use data from Task 2.2 as input for the modelling, both for natural strains of B. braunii, and for the modified yeast and algal (both C. reinhardtii and B. braunii) strains. For inference of underlying regulatory networks, we will apply statistical and multivariate data analysis tools such as k-means, self-organising maps, Network- and Principal Component Analysis, and Multi-Dimensional Scaling, using existing routines in R and MATLAB. Subsequently, we will apply pair wise transcript-protein and transcript-metabolite correlation analysis to determine correlation between transcript and protein levels, and transcript and metabolite levels. To determine whether the data obtained are in agreement with the observations made using different experimental conditions, we will focus in on (putative) functions that have been implicated in biosynthesis of the compounds of interest, such as acetyl-CoA carboxylase or glycerol-3P acyltransferase for hydrocarbon production, or glycosyl transferases for cell wall polysaccharide biosynthesis. We will establish whether they show either positive or negative correlations with expression, protein and metabolite levels. The information obtained will be integrated into a metabolic pathway model in order to detect rate-limiting steps and identify possibilities for yield optimisation. Reverse engineering approaches based on Boolean-Probabilistic Boolean logic, Multiple Regression Models and other expression-based network inference methods, which capture probabilistic multivariate statistical dependencies between the various cellular factors in these networks, will generate hypotheses for underlying circuitry . Having identified these in natural strains of B. braunii, they will be used to develop metabolic engineering strategies for C. reinhardtii. Analysis of these modified strains will then provide the means to verify, refine or dismiss the models produced. We will also use (meta-)genomic, thermodynamic, biochemical and strain-specific information to generate genome-scale constraint-based models of the metabolism and transport B. braunii as done previously for other organisms, drawing on our recently developed computational workflow for data handling and model generation (TOBIN, and Microme.eu) . Experimental validation of the model will be done through experiments in specialized photobioreactors as described above in WP3. Through various techniques (such as Flux Balance, Flux Variability and Extreme Pathways Analysis) and by integrating these with the omics analyses and reverse engineering procedures, the constraint-based models will help in predicting the specific cellular phenotypes under the particular environmental conditions to be tested, will define the organisms global metabolic space, network structural properties and flux distribution potential, enabling to study the consequences of alterations in the genotype, to gain insights into the phenotype-genotype relation, to generate testable hypotheses and to rationally design experiments. Task 2.4 Verification of candidate genes for hydrocarbon and polysaccharide pathways from B. braunii (UCAM, UNIBI, UMUE) This task is aimed at establishing the function of candidate genes identified in Tasks 2.1 and 2.2, and also to test predictions from the modelling in Task 2.3. It will be highly dependent on the genome information from Task 2.1, and the product-testing that is the major focus of WP4. The outcomes will enable evaluation of different strategies for metabolic engineering, with emphasis on generating strains with a reduced complexity of hydrocarbons, to facilitate downstream processing and avoid unnecessary waste streams. Candidate genes will be expressed in a heterologous host to enable their function to be tested in a clean background. We will use C. reinhardtii for these experiments because each offers particular advantages. It is important to characterize these candidate genes in a photosynthetic context. Moreover, the fact that C. reinhardtii is unicellular green alga like B. braunii will offer insights in regard to product yield and downstream processing issues that will be addressed in WP3. Subtask 2.4.2 Testing candidate genes in C. reinhardtii (UCAM, UNIBI, UMUE) Codon-optimised synthetic genes will be cloned into a C. reinhardtii expression vector under control of the strong promoter PSAD. The proteins will be expressed as HA-fusion proteins to facilitate detection of expressed protein. Targeting sequences such as for the chloroplast or endomembrane system will be included, although if incorrect targeting was observed, then they would be substituted by endogenous C. reinhardtii targeting signals.
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Enzyme activity will be assessed in cell-free extracts as above. If necessary, an inducible promoter system, such as the Cu-inducible promoter , or thiamine-pyrophosphate riboswitch , will be used to avoid problems of constitutive expression if the enzymes should prove toxic. Task 2.5 Development of a green microalgal platform for hydrocarbon and polysaccharide production (UCAM, UNIBI, UMUE, DLO-FBR) We want to develop photosynthetic microalgae as expression platforms because they can harness solar energy, so there is no need for a supply of fixed carbon. Moreover, since the processing methodology in WP3 is aimed at optimizing production in B. braunii, it will be important for the integration of the project to explore the potential of improving production of the hydrocarbons and sugars in this organism. At the same time, the gene discovery in the earlier tasks will facilitate expression in other microalgal hosts that are more readily manipulated genetically Subtask 2.5.1 Production of hydrocarbons and polysaccharides in C. reinhardtii (UCAM, UMUE, UNIBI) We will attempt to reconstitute the complete hydrocarbon pathway in C. reinhardtii, using data from Tasks 2.4 to inform our strategy. It should be possible to express 2-4 genes in the nucleus of this alga, but this is limited by the number of different promoter and 3 controlling elements available, since the presence of multiple copies of a sequence can lead to silencing. Moreover, because transformation results in random insertion into the nuclear genome it is necessary to screen large numbers of transformants to find one with high level expression of the transgene. Fortunately there is an excellent genetic system in this alga, and so for each gene we want to express, we will generate single transformants, and then combine the transgenes by crossing. In addition, we will also explore the possibility of expressing one or more of the proteins in the chloroplast, where homologous recombination means stable and predictable levels of expression. Depending on the success with hydrocarbon pathway reconstruction, we will explore avenues to produce polysaccharides in C. reinhardtii. One potential difficulty with this is that the cell wall of C. reinhardtii is proteinaceous with little or no carbohydrate. However, we will test the efficacy of expressing candidate glycosyl transferases, and test for the presence of the desired hemicellulosic compounds using commercially available antibodies. Subtask 2.5.2 Molecular tools for metabolic engineering in microalgae (UCAM, UNIBI) Robust and reproducible metabolic engineering of microalgal strains requires the further optimisation of existing transformation technologies to improve efficiencies, allow the targeted integration of transgenes and facilitate the introduction of entire metabolic pathways. We will explore the following approaches for C. reinhardtii metabolic engineering in the first instance, but they can also be applied to B. braunii if we are successful in developing a robust transformation methodology in Subtask 2.6.4. Advanced transformation methodologies will be investigated to assist the integration of transgenes and increase transformation efficiencies, including the use of integrases, activated transposon complexes and I-Sce1 restriction enzyme mediated integration. Meganuclease (Cellectis) site specific integration or similar technology will be evaluated for its ability to generate B. braunii lines that reproducibly integrate transgenes at a defined locus with no fitness cost or variability in expression. At Cambridge, a project is about to commence to develop modular gene expression components including promoters (constitutive and inducible), introns, codon optimized genes, epitope tags and terminators. It will follow the BioBrick model (http://bbf.openwetware.org/) to allow rapid and interchangeable assembly of expression vectors, thus providing a high throughput research tool for the combinatorial analysis of candidate genes and regulatory elements. Once the parts (called PhycoBricks) come on stream, they will be available for use to optimize expression of the key candidate genes in Subtask 2.6.1. The same approach will be used to design appropriate expression systems for B. braunii, once transformation methodology is established in subtask 2.6.4. Subtask 2.5.3 Breeding tools for B. braunii (DLO-FBR) This task aims to develop a random mutagenesis and selection platform to improve growth and production characteristics of B. braunii. Optimized methods will be developed using chemical (e.g. NTG or EMS) or UV mutagenesis in order to obtain libraries of mutants from selected B. braunii strains. Aspects that will be taken into account are the lethal and mutagenic action of the various mutagens and the influence of repair phenomena. Mutant B. braunii libraries will then be subjected to screening procedures, which will be optimized to select for improved growth characteristics and/or improved productivity. Robotics connected to analysis equipment will be used to develop methods to screen a large number of mutants. Improved growth characteristics can for instance be monitored by absorbance measurements in time and improved productivity by specific staining techniques (e.g. in situ lipid staining by Nile red) or by other analysis techniques.
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Selected mutant B. braunii strains will further be analysed under standard growth conditions to confirm the effect and stability of the mutation. Task 2.5.4. Towards the development of an industrially-relevant microalgal expression platform for production of novel hydrocarbons and sugars (UCAM, UMUE, UNIBI) The development of transformation systems for microalgae is expanding all the time, and there are now three species, in addition to C. reinhardtii, for which routine expression of transgenes has become established and for which complete genome sequences are available. Phaeodactylum tricornutum, a marine diatom, is increasingly being considered as an expression platform for the introduction of novel pathways, since transformation is straightforward, and transgenes are stably expressed. Vectors and promoters for transformation are available, and RNA-silencing to reduce levels of endogenous genes has been demonstrated, providing the means to reduce competing pathways. Ostreococcus tauri is a marine picoeukaryote in the Chlorophyta, and thus in the same phylum as B. braunii. Vectors for transformation are available, and homologous recombination has been observed, opening the possibility of targeted knockout and knock ins for fine tuning of expression. Nannochloropsis oculata is a heterokont, and also exhibits homologous recombination. In particular this species is well established as a commercial algal production platform. This subtask will investigate expression of key candidate B braunii genes and a comparison made for stable expression, and product formation between these three possible microalgal platforms. Further refinement of the metabolic engineering will be by reference to the systems modelling in the earlier tasks. Person-Months per Participant Participant number
10
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Participant short name 1 DLO 8 LG 12 UCAM 15 WU 16 UNIBI 17 UMUE
11
Person-months per participant 62.00 89.00 85.00 45.60 65.00 46.20 Total 392.80

61
Deliverable Title Annotated genome and transcriptome of B. braunii Measurements of transcripts, proteome and metabolite profiles in B. braunii obtained under different Systems model on regulatory mechanisms for growth and hydrocarbon production Update Systems model on regulatory mechanisms for growth and hydrocarbon production
Lead beneficiary number 1 16
Nature
62
Dissemination 63 level CO CO
Delivery date
64
D2.1 D2.2
49.00 O 81.20 R
18 30
D2.3
76.00 O
CO
24
D2.4
19.00 R
CO
30

61
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Deliverable Title Update Systems model on regulatory mechanisms for growth and hydrocarbon production Mutagenised library of B. braunii and characterization of mutants Verification of a selected number of candidate genes in C. reinhardtii Establishment of molecular tools for metabolic engineering in C. reinhardtii
Lead beneficiary number 8 1 12 12 Total
Nature
62
Dissemination 63 level CO CO CO CO
Delivery date
64
D2.5 D2.6 D2.7 D2.8
13.60 R 10.00 R 67.00 R 77.00 R 392.80
48 30 30 48
Description of deliverables D2.1) Annotated genome and transcriptome of B. braunii: [month 18] D2.2) Measurements of transcripts, proteome and metabolite profiles in B. braunii obtained under different: [month 30] D2.3) Systems model on regulatory mechanisms for growth and hydrocarbon production: [month 24] D2.4) Update Systems model on regulatory mechanisms for growth and hydrocarbon production: Update for metabolically engineering strains on systems model pinpointing the main regulatory mechanisms for growth and hydrocarbon production. [month 30] D2.5) Update Systems model on regulatory mechanisms for growth and hydrocarbon production: Update for metabolically engineering strains on systems model pinpointing the main regulatory mechanisms for growth and hydrocarbon production. [month 48] D2.6) Mutagenised library of B. braunii and characterization of mutants: [month 30] D2.7) Verification of a selected number of candidate genes in C. reinhardtii: [month 30] D2.8) Establishment of molecular tools for metabolic engineering in C. reinhardtii: [month 48] Schedule of relevant Milestones Milestone 59 number MS3 MS4 MS5 MS6 Lead beneficiary number 15 16 12 1 Delivery date from 60 Annex I 12 30 30 30
Milestone name Metabolic pathway map (Flux model) Identification of genes for biosynthesis of PLS and HC, genome sequence and expression analysis Verification of candidate genes Identification of mutant strains of B. braunii with improved growth or higher PLS or HC
Comments

Schedule of relevant Milestones Milestone 59 number Lead beneficiary number 12 Delivery date from 60 Annex I 48
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Milestone name Introduction of transgenes into other microalgal species, and positive expression of these transgene
Comments
MS7

Project Number
1
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311956
Project Acronym
SPLASH
55 53
WP3
54
RTD
Process design: production & downstream processing
Objectives The main objective of this WP is to develop innovative cultivation and downstream concepts for improved growth, product enhancement and integrated recovery of exopolysaccharides and hydrocarbons. Specific Objectives Obtain insight into the relation between medium composition and process conditions on one hand and growth and product formation on the other hand for the microalgae Botryococcus braunii and the engineered Chlamydomonas reinhardtii with the aim of optimizing growth and steering production of hydrocarbons and polysaccharides Optimise outdoor cultivation of B. braunii (Task 3.2) Develop in situ continuous technologies to recover and separate the products (hydrocarbons, polysaccharides) Task 3.3 Develop a concept for an integrated industrial scale biomass production and product recovery facility for B.braunii (Task 3.4) Provide products for WP4 (Task 3.5) Description of work and role of partners Task 3.1 Optimization of growth and production of hydrocarbons and polysaccharides (WU, UHU) For the optimisation of growth and production conditions two different approaches will be followed being a statistical/empirical approach that will enable a fast optimization of the medium and conditions and a mechanistic approach, which will lead to a better understanding of B. braunii and engineered C. reinhardtii metabolism. Although this task does not depend on genome sequencing and annotation, information on metabolic capabilities of the algae as derived from the sequenced genome will aid in finding optimal media. Furthermore, data generated in this task (especially in the mechanistic approach) can be used for modelling (2.) and further improvement of the media and conditions. In addition to this, we will study the efficiency of nutrient supply to B. braunii and eventual mass transfer limitations due to the formation of colonies for growth enhancement Statistical/empirical approach. Relevant parameters will be identified based on literature and existing knowledge present in the consortium. These parameters will be studied in small batch cultures. Growth and product formation will be assessed. At first a large number of parameters will be studied using a genetic algorithm approach. In this approach more parameters can be taken along than in an experimental design. Batch cultures will be scored based on growth first. Samples will however be stored for later evaluation of the product formation. Random approaches like this are able to optimize media and conditions, however they are not able to find the exact optimum. Afterwards the data can be evaluated to study which parameters are important for growth and product formation. Since the random approach does not result in the exact optimum, the important parameters found in this way can next be further optimized in a design of experiments study. In an experimental design only a limited number of parameters can be taken along, because the number of experiments that need to be performed increases rapidly with the number of parameters. In this approach growth and product formation will be taken along. Optimised media and conditions found in the first two years can be directly used in tasks 3.2 and 3.4 Mechanistic approach. This approach involves the cultivation of the B. braunii race A in controlled short light path photobioreactors a in chemostat and/or turbidostat mode. This allows for measurement of the consumption rate of nutrients and the

production rate of products and biomass components. These data can be used directly to improve the medium. In addition they can be used in combination with a stoichiometric model to calculate flux distributions. Next the effect of culture parameters on the flux distribution can be studied in different chemostats/turbidostats. For example, the effect of growth rate can be studied, which is important for estimating the energy parameters of the strains. Also different strains can be compared in this way and the effect of metabolic engineering approaches can be studied. In steady state also gene expression, proteomics and metabolomics analysis can be done. Samples will be taken and analysed in WP2. In combination with changes in flux distributions this will give information on the control and regulation of metabolism. This information can be used to improve the models developed in task 2.3 and 2.5. Furthermore, since in task 3.2 also a flat panel reactors will be used for B. braunii, results can be translated and compared to data obtained in that task. Understanding efficiency of nutrient supply in B. braunii cultures. In order to validate the existing hypothesis that slow growth of B. braunii is due to the formation of colonies and consequent light or nutrient limitation, different mixing rates in the photobioreactors will be tested. We expect within these experiments that different colonies sizes will be formed leading to different degrees of mass transfer limitation. Task 3.2 Outdoors control strategies and biomass production of B. braunii (BIT, FBR, UHU, PAQ) Control strategies This task will address the sustainability and dynamics of Botryococcus cultures with respect to growth, product formation and performance under simulated and real outdoor conditions, e.g. due to seasonal changes, light/dark, temperature effects, wall growth, clumping, foaming, process energy requirements etc. Under outdoor conditions, the daily solar cycles determine the main algae growth conditions in the photobioreactors: light and temperature regimens. While temperature can be controlled, light availability becomes the dominant factor determining the productivity. Results from the mechanistic studies will be used to design, optimize and analyze outdoor pilot scale production. In particular, we are interested in the effect of changing light intensities on productivity of hydrocarbons and polysaccharides. The quantity of solar energy consumed by algae cultures during daytime and as a function of seasons directly influences growth and overall performance. We intend to develop dynamic process strategies in such a way that optimal productivity is achieved during the entire day. Biomass density, medium composition (e.g. nitrogen deficiency), temperature and harvest mode are possible parameters to be considered in such strategy We will use small scale photobioreactors and simulate outdoor conditions both at UHU and FBR. In addition experiments will be done under real outdoor conditions. An outdoor photobioreactor of 25 m2 will be built in AlgaePARC (Algae Production and Research Centre), which is a recently subsidised project from Wageningen UR, making therefore use of the already existing infrastructures. The reactor will be a flat panel reactor which has proven so far to be one of the most efficient systems. In the first year of the project the reactor set up will be done. B. braunii will be cultivated in this photobioreactor during the second and third year of the project with an accurate online measurements and control of a wide range of metabolic and environmental parameters (air flow in, CO2 flow in and out, O2 out, pH, temperature, biomass concentration, and light intensity). Operation conditions (flow patterns, air flow rate, harvesting and feeding patterns) will be set and optimised based on the results obtained in task 3.1. The resulting biomass and products (hydrocarbons and polysaccharides) will be used in WP4. We will evaluate outdoor cultivations before demonstration in WP5, with the objective to develop and optimised design and production strategies. Biomass production for supply of hydrocarbons and polysaccharides In parallel to these activities, BIT will continuously produce B. braunii to supply biomass and product for the following workpackages (WP4) in a tubular photobioreactor system, which will provide an additional system for comparison in terms of overall volumetric (g/L/day) and areal (g/m2/day) biomass and product productivities. The production volume is 5.0 m3, the area covered by tubes is 100 m2. We expect an annual production of either 800 kg (non-limited biomass) or 400 kg nutrient-limited, high lipid biomass. This activtiy will be performed in close collaboration with tasks 3.1 and 3.2 and optimisation strategies will be implemented immediately upon development. Time course of biomass, hydrocarbon and polysaccharides production will be gathered in monthly data sheets with several production parameters: operation mode, volumes harvested volumes, biomass production, biomass dry weight, culture cell density and biomass dry weight. Table 3.1 Algal product requirements for polymer development and testing in the SPLASH project Volumes (L) or mass (kg) /year for lab scale experiments Volumes (L) or mass (kg) for pilot scale experiments Hydrocarbons for green nafta 1 (L) 10 (L)
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Glucose 0.5 (kg) 10 (Kg) Galactose for adipic acid 1 (kg) 25 (Kg) Fucose and Rhamnose for furanics 1 (kg) 25 (kg) Task 3.3 Development of in situ DSP methods (DLO-FBR, PDX, EGE, VFT) The key objectives of this task are: To conduct an R&D study of extraction method requirements for extraction of hydrocarbons and polysaccharides from Botryococcus by investigation of all the major variables and design and development of optimum extraction mechanisms, system and control To design and construct small scale, pilot scale and in-situ extraction capabilities In this task we will study four different routes with different technologies for in situ separation of the cells, extraction and separation of hydrocarbons and polysaccharides, which will be compared in terms of efficiency of product extraction, energy requirements and costs. These routes will be studied in parallel and depending on results, will be integrated with upstream cultivation. The experimental work shall prove the applicability and enable the scale-up. The material here produced (hydrocarbons and exopolysaccharide) will be used in WP4. The profiling of hydrocarbons and exopolysaccharides will be done in WP4. Route 1 - Supersonic flow fluid processing This route includes the implementation of industry proven supersonic flow fluid processing an existing and proven fluid processing Reactor technology for the separation of the hydrocarbons and polysaccharides. R&D evaluation studies, including the development of a small scale R&D test rig will initially be done at PDX Huntingdon, United Kingdom. A series of tests will be conducted, assessing the importance of known key variables and possible Reactor system configurations and conditions, with the aim of finding the optimum balance of best extraction with least cell damage. Algae provision and sample analysis will be carried out at FBR, the Netherlands. Initiate interface with the upstream and down-stream process stages to understand their requirements. At R&D scale it will be demonstrated what extraction capabilities are achievable and how this would impact upstream and downstream processes. A scaled-up pilot rig will be developed and installed at FBR, The Netherlands. This will interface with the upstream and downstream processes. The pilot rig will include steam provision, the optimised Reactor system configuration and settings identified earlier in the evaluation studies, and a control system. Lastly we will demonstrate the operation of the in-situ up-scaled pilot rig and optimise the performance of the extraction process. In particular the interoperability between the extraction stage with the upstream and downstream processes. This will be in close collaboration with partners from WP4. The capability of the optimised process will be demonstrated in WP5 and the options for further up-scaling and commercialization of the technology will be analysed and identified. Route 2 - Milking Organic Solvents A continuous in situ extraction system for hydrocarbons will be developed (milking). Successful milking is dependent on a various of factors which will be addressed here and in other work packages: - product related: the function and metabolic pathways for the production of hydrocarbons in the cell will be addressed in WP2 while the location and way of accumulation in the cell will be addressed in this task - cell related: the structure and composition of the wall (literature) and physiological aspects of the cell (Task 3.1) will affect the efficiency of the method. - solvent related: biocompatibility, molecular structure of the solvent and solubility of the product in the solvent will be addressed in this task. The cellular mechanisms involved in the milking process need to be understood at a cellular level (the milking process is directly related to the production pathway which, in turn, is related to the factors that enhance the production of hydrocarbons addressed in WP2), and the tools for reactor scale up have to be developed. Supercritical CO2 As an alternative to the extraction with hexane near-critical or supercritical carbon dioxide can be applied. The supercritical fluid extraction of water based products has been demonstrated on the removal or alcohol from wine or the recuperation of aroma compounds in the production of concentrated fruit juices. The direct extraction of algal cultures has so far not been studied but seems feasible. However, the application needs research and development activities to minimize the impact of the extraction conditions on the living algal culture. The impact of pressurization and depressurization of the culture must be fundamentally investigated in collaboration with experts on cultivation of algae. Another challenge lies in the seamless integration of the extraction system with the bioreactor. Here, sufficient data of the phase behaviour of the key substances in the complex matrix
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water-algae-hydrocarbons must be determined. The experimental work shall prove the applicability and enable the scale-up. Comparison of the process economy and sustainability with the liquid-liquid extraction with hexane should round up the investigations. If successful, the developed supercritical extraction method will be used to produce material for WP5. The profiling of hydrocarbons will be done in WP2. In this route, hydrocarbons will be firstly separated and then the cells will be separated from the culture broth (with exopolysaccharides) via microfiltration. Route 3 - Combined Micro-Sieve Extraction The use of a Combined Micro-Sieve Extraction is a technique for product recovery from fermentation broths. The whole medium including the alga cells will flow over the micro-sieve. The alga cells will retain on the micro-sieve while the filtrate will be recycled back to the photobioreactor. In a second step the exopolysaccharides are extracted of the cells and collected for further processing. The next step extracts and collects the Hydro Carbons by a solvent extraction. After a final washing the extracted cells are rinsed of the micro-sieve and recycled back to the photobioreactor. The collected fractions of the exopolysaccharides and the hydrocarbons in most of the cases needs to be processed further in order to achieve the desired purity. Different process parameters can be varied to optimize the process. One can think about the selection of the micro-sieves, batch sizes, selection of solvents, extraction times etc. Route 4 - Rotary vacuum-drum filter An alternative method for in-situ product recovery is a rotary vacuum drum filter. This set-up consists of a drum rotating in a tub of liquid to be filtered. The drum rotates through the liquid coming out of the photobioreactor and the vacuum sucks liquid and solids onto the drum pre-coat surface, the liquid portion is "sucked" by the vacuum through the filter media to the internal portion of the drum, and the filtrate pumped away. The cells on the outside of the drum can then undergo a second separation and purification step. The cells are washed and the exopolysaccharides are extracted through the drum. The solids (cells) still adhering to the outside of the drum are then rinsed on the filter with an organic solvent, the HC can already be separated from the cells in this stage. Possibly this can accelerate the further purification of the achieved mixture of products. It has to be noted at this point that special attention needs to be given to the selection of the organic solvent, since the cells should not be harmed by the solvent treatment. Process parameters, which can be varied is the turning velocity of the drum, the under pressure and the temperature (which also influence the viscosity of the feed) as well as the solvents being used. The advantage of this technique is different extraction techniques can be combined in this system. Common methodologies to all routes Depolymerisation of exopolysaccharides The exopolysaccharides recovered will be depolymerized into their constituting monosaccharides by chemo-enzymatic methods. Subsequently, the various monomers will be further purified based upon differences in solubility and crystallization behaviour. This will result in significant amounts of rhamnose/fucose and galactose, which will be used in WP4 Common methodologies to all routes Depolymerisation of exopolysaccharides The exopolysaccharides recovered will be depolymerized into their constituting monosaccharides by chemo-enzymatic methods. Subsequently, the various monomers will be purified based upon differences in solubility and crystallization behaviour. This will result in significant amounts of rhamnose/fucose and galactose, which will be used in WP4 Task 3.4 integration and optimization of cultivation and DSP processes (DLO-FBR, PDX, PAQ, EGE) Specific objects of this task: Design, construct, install a scaled-up in-situ integrated algae extraction system Coordinate and integrate (both scientific and engineered) upstream production and downstream separation processes Analyse and identify the specific capabilities and limitations of production, extraction and separation and develop a synergistic system solution Analyse and provide recommendations for up-scaling and commercialization of the technology Task 3.5 Biosafety (UCAM, UNIBI, DLO-PRI, WU, UMUE, DLO-FBR) A Biosafety Officer will be appointed from among the staff involved in the Project. He/she will be responsible for drawing up a plan to ensure that all partners comply with the Biosafety regulations required by EU directives 219
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& 90, and local Health and Safety regulations. A database of all strains of algae generated during the Project will be set-up and maintained by the Biosafety Officer, and a DNA profile for each strain established to enable them to be identified and distinguished from one another. For the duration of the project, GM strains will be grown exclusively in enclosed photobioreactors, with suitable containment measures, and after official notification to the appropriate local government agency. In addition, regular barcoding analyses will be carried out where these are grown in order to assess the potential for contamination of the environment. All outside cultivation will be with non-GM strains, but mutagenized strains will be assessed for their potential risk, and similar barcoding will be used to determine the extent of escape, if any. At the same time, we will look to develop strains that are compromised in some aspect, so that they are less fit than native species. Auxotrophic mutants unable to synthesise key cellular building blocks such as amino acids are one possibility, although the supply of nutrients may mean unacceptable costs for cultivation. A better choice might be mutants lacking nitrate reductase (nit-), selected by their ability to live on chlorate (converted to toxic chlorite by nitrate reductase), since these could not survive in the environment without a supply of fixed nitrogen. Alternatively, non-starch producing strains, equivalent to the C. reinhardtii sta6, are unable to survive prolonged dark periods without a carbon source. In addition, strains of B. braunii require several vitamins including vitamin B12, which has been shown to be supplied by heterotrophic bacteria in return for photosynthate. Accordingly, since any outdoor cultivation is likely not to be axenic, it may be possible to contain the algal growth by treatment with antibiotics to kill the vitamin-supplying bacteria. Person-Months per Participant Participant number
10
WT3:
Participant short name 1 DLO 4 PAQ 5 BIT 6 VFT 9 PDX 12 UCAM 14 UHU 15 WU 16 UNIBI 17 UMUE 18 EGE
11
Person-months per participant 65.30 24.00 36.00 1.00 46.70 1.00 106.60 43.20 1.00 1.00 34.30 Total 360.10

61
Deliverable Title Optimized growth and production of hydrocarbons and polysaccharides Optimised outdoor process Products for WP4 as needed
Lead beneficiary number 15 14 5
Nature
62
Dissemination 63 level CO CO CO
Delivery date
64
D3.1 D3.2 D3.3
80.00 R 116.30 R 36.00 O
30 48 48

61
WT3:
Deliverable Title Process for continuous product recovery of hydrocarbons and polysaccharides from Botryococcus Concept for industrial scale biomass production and product recovery Biosafety
Lead beneficiary number 9 4 1 Total
Nature
62
Delivery date
64
D3.4 D3.5 D3.6
102.20 R 19.60 O 6.00 R 360.10
48 48 30
Description of deliverables D3.1) Optimized growth and production of hydrocarbons and polysaccharides: [month 30] D3.2) Optimised outdoor process: [month 48] D3.3) Products for WP4 as needed: [month 48] D3.4) Process for continuous product recovery of hydrocarbons and polysaccharides from Botryococcus: [month 48] D3.5) Concept for industrial scale biomass production and product recovery: [month 48] D3.6) Biosafety: [month 30] Schedule of relevant Milestones Milestone 59 number MS8 MS9 Lead beneficiary number 15 1 Delivery date from 60 Annex I 30 42
Milestone name Optimised media and process conditions for growth and production Long term cultivation and in situ or in-line continuous extraction of HC and PLS
Comments

Project Number
1
WT3:
311956
Project Acronym
SPLASH
55 53
WP4
54
RTD
Product development and testing
Objectives The main objective in this work package is to develop processes to convert the hydrocarbons and polysaccharides into the different monomers and polymers. Specific Objectives Development of a cracking process to convert hydrocarbons to green naphtha Development of a cracking process to convert green naphtha to ethylene and propylene Hydrolysis of algae polysaccharides into monomeric sugars Separation of monomeric sugar mixture into individual sugar fractions Conversion of galactose to adipic acid Conversion of fucose and rhamnose to 1,4-pentanediol Conversion of glucose to 2,5-FDCA Conversion of adipic acid and 1,4-pentanediol into polyesters and polyamides Conversion of 2,5-FDCA to PEF Testing of polyesters for the production of yarns Description of work and role of partners Task 4.1 Analytics of lipid fraction produced by B. braunii (FRAUNHOFER) Hydrocarbons: the hydrocarbon fraction produced in WP3 will be characterised to determine the hydrocarbon profile (type and abundance) using GC-MS and LC-MS analysis. Emphasis will be on composition, C/H/O/P distribution, by-product profile, trace element distribution and minor-compound composition. Task 4.2 Development of a cracking process to convert hydrocarbons to green naphtha and conversion of green naphtha to ethylene and propylene (FRAUNHOFER). The hydrocarbon fraction of B. braunii can be regarded as a heavy gas oil, containing high-boiling-point hydrocarbons of molecular weight 400 Daltons and higher. In petrochemical refineries, similar oil fractions are treated in a process called catalytic cracking (e.g. FCC), by which the long carbon chains are cleaved to shorter-chain products at high temperature and moderate pressure, with a fluidized powdered catalyst (Figure10). The shorter-chain products will be analysed by GC-MS. The products of this process are suitable for steam cracking to base chemicals. Hence, hydrocarbons from B. braunii may serve as a sustainable source of green naphtha for the chemical industry. In EU-SPLASH, the hydrocarbons from B. braunii will be subjected to small-scale catalytic cracking and steam cracking in order to transform them into ethylene and propylene (FRAUNHOFER). Task 4.3 Hydrolysis of algae polysaccharides into monomeric sugars (DLO-FBR) The polysaccharide fraction produced in WP3 will be characterized. The polysaccharides will be depolymerized into their constituting monosaccharides by chemo-enzymatic methods. Task 4.4 Separation of monomeric sugar mixture into individual sugar fractions (DLO-FBR) The various monomers will be separated into individual sugar types and purified based upon differences in solubility and crystallization behaviour. This will result in significant amounts of rhamnose/fucose and galactose isolated from the algae polysaccharides. These monosaccharides will be analysed by NMR, FT-IR and GC- or LC-MS.

Task 4.5 Conversion of galactose to adipic acid (DLO-FBR, RHO) Galactose, the most abundant monosaccharide produced by depolymerisation of the B. braunii polysaccharides, is an interesting building block for the production of reactive carbohydrates, such as galactose dialdehyde (galacto-hexodialdose, GALA) and galactaric acid. These compounds belong to a larger family of materials known as oxidized sugars, and represent what we believe to be a significant market opportunity. They can be used as cross-linking agents and for the production of biobased nylons, for the synthesis of hyperbranched polyesters, as well as biosurfactants. Recently, Food & Biobased Research (DLO-FBR) has developed a chemo-enzymatic process for the synthesis of glucaric acid starting from glucose that will be applied for the conversion of galactose into galactaric (mucic) acid. The success of the enzymatic approach for production of galactaric acid will represent a technological breakthrough and generate data that could be commercially exploited if the biocatalysed route can be carried out cost effectively. In EU-SPLASH, galactose from B. braunii exopolysaccharides will be chemo-enzymatically converted to galactaric acid. Furthermore, galactaric acid will be converted to adipic acid by catalytic hydrogenolysis. Adipic acid (1,6-hexanedioic acid) is an important building block for nylons and other condensation polymers, with a global annual market volume of more than two million tonnes. It is currently derived from benzene. The partner RHO will support the DLO-FBR investigations on conversion of galactose into carboxylic aliphatic di-acids with a priority on adipic acid, by providing know how on adipic acid analysis and quality requirements for polyamide synthesis. Task 4.6 Conversion of fucose and rhamnose to 1,4-pentanediol (DLO-FBR) Rhamnose and fucose both are excellent starting materials for the subsequent chemoenzymatic synthesis of 5-methyl furfural. This approach has the potential to lead to both a more environmentally benign as well as cheaper route to 5-methylfurfural. Decarboxylation of 5-methylfurfural and subsequent hydrogenation of the resulting 2-methylfuran leads to 2-methyltetrahydrofuran (MeTHF). Hydrolysis of MeTHF results in 1,4-pentanediol, which will be used as a diol building block for adipic acid based polyesters. This route to 1,4-pentanediol takes less steps than the alternative route starting from fructose( HMF levulinic acid gamma-valerolactone 1,4-pentanediol). Task 4.7 Conversion of glucose to 2,5-FDCA (AVT) Efficient methods for the dehydration of extracellular polysaccharides resulting from B. braunii will be developed by the partner AVT (SME). All sugars will be assessed for furanics production by Avantiums patented dehydration process into furanic components. Targets and boundary limits for dehydration of unusual carbohydrates into furan-derivatives The objective of this activity is to define the targets and boundary limits for the catalytic performances (productivity, selectivity, stability, sensitivity to minor components, range of operative conditions, etc.) of known catalysts and novel multifunctional catalysts developed. Testing of the multifunctional catalysts Screening and ranking of the catalysts using fucose and rhamnose as substrates. The tests will be made in a flow reactor. Based on the criteria formulated earlier, selected catalysts will be identified for the (i) analysis of the optimization of the reaction conditions, (ii) evaluation of the performances with large volume reactors, also to prepare (ii) the evaluation of the performances using real feeds, (iii) the analysis of the catalyst stability and sensitivity to feed components. The catalysts after reactivity tests are analysed as described in the previous activity. Task 4.8 Conversion of adipic acid and 1,4-pentanediol into polyesters and polyamides (DLO-FBR, RHO) Subtask 4.8.1. Conversion of bioadipic acid into polyesters (DLO-FBR) - Small scale melt polymerization (50 gram scale) of adipic acid and 1,4-pentanediol with various catalysts in order to obtain polyesters without discolouration. Comparison of this polymer with solution polymerization with regard to molecular weights. Evaluation of the solid state post condensation reaction in order to get a better insight of molecular weight build up as function of time and temperature. - Characterization of polyesters on molecular weight, thermal material properties and mechanical properties. Mechanical properties will be evaluated as function of molecular weight. - From the most less discoloured polyester, solid state post condensation will be performed at large scale to obtain sufficiently high molecular weight polyester suitable for fibre spinning. - Small scale polymerization of 2,5-FDCA, adipic acid and 1,4-pentanediol with various ratios diacids and with various catalysts in order to obtain co-polyesters with various compositions. - Characterization of co-polyesters on molecular weight, composition, thermal- and mechanical properties.
WT3:

- Systematic research into polyester composition and polyester degradation relationship. - Polymerization of co-polyester with the most promising composition and subsequent solid state post condensation in order to get a co-polyester with sufficiently high molecular weight suitable for fibre spinning. - The O2, CO2 and H2O permeability of the most promising polyesters and co-polyesters will be evaluated after sheet extrusion. Sub task 4.8.2. Conversion of bioadipic acid into Polyamides (RHO) - Small scale melt polymerization (100 gram scale) of adipic acid and various aliphatic diamines to assess the polymerization ability of the bioadipic acid. Characterization of polyamides on molecular weight, and thermal material properties. - Lab Pilot scale melt polymerization (3kg scale). This polymerization scale will allow obtaining more material, necessary for first trials of injection molding and mechanical properties evaluation. Other properties like coloration, rheology, melt stability will also be investigated. - Finally, the obtained biopolyamides will be positioned comparatively to the oil based ones with the available results. Task 4.9 Conversion of 2,5-FDCA to PEF films (AVT) - Definition and small-scale experimental synthesis experiments of grams of PEF films with different molecular masses, crystallinity etc. at Avantium. We strive to create 10 variations on the PEF compounds, however exact numbers are yet to be determined and adjusted in progress meetings. - Systematic research into characterization and material properties of found PEF polymer variation versus current PET polyesters, including (high!) molecular weights, melting points, glass transition temperatures, colors, transparencies, degradation temperatures, reactions with acids, bases, solvents, and moist, tensile strengths and modulus, elongations, response to UV-light (through accelerated aging), etc. This will require physical and spectroscopic assessments using NMR, GPC, HPLC, XRD, UV-Vis, DSC, TGA. - Techno-economical specifications of the most promising PEF polymers Task 4.10 Testing of polyesters for the production of fibers (LEP) The use of the polyesters from task 4.8 and PEF from task 4.9 will be investigated on pilot scale. The processing properties of these bio-based polymers will be studied by means of a cast-film extrusion process. The polymer material will be extruded into a thin flat film as mono-material and/or blends and subsequently after slitting in small tapes, subjected to single or multiple stretching stages. Optionally, the stretched tapes can be fibrillated and/ twisted into yarns. During these extrusion trials conclusions can be drawn about the process ability of these bio-based materials. The resulting tapes can be tested for tensile properties to determine strength, elongation, and stiffness. In addition, shrinkage, abrasion sensitivity, UV sensitivity and burn behavior can be evaluated. The process ability of the bio-based polymers and the mechanical properties of the prepared tapes will be directly compared to the process ability of traditionally produced polymers from the same class and tapes made thereof. By doing so, conclusions can be drawn about the feasibility of using these bio-based polymers in the production of tapes and yarns. Also by studying abrasion, UV and burn behavior possibly positive specific material properties can be recognized Person-Months per Participant Participant number
10
WT3:
Participant short name 1 DLO 7 AVT 11 FRAUNHOFER 19 LEP 20 RHO
11
Person-months per participant 37.00 37.00 12.20 1.40 11.30 Total 98.90

61
WT3:
Deliverable Title Analytics of lipid fraction produced by B. braunii Cracking process to convert hydrocarbons to green naphtha and further to ethylene and propylene Hydrolysis of algae polysaccharides into monomeric sugars Separation of monomeric sugar mixture into individual sugar fractions Conversion of galactose to adipic acid Conversion of fucose and rhamnose to 1,4-pentanediol Conversion of glucose to 2,5-FDCA Conversion of adipic acid and 1,4-pentanediol into polyesters and polyamides Conversion of 2,5-FDCA to PEF films Report on suitability of polyesters for the production of fibers
Lead beneficiary number 11 11 1 1 1 1 7 20 7 19 Total
Nature
62
Dissemination 63 level CO CO CO CO CO CO CO CO CO CO
Delivery date
64
D4.1 D4.2 D4.3 D4.4 D4.5 D4.6 D4.7 D4.8 D4.9 D4.10
8.00 R 4.20 R 5.00 R 7.00 R 17.30 R 12.00 R 25.00 R 9.00 R 10.00 R 1.40 R 98.90
30 36 24 30 30 30 36 36 48 48
Description of deliverables D4.1) Analytics of lipid fraction produced by B. braunii: [month 30] D4.2) Cracking process to convert hydrocarbons to green naphtha and further to ethylene and propylene: [month 36] D4.3) Hydrolysis of algae polysaccharides into monomeric sugars: [month 24] D4.4) Separation of monomeric sugar mixture into individual sugar fractions: [month 30] D4.5) Conversion of galactose to adipic acid: [month 30] D4.6) Conversion of fucose and rhamnose to 1,4-pentanediol: [month 30] D4.7) Conversion of glucose to 2,5-FDCA: [month 36] D4.8) Conversion of adipic acid and 1,4-pentanediol into polyesters and polyamides: [month 36] D4.9) Conversion of 2,5-FDCA to PEF films: [month 48] D4.10) Report on suitability of polyesters for the production of fibers: [month 48]

Schedule of relevant Milestones Milestone 59 number MS10 MS11 MS12 MS13 Lead beneficiary number 11 1 7 20 Delivery date from 60 Annex I 30 24 36 36
WT3:
Milestone name Process for cracking of B. braunii hydrocarbons to base chemicals Process for conversion of fucose and rhamnose to 1,4-pentanediol Conversion of 2,5-FDCA to PEF Process for conversion adipic acid and 1,4-pentanediol into polyesters and polyamides
Comments

Project Number
1
WT3:
311956
Project Acronym
SPLASH
55 53
WP5
54
DEM
Process demonstration at pilot scale
Objectives The main objective of this WP is to demonstrate the capability of the optimised process (cultivation, integrated extraction and product separation of exopolysaccharides and hydrocarbons), at pilot scale under industrial representative conditions, based on the technology and methodologies developed in the previous WPs. Specific objectives Demonstration and proof of concept of continuous production and in line extraction and separation of hydrocarbons and polysaccharides from Botryococcus at pilot scale. Demonstration and proof of concept of production of polymer based chemicals (polyesters, copolyesters, polyamides, ethylene and propylene, and PEF (a substitute for polyethylene terephthalate, PET) from algal hydrocarbons and exopolysaccharides, at pilot scale Demonstration of suitability of the above products for the production of fibers. Description of work and role of partners Task 5.1 Demonstration of optimised process: Cultivation and integrated downstream processing for hydrocarbons and polysaccharides at pilot scale (PAQ, DLO-FBR, PDX) In this task we will implement the optimised process and control strategies for upstream, extraction technologies and downstream processes to demonstrate performance and identify options for further up-scaling and commercialisation of the technologies. If required the system will be reassembled in an optimised configuration. The integrated pilots will be run at AlgaePARC on a demonstration scale based on the results obtained in WP3 which will be directly implemented in the run, and in this way be validated. The scale will be 25 m2 ground area for the photobioreactor production meaning a production of about 150 Kg dry weight biomass per year Task 5.2 Conversion of hydrocarbons and polysaccharides into biopolymers (AVT, FRAUNHOFER, RHO, LEP) In this task hydrocarbons and polysaccharides will be converted in green naphtha, polyesters, copolyesters and polyamides at pilot plant facilities of the partners FRAUNHOFER, RHO and AVT and further tested by LEP in fibers. The partner RHO will investigate the production of large batches of polyamides 6.6 at plant pilot scale (80-100kg per batch) for further evaluation on semi-industrial equipment of compounding and injection molding. Task 5.3 Technology and product commercialization (PAQ, AVT, VFT, PDX, RHO) In this task we will perform an analysis and identification of the options for further up-scaling and commercialization of the technology. There will be a close collaboration with WP6 (activity 6.4 related to market analysis) and WP7 (Dissemination, exploitation and intellectual property management) to provide a direct path to quickly and effectively commercialisation of the different technologies through the international licensors network of PAQ, PDX and AVT. Person-Months per Participant Participant number
10
Participant short name 1 DLO
11
Person-months per participant 3.00

Person-Months per Participant Participant number
10
WT3:
Participant short name 4 PAQ 6 VFT 7 AVT 9 PDX 11 FRAUNHOFER 19 LEP 20 RHO
11
Person-months per participant 14.90 1.00 7.00 2.50 2.80 1.00 15.00 Total 47.20

61
Deliverable Title Validation of experimental data resulting from process optimisation Conversion of hydrocarbons and sugars into biopolymers Technology, scale up and product commercialization
Lead beneficiary number 4 20 4 Total
Nature
62
Delivery date
64
D5.1 D5.2 D5.3
17.90 D 21.80 D 7.50 D 47.20
44 42 48
Description of deliverables D5.1) Validation of experimental data resulting from process optimisation: [month 44] D5.2) Conversion of hydrocarbons and sugars into biopolymers: [month 42] D5.3) Technology, scale up and product commercialization: [month 48] Schedule of relevant Milestones Milestone 59 number MS14 Lead beneficiary number 4 Delivery date from 60 Annex I 36
Milestone name Upstream and downstream integrated demonstration facility
Comments

Project Number
1
WT3:
311956
Project Acronym
SPLASH
55 53
WP6
54
RTD
Process integration: Sustainability assessment and market analysis
Objectives The overall objective of WP6 is to integrate the different steps in the process, optimise the process chain and assess the economic, environmental and social impacts of the entire process. Description of work and role of partners Task 6.1 Conceptual process design (CERTH, PAQ) As a basis for the environmental and the economic assessment, mass and energy balances for the algae and yeast based systems will be drawn up by application of conceptual process design. The flow sheet for production process will be based on experimental data from within this project. Flow sheets will be prepared for the production and separation of exopolysaccharides and hydrocarbons and their subsequent conversion into the products of interest. Variants of the flow sheets will be prepared in order to reflect options offered by making use of different microorganisms, different workup methods and varieties in subsequent processing. By close interaction within the project team, the relevant combinations will be chosen, resulting in a limited number of distinct flow sheets. The ultimate goal of the digital integrated simulator will be the development of modeling and design software tools to create a multi-dimensional, multi-scalable digital representation of the entire product-process plant, including downstream processing across the product-process life cycle. The digital platform will be utilized for optimal operation of biorefinery processes using dynamic control, multivariate optimization and performance monitoring methods, to ensure maximum economic and societal benefit through minimizing the energy resources, as well as the cost involved in supply chain operations intrinsic to biorefining. Model-based optimization with a particular emphasis on the design of optimal trajectories for the individual processes will be particularly suitable for provision of complex limiting operating conditions for desired product formation. Holistic optimization of the integrated microalgal-based process targeting the increase of the products yield, the upscaling modeling as well as the overall technology sustainability performance will be carried out Task 6.2 Techno- economic model: feasibility and optimisation (CERTH, PAQ, DLO-FBR) The information generated throughout the execution of the project will be used to carry out a detailed techno-economic feasibility analysis of the proposed technology. In addition the energetic efficiency of the integrated process and its economic feasibility will be assessed. The energetic analysis will request performing mass and energy balances to each step of the process. In this task, the techno-economic evaluation of the proposed process will be done. Moreover, a detailed sensitivity analysis will be conducted to determine the parameters that may have the largest effects on the results and to determine the impact of the estimated techno-economic data as well as its variations on the conclusions. Variables included in the sensitivity analysis will be chosen to reflect system areas that have inherently more unknowns in the techno-economic data as well as areas in which variations will likely occur during normal process operations. Each parameter will be changed independently of all others so that the magnitude of its effect on the base case will be properly assessed. Therefore, no single sensitivity case will reflect the best or worse-case scenarios for this complex integrated system. However, any dependence that other variables may have on the variable being changed will be taken into account. The output of the extensive sensitivity analysis performed in this task will be properly incorporated in the LCA model. Economic performance parameters will be calculated and will provide input for the final assessment of the sustainability performance. Task 6.3 LCA- Life Cycle Assessment (OWS)

In this task we will assess: the hot spots in the production chain for biopolymers via one renewable process pathways: green microalgae (B. braunii and C. reinhardtii). the environmental impact and burden during production of the biobased polymers and compared with conventional production processes from petrochemicals Employing the outputs of the other WPs, the whole process will be subjected to a sustainability assessment. Subtask 6.3.1: Quantify all relevant economic, environmental and social elements, employing standard methodologies, such as life cycle assessment and cost benefit analyses. Subtask 6.3.2: A complete environmental sustainability assessment will be carried out, comparing sustainability to conventional systems for biopolymers and product services. Subtask 6.3.3: Quantify most important environmental impacts and benefits (e.g. greenhouse gas (GHG) emissions, fossil energy consumption, water consumption, land-area demand and land use change, competition for food and biomass resources, the impact on water use and quality, changes in land-use, net balance of greenhouse gases (CO2, CH4 and N2O), and potential toxicological risks) for each step in the value chain including: cultivation of algal biomass, its transport to conversion centers, conversion to biofuels, disposal of wastes if any, transport to users, and use in a final energy product. Subtask: 6.3.4: Perform economic analysis including socio-economic and market aspects. The economic assessment will consider competition for price impacts on food, feed, costs of transportation, capital and operating costs for algal cultivation and conversion, expected final costs of products, and market prices for alternative products or means for supplying equivalent services. Subtask 6.3.5: Overall sustainability assessment based on the results of the analyses of the economic, environmental and social aspects, using an expert system to balance and to weigh all the different aspects in a multi criteria assessment. Subtask: 6.3.6: Link back the results to process and product development in WPs 2 5 . All partners will be involved in the inventory and assessment of the LCA, for which the support will be strongly requested. Existing and evolving sustainability requirements for the chemicals sector will be monitored and translated into specific LCA requirements. Task 6.4 Economic assessment and market analysis (VFT) The economic assessment will also be based on the material balances and will result in estimated process costs for various process configurations and considering different boundary conditions (e.g., with regard to chemical and polymers prices). Extensive use will be made of the experience gained in previous projects . The following activities will take place in this task: 1. Identification of potential markets for chemicals derived from Botryococcus 2. Listing of current product references in each market: product type, requirements, specs, SWOT, etc. 3. Identification of the potential of the exopolysaccharides and hydrocarbons in their native stage and evaluation of the market opportunities. 4. Quantification of potential markets 5. Analysis of the potential for market penetration 6. First shortlist of commercial targets: product definition, specs, potential market size... 7. Re-iteration according to results obtained in WP4 8. Positioning of the products 9. Edition of a marketing plan for SPLASH Activities 1-4 will start at the beginning of the project and will be completed within the first 6 months. This will allow the research groups involved in WP1-5 to have as soon as possible first documented targets for their research. Task 5 implies a fine-tuning / adaptation of the first conclusions of activities 1-4 based on the results obtained in WP1-5. and will be updated throughout the project. Activities 6-7 are scheduled at the end of the project.
WT3:

With this project we expect to have new molecules available supporting the emergence of the bio-based economy. This covers innovations on the levels of Product design (new products) Product functionality (new functionalities) Production process (making those products economically available) Through VFTs sister company, Orineo, VFT has the resources and channels to valorise effectively these new products. Task 6.5 Standardisation (VFT) Technical standards will be developed and implemented to the products This is required to ensure quality and consumer information on the new products.The goals of standardization can be to help with independence of single. Task 6.6 Social aspects (VFT) Social aspects studied will include: 1. the extent to which the new algae-based systems change the demand for agricultural land and water. 2. to the extent possible, an assessment of the contribution of employment and analysis of economic growth (see below). To summarize the intermediate deliverables, this work will quantify the technical, economic, environmental and as far as possible the social performance of the various product systems for the short, medium and longer term and offer a comparison with conventional chemicals and their recent bio-based competitors if any. The most critical steps in the process chain will be identified and options for improving the process will be evaluated. Sensitivity analyses will also be performed for critical aspects identified in the process. Person-Months per Participant Participant number
10
WT3:
Participant short name 1 DLO 2 CERTH 3 OWS 4 PAQ 6 VFT 7 AVT
11
Person-months per participant 2.00 37.50 14.90 9.80 8.30 4.90 Total 77.40

61
Deliverable Title Conceptual design and integrated processes Update conceptual design and integrated processes Update conceptual design and integrated processes Techno-economic model and sensitivity analysis
Lead beneficiary number 2 2 2 2
Nature
62
Dissemination 63 level CO CO CO CO
Delivery date
64
D6.1 D6.2 D6.3 D6.4
13.60 R 3.90 R 2.90 R 28.90 R
12 30 48 30

61
WT3:
Deliverable Title Update techno-economic model and sensitivity analysis Report on cradle-to-factory-gate LCA Update report on cradle-to-factory-gate LCA Update report on cradle-to-factory-gate LCA Report on economic assessment and market analysis Report on standardisation Report on social aspects
Lead beneficiary number 2 3 3 3 6 6 6 Total
Nature
62
Dissemination 63 level CO CO CO CO CO CO CO
Delivery date
64
D6.5 D6.6 D6.7 D6.8 D6.9 D6.10 D6.11
3.90 R 4.90 R 5.00 R 5.00 R 4.30 R 2.00 R 3.00 R 77.40
48 24 30 48 42 48 42
Description of deliverables D6.1) Conceptual design and integrated processes: [month 12] D6.2) Update conceptual design and integrated processes: [month 30] D6.3) Update conceptual design and integrated processes: [month 48] D6.4) Techno-economic model and sensitivity analysis: [month 30] D6.5) Update techno-economic model and sensitivity analysis: [month 48] D6.6) Report on cradle-to-factory-gate LCA: [month 24] D6.7) Update report on cradle-to-factory-gate LCA: [month 30] D6.8) Update report on cradle-to-factory-gate LCA: [month 48] D6.9) Report on economic assessment and market analysis: [month 42] D6.10) Report on standardisation: [month 48] D6.11) Report on social aspects: [month 42] Schedule of relevant Milestones Milestone 59 number MS15 MS16 Lead beneficiary number 2 3 Delivery date from 60 Annex I 30 30
Milestone name Optimal process chain and operating policies for improved product yield First report on environmental and economic performance
Comments

Project Number
1
WT3:
311956
Project Acronym
SPLASH
55 53
WP7
54
OTHER
Dissemination, exploitation and intellectual property management
Objectives This WP aims at dissemination exploitation and technology transfer of project results to the relevant stakeholders including policy makers, industry and society including the management of intellectual property rights (IPR). Description of work and role of partners Effective dissemination and exploitation includes a direct communication between project partners, between science and potential stakeholders (including end-users) from the beginning of the project. The idea of the dissemination in the SPLASH project is to develop concise information and awareness to all relevant stakeholders in Europe and abroad. The strategy will include internal communication within the SPLASH project and external communication to the academic and research communities, industrial networks and the general public. The Dissemination and Exploitation officer (DEO) is the leader of WP7. There will be very intensive communication between the DEO and PC. The DEIPAB will advise and report to the PMT on issues regarding project strategy and optimization of applicability and exploitation of the scientific and technological Project results. One of the first tasks of the DEO) will be the preparation of an overall communication and dissemination plan. This will include internal and external dissemination tasks, exploitation, and management of intellectual property. Task 7.1 Internal communication (Nova, DLO-FBR) For internal communication, it will be important that all partners will receive the information and (intermediate) results they need in order to do an optimal job. Therefore nova will set up a website for internal management. The internal communication will be implemented in a platform where access is restricted to the consortium partners and designated EC officials. Inter-group trainings for the project staff and PhD students shall support and ensure the transfer of knowledge amongst them. In addition nova will support the management in their communication process. Task 7.2 External communication (Nova, PNO, LG, DLO-FBR) The projects research partners will disseminate the scientific and technological knowledge developed during the SPLASH project to the academic community and other stakeholders. Primarily the industrial partners will disseminate commercial benefits of the novel technologies to end users and key decision makers. The industrial partners will use their existing links to the appropriate industrial, trade and professional organisations and associations. For this the following types of dissemination media have been chosen to ensure optimum communication with the end users and other stakeholders. In all dissemination activities it will clearly be stated that the project is funded by the European Commissions Seventh Framework Programme for Research and Technological Development and, subject to approval by the EC, the FP7 logo used on all electronic and physical dissemination material. Project Website The consortium will create a project website under the lead of nova-Institute for internal management (see above) and external information of the project. The public areas will attend for the dissemination of information on the project to the consortium, the wider academic and industrial communities and the general public. The website will also be used to promote the upcoming project events. Pre-market Stimulation Activities

A series of activities for the project consortium will be organised to support pre-market dissemination of the project results targeted at the technology evolved in the SPLASH project and the opportunities in using microalgae and metabolic engineered yeasts as a source for oils and polysaccharides for the production of chemicals. Press releases will be made available on the project website and distributed to trade, technical and scientific journals and brochures will be prepared for detailing benefits and opportunities of outputs from the project. Training, Exhibitions, Conferences The consortium will actively promote the technology and project by attending international exhibitions and conferences focussed on industrial biotechnology and the use of microalgae. The industrial and academic partners will use exhibition stands on national and international levels to promote the EU-SPLASH developments to the industry and end users whilst nova and the research partners will deliver presentations at conferences describing their activities and results obtained during the project to target the scientific and technical communities. Additionally Nova will establish an international conference on the use of microalgae in the industrial biotechnology to promote the results of this project as well as to get in contact with other projects in this field and to find partners for the exploitation of the technical innovations. For graduate students and researchers, a summer school will be implemented to spread and promote the developments of the SPLASH project. Articles in Peer Reviewed Journals, Industrial Journals and Trade Magazines In order to disseminate the SPLASH project results into the scientific community, the research organisations in the consortium will author scientific articles describing the scientific and technical achievements of the SPLASH project for publication in peer-reviewed journals focussed on industrial biotechnology and related research areas. When possible, the research organisations will use green (self-archiving) open access to deposit peer reviewed research and final manuscripts arising from their activities in the SPLASH project, in line with the ECs Open Access Pilot initiative and allowing the scientific community greater access to the knowledge generated in the project. In order to stimulate interest in the take up and dissemination of the SPLASH developments with the industrial sector, the projects industrial partners will place article publications in relevant trade journals. EC Dissemination Routes The consortium partners will ensure that the excellent dissemination tools provided by the EC for dissemination to European citizens, industry and the scientific community will be utilised in the project. In particular dissemination material will be prepared by the consortium partners which in turn will be included on the CORDIS website. Details of the project will be made available for articles to be included in European electronic and paper publications such as research*eu. Accompanying measures In addition to the specific aforementioned dissemination routes, we will also use an assessment of the following measures to target the wider general public: Press releases and interviews to newspapers and magazines; DVD / CD ROMs containing project summaries / demonstrations; Web 2.0 (Twitter, scientific platforms, iTunes U), Wikipedia for project related topics Brochures, posters and leaflets; The partners existing customer networks; National initiatives and regional government bodies. Task 7.3 Exploitation (VFT, PAQ, PNO, OWS, AVT, LG, PDX, DLO-FBR) In the first phase of the project exploitation issues and targets will be defined. SPLASH has a strong industrial component with major industrial participation; 2 large companies as end users and 8 SMEs which cover the entire chain of the process. The academic partners and research institutes have critical roles in improvement of algal strains, genetic tools, cultivation and harvesting equipment and know-how, biorefining and valorization. This will create opportunities for each of the partners as reflected by their interest in the results of SPLASH. In the second year an Exploitation Business Plan will be developed by the DEO and PC, with input of the SEAB . This plan will give an overview of potential exploitable results, already foreseen by the participants, potential markets, and foreseen profits for the potential end-users. Also in the final year of SPLASH concepts for follow-up projects will be developed. These may be demonstration projects, further scientific and development programs, or programs aimed at specific issues coming up during SPLASH all aiming at the exploitation of SPLASH projects results in European practice.
WT3:

Task 7.4 Intellectual Property Management (VFT, DLO-FBR) During the first phase of the project a full update of the scientific state of the art and IP positions will be generated in collaboration with all partners. The resulting overview will be continuously updated. In addition potential competitive or complementary actions will be reviewed. Also, the impact on commercialization of project results and exploitation of these actions will be identified and dealt with in further progress of the project. All details relating to IP issues will be properly addressed in a consortium agreement (CA), which will be finalized during the contract negotiations. The FP8 Consortium Agreement of DESCA will be used as model for the CA. This agreement will provide the legal framework within which all partners can work freely, thus maximizing opportunities for effective collaboration and exploitation. Section 2.1 outlines the management structure for coordination, quality control and decision making in regard to the research being performed and the IP that is generated. Management of IP in the first instance is the responsibility of the partner who generates it. To ensure maximum use is made of all IP generated the following issues will be addressed in the CA to be agreed on: 1) Personal responsibility to identify potential IP generated by their research activities and to refrain from public disclosures until the necessary IP protection measures have been implemented 2) A mechanism to safeguard against accidental disclosure of valuable IP will be agreed on. This may include an evaluation of manuscripts, poster and oral presentation abstracts prior to publication. Such an evaluation will be performed within a short time frame (if possible less than two weeks) to avoid delays in publication of material. 3) Patent protection Partners are free to protect any inventions created during the SPLASH project by filing a patent, either independently or with input from a(n) (industrial) partner. Alternatively, if a partner decides not to patent an invention it should be offered to another partner with the full knowledge of the EO and the PC. Budgets for the costs of IP protection (patents) and mechanisms for drawing up patents and filing will be agreed on in the CA, but are the responsibility of the individual partners. 4) Access rights to IP will be agreed on in the CA. A three-step approach will be adopted for the exploitation of IP generated in the course of SPLASH. 1) Given the ability of the industrial partners to transfer SPLASH technology into the marketplace, the best strategy for exploitation is through these channels 2) In the event of IP being generated by SPLASH that is not adopted for exploitation by any of the industrial partners, then the PC and DEO (and the commercialization manager from the relevant partner institutes) will approach other industrial partners associated with related projects, or identified in the course of the SPLASH project. 3) If an exploitation route is not established through 1) or 2) than the PC and DEO will approach the extended audience. Person-Months per Participant Participant number
10
WT3:
Participant short name 1 DLO 3 OWS 4 PAQ 6 VFT 7 AVT 8 LG 9 PDX 10 NOVA 13 PNO
11
Person-months per participant 2.00 2.00 2.00 3.00 2.00 6.40 3.00 35.00 6.00 Total 61.40

61
WT3:
Deliverable Title Communication and dissemination plan Scientific state of the art and IP positions Update on scientific state of the art and IP positions Update on scientific state of the art and IP positions Public website and newsletter, first press release on the project scopes Project leaflet on several languages Update project leaflet on several languages Project brochure Update project brochure Concept for an International conference on industrial microalgae biotechnology Exploitation business plan Update exploitation business plan Pre-market stimulating activities Concept for summer school Report on compliance of the SPLASH concept with stakeholders requirements
Lead beneficiary number 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Total
Nature
62
Dissemination 63 level CO CO CO CO PU PU PU PU PU PU CO CO PU PU CO
Delivery date
64
D7.1 D7.2 D7.3 D7.4 D7.5 D7.6 D7.7 D7.8 D7.9 D7.10 D7.11 D7.12 D7.13 D7.14 D7.15
5.50 O 4.30 R 2.60 O 1.10 O 8.00 O 3.50 O 2.40 O 3.50 O 1.50 O 5.00 O 9.00 O 7.00 O 3.00 O 3.00 O 2.00 R 61.40
3 12 30 48 6 12 30 30 48 12 30 48 42 24 48
Description of deliverables D7.1) Communication and dissemination plan: [month 3] D7.2) Scientific state of the art and IP positions: [month 12] D7.3) Update on scientific state of the art and IP positions: [month 30] D7.4) Update on scientific state of the art and IP positions: [month 48] D7.5) Public website and newsletter, first press release on the project scopes: [month 6] D7.6) Project leaflet on several languages: [month 12] D7.7) Update project leaflet on several languages: [month 30] D7.8) Project brochure: [month 30] D7.9) Update project brochure: [month 48] D7.10) Concept for an International conference on industrial microalgae biotechnology: [month 12]

D7.11) Exploitation business plan: [month 30] D7.12) Update exploitation business plan: [month 48] D7.13) Pre-market stimulating activities: [month 42] D7.14) Concept for summer school: [month 24] D7.15) Report on compliance of the SPLASH concept with stakeholders requirements: [month 48] Schedule of relevant Milestones Milestone 59 number MS17 MS18 MS19 MS20 Lead beneficiary number 10 10 10 10 Delivery date from 60 Annex I 6 30 42 36
WT3:
Milestone name Website set-up Layout, print and dissemination of the leaflet to key stakeholders and general public International conference finished successfully Summer school finished successfully
Comments
List of Milestones
Project Number
1
WT4:
311956
Project Acronym
SPLASH
List and Schedule of Milestones Milestone 59 Milestone name number MS1 MS2 MS3 Kick-off Meeting Robust project management Metabolic pathway map (Flux model) Identification of genes for biosynthesis of PLS and HC, genome sequence and expression analysis Verification of candidate genes WP number WP1 WP1 WP2
53
Lead beneficiary number 1 1 15
Delivery date 60 from Annex I
Comments
1 Report 48 12 Contractual deliverables met on time and in full
MS4
WP2
16
30
MS5
WP2
12
30
MS6
Identification of mutant strains of B. braunii with improved WP2 growth or higher PLS or HC Introduction of transgenes into other microalgal WP2 species, and positive expression of these transgene Optimised media and process conditions WP3 for growth and production Long term cultivation and in situ or in-line WP3 continuous extraction of HC and PLS Process for cracking of B. braunii hydrocarbons to base chemicals WP4
30
MS7
12
48
MS8
15
30
MS9
42
MS10
11
30
MS11
Process for conversion of fucose WP4 and rhamnose to 1,4-pentanediol Conversion of 2,5-FDCA to PEF Process for conversion WP4 WP4
24
MS12 MS13
7 20
36 36
List of Milestones
Milestone 59 Milestone name number adipic acid and 1,4-pentanediol into polyesters and polyamides MS14 Upstream and downstream WP5 integrated demonstration facility Optimal process chain and operating WP6 policies for improved product yield First report on environmental and economic performance Website set-up Layout, print and dissemination of the leaflet to key stakeholders and general public International conference finished successfully Summer school finished successfully WP6 WP7 4 36 WP number
53
WT4:
Lead beneficiary number
Delivery date 60 from Annex I
Comments
MS15
30
MS16 MS17
3 10
30 6
MS18
WP7
10
30
MS19 MS20
WP7 WP7
10 10
42 36
Tentative schedule of Project Reviews

Project Number
1
WT5:
311956
Project Acronym
SPLASH
Tentative schedule of Project Reviews Review Tentative Planned venue 65 timing of review number RV 2 30 Wageningen, The Netherlands Comments, if any Intern mid-term review
Project Effort by Beneficiary and Work Package

Project Number
1
WT6:
311956
Project Acronym
SPLASH
Indicative efforts (man-months) per Beneficiary per Work Package

Beneficiary number and short-name 1 - DLO 2 - CERTH 3 - OWS 4 - PAQ 5 - BIT 6 - VFT 7 - AVT 8 - LG 9 - PDX 10 - NOVA 11 - FRAUNHOFER 12 - UCAM 13 - PNO 14 - UHU 15 - WU 16 - UNIBI 17 - UMUE 18 - EGE 19 - LEP 20 - RHO Total
WP 1 27.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 27.30
WP 2 62.00 0.00 0.00 0.00 0.00 0.00 0.00 89.00 0.00 0.00 0.00 85.00 0.00 0.00 45.60 65.00 46.20 0.00 0.00 0.00 392.80
WP 3 65.30 0.00 0.00 24.00 36.00 1.00 0.00 0.00 46.70 0.00 0.00 1.00 0.00 106.60 43.20 1.00 1.00 34.30 0.00 0.00 360.10
WP 4 37.00 0.00 0.00 0.00 0.00 0.00 37.00 0.00 0.00 0.00 12.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.40 11.30 98.90
WP 5 3.00 0.00 0.00 14.90 0.00 1.00 7.00 0.00 2.50 0.00 2.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 15.00 47.20
WP 6 2.00 37.50 14.90 9.80 0.00 8.30 4.90 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 77.40
WP 7 2.00 0.00 2.00 2.00 0.00 3.00 2.00 6.40 3.00 35.00 0.00 0.00 6.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 61.40
Total per Beneficiary 198.60 37.50 16.90 50.70 36.00 13.30 50.90 95.40 52.20 35.00 15.00 86.00 6.00 106.60 88.80 66.00 47.20 34.30 2.40 26.30 1,065.10
Project Effort by Activity type per Beneficiary

Project Number
1
WT7:
Part. 14 UHU
311956
Project Acronym
SPLASH
Indicative efforts per Activity Type per Beneficiary Activity type Part. 1 DLO Part. 2 CERTH Part. 3 OWS Part. 4 PAQ Part. 5 BIT Part. 6 VFT Part. 7 AVT Part. 8 LG Part. 9 PDX Part. 10 Part. 11 Part. 12 NOVA FRAUNHO UCAM Part. 13 PNO
1. RTD/Innovation activities WP 2 WP 3 WP 4 WP 6 Total Research 62.00 65.30 37.00 2.00 166.30 0.00 0.00 0.00 37.50 37.50 0.00 0.00 0.00 14.90 14.90 0.00 24.00 0.00 9.80 33.80 0.00 36.00 0.00 0.00 36.00 0.00 1.00 0.00 8.30 9.30 0.00 0.00 37.00 4.90 41.90 89.00 0.00 0.00 0.00 89.00 0.00 46.70 0.00 0.00 46.70 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12.20 0.00 12.20 85.00 1.00 0.00 0.00 86.00 0.00 0.00 0.00 0.00 0.00 0.00 106.60 0.00 0.00 106.60
2. Demonstration activities WP 5 Total Demo 3.00 3.00 0.00 0.00 0.00 0.00 14.90 14.90 0.00 0.00 1.00 1.00 7.00 7.00 0.00 0.00 2.50 2.50 0.00 0.00 2.80 2.80 0.00 0.00 0.00 0.00 0.00 0.00
3. Consortium Management activities WP 1 Total Management 4. Other activities WP 7 Total other Total 2.00 2.00 198.60 0.00 0.00 37.50 2.00 2.00 16.90 2.00 2.00 50.70 0.00 0.00 36.00 3.00 3.00 13.30 2.00 2.00 50.90 6.40 6.40 95.40 3.00 3.00 52.20 35.00 35.00 35.00 0.00 0.00 15.00 0.00 0.00 86.00 6.00 6.00 6.00 0.00 0.00 106.60 27.30 27.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Project Effort by Activity type per Beneficiary

Activity type Part. 15 WU Part. 16 UNIBI Part. 17 UMUE Part. 18 EGE Part. 19 LEP Part. 20 RHO Total
WT7:
1. RTD/Innovation activities WP 2 WP 3 WP 4 WP 6 Total Research 2. Demonstration activities WP 5 Total Demo 3. Consortium Management activities WP 1 Total Management 4. Other activities WP 7 Total other Total 0.00 0.00 88.80 0.00 0.00 66.00 0.00 0.00 47.20 0.00 0.00 34.30 0.00 0.00 2.40 0.00 0.00 26.30 61.40 61.40 1,065.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 27.30 27.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 15.00 15.00 47.20 47.20 45.60 43.20 0.00 0.00 88.80 65.00 1.00 0.00 0.00 66.00 46.20 1.00 0.00 0.00 47.20 0.00 34.30 0.00 0.00 34.30 0.00 0.00 1.40 0.00 1.40 0.00 0.00 11.30 0.00 11.30 392.80 360.10 98.90 77.40 929.20
Project Effort and costs

Project Number
1
WT8:
311956
Project Acronym
SPLASH
Project efforts and costs Estimated eligible costs (whole duration of the project) Beneficiary number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Beneficiary short name Effort (PM) 198.60 37.50 16.90 50.70 36.00 13.30 50.90 95.40 52.20 35.00 15.00 86.00 6.00 106.60 88.80 66.00 47.20 34.30 2.40 26.30 Total 1,065.10 Personnel costs () 1,287,676.00 89,250.00 103,445.00 502,174.00 133,200.00 93,100.00 441,150.00 622,345.00 472,880.00 201,250.00 80,475.00 314,932.00 44,352.00 257,938.00 488,400.00 310,200.00 221,840.00 34,300.00 7,358.00 162,324.00 Subcontracting () 25,000.00 0.00 0.00 10,000.00 5,000.00 5,000.00 5,000.00 10,000.00 10,000.00 35,000.00 0.00 5,000.00 0.00 0.00 10,000.00 5,000.00 5,000.00 0.00 1,500.00 0.00 Other Direct costs () 457,459.00 29,830.00 20,186.00 148,654.00 200,000.00 18,288.00 119,514.00 77,960.00 412,614.00 14,000.00 7,528.00 71,758.00 7,831.00 8,789.00 161,185.00 75,957.00 150,303.00 32,250.00 12,155.00 151,363.00 Indirect costs OR lump sum, flat-rate or scale-of-unit () 721,088.00 80,325.00 70,343.00 248,276.00 199,920.00 66,832.80 336,398.40 420,183.00 177,098.80 129,150.00 88,555.00 232,014.00 31,046.00 160,036.20 405,372.00 231,694.20 223,285.80 39,930.00 1,988.00 62,737.40 Total costs 2,491,223.00 199,405.00 193,974.00 909,104.00 538,120.00 183,220.80 902,062.40 1,130,488.00 1,072,592.80 379,400.00 176,558.00 623,704.00 83,229.00 426,763.20 1,064,957.00 622,851.20 600,428.80 106,480.00 23,001.00 376,424.40 12,103,986.60 Total receipts () Requested EU contribution ()
DLO CERTH OWS PAQ BIT VFT AVT LG PDX NOVA FRAUNHOFER UCAM PNO UHU WU UNIBI UMUE EGE LEP RHO
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1,959,154.00 149,553.00 150,622.00 471,402.00 404,840.00 143,015.00 612,657.00 866,088.00 808,895.00 379,400.00 124,279.00 469,028.00 83,229.00 320,072.00 801,217.00 468,338.00 451,571.00 79,860.00 11,500.00 188,212.00 8,942,932.00
5,868,589.00 131,500.00 2,177,624.00 3,926,273.60 311956 SPLASH - Workplan table - - Page 48 of 49
Project Effort and costs
WT8:
1. Project number The project number has been assigned by the Commission as the unique identifier for your project. It cannot be changed. The project number should appear on each page of the grant agreement preparation documents (part A and part B) to prevent errors during its handling. 2. Project acronym Use the project acronym as given in the submitted proposal. It cannot be changed unless agreed so during the negotiations. The same acronym should appear on each page of the grant agreement preparation documents (part A and part B) to prevent errors during its handling. 53. Work Package number Work package number: WP1, WP2, WP3, ..., WPn 54. Type of activity For all FP7 projects each work package must relate to one (and only one) of the following possible types of activity (only if applicable for the chosen funding scheme must correspond to the GPF Form Ax.v): RTD/INNO = Research and technological development including scientific coordination - applicable for Collaborative Projects and Networks of Excellence DEM = Demonstration - applicable for collaborative projects and Research for the Benefit of Specific Groups MGT = Management of the consortium - applicable for all funding schemes OTHER = Other specific activities, applicable for all funding schemes COORD = Coordination activities applicable only for CAs SUPP = Support activities applicable only for SAs 55. Lead beneficiary number Number of the beneficiary leading the work in this work package. 56. Person-months per work package The total number of person-months allocated to each work package. 57. Start month Relative start date for the work in the specific work packages, month 1 marking the start date of the project, and all other start dates being relative to this start date. 58. End month Relative end date, month 1 marking the start date of the project, and all end dates being relative to this start date. 59. Milestone number Milestone number:MS1, MS2, , MSn 60. Delivery date for Milestone Month in which the milestone will be achieved. Month 1 marking the start date of the project, and all delivery dates being relative to this start date. 61. Deliverable number Deliverable numbers in order of delivery dates: D1 Dn 62. Nature Please indicate the nature of the deliverable using one of the following codes R = Report, P = Prototype, D = Demonstrator, O = Other 63. Dissemination level Please indicate the dissemination level using one of the following codes: PU = Public PP = Restricted to other programme participants (including the Commission Services) RE = Restricted to a group specified by the consortium (including the Commission Services) CO = Confidential, only for members of the consortium (including the Commission Services)
Restreint UE = Classified with the classification level "Restreint UE" according to Commission Decision 2001/844 and amendments Confidentiel UE = Classified with the mention of the classification level "Confidentiel UE" according to Commission Decision 2001/844 and amendments Secret UE = Classified with the mention of the classification level "Secret UE" according to Commission Decision 2001/844 and amendments 64. Delivery date for Deliverable Month in which the deliverables will be available. Month 1 marking the start date of the project, and all delivery dates being relative to this start date 65. Review number Review number: RV1, RV2, ..., RVn 66. Tentative timing of reviews Month after which the review will take place. Month 1 marking the start date of the project, and all delivery dates being relative to this start date. 67. Person-months per Deliverable The total number of person-month allocated to each deliverable.
311956 SPLASH
Contents
B1. Concept and objectives, progress beyond state-of-the-art, S/T methodology and work plan ............................2 B1.1 Concept and Objectives .....................................................................................................................................2 B1.2 Progress beyond the state of the art.................................................................................................................8 B1.3 S/T methodology and associated work plan ..................................................................................................18 B2. Implementation ..................................................................................................................................................24 B2.1 Management structure and procedures ....................................................................................................24 B2.2 Individual participants ................................................................................................................................30 B2.3 Consortium as a whole ...............................................................................................................................45 B2.4 Resources to be committed........................................................................................................................49 B3. Impact .................................................................................................................................................................51 B3.1 Contribution of this project to the expected impacts listed in the work programme ...............................51 Other national or international research activities ....................................................................................................57 B3.2 Dissemination and exploitation of project results, management of intellectual property ........................57 B4. Ethics Issues ........................................................................................................................................................62 B5. Consideration of gender aspects ........................................................................................................................64
FP7-KBBE.2012.3.4-02
SPLASH
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B1. Concept and objectives, progress beyond state-of-the-art, S/T methodology and work plan B1.1 Concept and Objectives Introduction Around the world steps are being taken to move from todays fossil based economy to a more sustainable economy based on biomass. The transition to a bio-based economy has multiple drivers; an over dependency of many countries on fossil fuel imports, the anticipation that oil, gas, coal and phosphorus are finite sources of energy and chemicals; the need for countries to diversify their energy sources, the global issue of climate change and the requirement to reduce atmospheric greenhouse gases, and the need to stimulate regional and rural development. The majority of organic chemicals and polymers are based on fossil raw materials, predominantly oil and gas. Non-energy applications account for 16% of crude oil1. Global petrochemical production is estimated at around 330 million tonnes. Primary output is dominated by a small number of key building blocks, which are mainly converted to polymers and plastics. This production requires large amounts of fossil fuels as feedstock and generates massive amounts of CO2. From a technical point of view almost all industrial materials made from fossil resources could be substituted by their bio-based2 counterparts. The historic low price of fossil feedstocks has restricted commercial production of biobased chemicals and polymers, current global production (excluding biofuels) is around 50 million tonnes3. The recent increase in oil prices and consumer demand for sustainability has now opened new windows of opportunity for bio-based chemicals and polymers. Industry is increasingly viewing chemical and polymer production from renewable resources as an attractive area for investment. Industrial biotechnology can enable a shift towards a biobased economy. The EU funded BREW project, which was based on independent analysis and expert input from industry representatives, analysed the market potential for group of bulk chemicals produced from renewable resources using biotechnology; projections were made that with favourable market conditions the production of bulk chemicals from renewable resources could reach 113 million tonnes by 2050, representing 38% of all organic chemical production4 with a reduction of energy consumption and an estimated GHG emission reduction of 282668 MtCO2e. SPLASH will broaden the range of platform biochemicals and biopolymers produced by biotechnological routes by making use of an algal species (Botryococcus braunii) with a unique ability to produce, as the main biomass component, compounds (long chain hydrocarbons) that can be used as a substitute for typical petrochemical raw materials and components as well as specific exopolysaccharides a resource for new functionalities and applications. These compounds can be obtained in high purity with innovative and simplified downstream processing, hence more economical and with lower energy requirement. Microalgae The fact that microalgae are identified with high potential for the production of biochemicals can easily be justified. Microalgae are very attractive for the purpose of producing energy-rich molecules as they are photosynthetic organisms that can live in various aqueous environments, such as saline or seawater. This gives them a low water footprint and moreover they do not have to compete with cultivated farmland. Although they are not superior to higher plants concerning photosynthetic efficiency, microalgae do have high growth rates and they provide much higher oil yields than higher plants such as palm, soybean or rapeseed oil, and do not produce lignocelluloses. Microalgae do not only use sunlight as energy source, but they are also very efficient in using fertilizers and waste streams as nutrient source. They could be used to clean these streams by removal of nitrogen and phosphate and use flue gas as source for carbon dioxide (1 kg of dry algal biomass utilises about 1.8 kg of CO2).
1 Shen, Li, Haufe, Juliane and Patel, Martin K.2009. s.l. : Utrecht University 2,3 Bio-based products chemicals and materials (pre-norm CEN/BT/WG 209: biobased product = product wholly or partly bio-based (=derived from biomass)) include all kind of bio-based chemicals, bio-based plastics and additives biodegradable and durable, biocomposites 4 Patel, Martin, et al. 2006.s.l. : Utrecht University.
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Up to now the commercial application of microalgae has primarily concentrated on high-value compounds (>10/kg), such as food and feed supplements, natural colorants, PUFAs, and cosmetic ingredients. Global production volume of algal derived products is still less than 5,000 ton/year. In order to enable the use of microalgae for the production of biochemical and biopolymers, the production volume should be increased and costs should be reduced. This requires new knowledge and technological developments. The intention of SPLASH is to develop knowledge, genes, strains, protocols, etc. needed to allow industrial production with algae on a much larger scale, analogous to Industrial Biotechnology5 with yeast, fungi and bacteria, for the production of biopolymers. For example, the scale of production of Industrial Biotechnology with microorganisms is up to 1 million ton/year 6 for several medium-value molecules (lysine, glutamic acid, citric acid and lactic acid with a value of 1-4/kg). Still higher production volumes are attained for slightly lower priced commodities such as bio-ethanol (55 million tonnes worldwide and 2.9 million tonnes in 20097 at a market value of about 0.6/kg). The aim of SPLASH is to develop the required knowledge and technology for the industrial production of platform biopolymers using algae as a renewable raw material and/or gene source. Why Botryococcus braunii? B. braunii is a green alga, widespread in freshwater and brackish lakes, that is resistant to a number of stress conditions. It is one of only a few known species that can accumulate large amounts of hydrocarbons (CxHy). The green algal genus B. braunii is also known for its unique and outstanding capacity to produce and excrete high quantities of long-chain hydrocarbons as well as an interesting group of polysaccharides that can be converted into platform biochemicals. SPLASH will focus on B. braunii races A and B. Race A produces C23C33 odd numbered nhydrocarbons, mono-, tri, tetra-, and pentaenes8, which can constitute up to 61% of the dry cell mass. The B race produces polyunsaturated and branched C30C37 terpenoid hydrocarbons which can accumulate to very high levels (2686% on dry weight)9. These hydrocarbons can replace fossil based hydrocarbons and therefore have a wide range of potential applications in the chemical and biopolymer industry. In addition to hydrocarbons, B. brauni produces extracellular (exo) polysaccharides both as cell wall component and as soluble exudate into the fermentation medium. The sugar composition of the polysaccharides is galactose as main component and the remaining fraction being mostly fucose, rhamnose, arabinose, xylose and glucose for some strains. Besides this, little is known about B. braunii polysaccharides even though very large amounts are released into the growth medium (0.25 to 1 g/L has been reported10). Galactose can be used for the production of adipic acid, and fucose/rhamnose can be used in the production of furan-based chemicals (one of the top 12 US DoE appointed biomass chemicals of the future). Another unique and advantageous feature of B. braunii is that the cell wall matrix acts as a sponge for the accumulation of the hydrocarbons, which can be up to 95% located in the outer wall of the cell. In addition B. braunii exudes the polysaccharides to the medium. The hydrocarbons and polysaccharides can therefore be easily extracted from the algal cell wall and medium culture, without impairing cell viability. Knowledge is presently limited for Botryococcus and litte is known about its genetics, life cycle, metabolic diversity. In addition its has a slow growth rate. SPLASH aims at acquiring this knowledge and go one step further to gene manipulation in order to increase product specificity and productivity.
5 Industrial Biotechnology often referred to as White Biotechnology, is the industrial process to convert sugar via microbial fermentation or enzyme catalysis into chemicals, food and feed additives, pharmaceuticals and fuels. In the present Project Industrial Biotechnology (Industrial Biotechnology) with algae is the conversion of CO2 into hydrocarbons and Polysaccharides for platform biochemicals. 6 Industrial biotechnology. http://www.springerlink.com/content/w000241l02j83316/fulltext.pdf 7 www.epure.org 8 Gelpi, E., Schneider, H., Mann, J., Oro, J. 1970. Phytochemistry. 9, 306. 9 A.C. Brown, B.A., Knights and C. Ellisie. 1969. Phytochemistry 8, pp. 543547. 10 Allard B. and Casadevall,E. 1990. Phytochemistry 29, 1875-1878.
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311956 SPLASH Because in many approaches to metabolic engineering, particularly with microorganisms, it has often proved easier and more effective to introduce a novel pathway into a nave host than to manipulate it in the original organism and since there is at the moment very little knowledge on Botryococcus, the green microalga Chlamydomonas reinhardtii is included as potential production platform in its own right, and as a platform to test unique hydrocarbon and polysaccharide-producing genes from B. braunii. C. reinhardtii has been chosen because it is a photosynthetic unicellular green algae (like Botryococcus) and the only one for which transformation protocols are available. In addition there is extensive knowledge on the metabolism and physiology of this strain and the only algae genome-scale model available is for C. reinhardtii.11. Overall project concept and objectives The overall concept of SPLASH is to produce biopolymers by using B. braunii as a producer and/or gene source for hydrocarbon and polysaccharide-producing genes. B. braunii and C. reinhardtii (both green microalgae) will be developed as production platforms. In addition, C. reinhardtii will be used to test candidate hydrocarbon-producing genes identified from B. braunii. An integrated downstream process for product recovery and further conversion of the hydrocarbons into polyolefin building blocks ethylene and propylene, and conversion of polysaccharides into adipic acid, 2,5-furandicarboxylic acid (2,5FDCA) and 1,4-pentanediol, will be developed. These dicarboxylic and diol building blocks will be further polymerised into (co-)polyesters PEF (a substitute for PET) and PPeAF, and tested in the production of polymer fiber applications and packaging, respectively (Figure 1). SPLASH comprises the complete chain of knowledge, from the development of enabling technologies from systems biology to product development, sustainability assessment and market analysis, not only needed for a successful development of the technology but also for commercial implementation in the medium term. The whole process chain will be integrated and assessed in terms of economics and sustainability.
Figure 1. Concept for the production of biopolymers from a range of new platform algal biochemicals.
In SPLASH, three development paths (see Figure 2) are followed to reach the main objective of the project, namely the development of at least one production platform for the sustainable production of algal hydrocarbons and (exo) polysaccharides, combined with an integrated downstream process for product recovery, isolation and conversion into biopolymers. 1. Development of production platforms based on compounds from the natural B races of B. braunii . This path includes the optimization of medium and process conditions to get an optimal growth rate and hydrocarbons and polysaccharide production. New growth and cultivation methods will allow the
11 Boyle, N.R., and Morgan, J.A., 2009. BMC Systems Biology, 3, 4
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311956 SPLASH natural and recalcitrant B. braunii to grow to higher densities and permitting the production of higher amounts of hydrocarbons and polysaccharides needed for further product development and testing. The path also includes the development of procedures for innovative and low-cost in situ continuous extraction and separation of hydrocarbons and polysaccharides, conversion into chemicals and scale up of production of one or more of the production platforms to allow product development. 2. Development of mutants using random mutagenesis and selection. Mutants with reduced metabolic complexity and/or higher growth rates are generated, which next follow the same path as path 1 with regard to process design, downstream processing and scale-up. However, since the wild-type strains are available from the start, considerable knowledge on optimal medium, process and extraction conditions will be available for the mutants made in this path but also in the next path (3), and it is likely that no or only slight modifications to the process design and DSP established for the wild type will be needed. 3. Development of production platforms based on genetically engineered microalgae. We will carry out fundamental omics-based research, including genome sequencing, to understand the molecular biology and metabolism of the natural hydrocarbon-producing B. braunii. We will use an integrated approach using state-of-the-art genomics, metabolomics, proteomics and fluxomics, in order to gain the most comprehensive insight into complex cellular processes and biosynthetic pathways controlling hydrocarbon composition and accumulation. Such an integrated approach will reveal relationships between genes, transcripts, enzymes and metabolites that may not be revealed by classical approaches12,13. From these relationships, metabolic engineering strategies will be developed. To be able to implement these strategies, SPLASH also focuses on the development of effective and stable transfection protocols for B. braunii, leading to mutants producing specific hydrocarbons and polysaccharides. Further, establishment of ectopic over-expression of B. braunii genes in C. reinhardtii, for which transformation protocols are available, will enable the identification of critical hydrocarbon and polysaccharide-producing genes, and will lead to establishment of this microorganism as possible alternative production platform for specific hydrocarbons. Promising engineered lines will then enter the process design, DSP and scale-up part, similar to the mutants generated in path 2. All three paths end with the development and testing of a new generation of platform biochemical building blocks and biopolymers based on hydrocarbons and polysaccharides from microalgae.
Figure 2. Overview of the SPLASH project concept. The overall objective is to develop an industrial platform for production of hydrocarbons and exopolysaccharides. These will be converted into polyesters, copolyesters: polyethylene furandioate (PEF,a substitute for PET-polyethylene terephthalate), polyolefin building blocks ethylene and propylene, and polyamides, and will be
12 Joyce and Palsson .2006 13 Oksman-Caldentey and Saito.2005
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tested in the production of yarns and packaging materials. Three routes are followed to establish production platforms for different hydrocarbons. Route (r1) starts from the natural B. braunii. Route (r2) also starts from natural B. braunii strains, but using mutagenesis to identify strains with enhanced growth and loss of metabolic functions, thus reducing the metabolic complexity of B. braunii. The third route (r3) uses metabolic engineering for ectopic expression of hydrocarbon and polysaccharide genes in C. reinhardtii (Z) of B braunii where metabolic engineering targets are obtained from WP2 (X), metabolomics of the generated mutants in WP2 (Y).
This project is fully compliant with the requirements and objectives of this call. The whole process chain will be considered in the project: strain improvement, gene transfer, growth and production optimization, development of in situ continuous extraction and separation methods, product isolation, conversion into biopolymers and scale up to pilot for demonstration. For the development of a pilot scale production platform the project will take advantage of the recently funded Algae Production and Research Centre 14 (AlgaePARC) in Wageningen UR (The Netherlands). AlgaePARC is an excellent, international applied research centre aiming at filling the gap between fundamental research on algae and full-scale algae production facilities. Within SPLASH a, pilot-scale photobioreactor will be set up at AlgaePARC in order to evaluate the system stability, biomass productivities and in situ extraction at pilot scale, and use it to link fundamental research with full-scale algae production facilities. This will aid the development of industrial-scale production and extraction concepts. Implementation will be into existing markets that presently use fossil feedstock. The specific project objectives are: 1. Produce polyesters PEF (a substitute for PET-Polyethylene terephthalate) and PPeAF and polyolefin precursors ethylene and propylene from the hydrocarbon and (exo)polysaccharide fractions of the green microalga B. braunii (WP4 & WP5). 2. Use comparative genomics (genome sequencing, transcriptomics, proteomics and metabolomics) to identify genes for enzymes involved in hydrocarbon and polysaccharide biosynthesis, and their regulation (WP2). 3. Develop both non-GM and GM methods for selection and improvement of the B. braunii production platform (WP2). 4. Use transformable green alga Chlamydomonas reinhardtii as host for testing metabolic engineering approaches, including a selection of hydrocarbon producing genes from B. braunii (WP2). 5. Optimize cultivation methods to get enhanced growth (at least 2 times higher) , and production of selected hydrocarbons and polysaccharide by B. braunii and C. reinhardtii (WP3). 6. Scale up cultivation of B. braunii to produce hydrocarbons and polysaccharides during the scope of the project (WP3), at quantities enough (about 60 kg sugars and 20 kg hydrocarbons) to allow development and testing of new polymers (WP4 & WP5). 7. Develop and scale up to pilot in situ continuous extraction and separation methods for hydrocarbons and polysaccharides (WP4 & 5). 8. Develop drop-in polymer building blocks from the hydrocarbon fraction (WP4). 9. Develop a range of industrial products from the polysaccharide fraction: adipic acid and furanic chemicals for the production of polyamides and polyesters (WP4). 10. Demonstrate applicability of products generated in polymer fibers and packaging (WP4 &5) 11. Assess the economics and sustainability of the integrated process (WP6) 12. Ensure the transfer of knowledge and exploitation of the results of SPLASH by industrial users (WP1 and WP7). 13. Train young researchers in the field of biorefinery and microalgae biotechnology (WP7) European challenges addressed by the project The energy and chemical industries are Europe's largest and most competitive industries. More specifically, the chemical industry is Europe's third largest manufacturing industry, employing 1.7 million people directly, with up to 3 million jobs depending on it. Besides several leading global (petro)chemical companies, this industry also comprises around 36,000 Small- and Medium-sized Enterprises (SMEs).
14 http://www.algae.WU.nl/UK/projects/Algae+Pilot+Production+and+Development+Centre/ FP7-KBBE.2012.3.4-02 SPLASH
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311956 SPLASH Compared to other industrial sectors, the importance of the chemical, material and energy-related industries is likely to grow, because growth of the production and demand for e.g. transportation fuels, bulk chemicals and fine chemicals/pharmaceuticals already outpaces that of most other bulk materials. Whereas in 2000 the world production of base chemicals (mostly used for the production of a variety of polymeric materials) was about 200 million tons, in 2006 this was already 300 million tons 15. This production of base chemicals for polymeric materials requires large amounts of fossil feedstocks and generates massive amounts of CO2. It has become increasingly clear that continued reliance on fossil fuel energy resources is unsustainable, owing to both depleting world reserves and the greenhouse gas emissions associated with their use. Therefore, there are intensive research initiatives aimed at developing alternative renewable and potentially carbon-neutral feedstocks for the chemical and energy-related industries. A significantly increased use of renewable feedstocks in these industries would not only avoid the impact of global warming, it would also significantly reduce Europes dependence on foreign crude oil imports. SPLASH addresses several European action plans and political measures that have been put forward in the last few years concerned with socio-economic, geo-political and evironmental challenges such as: (1) actions to continue and stimulate R&D by building a European Knowledge Based Bio Economy; (2) decrease of CO2 emission (Kyoto Protocol; Copenhagen Climate Summit); (3) actions to broaden the carbon-resource basis from fossil to natural resources. SPLASH aims to exploit one of the unique strengths of algae, the ability to produce hydrocarbons using energy from photosynthesis. These hydrocarbons mimic the properties of typical petrochemical feedstocks, and are therefore excellent starting material for biopolymers, fuels and base chemicals. SPLASH will therefore enable the European industry to become less dependent on fossil fuels and to reduce the pressure on the environment. In addition to industrial applications, SPLASH will provide fundamental information about the biology, the physiology and metabolic diversity of hydrocarbon and polysaccharide-producing microalgae, and a considerable number of molecular resources that will be extendable to microalgal species in general. Moreover, the interdisciplinary nature of SPLASH will ensure the training of highly skilled researchers and support personnel, who will provide the future workforce to underpin economic and environmental developments in the marine and maritime sectors. The involvement of industry as key members of the Consortium will foster intimate and effective cross-sectoral integration and will improve and enable knowledge transfer. Outputs from SPLASH will be (i) infrastructure for exploitation of microalgae; (ii) development of new products and biobased production technologies; (ii) substitution of conventional petrochemical products with renewables, thus contributing to mitigation of CO2 emissions; (iii) highly skilled personnel with expertise in algal microbiology, microalgal cultivation and processing systems biology, and algal molecular biology and physiology; (iv) knowledge transfer into the several SMEs associated with the Project.
15 Biomass for the Dutch Chemical Industry. Opportunities for agriculture. R. Blaauw; J. van Haveren; E. L. Scott; H. L. Bos. FP7-KBBE.2012.3.4-02 SPLASH
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311956 SPLASH B1.2 Progress beyond the state of the art Table 1. Resume of the progress beyond state of the art State-of-the-art Systems Biology & Strain Engineering (Post-) genomics and systems Only for C. reinhardtii without modelling and integration (WP2) covering all cellular levels Development of a genetic Inexistence for Botryococcus engineering platform (WP2) Metabolic engineering (WP2) Never applied to Botryococcus
Progress beyond state-of-the-art Integrated approach applied to a new alga: Botryococcus braunii Genetic engineering platform for Botryococcus braunii Development of metabolic engineering strategies to increase the production and obtain desired hydrocarbon and polysaccharide profiles in Botryococcus
Process design, integrated downstream processing and scale-up Scattered data on effect of Biomass and product optimisation culture parameters on growth
in indoors (WP3) Biomass and product optimisation outdoors (WP3) Very limited data on product formation
Systematic approach on effect of culture parameters on growth Validation of results indoors New solvent (supercritical fluids) and experiments done under well-defined controlled conditions. First time applied to microalgae Ethylene and propylene from algal derived naphtha Production of adipic acid from algal derived sugars PEF as a replacement of PET with exceptional material properties possibility to extend applications. Polymerization and processability of PEF has never been done Production of 1,4-pentanediol from algal sugars via a new route with fewer steps
No data available for Botryococcus Always done with organic solvents. Lack of well-defined Milking (WP3) process conditions in experiments with Botryococcus Continuous solid liquid Rotary Vacuum Drum Filter (WP3) separation Product development Ethylene and propylene from Hydrocarbons (WP4) fossil derived naphtha Production of adipic acid from Polysaccharides (WP4) benzene (fossil origin) PET production with fossil fuels Production of 1,4-pentanediol from sugars
Polysaccharides (WP4)
Polysaccharides (WP4)
Introduction Microbial fermentations have been used for production of fermented food and beverages since ancient times. In 1920 microbial fermentation was introduced for the production of citric acid, the first large scale industrial production process of a chemical compound based on microbial fermentation. With the development of genetic engineering in the 1970s it became possible to produce compounds that are not native to microbes, such as pharmaceutical proteins like human insulin and human growth hormone using fermentation technology. Genetic engineering also allowed the transformation of microbes into cell factories for the production of chemicals through so-called metabolic engineering, a field dedicated to design microbial metabolism efficiently to convert cheap raw materials like glucose and sucrose into higher-value chemicals. With the further development of genomics and -omics analysis and advanced modelling tools in the field of systems biology it has become possible to perform very detailed phenotypic characterization of microorganisms that can serve as efficient cell factories for the production of fuels and chemicals. Thus, the last 10 years have witnessed a substantial technology push
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311956 SPLASH in terms of cell factory design, and with the recent desire to develop more sustainable processes for the production of fuels, chemicals and materials, the fuel and chemical industry is trying to exploit these technological developments. The net result is the development of what is generally referred to as industrial biotechnology. There are already several examples of how the fuel and chemical industry is trying to develop novel bioprocesses that can change from the use of mineral oil as the primary feedstock to the use of agricultural based products: 1 Dupont, one of the largest chemical companies in the world, has launched a process for production of 1,3-propanediol using a recombinant Escherichia coli. 1,3-Propanediol is used as one of the key chemicals in the production of the polymer Sorona that is used for the manufacturing of fabrics, carpets and a wide range of plastic based materials. The process was developed through close collaboration of Dupont, Genencor and Tate&Lyle. 2 DSM has launched a completely biotechnological route for production of the antibiotic cephalexin, which was earlier produced by chemical conversion of penicillin. 3 BASF has launched a completely biotechnological route for production of the vitamin riboflavin, which is used as feed additive. The earlier process relied on several chemical synthesis steps. The biotech route resulted in both a reduction in raw materials and energy usage. 4 Dupont has entered into a joint venture with British Petroleum (BP) on developing biobased production of butanol as a sustainable biofuel. 5 Dupont has also entered into another joint venture with Danisco for the development of a secondgeneration bioethanol production plant that will rely on the use of lignocellulosics as raw materials for ethanol production. BP and Verenium have formed another joint venture with the same target (using a different technology though). 6 ExxonMobil has entered into a joint venture with Synthetic Genomics to develop a novel microalgae-based process for the production of biodiesel. 7 Novozymes has entered a joint venture with Cargill with the objective to develop a biobased process for the production of 3-hydroxypropionic acid (3-HPA), which is to be converted to acrylates for the production of a range of personal care products, e.g. diapers and other hygienic products by the Swedish company SCA. The Swedish company Perstorp recently received a grant from Vinnova to develop a biobased process for production of 3-HPA. These and many other examples clearly demonstrate two key points: 1) the large chemical, material and fuel companies are turning to biotechnology as the solution to develop sustainable processes for the production of fuels and chemicals, and 2) most novel processes are developed through close collaboration/joint ventures involving two or more companies, and often also involving academia as a provider of novel technologies. The reason for the latter is that the development of a novel bioprocess requires a wide range of competences, and rarely are all these present within a single company or research group. Traditional chemical companies hold the necessary engineering competence required for scale-up and plant construction, but they often lack competence on the biotechnological aspects. There is, therefore, an increasing demand for the collaboration between academia and SMEs to provide the necessary competence base and ensure training of workforce to meet societal demand in industrial biotechnology. The challenge is not only to substitute limited fossil resources with undepletable renewable resources, but also to do this with feedstocks having high productivity and requiring a minimum of arable land, and resources such as water and phosphate, so as to avoid competition with food production and destruction of biodiversity. Current bio-resources derived from terrestrial crops such as sugarcane, sugar beet, maize and rapeseed place an enormous strain on world food markets, contribute to water shortages and speed up the destruction of the worlds forests. Advanced materials, chemicals and biofuels derived from lignocellulosic agriculture and forest residues and from non-food crop feedstocks address some of the above problems. However, there is concern over competing land use or required land use changes. Therefore, based on current knowledge and technology projections, even more advanced materials, chemicals and biofuels specifically derived from microalgae are considered to be a technically viable alternative resource that is devoid of the major drawbacks associated with terrestrial feedstock sources.
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Despite their potential as a resource for exploitation, microalgae are not nearly as well understood as other microorganisms that have found a role in todays biotechnology industry. This is due in part to the relative small size of the scientific community involved in the field. As a result, there is a significant backlog for algal biotechnology compared to what is available for industrial biotechnology. For the target genus Botryococcus in particular, knowledge is limited, and more fundamental research will need to be done to understand its genetics, life cycle, metabolic diversity and slow growth rate. Furtherm ore, stable transformation and expression systems are currently missing for Botryococcus. A part of SPLASH will be dedicated to tackling these issues, and the development of successful transformation protocols to genetically engineer Botryococcus will represent a major breakthrough for algal biotechnology. Progress in the field of microalgae will represent major gains for the chemical , material and energy industry sectors, and the development of the Agribusiness sector for microalgae production and processing will lead to an increase in job opportunities. In turn, the need to train professionals in the field will have a positive benefit for the algal scientific community. (Post-) Genomics and systems modelling and integration (Post-)genomics Genomic approaches have revolutionized our understanding of life processes in cells, and made an enormous contribution to the successful implementation of many industrial biotechnological platforms with microbes. With the advent of algal genome sequencing, it is clear that the same holds true for these organisms, and many novel metabolic pathways have been identified and elucidated through the lens of genomics16. As a complement to the sequence information, omics technologies have provided insights into many important aspects. Transcriptomics, including microarrays and deep sequencing, are already well developed in C. reinhardtii as exemplified by numerous publications10. Similarly, proteomics, using highly sensitive mass spectrometry techniques, has become a prime tool in the analysis of protein identity, protein localization and protein dynamics in algal model systems17. Both approaches can be established for any species with good genomic sequence information. In contrast, metabolomics can be carried out without knowledge of genome sequence. Both anonymous profiling, for example with 1HNMR, or GC-MS have been used successfully to study metabolism in tomato fruit, or production of volatiles after pest attack. Alternatively, targeted metabolite studies focus on a group of compounds, such as oil composition in oilseed crops. So, how will we advance the state-of-the-art? Data analysis and integration With the large amounts of data generated by the omics techniques, it is essential to take a systems approach and build appropriate models, particularly of metabolic fluxes. Such models have been generated for several organisms, but for algae the only genome-scale model available is for C. reinhardtii.18 However, this model has not been experimentally validated yet. In summary, the individual omics techniques have been developed and applied for model algae. However, the integration of the different levels into a systems approach is very limited and most work is done for C. reinhardtii. To take this a step further, a fundamental aspect of the SPLASH project, which will underpin the entire programme of work, is to apply a systems biology approach to B. braunii hydrocarbon and polysaccharide synthesis, including obtaining the genomic DNA sequences, and further genomic approaches such as transcriptomics, proteomics and metabolomics. With these tools in hand, we will use a combination of labelling techniques, thermodynamic analysis and gene expression analysis to build systems models of hydrocarbons and polysaccharides metabolism in B. braunii. This will allow us to gain the most comprehensive insight into complex cellular processes and biosynthesis pathways underlying the physiological and genetic basis of differential growth rate and hydrocarbon and polysaccharide composition accumulation and excretion. Such an integrated approach (Figure 3) offers possibilities to reveal relationships between genes, transcripts, enzymes and metabolites that may not be revealed by classical approaches. This will provide the essential foundation on which to devise
16 Grossman, A.R. 2007. Adv Exp Med Biol.616:54-76. 17 Rolland et al., 2009 18 Boyle, N.R., and Morgan, J.A., 2009. BMC Systems Biology, 3, 4 FP7-KBBE.2012.3.4-02 SPLASH
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311956 SPLASH metabolic engineering strategies to modify hydrocarbon and polysaccharide production in this alga, or other platforms.
Figure 3 Research Strategy: integrated & iterative
Development of a genetic engineering platform There are currently no published reports on the identification or generation of genetically altered strains of B. braunii. In biotechnology, random mutagenesis is a classic technique for strain improvement with a long history of success, especially suitable for improving complex processes like growth or multi step metabolic routes. A striking example is the improvement by at least 4,000 fold of penicillin titers during production. Also in algal research there are examples of improved properties of strains after using a mutation and selection procedure. For instance, astaxanthin-hyperproducing mutants of the green algae Haematococcus pluvialis19 and strains of C. reinhardtii with oxygen-tolerant hydrogen production20 have been obtained by mutagenesis, as well as mutants of C. reinhardtii defective in amylose biosynthesis21. The partner DLO-FBR has successfully applied these procedures for screening of lipid producing yeasts and fungi and for selection of PHA or PHB accumulating bacteria. The alternative approach to generate genetically altered strains is genetic modification. Currently there are about 10 different algal species that can be transformed, notably the green alga C. reinhardtii. It is possible to transform readily the nucleus, where the transgene is inserted randomly, and the chloroplast, where integration is by homologous recombination. 22 In addition, there is a substantial range of molecular tools available including well-developed selection markers, overexpression systems, and RNAi technology for down-regulation of target nuclear genes. Molecular tools for modification of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana are being developed, although they are still limited. Several other species have been successfully transformed, including Nannochloropsis oculata,23 but it is true to say that sophisticated metabolic engineering where several genes are overexpressed or down-regulated in a single organism - is currently only really possible with C. reinhardtii. In this project mutagenesis and selection procedures will be developed for B. braunii with the aim to generate mutants with desirable characteristics, in particular enhanced growth, and production of hydrocarbons and polysaccharides, but also auxotrophic mutants that might be useful as hosts for transformation. Furthermore, potential transformation systems for B. braunii will be investigated by a
19 Chen, Y., et al., 2003. Biotechnol. Letters, 25, 527-529. 20 Flynn, T., et al., 2002. Int. J. Hydrogen Energy, 27, 1421-1430. 21 Delrue, B., et al., 1992. J. Bacteriol., 174,3612-3620. 22 Walker, T.L., et al., 2005. J. Phycol., 41, 1077-1093. 23 Chen, H.L., et al., 2008. H.J. J. Phycol., 44, 768-776. FP7-KBBE.2012.3.4-02 SPLASH
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311956 SPLASH systematic analysis of different DNA delivery systems, regulatory elements and reporter genes, generated from the genomic studies. In parallel, further development of algal transformation protocols for metabolic engineering will be actively developed. These transformation systems can then be used for metabolic engineering purposes. Metabolic engineering Over the last 10 years or so, there has been increasing emphasis on using engineering design principles for manipulating metabolism, the so-called synthetic biology approach, which requires the appropriate toolkit, and extensive genomic information. In addition, detailed knowledge of endogenous regulatory mechanisms is necessary, including the production of starter substrates, subcellular location of the enzymes, and final destination of the product. To characterize efficiently genes identified in genomics programmes it is now routinely the case that there is a set of modular gene expression components (incorporating the appropriate regulatory elements) that can be custom assembled using multisite recombination technology for use in the appropriate organism. There have been some attempts at metabolic engineering in algae, mainly concentrating on increasing the levels of isoprenoids such as -carotene, by manipulation of the final enzymes of the carotenoid pathway24. However, attempts to engineer algal metabolism are very limited. In contrast, metabolic engineering in both bacteria and yeast is well-established and sophisticated, employing the use of multiple genes singly or in operons, together with enhancement of precursor synthesis and/or deletion of competing pathways. One particularly successful project in microorganisms resulted in production of large amounts of short-chain isoprenoids, at the expense of longer molecules. The first strategy that was tried was to inhibit the downstream enzymes, such as farnesyl PP synthase, so that C10 compounds were favoured, but in yeast and E. coli, this proved less successful than overexpression of terpene cyclases to siphon-off intermediates. Expression of the geraniol synthase gene (GES) from sweet basil in yeast resulted in synthesis of large amounts of this C10 isoprenoid, and its secretion into the medium.25 In E. coli, production of isoprenoids was found to be limited by the availability of the initial precursor isopentenyl pyrophosphate (IPP), but could be overcome by introduction of the complete yeast IPP biosynthesis pathway via mevalonate, comprising eight different genes.26 In this project we will develop metabolic engineering strategies to increase the production and obtain desired hydrocarbon and polysaccharide profiles. Different strategies can be applied ranging from overexpression of genes, gene knock out, or altering enzyme function in the form of, for example, substrate specificity and affinity or removing feedback inhibition. The target genes and the strategy applied will be based on the information obtained in WP2 (Tasks 2.2 and 2.3) especially the modelling using systems biology approaches. B. braunii will be used as a producer and/or gene source for hydrocarbon- producing genes and polysaccharides. B. braunii (microalgae), and C. reinhardtii will be developed as production platforms. Candidate genes will be expressed in a heterologous host to enable their function to be tested in a clean background. We will use C. reinhardtii for these experiments because each offers particular advantages; it is important to characterize these candidate genes in a photosynthetic context. Moreover, the fact that C. reinhardtii is unicellular green alga like B. braunii will offer insights in regard to product yield and downstream processing issues that will be addressed in WP3. The function of the candidate genes will be verified C. reinhardtii, before designing an appropriate engineering strategy to be carried out in both these model organisms. We will also investigate transformation protocols for B. braunii as a first step for metabolic engineering in this species (Task 2.6). Process design, integrated downstream processing and scale-up Biomass and product formation Biomass and product optimisation indoors
24 Raja et al., 2007. Appl. Microbiol. Biotechnol., 74, 517523. 25 Oswald, M., et al., 2007. FEMS Yeast Res., 7, 413-421. 26 Martin et al., 2003. Nature Biotech., 21, 796-802. FP7-KBBE.2012.3.4-02 SPLASH
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311956 SPLASH Much work has already been done on establishing optimal growth conditions for B. braunii.27 Factors studied include CO2, nitrate, nitrite, ammonia, phosphorous, pH, salinity, and light conditions. However, the maximum growth rate attained is still relatively low at about 1 doubling in 2 days. (=0.35 day-1). Furthermore, the environmental conditions also affect the products that are formed.22 For example, upon an increase in salinity the fatty acid distribution in the polar lipids changed towards more polyunsaturated fatty acids for strain A. In comparison with lipids, little is known about B. braunii polysaccharides, even though very high amounts are released into the growth medium. The production of extracellular polysaccharides has been reported in the three different races of B. braunii (A, B and L) and it ranges from 0.25 to 1 g/l of medium.28 In all the strains galactose is the main component of the polysaccharides. Besides this, fucose, rhamnose, glucose and two unusual sugars (3-O-methyl fucose and 3-O-methyl rhamnose) are also present in significant amount in the extracellular polysaccharides of some of the strains examined. However, the effects of process conditions on polysaccharide formation have not been studied. In summary, although much work has been done on the relation between process parameters and growth and product formation, the data are still, scattered and studies on interaction effects of different parameters as well as on polysaccharide production are limited. In this project the effect of relevant culture parameters, including media composition, light and cultivation parameters, on growth and product formation will be systematically studied using experimental design and random optimisation methods. Furthermore, we will elucidate efficiency of nutrient supply in order to validate the existing hypothesis that slow growth of B. braunii is due to the formation of colonies and consequent light and/or nutrient limitation. Cultivation strategies to reach high biomass concentrations and make use of this biomass for prolonged times will be developed. In this process the cells are used as a photocatalyst for the continuous production of hydrocarbons and polysaccharides. Ideally, in such a process the major part of supplied photosynthetic energy is converted directly into the products of interest, which are then excreted by the cells and can be recovered continuously.
Figure 4 AlgaePARC. The opening of this facility took place in June 2011. 2 2 There are 4 different production systems of 24 m each and 3 of 2.5 m each.
27 Banerjee A., et al., 2002. Critical Reviews in Biotechnology, 22, 245279.

28
Allard, B. and Casadevall, E., 1990. Phytochem., 29, 1875-1878.
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311956 SPLASH Biomass and product optimisation outdoors For sustainable production, microalgae should use sunlight. The quantity of solar energy consumed by algal cultures during the day, and as a function of the seasons, directly influences growth and overall performance. In particular, in SPLASH we are interested in the effect of changing light intensities and temperature on productivity of hydrocarbons and polysaccharides. Photosynthetic efficiencies reached outdoors range from 1 to 3 % depending on the cultivation system and operation mode29, while the theoretical maximum efiiciency is 9%. There is, without doubts, a large area for improvement. We intend to develop dynamic process strategies in such a way that optimal productivity is achieved and maintained throughout the entire day. Biomass density, medium composition, temperature, harvest and mixing mode are possible parameters to be considered in such strategies. In SPLASH we will use the infrastructure of a new algal pilot facility called AlgaePARC, from the partners DLO-FBR and WU (Figure 4). This has different cultivation systems of 24 m2 each (ranging from 500 to 5 m3) that can be used to test results obtained under artificial light at bench scale, and develop process strategies in an industrial-like environment, therefore facilitating process scale-up.
In situ recovery of hydrocarbons and polysaccharides and separation Different continuous and/or extraction and innovative separation technologies and methodologies for recovery of hydrocarbons and polysaccharides will be tested, first individually and at a later stage, integrated with the cultivation process. A technical, economical and sustainability assessment of the different routes will be performed in order to choose the best system/process to be scaled-up and demonstrated at pilot-scale. Integration with the cultivation system at pilot scale in AlgaePARC will also be done. Supersonic flow fluid processing This includes, the implementation of industry-proven supersonic flow fluid processing, an existing and proven fluid processing Reactor technology for the separation of the hydrocarbons and polysaccharides (Figure 5). The Reactor system is a fluid/fluid reactor that utilizes the injection of supersonic steam to initiate a thermofluid interaction with the process fluid, achieving homogenous disruption, agitation, heating and mixing. These controllable forces are achieved fluidically with no moving parts, allowing flexibility and durability. The energy intensity of the fluid processing forces can be controlled, with the goal being to find a balance between the forces required for extraction, whilst minimizing the damage to the algae cells. The extraction method will be developed so that it is integrated to the upstream production and downstream separation processes.
Figure 5. Supersonic fluid processing
Botryococus is a mat forming algae. The algal cells exude a polysaccharide into their local environment to modify their local conditions as a protection mechanism. The result is a gel or slime mat around cell colonies. The lipid is held in the walls of the algae, but can be produced at a level that allows it to be excreted through the wall and sit in the polysaccharide matrix around the cells. The goal is to milk the cells and return them to culture for further lipid and polysaccharide production, so careful consideration will be made about the process conditions. Based on findings from previous commercial studies on other algae strains, the cell membranes of the algae are expected to be increasingly sensitive to temperature and there is a risk of disruption above approx. 40C. In passing through the low pressure zone of the Reactor system. Accordingly, all of the vapour pressures, boiling, and melt points will be shifted down for limited time at high speed. The study will include an investigation into whether this phenomenon can be tailored to maximise the separation of the hydrocarbons from the cell walls, whilst minimising cell
29
Wijffels, R.H.; Barbosa, M.J. (2010) An Outlook on Microalgal Biofuels. Science. 329: 796 - 799.
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311956 SPLASH wall/cell membrane damage. It will be the first time that Supersonic flow fluid processing is applied to microalgae cultivation with this aim. Milking The continuous extraction of products (milking) from microorganisms has been reported previously by Frenz et al.30 In their method, the cells were harvested, separated and then contacted with the organic phase for extraction, after which the cells were returned to the bioreactor. More recently, a new method was developed for milking -carotene from Dunaliella salina in a two-phase bioreactor.31 In this technique, the -carotene is extracted selectively via continuous re-circulation of a biocompatible organic solvent through the aqueous phase containing the cells. In SPLASH this concept will be further explored and a continuous in situ extraction system for hydrocarbons will be developed. Both organic solvents and supercritical CO2 will be tested for extraction. In addition to hydrocarbons, extracellular polysaccharides will be separated from the broth and further converted into biopolymers. Rotary vacuum drum filter This set-up consists of a drum rotating in a tub of liquid to be filtered (Figure 6). This is a state of the art technology but for the first time it will be adapted to microalgae in an innovative concept in which the cells and products can undergo in cascade several types of separation and purification steps; cell separation from the medium, rinsing the cells with organic solvent to extract the hydrocarbons (while maintaining the cells alive) and further separation of the liquid phase.
Figure 6. Rotary Vacuum Drum Filter
Product development Greening the chemical and material industry B. braunii has the remarkable capacity of producing large amounts of hydrocarbons and polysaccharides. To our knowledge, the only products used outside the pharmacy sector are only some exopolysaccharides from Chlamydomonas mexicana as soil conditioner32 and from Porphyridium cruentum.33 Altogether about 70 scientific papers have been published about the structure of some of these cell wall materials. The search for applications of chemicals produced by micro-algae has barely started, and no routes toward bio-based polymers derived from B. braunii constituents seems to have been reported so far. On the fuel and oil side, a single recent June 2009 patent (WO 2009/071629 A1) describes the use of B. braunii races B for the production of biolubricants. Consequently, there is a vast empty field for developing new products. In this project our research is aimed at developing new renewable commercial product lines for the polymer industry. Hydrocarbons The type of hydrocarbons produced by B. braunii depends on the race. Hydrocarbons are the major hydrocarbon type present in B. braunii race A, hence the A. The hydrocarbons are linear olefins with chain lengths ranging from 23 to 33 carbon atoms. For race B, the major hydrocarbons are so-called botryococcenes, which are C30C37 triterpenoids, and C31C34 methylated squalenes. Finally, Race L produces a single tetraterpenoid hydrocarbon, lycopadiene (Metzger & Largeau, 2005).34 The current feedstock for all petroleum-derived chemicals is a light oil distillate called naphtha. By subjecting naphtha to a process called steam cracking, C2C4 olefins are produced which serve as the primary feedstock for other chemicals: ethylene, propylene, C4 olefins. The chemical industry is actively
30 Frenz, J. et al., 1989. Enzyme Microb. Technol., 11, 717724. 31 Hejazi, M.A. and Wijffels, R.H., 2004. Biotechnol. Bioeng., 85, 475481.
32 Koren, K. M. and Rayburn, W. R., 1984. J. Phycol., 20, 253. 33 Anderson, D. B. and Eakin, D. E., 1985. Biotechnol. Bioeng. Symp., 15, 533 34 Metzger, P. and Largeau,C., 2005. Appl. Microbiol. Biotechnol. 66, 486496.
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311956 SPLASH searching for green alternatives for naphtha, since this would allow them t o keep using the existing chemical factories and infrastructure. The hydrocarbon fraction isolated from B. braunii can be regarded as a heavy gas oil, containing highboiling-point hydrocarbons of molecular weight 400 Daltons and higher. In petrochemical refineries, similar oil fractions are treated in a process called fluid catalytic cracking (FCC), by which the long carbon chains are cleaved to shorter-chain products at high temperature and moderate pressure, with a fluidized powdered catalyst. The products of this process are suitable for steam cracking to base chemicals. Hence, hydrocarbons from B. braunii may serve as a sustainable source of green naphtha for the chemical industry. In SPLASH, the hydrocarbons from B. braunii will be subjected to small-scale FCC and steam cracking in order to transform them into existing C2C8 base chemicals. Since the algae hydrocarbons are an unconventional, new feedstock for the FCC cracking process, adaptation of reaction conditions and catalysts may be required to obtain green naphtha with the right product mix for further steam cracking. This is the task of the partner FRAUNHOFER. The focus will be on the production of ethylene and propylene as building blocks for polyolefins. The polymerization of algaebased ethylene and propylene is not expected to be different from the existing polymerization process, and will therefore not be pursued in the project. Polysaccharides Galactose, the most abundant monosaccharide produced by depolymerisation of the B. braunii polysaccharides, is an interesting building block for the production of reactive carbohydrates, such as galactose dialdehyde (galacto-hexodialdose, GALA) and galactaric acid. These compounds belong to a larger family of materials known as oxidized sugars, and represent what we believe to be a significant market opportunity. They can be used as building blocks for the production of biobased nylons, polyesters and polyamides. In SPLASH, galactose from B. braunii exopolysaccharides will be chemoenzymatically converted to galactaric acid. Subsequently, galactaric acid will be converted to adipic acid by catalytic hydrogenolysis. (1,6-hexanedioic acid) is an important building block for nylons and other condensation polymers, with a global annual market volume of more than 2 million tonnes. It is currently derived from benzene. Glucose from B. braunii will be thermally dehydrated to 5-hydroxymethylfurfural (HMF) and subsequently oxidized to 2,5-furandicarboxylic acid (2,5-FDCA) which will be further converted into PEF films. This conversion is expected to cause a major stir in the polymer industry. The partner AVT has 15 patent families on the production and usage of furanics for biofuels and Biopolymers applications and has already impressed the scientific community by exploring, funding, and establishing a FDCA pilot facility at Chemelot. The combined efforts with end users on PEF films will hold radical innovations and a major breakthrough beyond the international state of the art on: Synthesis of a completely biobased engineering polyester with: o very favorable Non-Renewable Energy Utilization (NREU) and GHG emissions, o exceptional material properties making it possible to extend current PET applications, o competitive cost price opportunities, enabling to create a unique market segment amidst fossil and (semi) bio-based chemicals. o the polymerization chemistry and processability of the novel PEF-film system has never been studied before and constitutes a true scientific innovation. The project aims to provide sound information, data and properties on the newly developed polyester films. New polymer processing techniques which will lead to novel material properties, especially film drawing of PEF to produce a transparent sheets. New applications can be developed currently outside the reach of polyesters such as for instance when a high transparency is required in thick sheets of polymeric material (food storage containers, dishes, reusable bottles, jars, optical protection devices). In this proposal PTA is replaced by FDCA. This replacement will increase the Tg of the resulting polymer bringing applications above 80C within reach! Exploration of PEF engineering polyesters versus established PET developments.
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311956 SPLASH Rhamnose and fucose are both excellent starting materials for the subsequent chemoenzymatic synthesis of 5-methylfurfural. Whereas this compound is a promising platform chemical for the production of flavours, biofuels and solvents, in SPLASH, 5-methylfurfural will be converted to 1,4pentanediol. This diol will be used as a monomer for the preparation of bio-based polyesters, by polymerizing it with other building blocks pursued in the project. Although 1,4-pentanediol can be prepared from sugars via levulinic acid,35 the new route proposed in SPLASH via 5-methylfurfural contains fewer steps. The conversion of extracellular carbohydrates to bio-polymer building blocks requires the development of efficient methods for e.g. thermal dehydration, oxidation, decarboxylation, hydrogenation and hydrolytic (tetrahydro)furan ring cleavage. Effective thermal dehydration technology will be developed by SME partner Avantium. All sugars will be assessed for furanic production by Avantiums patented dehydration process into furanic components, to allow screening for effective heterogeneous sugar dehydration catalysts, using high-throughput experimentation technologies, as well as analytical GC and HPLC techniques with regard to sugar and furan analysis. The technology developed by AVT for converting sugars to dehydrated sugars under methanolic conditions, which is protected by various patent applications, will be further extended to sugars like rhamnose, fucose and galactose, and impure galactose-containing streams derived from the polysaccharide part of Botryococcus. A critical evaluation of the technical and economic feasibility of the technology proposed will be performed in WP 6, and when results recommend it, we will present this technology to leading companies in the field (i.e. Frutarom, AkzoNobel) aiming to demonstrate the feasibility at industrial scale. Product testing In this project polymers prepared from bio-based building blocks will be studied. In order to be able to replace fossil based polymers by polymers based on renewable resources in a successful way, these polymers must be intensively studied on their process ability in existing extrusion technologies. In addition the properties of the prepared polymer products from these materials, e.g. tapes and yarns must be investigated to be able to have a fair comparison between renewable and fossil based polymers. This is an important task to perform in order to prevent a decrease in material performance in a variety of applications upon moving to bio-based polymers. The testing of these polymers is necessary and unavoidable. In addition, the preparation of these polymers from bio-based building blocks could give us the opportunity to study the relationship between structure and performance by fine tuning the polymer structures. This project could be the first step to investigate using different varieties and ratios of biobased building blocks and their effect on the mechanical properties of the resulting materials. Furthermore, going from traditional polymers to bio-based polymers opens a new route to materials which could show a positive change in certain material properties. One could think of UV resistance, abrasion sensitivity and burn behaviour.
35 Mehdi, H.; Fabos, V.; Tuba, R.; Bodor, A.; Mika, L. T.; Horvath, I. T., 2008. Top. Catal., 48, 4954. FP7-KBBE.2012.3.4-02 SPLASH
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B1.3 S/T methodology and associated work plan Overall strategy of the work plan The aim of SPLASH is to develop the required knowledge and technology for the industrial production of platform biopolymers using algae as a renewable raw material and/or gene source. Key enabling technologies for establishing Industrial Biotechnology with algae are metabolic pathway engineering, fermentation science, innovative downstream processing and product design. A key technology in the successful application of metabolic engineering is the availability of a well annotated genome and quantitative tools or genome-scale metabolic models that permit manipulation of the genome. All these enabling tools are addressed or subject to further development in SPLASH. In addition to enabling tools, a robust production organism is essential for industrial production as well as innovative low-cost downstream processes. A four year programme of work has been prepared to maximise the likelihood of success of the project in meeting all of the objectives laid out in section 1.1.4. Each work package and task covers a specific activity and has been prepared in such a way to maximise the likelihood of success and minimise risks. Each of the RTD and DEM work packages directly involves the meeting of one or more of the projects scientific, technical or economic objectives, MGT and OTH work packages ensure the wider delivery of the project. The overall WP scheme of the SPLASH project is presented in Figure 7.
Figure 7. Anticipated process scheme and partners distribution per WP. WP leaders are in bold, underlined and with a darker background (WP 1, 2 and 3) or darker letter colour (WP 5, 6, 7).
Products of WP2 are concepts for enhancement of overall biomass yield, production of hydrocarbons and polysaccharides. These concepts will be translated to applied research and will be tested in photobioreactors (WP3). WP3 includes applied research, such as optimisation of cultivation parameters, analysis of the robustness and stability of the systems, development of process control strategies, scale-up and production of hydrocarbons and polysaccharides for WP4. In line product recovery methodologies and technologies will be developed in WP4. The hydrocarbons and polysaccharides recovered from the pilot and demonstration plant(s) will be the starting material for refinery, further isolation and conversion of hydrocarbons and polysaccharide to biopolymers and test in a final product (WP4). WP3 and WP4 will be up-scaled in demonstration activities to test and validate the production process at industrial similar conditions (WP5). During the entire project there will be a pilot production facility (WP3) dedicated to produce biomass, hydrocarbons and polysaccharides required for the development of downstream processes (WP3) and product development (WP4). In the end overall integrated process design should lead to optimisation of the total process (WP6). Full life economic and environmental sustainability will be advanced and assured (WP6). For instance, by using algae as a photocatalyst and focusing on extracellullar products we will reduce nutrient consumption, water usage and the need for additional steps such as breaking the cells and biomass pre-treatment. Such issues and the possibilities for embedded recovery and reuse of materials and energy to provide a self-contained system will be assessed.
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311956 SPLASH Timing of the different WPs and their components

Task A ct. no Type WP 1 M NG T1 .1 T1 .2 T1 .3 RTD C o o rdina t io n Kick-o ff meeting P ro cess management tasks Financial/administrative management F ro m s ys t e m s bio lo gy t o s t ra in e ngine e ring Reco nstructio n o f Geno me sequence o f B . braunii A nalysis o f metabo lic pathway regulatio n in B braunii Data analysis, reverse engineering and systems mo dels in B . braunii Verificatio n o f candidate genes fo r HC and EP S pathways fro m B . braunii Develo pment o f a green micro algal platfo rm fo r HC and EP S pro ductio n P ro c e s s de s ign: P ro duc t io n a nd do wns t re a m pro c e s s ing Optimizatio n o f gro wth and pro ductio n o f hydro carbo ns and po lysaccharides Outdo o rs co ntro l strategies and bio mass pro ductio n o f B . braunii Develo pment o f in situ DSP metho ds Integratio n and o ptimizatio n o f cultivatio n & DSP pro cesses B io safety P ro duc t de v e lo pm e nt a nd t e s t ing A nalytics o f lipid fractio n pro duced by B . braunii Develo pment o f a cracking pro cess to co nvert HC to green naphtha and to ethylene and pro pylene Hydro lysis o f algae po lysaccharides into mo no meric sugars Separatio n o f mo no meric sugar mixture into individual sugar fractio ns Co nversio n o f galacto se to adipic acid Co nversio n o f fuco se and rhamno se to 1 ,4-pentanedio l Co nversio n o f gluco se to 2,5-FDCA Co nversio n o f adipic acid and 1 ,4-pentanedio l into po lyesters Co nversio n o f 2,5-FDCA to P EF films Testing o f po lyesters fo r the pro ductio n o f fibers D e m o ns t ra t io n a t pilo t s c a le Demo nstratio n o f o ptimised integrated pro cess at pilo tscale Co nversio n o f hydro carbo ns and po lysaccharides into bio po lymers Techno lo gy and pro duct co mmercializatio n P ro c e s s int e gra t io n: e c o no m ic s , s us t a ina bilit y a nd m a rk e t a na lys is Co nceptual pro cess design Techno - eco no mic mo del: feasibility and o ptimisatio n LCA A ssessment Eco no mic assessment and market analysis Standardisatio n So cial aspects D is s e m ina t io n, e xplo it a t io n a nd int e lle c t ua l pro pe rt y m a na ge m e nt Internal co mmunicatio n External co mmunicatio n Explo itatio n Intellectual P ro perty M anagement M M M M M M M
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Figure 8. Gantt chart of SPLASH, the symbols represent: x = consortium agreement, = meeting (kick -off, project management team or general assembly meeting), M = milestone, = deliverable,
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Interdependencies of project components
Figure 12. Pert diagram showing the interdependencies of Work packages and Tasks.
Significant risks and their contingency plans In the following section an overview of the risks within the different work packages will be given including their contingency plans. For further details on risk assessment in this project see 2.1.7.4. Technological and scientific risks WP2 Strain development One of the objectives of WP2 is to generate modified algal strains that produce increased amounts and specific profiles of hydrocarbons and exopolysaccharides (galactose, rhamnose, fucose and glucose). Our approach is to do this by non-GM mutagenesis on the one hand, (loss-of-function), and develop transformation protocols (generally gain of function) on the other hand. Using these approaches will mitigate some of the risk. If the transformation process is not successful we will still be able to test candidate genes obtained from B. braunii and metabolic engineering strategies in the green alga Chlamydomonase and develop these into a platform for production of B.Braunii hydrocarbons and polysaccharides. Chamydomonas is a well-studied green microalgae with existing transformation protocols. WP3 Process design: Cultivation and Downstream Processing The first objective of WP3 is to develop a continuous integrated cultivation and downstream process for Botryococcus for hydrocarbons and polysaccharides production and recovery. In this WP3 there are multiple risks.
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311956 SPLASH The first risk is that further optimization of growth rate is simply physiologically not possible. This risk can be mitigated by developing cultivation methods that can maintain a high biomass density and use this biomass for as long as possible by applying continuous extraction. The second risk is that not enough hydrocarbons and polysaccharides are produced for the development of downstream processes during cultivation optimisation experiments. To mitigate this risk the partner BIT has the sole purpose of producing Botryococcus starting at month 1 of the project in an outdoor horizontal tubular photobioreactor (5.0 m3) located in the south of Spain, where productivities are higher. Expected annual production of either 800 kg (non-limited biomass) or 400 kg nutrient-limited, high lipid biomass. If everything fails partner from WP4 (product development) can start their experiments by using commercially available sugars for the production of adipic acid for polyamides / polyesters and furanic chemicals for PEF (substitute for PET) production. The third risk is that B. braunii cannot withstand continuous extraction resulting in rapid cell death and loss of productivity. To mitigate this risk we will test in parallel four different routes for extraction and separation of the fractions of interest. In the remote case that none of them show to be feasible, a two-step process should be developed with a separate growth and production/harvest reactor. If continuous extraction is not possible at all, batch wise harvest can be applied. Furthermore, the need to use organic solvents for continuous in situ extraction of the product which brings an additional challenge to scale up will be further minimized by testing as well supercritical CO2 as a clean solvent for extraction of hydrocarbons and the supersonic flow fluid processing Reactor developed by partner PDX. In case the cells need to be destroyed in order to extract the hydrocarbon fraction, additional components will be extracted from the remaining biomass such as polyacetals and polyaldehydes which have a potential commercial value. WP4 Product development and testing The objective of WP4 is to test the suitability of hydrocarbons and polysaccharides obtained in WP3 for the production of new renewable building blocks for bio-based polymers. The major risk associated with WP4 is the lack of product resulting from WP3, although with the measures taken for the risks in WP3 it seems unlikely that there will not be enough material available for testing. Nevertheless, if this is the case, development of adipic acid, 2,5-FDCA and 1,4-pentanediol will still be made by using commercial sugars (galactose, fucose, glucose and rhamnose) from independent sources as a starting material . Another, unlikely, risk is the unsuitability of hydrocarbons and polysaccharides produced by Botryococcus for the production of one or more renewable biopolymers proposed in this proposal. To minimize this risk, SPLASH focuses on several different renewable monomers/polymers. If one or more products seem not to be feasible, the focus will be shifted to one of the other chemicals/polymers. WP5 Process demonstration at pilot scale This work package aims at demonstrating the findings and developments in WPs 3 and 4 at pilot scale. Therefore, the major risks that could take place are similar to those in WP3 and 4 and have been previously addressed. WP6 Process integration: economics, sustainability and market analysis In WP6 several different studies will be performed in which different risks can be thought of: Environmental risks: Although not expected, the use of microalgae for sustainable bulk chemical production could have an adverse effect on the environment. This risk is actually quite low as it is known that microalgae are well suited for uptake CO236, can be grown in closed systems decreasing therefore the risk of impact on local ecology and in places not suitable for agriculture, avoiding competition with food production. In addition the water consumption is very low in comparison to agricultural crops. An LCA study will be performed to analyse in detail the impact of the process proposed in SPLASH on the environment. Economic / Market risks: There are several risks that could prevent wider implementation of microalgae use for bulk chemical production. First of all production should become cost competitive with at least other biobased chemicals and even better with petrochemical products.
36 Brown, L., Zeiler, K. (2003) Aquatic biomass and carbon dioxide trapping. Energy and conversion management. 31:1005-1013. ANNEX 1 SPLASH 21 | P a g e
311956 SPLASH A good prospect for commercialization of biobased bulk chemicals is the expectation that fossil resources will become less available and will increase in price. This improves the market position of biobased chemicals maybe even to a point that many subsidies for biobased products can be abolished. The development of policies on biobased chemicals by the EC in similarity to what has been done for biofuels37 would positively stimulate the development and introduction of bulk chemicals from microalgae and other renewable sources on the market. Large Research projects are underway to decrease production costs of microalgae cultivation and turn it into a competitive feedstock for bulk products38. Developments in these projects will have an impact in the competitiveness of the process in SPLASH. These developments will be closely monitored during the project and considered in the technoeconomic study (WP5). Risk management To identify and manage risk during the project, a risk register will be maintained by the project coordinator (PC) Maria Barbosa (DLO-FBR) who has an overview of the complete project and its progress and will provide appropriate level of contingency planning to address risks associated with this project. This register will record issues that challenge the achievement of milestones and project deliverables. At each 6 month project meeting, the risks, likelihood of occurrence and, severity can be discussed with all partners to determine the best remedial course of action. New risks will probably be identified during the course of the project and will be included within the register. If any high risk issues are identified between meetings, the PC will notify the partners and arrange working party meetings to discuss the risks and ensure expedient corrective action is undertaken. This register will be the primary working document for managing risks within the project, the action column will be updated as the project progresses. Risk Likelihood (1-5) Effect (1-3) Risk factor (1-15) Contingency plan
WP2 Transformation of Botryococcus fails
WP3 Improvement of growth rate is physiologically not possible WP3 Botryococcus cannot withstand continuous extraction WP3 Use of organic solvents for continuous in situ extraction of the product brings an additional challenge to scale up WP4 There are not enough
37
Botryococcus genes responsible for product formation will be expressed in the green algae Chlamydomonas. In addition nonGMO mutagenesis will as well take place in SPLASH Development of cultivation methods that can maintain a high biomass density and use this biomass for as long as possible by applying continuous extraction. Several different continuous processes will be tested so risks are spread. If none of them works, batch recovery of products can be applied. Supercritical CO 2 will be tested as a clean solvent for extraction of hydrocarbons and additional continuous extraction processes will be as well tested such as the supersonic flow fluid processing Reactor. The partner BIT is dedicated to biomass production at pilot scale (5
Directive 2009/28/EC of 23 April 2009 on the promotion of the use of energy from renewable sources and amending and subsequently repealing Directives 2001/77/EC and 2003/30/EC 38 AlgaePARC (www. algaePARC.com); FP7-ENERGY-2010-2 demonstration projects: InteSusAl, All-gasoil and BIOFAT
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311956 SPLASH hydrocarbons and polysaccharides for product development WP5 Hydrocarbons and polysaccharides produced by Botryococcus are not suitable for product development m3) in south Spain. Sugars will be bought to allow product development and testing The risk is minimized by focusing on different renewable polymers. If one or more products seem not to be feasible, the focus will be shifted to another product. Microalgae can be grown nonarable land and in closed photobioreactors. They do not compete with food or represent a danger to local ecology. They are also able to take up large amounts of CO2 making this risk not seen as a threat. An LCA will be performed in the project to address this issue. Large Research projects are underway to decrease microalgae cultivation costs39. In addition sustainability and the prices of fossil-based products will play a major role in bringing (or not) microalgae bulk chemicals into the market. Data on costs and energy requirement of the downstream are very scarce. In SPLASH information and data will be gathered on both the upstream and downstream. SPLASH will provide a new insight on the potential for short, mediumor long term economic viability.
WP6 Microalgae for the production of bulk chemicals has negative environmental consequences.
WP6 Microalgae for bulk chemical production is not cost competitive with conventional fossil based sources
39 AlgaePARC (www. algaePARC.com); FP7-ENERGY-2010-2 demonstration projects: InteSusAl, All-gasoil and BIOFAT ANNEX 1 SPLASH 23 | P a g e
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B2.
Implementation
B2.1 Management structure and procedures For a successful execution and management of SPLASH, an adequate and efficient management structure has been created that corresponds well to the size and complexity of the project. Several elements were taken into account when designing the management structure, such as appointing a Project Coordinator with extensive experience in both academic and private sectors, clear reporting and communication lines and frequencies; and a central role of the Project Management Team and Daily Management Team (including Project Coordinator) aimed at a successfully supporting the consortium in reaching its goals. The management structure will also be established in the Grant Agreement with the Commission as well as in the Consortium Agreement (CA), to be finalised during the contract negotiations. The FP7 Consortium Agreement DESCA will be used as model for the CA. Simplicity, flexibility and transparency are key factors to the management of the present project. The overall project management structure is presented in Figure 13.
Figure 13: Project management structure. (Double arrow = strong interaction; single arrow = advice; dashed arrow lines = membership)
The project consists of the following management and advisory bodies, described further in separate sections: 1. General Assembly (GA) 2. Project Coordinator (PC) 3. Daily Management Team (DMT) 4. Project Management Team (PMT) 5. Scientific Advisory Board (SAB) 6. Dissemination, Exploitation and Intellectual Property Advisory Board (DEIPAB) General Assembly (GA) The GA is the ultimate decision-making body and will be responsible for monitoring the project implementation, taking major strategic decisions and determining the long-term strategy and direction of the project. All project partners are represented at the GA, chaired by the PC. The GA will meet at least
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311956 SPLASH once a year to review the overall project progress. The GA shall have the following decision powers (to be defined in greater detail in the Consortium Agreement): Approval of major strategic decisions Approval of the long-term detailed work programmes, as implemented during the progress of this project Approval of the scientific and the financial reports for the European Commission Approval of any new contractors entering into the EU contract and the Consortium Agreement Agreeing upon the proposed budget allocation in accordance with the EU contract and proposing reallocations of any partner budgets Making proposals to the project partners for any review and/or amendment of the terms in the EU contract Agreeing upon proposals made by the PMT on defaulting parties Deciding upon the change and exchanges of tasks between project partners and propose respective amendments in Annex I of the EU contract Project Coordinator (PC) In SPLASH, Dr. Maria Barbosa will be the Project Coordinator. She has ample experience in managing R&D projects in the field of microalgae biotechnology as well as managing funding research programs (at the European Molecular Biology Organization EMBO). She is presently Director of AlgaePARC (Algae Production and Research Centre), an 8 million project subsidised by the Dutch government and 18 different industrial partners. The Project Coordinator carries, on behalf of the beneficiaries of the consortium, the responsibility for the overall coordination of the project activities, including maintaining close contacts with the various work package leaders. This includes the overall coordination of the technical activities of the project and the overall legal, contractual, ethical, financial and administrative management. The PC will be responsible for the on-time delivery of the project deliverables and exploitation and dissemination of the results, not only during but also after the project has finished. The PC will also act as an intermediary between the Commission and the project. The PC will be assisted by a legal and a financial manager, a representative of the WUR (Wageningen University & Research centre) Project Support Office, and a project manager. Together, they form the Daily Management Team (DMT). The PC will also be member of the DEIPAB and will chair the SAB and the GA. Additional tasks and responsibilities of the PC include: negotiating the project with the Commission, including finalization of the Consortium Agreement ensuring that contractual obligations are fulfilled and the scheduled deliverables of project produced (including the annual progress and financial reporting to the Commission) setting up the administrative procedures for the project and for the reporting to the Commission calling and drafting an agenda for the biannual meetings of the PMT chairing the biannual meeting of the PMT and preparing the Minutes implementing the decisions of the PMT facilitating project implementation in general; monitor progress and prepare contingency plans if necessary making daily decisions required for the project implementation reporting to, and maintaining contact with, the Commission at the required frequency monitoring dissemination of results toward different target groups, filing of patents and other exploitation Details of the PC responsibilities will be further elaborated in the Consortium Agreement.
Daily Management Team (DMT) The DMT has a daily responsibility for the monitoring and reporting of the project progress, execution of annual financial auditing of the consortium partners, proper execution of the matters decided by the PMT, communication with all stakeholders, and all legal, financial and administrative matters. The DMT is composed of:
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311956 SPLASH the PC, who chairs the DMT: Maria Barbosa a representative of the WU Project Support Office a representative of the coordinators financial department: Ruben Deursen a representative of the coordinators legal department: Claire Gemert Beaumont a project manager: Lolke Sijtsma All DMT members have a long track record in coordinating or assisting of national and international projects, including EU projects, and are highly professional regarding the assigned activities and responsibilities. Project Management Team (PMT) The PMT is the overall scientific management body of this project. The PMT consists of the seven WP leaders (WPL) and the PC. The WPL from WP7 is also the Dissemination and Exploitation officer (DEO) who is in close contact with the PC as they both have an important role in the communication, dissemination and exploitation of project results. The PMT will be assisted by the DMT and advised by the members of the two Advisory Boards. The main tasks of the PMT are to define the project strategy, monitor progress, and advise and decide on major project revisions, exchanges of tasks & budgets, intellectual property, dissemination strategies, communication, interaction with other activities and political issues. The PMT will meet regularly but at least twice a year. Where necessary and appropriate, additional meetings can be held. The PMT has the power to make short-term decisions on a daily basis and to execute long-term more strategic decisions (to be defined in greater detail in the Consortium Agreement). Decisions will normally be taken in consensus. If this cannot be realised, the principle of majority of votes will apply. In the case a matter cannot be resolved by majority voting, the vote of the Project Coordinator will count twice. In such case the Project Coordinator will first gain advice from the EC scientific officer. Work Package Leaders (WPL) This project consists of seven work packages, each having one or more active participants assigned with specific tasks. Each work package has a WPL who is also a member of the PMT. Each WPL has the task to present the status and progress of their individual work package to the PMT and report back on this to the participants of their own work package. The minutes prepared by the PC or the DEO will be made available through each WPL. The WPL have proven track records regarding the specific topic(s) of their work package and lead international activities successfully. They are responsible for the management and technical coordination of their WP on a daily basis and they will translate decisions of the PMT into daily (management) tasks, organise call meetings with the WP participants when necessary and report results and potential critical issues to the PMT. The WPL are responsible for the progress and reports of their respective WP participants. Additionally, the WPL will remain in contact with all WP participants (in and outside their own WP) and ensure the flow of information from both inside and outside of the WP. Additional responsibilities of WPL include: Design of work programme for their WP Assignment of task to participants of their WP Progress monitoring of milestones and expected outcomes of the WP Quality control of review activities within the WP Preparation of the six monthly interim and annual consolidation reports of their WP Organisation of WP meetings to ensure proper execution of their WP work programme Stimulation of interaction with other WPs, together with the PC and DEO Preparation of the WP meeting minutes and their distribution
Work package leader WP7: Dissemination & Exploitation Officer (DEO) The DEO has additional responsibilities besides the leadership of WP7. These additional responsibilities are mainly focused on overall communication, preparation of a dissemination plan, preparation of a business plan, and management of intellectual property. The DEO has a strong interaction with the PC and other WPLs. The DEO is also a member of the DEIPAB. The responsibilities of the DEO are in detail described in WP7.
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311956 SPLASH Scientific Advisory board (SAB) The SAB is appointed by the PMT to study and advise on strategic matters regarding the scientific scope and direction of the project. If necessary, they will advise about both long-term and short-term scientific strategies of the project, taking new developments and insights into account. The SAB is composed of independent scientific experts who do not take part in the SPLASH project. The SAB members will be appointed by the PMT at the start of the project on criteria such as: excellence in (microalgae) metabolism, production, processing and (chemical) conversion. These scientific experts and are expected to represent different EU countries and disciplines and also relevant regions outside Europe. Dissemination, Exploitation and Intellectual Property Advisory board (DEIPAB) Besides gaining knowledge, implementation of this new gained knowledge is an important goal of the project. For this purpose, it is important to ensure communication with key stakeholders in the production chain to obtain feedback and implement the results. The DEIPAB will advise and report to the PMT on issues regarding project strategy and optimization of applicability and exploitation of the scientific and technological results of the project including Intellectual Property. Project members active in the DEIPAB are the PC, the DEO and for each industrial partner one representative of their management board and an EU scientific officer from the three recently funded EU demonstration projects for microalgae cultivation at a scale of 10 ha (InteSusAl, BioSolar and AlgaePARC). Both the SAB and DEIPAB will be active as long as the duration of the specific tasks that have to be studied.
Management Procedures Management of information exchange The PC is responsible for the information exchange with partners. The PC will keep project participants fully informed about the project status, technical issues, work programme planning and other issues that are important and relevant. These activities include organisation of project meetings, telephone, video and e-mail communication, progress reports, a dedicated project website accessible for all partners. Project results, deliverable reports, minutes of GA, PMT and WP meetings and other news will be published on this website. In addition, an electronic newsletter will appear twice a year, with a special annex for project partners. The project site will further serve as a web-based management tool. Furthermore, the consortium will exchange information at meetings and workshops. The following meetings and reports are foreseen: Overview of meetings Responsible Frequency Participants body DMT DMT Weekly 2/year DMT members All project participants (GA)
Purpose of the meeting DMT meeting Daily management Consortium meeting Exchange information & overall integration Presentation of results PMT meeting Monitoring progress Research, IP and dissemination strategy Approval progress report to be submitted to the EC WP meeting Management & progress evaluation Inter-WP workshops
PMT
2 / year
PMT members
WPL
2 / year
WP members
Overview of progress reports

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311956 SPLASH Responsible body All participants WPL WPL WPL PC PC Name of Report Progress tracking report: action points WP Summary progress report WP interim progress report WP consolidated annual progress report Project consolidated annual report All reports Report to WPL PMT PMT PC EC All participants Frequency 3 months 3 months 6 months Annual Annual As soon as available
Management of Intellectual Property (IP) and publications Intellectual Property Rights, Access Right, Confidentiality, Liability and Indemnification will be governed by the relevant stipulations of the Grant Agreement and addressed in the CA for which the DESCA Simplified FP7 Model Consortium Agreement will be used. The provisions will be in accordance with EU regulations. The PMT will oversee all activities related to exploitation, including the consulting and marketing concepts, the Technology Implementation Plan (TIP), etc. The TIP will be drawn up by the PMT and will detail how the results of the research and development will be exploited. All foreground knowledge produced during the project will be assessed by the PMT in terms of need for IP protection before it is further disseminated. For that purpose, all results presented in reports or at project meetings will be deemed confidential to the consortium and all its employees and (sub)contractors (including the Commission) until it is either properly protected or decided to be free for public dissemination by the partners(s) that own the result and by the PMT. The PMT, through consultation with the owning party, the DEIPAB and the WUR Support Office, will decide, if necessary, to make a division between the parts of the reports that are confidential and those that are open to the public. All other responsibilities and roles of the different Participants will be described in the Consortium Agreement (CA). Decision-making structures The PC has the final responsibility for project management decisions. Daily progress decisions and issues are communicated to the PMT and all participants will be informed, where necessary, by email, fax or phone. The more major day-to-day issues will be discussed in PMT meetings and the major strategic decisions to determine the long-term strategy and direction of the SPLASH project will be prepared by the PMT and agreed upon in the annual GA meeting. The PC will be responsible for the agendas for the PMT and GA meetings and send these to the PMT and GA members two weeks in advance of the relevant meeting. All members may request to add an issue on the agenda until two days before the meeting and minutes will be made available to all participants via the WPL, the website or email. An extra PMT or GA meeting will be organised in case issues requiring urgent consideration come up. During meetings of this project (this counts for all meetings of the consortium but most likely counting for PMT and GA) decisions will be taken by consensus, but where this is not feasible, the principle of majority voting will apply. If a matter cannot be resolved by majority voting, the chairman will have the casting vote (in most cases the PC) in such case the PC will first gain advice from the EC scientific officer. Serious disputes As a general rule, the Consortium will follow a collaborative approach for avoiding conflicts. In the unlikely case that serious disputes should arise among project partners, conflict resolution procedures will be initiated whereby the PC will advise the GA to meet in an emergency session to discuss the conflict and reach a resolution by majority vote. The Consortium believes that any conflicts should be resolved as speedily as possible so that, within 21 days of notification by the PC of the requirement for an emergency procedure, the SC will meet in session. The quorum threshold for this meeting is set at 90%. The meeting will attempt to achieve full consensus on the resolution of the issue but, failing this, a majority vote will be taken to determine what resolution should be implemented. The PC is an advisor to the SC and therefore has no vote. In circumstances of persistent and serious conflict arising out of continuation of the project, the GA will instruct the PC to involve the EU project officer. In addition, the GA may seek external advice followed by a review of the situation, after which a collective decision in
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311956 SPLASH should be reached to implement a final remedy for the issues involved. In the most serious cases arbitration may lead to proceedings or a court. Risk assessment Risk assessment covers all project activities and aims at a timely response to issues that were not foreseen in the planning phase. It can be defined at two levels: scientific and non-scientific. Scientific and technological risks and their contingency plans are described in paragraph 1.3.3.7; any non-scientific risks, in particular with respect to project management, are inconsequential as the individuals in charge have ample experience with many (large) EU projects and have built up extensive knowledge in this field. Risk assessment starts with monitoring, communicating and evaluating the progress within the clearly specified WPs, Deliverables and Tasks. The status of all Tasks will be registered in a project progress database. Application of these procedures and tools will ensure that the PC and individual WPL at all times will have an overview of status of the project results. The WPL are responsible for planning and control of progress of the Deliverables and Tasks within their WP and will report its monitoring in a progress tracking report. At the start of the project, the PMT will provide planning and control tools that will help to evaluate the project progress every 3 months and if necessary will propose actions, including reallocation of resources when the deliverables of critical tasks are expected to be delayed. To allow rapid allocation of additional financial resources to delayed critical Tasks, 3% of the project budget will be set aside as emergency budget, controlled by the PMT. Dissemination and knowledge exchanges Dissemination and communication will have a central role in this project. The consortium will communicate its project results to a wide range of stakeholders (research community, policy-makers, interest groups, media, the public at large), using the most appropriate communication channels per target group. All participants will have their responsibility in these issues, in particular the DEO (WPL of WP7). The DEO is dedicated to issues related to Dissemination and Exploitation of results and responsible for the internal and external communication of the project. The dissemination activities of this project will be composed of the following elements: Construction and maintenance of a website that is accessible to all internal and external parties. The website will cover various publications, patents, press releases, training activities and workshops. Current and new information networks of the participants involved in this project will ensure that both project participants and those outside the project will be informed about the latest developments of science, application development and technology in the area of microalgae research their products and applications. A web-based system will accommodate all project-related files in one central domain. Access to confidential information is restricted by different levels of authorisation. Furthermore different modules will provide tools for optimal exposure of results to general public and interested parties. To support dissemination of project results, various publications will be made available such as press releases available to trade, technical and scientific journals, and brochures detailing benefits and output opportunities from the project. The research community will also author scientific articles on the scientific and technical project achievements, for publication in peerreviewed journals. Consortium members will participate in international exhibitions and conferences to present and promote project developments and outcomes. Additionally, a summer school will be made available to graduate students and researchers to promote project results. A detailed description of the dissemination and knowledge exchange activities can be found in the Work package description WP 7.
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B2.2 Individual participants P1a Stichting Dienst Landbouwkundig Onderzoek, Institute Food & Biobased Research (DLO-FBR) General FBR provides fundamental, strategic and applied research to enable rapid application of description academic knowledge to the market. The Business Unit Biobased Products has a strong focus on primary streams of biomass and bioresidues to produce sustainable industrial raw materials, green chemicals (added value products) and bioenergy. Together with WU, FBR has been able to establish a total research capacity with 15 PhD students and collaborations with about 20 companies in the field of microalgae. Presently, an International Algae Pilot Production and Development Centre (AlgaePARC) will be opened in collaboration on the observed demand from industry and agribusiness sectors. FBR has also considerable experience in setting up project consortia, driven both by industrial and scientific objectives. FBR is currently coordinator of a large Integrated Project in the 6th (NOVEL-Q) and the 7th Framework Programme (EU-PEARLS) and participant in many more. Given the importance of EC-funded projects, FBR supports coordinators, project leaders and work package leaders in a number of ways: (1) Europe helpdesk for procedural information (www.europadesk.nl); (2) Controllers specialized in financial handling of projects; (3) Staff members and/or management assistants to support project leaders with operational matters. Role WP1, WP2, WP4 and WP5 tasks addressing coordination, mutagenesis, microalgae cultivation, DSP product development and testing. Key Dr. M.J. Barbosa, Sr. scientist, bioprocess engineering project leader AlgaePARC, >10 persons years experience Dr. Dorinde Kleinegris, scientist , DSP microalgae, >5 years experience Dr Ben Langelaan, separation technology expert, >10 years experience Dr. Rolf Blaauw, senior chemistry scientist, >10 years experience Dr. Jacco van Haveren, programme manager and chemistry scientist, >10 years experience Prof. Gerrit Eggink, Professor Industrial biotechnology, >10 years experience Ing. Jan Springer, Molecular biologist, >10 years experience 1. Wijffels, R.H.; Barbosa, M.J. (2010) An Outlook on Microalgal Biofuels. Science. 329: Selected 796 - 799. publications 2. Norsker, N., Barbosa, M.J., Vermue, M, Wijffels, R.H. (2011). Microalgal productiona close look at the economics. Biotechnology Advances. 29 : 2427. 3. Wijffels, R.H., Barbosa, MJ. Eppink, M.H.M (2010) Microalgae for production of bulk chemicals and biofuels. Biofuels Bioproducts and Biorefining. 4:287 - 295 4. Haveren, J. van; Scott, E.L.; Sanders, J.P.M. (2007) Bulk chemicals from biomass. Biofuels Bioproducts and Biorefining. 2: 41 - 57. 5. Scott, E.L.; Haveren, J. van; Sanders, J.P.M. (2010) The production of chemicals in a biobased economy In: The biobased economy: biofuels, materials and chemicals in the post-oil era / Langeveld, J.W.A., Sanders, J.P.M., . - London : Earthscan. P1b Stichting Dienst Landbouwkundig Onderzoek, Plant Research International (DLO-PRI) General PRI has expert knowledge in all required areas including genome and transcriptome description sequencing, genome annotation, metabolomics, proteomics and analysis of regulatory networks. In the field of Genomics, PRI has applied key concepts like high-throughput analyses of DNA and RNA, and genome analyses and bioinformatics. PRI is the selected partner for proteomics analysis in the EU-SPLASH project with the aim to elucidate the regulation and biosynthesis of hydrocarbons in B. braunii. Role WP2 genomics and transcriptomics Key Dr. Sander Peters, Researcher, project leader Genomics, Bioinformatics, >10 years persons experience Dr. Roeland van Ham, Bioinformatics cluster leader Genomics, Bioinformatics, > 10
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311956 SPLASH years experience Dr. Twan America, Sr. Res., project leader Proteomics, > 10 years experience Dr. Ric de Vos, PhD Sr. Res. Metabolomics, >10 years experience Dr. Bert Schipper, Bsc Assistant Metabolomics, >10 years experience Dr. Jan Cordewener, PhD Researcher, project leader Genomics, Proteomics, >10 years experience Dr. Andries Koops, PhD Dept. leader Bioscience Gen., Metabolomics, Proteomics, >10 years experience Selected 1. America, A.H.P., and Cordewener, J.H. (2008) Comparative LC-MS: a landscape of publications peaks and valleys. Proteomics. 8:731-49. 2. De Groot, J.C., Fiers, M.W., van Ham, R.C. and America, A.H.P. (2008) Post alignment clustering procedure for comparative quantitative proteomics LC-MS Data. Proteomics. 8: 32-36 3. De Vos, R.C.H., Moco, S., Lommen, A., Keurentjes, J.J., Bino, R.J., Hall, R.D. (2007) Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nature Protocols. 2: 778-791. 4. Packo P. Lamers, Marcel Janssen, Ric C.H. De Vos, Raoul J. Bino and Rene H. Wijffels (2008) Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends in Biotechnology. 26: 632-638 5. Peters, S.A., Datema, E., Szinay, D., Van Staveren, M.J., Schijlen, E.G.W.M., Van Haarst, J.C., Hesselink, T., Abma-Henkens, M.H.C., Bai, Y., De Jong, H., Stiekema, W.J., Klein Lankhorst, R.M., and Van Ham, R.C.H.J. (2009) Solanum lycopersicum cv Heinz 1706 chromosome 6: distribution and abundance of genes of retrotransposable elements. Plant Journal. 58: 857-869. P2 Centre for Research and Technology Hellas/Chemical Process Engineering Research Institute (CERTH) General The Centre for Research and Technology - Hellas (CERTH), is one of the largest description research centres of Greece, founded in March 2000. It is a legal, private law, non-profit organization, under the supervision of the General Secretariat for Research and Technology (GSRT), of the Greek Ministry of Development. Its mission is to stimulate and promote industrial innovation by conducting fundamental and applied research that meets the specific technological needs of the Greek and European industries in the following areas: advanced production systems for petrochemical processes, reformulated fuels and hydrocarbons, catalytic processes, energy and environmental technologies and processes, advanced materials and nanotechnologies, polymerization processes, development of advanced software tools, design, optimization and control of industrial processes, renewable energy sources and natural resources utilization and aerosol and particle technologies. From 1985 to 2005, the Chemical Process Engineering Research Institute (CPERI) as a founding member of CERTH, participated successfully in more than 150 research projects and industrial contracts with a total budget of 30 MEURO, financed by the EU, the GSRT and Industry. These R&D projects were carried out in collaboration with 60 academic institutions, 35 research centres and 115 industries in Greece and the European Union. Role WP6 process integration, mathematical modelling, optimization and control of complex biorefinery processes. Key Dr. Vasileios Kanellopoulos, Chemical Engineer, Associated Researcher persons Dr. Sophia Parouti, Chemical Engineer, Associated Researcher Selected 1. Dompazis, G., Kanellopoulos, V., Touloupides, V., Kiparissides, C. (2008) publications Development of a Multi-scale, Multi-phase Multi-zone Dynamic Model for the Prediction of Particle Segregation in Catalytic Olefin Polymerization FBRs. Chem. Eng. Sci. 63: 4735. 2. Kanellopoulos, V., Gustafsson, B., Kiparissides, C. (2008) Gas-phase Oefin Polymerization in the Presence of Supported and Self-supported Ziegler-Natta Catalysts. Macr. Reac. Eng. 2: 240.
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P3 Organic Waste Systems N.V. (OWS) General Organic Waste Systems (OWS) is a stock company under Belgian law, constituted in description 1988. The company is mainly active in following areas: engineering, construction and operation of composting and biogasification plants, biodegradation and composting laboratory testing and waste management consulting services. OWS developed the anaerobic Dranco-process to treat several waste streams. In the last years, OWS also offers processes for energy crops, namely Dranco-Farm and BES reactor types. Moreover, OWS has a longstanding experience in the field of biodegradation and compostability testing. OWS was, and still is, very actively involved in the development of test procedures (and international standards) to evaluate the biodegradation of materials under different environments (compost, soil, anaerobic). Furthermore OWS has experience in Life Cycle Assessment studies in previous and current EU-projects. Role WP6 -Life Cycle Assessment Key persons Bruno De Wilde, Lab manager, engineer in agricultural engineering, specialization biotechnology at the State University of Gent, general management. Steven Verstichel, Lab project manager, engineer in agricultural engineering, specialization biotechnology at the State University of Gent. Aline Vercruyssen, Lab project manager, engineer in agricultural engineering, specialization biotechnology at the State University of Gent, LCA performer. Selected 1. EU QKL5-CT-2000-00799 Biopack: biodegradability and compostability testing; publications/ LCA; economical evaluation of different waste treatment scenarios. previous 2. EU FP7-KBBE-2007-212239 Forest Resource Sustainability through Bio-Basedexperience Composite Development: LCA, biodegradability, compostability and anaerobic related the digestion. task 3. EU FP7-ENV-2008-227078 Development and application of a standardized methodology for the Prospective Sustainability assessment of Technologies: case study. P4 Paques (PAQ) General For over 25 years, Paques has developed and produced high-quality, cost-effective description purification systems for water, gases and air using innovative biotechnology. Novel purification processes have been brought onto the market for removing organic and inorganic compounds, such as sulphate, sulphur, nitrate and metals. Paques is now the leader in the field of anaerobic purification plants. The added value of the treatment process is most important, such as the re-use of water, the generation of energy by the conversion of residual organic compounds and the reclamation of valuable substances from the wastewater. This way, Paques bridges the gap between environmental responsibility and economic progress. Paques has >40 patents and over 1.200 Paques installations have been built worldwide. About 90% of the turnover of Paques is realised outside The Netherlands. With in-house research facilities for conceptual and applied research, a piloting group, engineering (mechanical, process, E&I), technology, sales and manufacturing of key components, Paques encompasses the complete chain to develop, scale-up, commercialise and construct technology on a full-scale. Finally, during the whole existence of Paques, there has always been a close cooperation with national and international research organisations (WUR, Delft University of Technology, TNO, etc.), large industrial partners (i.e. Shell and Siemens water) and hundreds of end users. Role WP3 process optimisation: co-development, design, construction and installation of a pilot photobioreactor. Key persons Dr. Jacco Huisman, Technology Manager. Ing. Sjoerd Vellinga, Chief engineer. Dr. Erik van Zessen, Senior technology expert.
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311956 SPLASH Ing. Ton Jorna, Lead mechanical engineer and creative force behind many innovative bioreactor concepts, ensuring that reactor concepts are developed that can be realised effectively on a full-scale. Dr. Wobby Bosma, Senior researcher, >10 years experience in operating bench and pilot scale technology for development purposes. Carl Schultz, Marketing & business development, involved in the commercialisation of several technologies. Selected 1. Mierzejewski, M., Lamarre, D., Dijkman, H., Schouten, G., Benschop, A. (2004) publications/ Combined Anaerobic Removal of COD, N and S from Industrial Waste Water, 10th previous World Congress on Anaerobic Digestion, Aug. 29th Sept. 1st Montreal, Canada. experience 2. Bosma R., Zessen, E. van, Reith, J.H., Tramper, J., Wijffels, R.H. (2007) Prediction related to of volumetric productivity of an outdoor photobioreactor. Biotechn. Bioeng. 97 :1108the task 20. 3. Habets, L., Driessen, W. (2007) Anaerobic treatment of pulp and paper mill effluents status quo and new developments. Wat. Sci. & Techn. 55: 223230. 4. Huisman, J.L., Schouten, G., Schultz, C. (2006) Biologically produced sulphide for purification of process streams, effluent treatment and recovery of metals in the metal and mining industry. Hydrometallurgy. 83: 106-113 5. Paques has (co-)developed and scaled-up a large number of technologies that are now commercially and successfully marketed on a worldwide scale. Examples are BIOPAQ UASB, BIOPAQ IC, THIOPAQ, SULFATEQ, SHELL-PAQUES PROCESS, ANAMMOX, PHOSPAQ. P5 BioTopic (BIT) General BioTopic is a single-person consultancy company, registered in 2005 in Denmark. The description company has assisted pharmaceutical and biotechnology companies in the field of microalgal biotechnology and regulatory matters. Typical projects include assistance at approval of a micro algal polysaccharide for human food supplements, investigation of microalgal production potentials for polyunsaturated fatty acids, production manuals for various microalgae for aquaculture, carotenoid research overview, assistance with photobioreactor operation etc. BioTopic is operated by Niels-Henrik Norsker, who in 1990 established BioProces ApS, that developed and produced photobioreactor systems, microalgal growth media and starter cultures for aquaculture- and research institutions. The photobioreactor system is a medium scale (0.6 m3) continuous cultivation system, based on acrylic air lift reactors. BioProcess ApS was in 1997 restructured into BioProcess AS that developed a photo-fermentation process, based on the astaxanthin producing green alga, Haematococcus pluvialis. The system was based on stainless steel fermentors (3 and 10m3) that were operated continuously and completely bacteria free. In addition, Niels-Henrik Norsker has experience with development of photobioreactors and biofilm reactors through employments at Wageningen University (NL) and Alborg University (DK). Role WP3 Biomass production and process optimisation outdoors Key persons Ing. Niels-Henrik Norsker, Microalgae biotechnologist > 10 years experience Selected 1. Norsker, N.-H., Barbosa, M.J. (2011) Microalgal production - A close look at the publications/ economics. Biotechnology Advances. 29: 24-27. previous 2. Norsker, N. H., Barbosa, M. (2010) Microalgal Biotechnology in the Production of experience Nutraceuticals. Biotechnology in Functional Foods and Nutraceuticals. related to 3. Norsker, N.-H., Nielsen, P. H. (1995). Influence of oxygen on biofilm growth and the task potential sulphate reduction in gravity sewer biofilm. Water Science and Technology. 31: 159-167. 4. Norsker, N.-H. and J. G. Stttrup (1991) Technical and Biological Aspects of Continuous Micro algae cultivation. European Aquaculture Society, Special Publication. 15: 87-90.
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311956 SPLASH 5. Stttrup, J. G. and N.-H. Norsker (1994) The importance of dietary HUFA's for fecundity and HUFA content in the Harpacticoid Tisbe holothuriae Humes. Aquaculture. 125: 155-167. P6 Value for Technology (VFT) General VFT is a Belgian SME founded in 2003 by Philippe Willems. VFT provides business description development services related to renewable feedstocks and technologies related to the processing and valorisation of these feedstocks. To reach this target, VFT covers different business activities such as: technology brokering; feasibility studies; partner matching; edition of market reports and marketing plans; market introduction of new products; coordination of research activities. VFT is unique in its activity by always combining technical, commercial and financial aspects in its recommendation and specialising on renewable resource-based topics. In doing so, VFT contributes to the development of the bio-based economy. Typical customers are large enterprises, mid-caps, SMEs, branch organisations, etc. Role WP6 and WP7 exploitation and IP management. Key persons Philippe Willems has spent 15 years as business developer in the starch industry before starting up Value for Technology. In the past 4 years, he has been personally involved in several international algae projects. Philippe is also part of the team that took the initiative for the launch of the Flemish Algae Platform (VAP). He has been asked to share his views about the algae market at several occasions (among others in a workshop of the Aquafuel project; at a workshop organised by the engineering association, etc.). Philippe Willems is also co-founder of Orineo, a SME developing actively markets for its clients (also bio-based and market oriented focus), by positioning the products and organising sales. Selected 1. VFT has been deeply involved in algae projects for the last four years, including publications/ participation in a FP7 project BioAlgaeSorb; several feasibility studies on integrated previous algae production; partner matching on algae projects; marketing plans for algae experience biomass. Also relevant past projects on industrial biotechnology; bioplastics and related to biomaterials; search for bio-based alternatives to petrochemical-based binders; the task FISCH project on sustainable chemistry, etc. 2. VFT is partner in three European projects: Ecobinders (FP6 project, completed; VFT was part of the project coordination team); Bioref-Integ (FP7 project, completed; VFT was WP leader); BioAlgaeSorb (FP7, running) and is actively participating in the FISCH project on sustainable chemistry (now also referred to as SusChem Flanders). P7 Avantium (AVT) General AVT is a worldwide recognized leader (Cleantech Top 100 Company) in catalytic description biomass conversion into furanic building blocks and high-throughput technology. It has created an impressive patent portfolio in this area and is a proven adept of the open innovation model. From 2011 to 2015, these novel concepts will be demonstrated in a first of a kind pilot facility. Therefore, the research results of the SPLASH project can directly be evaluated on pilot scale. AVT has over 90 employees and employs a team of 65+ highly educated professionals (>40 PhDs) expert in biomass pre-treatment and conversion, polymer science, organic chemistry, catalysis, engineering of robotic systems, process engineering, statistics, analytics, cheminformatics and software development. AVT's employees are dynamic and enthusiastic people, open-minded, ambitious and professional. The company fosters creativity as well as a result-oriented, customer-oriented attitude. Critical infrastructure: AVT has proprietary hardware and software platforms to screen and evaluate catalysts for a broad range of applications in fixed-bed and batch operation. Research is executed following advanced high-throughput workflows. Role WP4 tasks addressing carbohydrate conversion into furanics. Key persons Dr. Ed de Jong ,VP Development at Avantium Technologies BV, Amsterdam. > 20 years
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311956 SPLASH proven expertise (more than 50 scientific papers and 5 patents) in biomass research and training, (bio-)chemical pre-treatment of lignocelluloses, chemical and enzymatic conversions of cellulose, hemicelluloses and lignin. Dr. Jan Kees van der Waal, Principle Scientist responsible for the heterogeneous catalysis and catalyst preparation and supports all projects within the program Furanics on both technical and scientific issues, >10 years experience. Dr. Etienne Mazoyer, catalyst chemist, years experience >4 years experience. Dr. Sarvat Iqbal, catalytic chemist, years experience >4 years experience. Selected 1. de Jong, E., van Ree, R., Sanders, J. P. M. and Langeveld H. (2010). Chapter 7 publications/ Biorefineries: Giving value to sustainable biomass use. In: The Biobased Economy: previous Biofuels, Materials and Chemicals in the Post-oil Era by Langeveld, Meeusen & experience Sanders (Eds). Earthscan Publishers, London, pp 111-130. related to 2. Pereira, S.R.M., Clerc, F., Farrusseng, D., van der Waal, J.C., Maschmeyer, Th. the task (2007) Optimisation methodologies and algorithms for research on catalysis employing high-throughput methods: comparison using the Selox benchmark. Combinatorial Chemistry & High Throughput Screening. 10: 149-159. 3. Gruter, G-J., E. de Jong (2009) Furanics: novel fuel options from carbohydrates. Biofuels Technology. 1:11-17. 4. Mazoyer, E., Trbosc, J., Baudouin, A., Boyron, O., Pelletier, J., Basset, J.M., Vitorino, M.J., Nicholas, C.P., Gauvin, R.M., Taoufik, M., Delevoye, L. (2011) Heteronuclear NMR Correlations To Probe the Local Structure of Catalytically Active Surface Aluminium Hydride Species on Alumina. Angewandte Chemie International Edition, In Press. P8 LifeGlimmer GmbH(LG) General LifeGlimmer GmbH is an SME incorporated in 2012 with the goal of providing to the description Biotechnological, Biomedical and Environmental Research Communities an efficient way to outsource Scientific Computing needs. Since its start, LifeGlimmer provides Softwareas-a-Service (SaaS) basis to a number of academic and industrial institutions. In addition to the IT-resource infrastructure and knowledge for genome-scale metabolic and dynamic modelling and reverse engineering, expertise includes data warehousing and visualization, data aggregation treatment and management, algorithm implementation, process scheduling, and on-the-cloud computing. This has been applied in a number of research settings, both in industry and academia (e.g. ERA-NET METAGUT project, http://www.pathogenomics-era.net/2ndJointCall/, or the ST-FLOW project). LifeGlimmer develops a wide range of biological models and provides in-house training. The training initiatives are performed in-house (on Entrepreneurship and Technology Development) or embedded in academic-training programmes (Graduate Colleges, Graduate Conferences, PhD training initiatives, annual Technology Courses, and Student Days) organized by collaborating partners, namely the Helmholtz Centre for Infection Research, Braunschweig, Germany, and the Wageningen University, both of which LifeGlimmer interacts with closely. Role WP2 tasks addressing Systems Biology Key persons Prof. Vtor Martins dos Santos, Founder, CSO, Systems & Synthetic Biology, Modelling > 15 years experience. Eng. Miguel Godinho, Co-founder and CTO, Systems architect, Scientific computing > 15 year experience. Dr. Ir. Peter Schaap, Bioinformatics, functional genomics, Shot-gun proteomics, > 25 years experience. Dipl. Sandra Placzek, Bioinformatician, Operations Research and Algorithmics over 4 years experience. Eng. Joao Cardoso, Bioinformatician with expertise on web-development and data integration. Eng. Sigrun May, Software Developer, Database development and Data Integration > 5
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311956 SPLASH years experience. Selected 1. Martins dos Santos, V.A.P., Damborski, J. (2010) Systems Biology at Work. Current publications/ Opinion in Biotechnology. 21: 498-50. previous 2. Puchaka, J., Oberhardt, M.A., Godinho, M., Regehardt, D., Timmi, K.N., Papin, J.A., experience Martins dos Santos, V.A. (2010) Genome-scale reconstruction and analysis of the related to Pseudomonas putida KT2440 metabolic network facilitates applications in the task biotechnology. PLoS Computational Biology. 4(10): e1000210. doi:10.1371/journal.pcbi.1000210. 3. Schneiker, S., Martins dos Santos, V.A., Bartels, D., Bekel, T., Brecht, M., Buhrmester, J., Chernikova, T.N., Denaro, R., Ferrer, M., Gertler, C., Goesmann, A., Golyshina, O.V., Kaminski, F., Khachane, A.N., Lang, S., Linke, B., McHardy, A.C., Meyer, F., Nechitaylo, .T, Puhler, A., Regenhardt, D., Rupp, O., Sabirova, J.S., Selbitschka, W., Yakimov, M.M., Timmis, K.N., Vorholter, F.J., Weidner, S., Kaiser, O., Golyshin, P.N. (2006) Genome sequence of the ubiquitous hydrocarbondegrading marine bacterium Alcanivorax borkumensis. Nature Biotechnol. 24 :9971004. *joint first-authorship, corresponding author 4. Ferreira de Oliveira, J.M., van Passel, M.W., Schaap, P.J., de Graaff, L.H. (2010) Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction. Appl Environ Microbiol. 76: 4421-9. P9 Pursuit Dynamics (PDX) General Pursuit Dynamics Plc (PDX) is a UK and Switzerland based company that develops and description provides innovative techniques and energy efficient solutions for various industrial applications through its proprietary fluid processing Reactor and atomising technologies. Since 2001, PDXs multi-disciplinary science and technology team has been involved in developing integrated fluid processing solutions to a range of industries, including biofuels and other renewable energy related industries. In 2005, PDX started developing its Reactor technology for producing bio-ethanol and has applied this technology successfully since at various US based corn-to-ethanol plants and since 2008, PDX has also been conducting research and development into the generation of both second generation (lignocellulosic) and third generation biofuels, including fuels from algae sources and biogas production in waste water treatment. This research utilised PDX Reactor technology and integrated use of enzymes and processing chemicals. Having gained substantial knowledge and research experience in third generation biofuels over recent years, PDX is now exploring further development of large scale integrated system industrial processes for third generation biofuels, together with commercial partners and various reputed European academic institutions such as the Universities of Cambridge, East Anglia, Cranfield, and Wageningen. Role WP3 and WP5: Applying PDX Reactor technology and other fluid processing expertise for developing disruption, extraction and separation solutions for hydrocarbons and polysacharides production and providing up-scaling and technology commercialisation expertise. Key persons Dr. Marcus Fenton, Chief Scientific Officer, > 16 years of experience managing commercial R&D. Dr. Michelle Gothard, Principal Scientist Biomaterials, >10 years of experience in plant material science and biopolymers. Dr. Olga Koralava, Senior Scientist Biomaterials, >10 years of experience in plant cell metabolism and function. Mr. Jeremy Pelczer interim CEO, PDX Board member, >15 years of experience in water industry. Selected 1. Developing an integrated Ethanol Reactor System (ERS) and whole cycle publications/ technology development, from R&D, through pilot scale testing, and culminating previous in up-scaling into commercial plant installs and acceptance in the corn-to-ethanol experience industry at various US plants.
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311956 SPLASH related the task to 2. Development of a new integrated cellulosic ethanol process. Currently conducting pilot scale testing with a US commercial partner (since March 2008). 3. R&D with academic and commercial partners into novel algae extraction scientific solutions and technologies (since February 2009). 4. R&D with academic and commercial partners for improved biogas production in waste water industry (since 2010).
P10 Nova-Institut (NOVA) General The nova-Institut GmbH was founded as a private and independent institute in 1994 and description it is located at the Chemiepark Knapsack in Huerth, which lies in the heartland of the chemical industry in Cologne. For over 15 years now, the Nova-Institut (Germany) has been globally active in communication, market research, industrial and political consultancy and project management. It uses and creates expert knowledge and innovative technologies to advance and develop the use of renewable resources as energy, material and products. With projects and surveys, market analyses, industrial and political consulting, network management, publications and conferences, the nova-Institut supervises and promotes a Raw Material Shift, moving away from fossil carbon carriers towards a sustainable raw material supply through renewable resources from field and forest. Our areas of specialist skills and competence include techno-economic evaluation, feasibility studies, market analysis, feedstock availability, economic analysis of area and usage competition and indeed the whole value added chain of resources. We concentrate on market research for bio-based materials like bioplastics, natural fibre composites and innovative wooden materials, and industrial biotechnology as well as on the communication and dissemination of project results. In the field of communication we concentrate on marketing support, dissemination of information via internet and print, preparation and organisation of workshops and conferences workshops and training to Web 2.0 applications, mainly Wikipedia. Nova is publisher of the news portal www.renewableresources.de, the Biowerkstoff-Report (a journal on bio-based materials). Role WP7 Dissemination and Exploitation Officer (DEO) is from NOVA Key persons Michael Carus, Managing Director of NOVA, and head of the division Renewable resources / market research. Christin Schmidt, head of the communication department is responsible for dissemination and communication strategies for internal and external Communication. Achim Raschka, leader of the biotechnology working group at nova-institut, is involved in different research programs concerning the material use of bio-based products and renewable resources, biotechnological and chemical-technical topics. Selected 1. Carus, M., Raschka, A., Piotrowski, S. BIOCORE - Biocommodity refinery. Project publications/ within the FP7-programme of the European Commission (FP7-241566), Feedstock & previous Techno-economic Evaluation, 2010-03 to 2014-02; experience 2. Carus, M., Raschka, A., Piotrowski, S. MouldPulp - Development of Durable, Fully related to Bio-Based Thermoplastic Composites from Bioplastics and Pulp Fibres for Injection the task Moulding Applications. Project within the WoodWisdom-Net Research Programme of the European Commission, 2011-01 to 2013-12; 3. Carus, M., Piotrowski, S. The development of natural fibre pellets for as reinforcement of (bio-) plastics injection moulding and extrusion. Funded by the Deutschen Bundesstiftung Umwelt (DBU), Osnabrck, 2010-04 to 2011-10; 4. Carus, M., Raschka, A., Piotrowski, S. The development of instruments to support the material use of renewable raw materials in Germany - Market volumes, structure and trends Policy instruments to support the industrial material use of renewable raw materials. Funded by Fachagentur Nachwachsende Rohstoffe e.V. (FNR) (FKZ: 22003908), 2008-04 to 2009-07;
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311956 SPLASH P11 Fraunhofer-Institute for Environmental, Safety and Energy Technology UMSICHT (FRAUNHOFER) Fraunhofer UMSICHT) is one of 57 institutes of the Fraunhofer-Gesellschaft fr General description angewandte Fraunhofer UMSICHT is a non-profit scientific / technical institution, which conducts contract research on behalf of industry, the service sector and government. The institute provides experimental lab-scale and pilot plant investigations, evaluates and optimises technical processes of prototype scale in the fields of environmental, safety, and energy technologies. Fraunhofer UMSICHT has intensified research activities concerning the usage of renewable resources, e.g. biofuels, biorefinery, bio-technology, bio-polymers, ORC-processes. The institute strives for the introduction of new and intelligent technologies, which save resources and energy but still operate economically. Experienced and interdisciplinary teams, consisting of engineers, scientists, economists, technicians and lab technicians, are well prepared and capable of tackling a broad range of challenging problems. Role WP4, product develoment and testing; WP5, demonstration Key persons Axel Kraft , head of the business unit biofuels. Dr. Anna Fastabend, chemical engineer Dr. Andreas Menne, chemical engineer 1. Danzig J, Fastabend A, Heil V, Kraft A, Meller K, Menne A, Unger C, Rossow S. Selected 2008. GREASOLINE - new technology for the conversion of waste fats to highpublications/ quality fuels: Co-operative Research Project No. 018109 - Publishable Final Activity previous Report (see also http://publica.fraunhofer.de/eprints/urn:nbn:de:0011-n-832678.pdf) experience 2. Peter S, Weidner E (inventors) 2006. Verfahren zur Umesterung von Fetten und len related to biologischen Ursprungs mittels Alkoholyse unter Verwendung spezieller the task Kohlensuresalze (Process for the transesterification of fats and oils of biological origin by means of alkoholysis using special carboxylic acid salts). DE102004044660A1; Filing date: 30.3.2006 3. Cinquemani C, Heil V, Jakob J, Weber, A (inventors) 2004. Verfahren zum Konvertieren von fett- oder lhaltigen Roh- und Abfallstoffen in Gemische mit hohem Kohlenwasserstoffanteil (Process for converting of raw materials and waste materials containing oil or fat in a composition containing hydrocarbons). EP000001489157A1; Publication date: 22.12.2004 4. Planned spin-off team Greasoline: Conceptual phase award winner of the German business plan contest Science4Life in March 2009. P12 Cambridge University, The Chancellor, Masters and Scholars of the University of Cambridge (UCAM) General The University is one of the oldest in Europe (established 1209), with a world-wide description reputation for outstanding academic achievement. Its scientific and technical research infrastructure is unrivalled in the UK, with a research grant income of 267m (300m) in 2008-2009. Prof. Alison Smith, a plant biochemist who has worked at the interface between biology and chemistry for over 25 years, with expertise in characterization and manipulation of metabolic pathways, particularly for high value products such as vitamins. Current UK-funded projects focus on developing synthetic biology tools for metabolic engineering of microalgae, which will be available for the project, and in understanding algal interactions with bacteria. Prof. Smith is a founding member of the Algal Bioenergy Consortium (ABC), an interdisciplinary group of biochemists, molecular biologist and chemical & process engineers, who bring an integrated approach to tackling the challenges of algal bioenergy production. The facilities available in Cambridge include all aspects of molecular biology, plant metabolite analysis (GC/MS, LS/MS, HPLCs), and algal growth at lab scale. Within the Department there is a bioinformatics facility and high capacity computer cluster. Role WP2 Gene expression, metabolic engineering Key persons Professor Alison Smith, project leader, metabolic pathways, >20 years experience Dr Krys Kelly, bioinformatician, >10 years experience Dr Beatrix Schlarb-Ridley, researcher & business liaison officer, >10 years experience ANNEX 1 SPLASH 38 | P a g e
311956 SPLASH Selected 1. Mochizuki, N., Tanaka, R., Grimm, B., Masuda, T., Moulin, M., Smith, A.G., Tanaka, A. publications/ and Terry, M.J. (2010) The cell biology of tetrapyrroles: a life and death struggle. previous Trends in Plant Science. 15: 488-498. experience 2. Stephenson, A.L., Dennis, J.S., Howe, C.J., Scott, S.A. and Smith, A.G. (2010) related to Influence of nitrogen-limitation regime on the production by Chlorella vulgaris of lipids the task for biodiesel feedstocks. Biofuels. 1: 47-58. 3. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D., and Smith, A.G. (2010) Biodiesel from algae: challenges and prospects. Curr Op Biotechnology. 21:277286. 4. Moulin, M., McCormac, A.C., Terry, M.J. and Smith, A.G. (2008) Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation. Proc Natl Acad Sci USA. 105: 15178 15183. 5. Croft, M.T., Moulin, M., Webb, M.E., Smith, A.G. (2007) Thiamine biosynthesis in algae is regulated by riboswitches. Proc Natl Acad. Sci USA. 104: 20770-20775. P13 PNO Consultants (PNO) PNO was set up in The Netherlands in 1984 and is a leading independent consultancy in General description 13 EU countries. PNO has 425 employees and supports its clients in analysing market needs, defining business-driven technology projects, developing dissemination strategies, building cross-national technical project partnerships. PNO supports industries, Public Administration and research organisations in the definition and implementation of their innovation strategies, the exploitation and dissemination of advanced technologies and management of international technology transfer networks. PNO also delivers advisory services to different innovation agencies and manages Lisbon Agenda related multi-stakeholder initiatives (European Grants Advisor-EUGAand the European Alliance on Skills for Employability). The PNO team has strong involvement in R&D activities conducted with highly qualified EU companies and research centres (University of Rome, Fraunhofer Institute, Finmeccanica, Microsoft, HP, Warsaw Technology University, Universidad Politcnica de Madrid, etc.). Role WP1: Support to coordinator project office (check financial and admin reporting before sending to EC) WP7: Dissemination Key persons Aya van den Kroonenberg, M.S. and Ph.D. in mechanical engineering, >10 years experience in innovation management, technical project development and market analysis in different sectors. Diana Hresko, Msc in Geography, post-graduate qualification in European Studies, Dissemination management, >8 years experience Specific competences in coordination of international projects and networks: Selected publications/ Coordinator of @HEALTH project (IST funded SSA, mentioned in ICT FP7 Work Programme as Basis for Technology Transfer action with Latin America on e-Health) previous experience Coordination support of eTen project Social Village (coordinator Province of Brescia) related to CRAFT AMI-SME (Exploitation and Dissemination responsible ) the task Coordinator of FP7-TRANSPORT SMART (Stimulating participation of SMEs through Regional Clusters) Partner in FP7 GUIDE complete database of funding opportunities for research and innovation. Coordinator of FP7 OSMOSIS (Security for SMEs; market analysis) Partner in FP7 ORECCA (CSA) market analysis for offshore energy
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311956 SPLASH P14 University of Huelva (UHU) General The group Biotechnology of Algae develops scientific activities in the field of applied use description of microalgal biomass and related products (lipids: carotenoids, PUFA and lipids for biodiesel) including enhancement of added-value metabolites accumulation, extraction techniques and processes in photobioreactors at a small scale. The group was created in 1995 and is located in the Faculty of Sciences, at the University of Huelva (Andalusia, Spain). In the past six years, the group has focused on investigating culture conditions, metabolic approaches and genetic modifications that enhance both algae growth and lipid accumulation in microalgae. The group currently researches microalgae from different habitats, including marine and acidophile microalgae. Additionally, Nannochloropsis, Dunaliella mutants obtained at our lab, Botryococcus braunii, Chlamydomonas acidophila and Chlorella sorokiniana are included among the species used for their research activities. The group is currently composed of 12 researchers, 6 of which are PhD students. Role WP3 Optimisation of outdoor microalgae cultures Key persons Prof. Carlos Vlchez is Associate Prof. in Biochemistry, about 40 publications in the field of microalgae biotechnology. Dr. Ins Garbayo assistant professor, about 30 publications in the field of microalgae biotechnology (immobilized cells, nitrate and nitrite consumption, nitrogen metabolism and carotenoid production). Selected 1. Cuaresma, M., Janssen, M., Vilchez, C., Wijffels, R.H. (2009) Productivity of Chlorella publications/ sorokiniana in a short light-path (SLP) panel photobioreactor under high irradiance. previous Biotechnology and Bioengineering, accepted. experience 2. Mogedas, B., Casal, C., Forjan, E., Vilchez, C. (2009) -carotene production related to enhancement by UV-A radiations in Dunaliella bardawil cultivated in lab reactors. the task Journal of Bioscience and Bioengineering, in press. 3. Garbayo, I., Cuaresma, C., Vilchez, C., Vega, J.M. (2008) Effect of abiotic stress on the production of lutein and -carotene by Chlamydomonas acidophila. Process Biochemistry. 43: 1158-1161. 4. Cuaresma, M., Garbayo, I., Vega, J.M., Vilchez, C. (2006) Growth and photosynthetic utilization of inorganic carbon of the microalga Chlamydomonas reinhardtii. Enzyme and Microbial Technology. 40: 158-162. 5. Leon, R., Vila, M., Hernanz, D., Vilchez, C. (2005) Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems. Biotechnology and Bioengineering. 92:695-701. P15 Wageningen University (WU) General Within the Bioprocess Engineering Group (BPE), research is done on development of description novel biotechnological processes for production of pharmaceuticals, healthy food ingredients, bulk chemicals and biofuels. BPEs challenge is to produce high quality biobased products in a sustainable and economical way. In this way, exhaustion of natural resources is prevented and the chance that newly developed technology is implemented in (bio)technological industry is increased. The group is headed by Prof. Dr. Ir. R.H. Wijffels. BPE has over 10 years experience with the cultivation of algae and has a solid scientific basis in the expertise of bioreactor design, medium optimisation and metabolic flux modelling in this area of application. The total number of PhD students in this area is now 20 and 3 assistant professors and a PostDoc from our staff are working in this area. We collaborate already with many companies and are involved in many large research programs Role WP3 medium and process optimisation, WP2 metabolic modelling Key persons Prof. Rene Wijffels, head of the Bioprocess Engineering Department Dr Dirk Martens, Assistant Professor, Metabolic Flux Analysis Dr. Marcel Janssen, Assistant professor, coordinator microalgae research programs
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311956 SPLASH and specialist in photobioreactor design and fundamental aspects of photosynthesis in microalgae Dr Packo Lamers, Post Doc, Metabolomics and Metabolic Flux Analysis Selected 1. Baart, G.J.E., Zomer, B. , Haan, A. de, Pol, L.A. van der, Beuvery, E.C., Tramper, J. publications/ and Martens, D.E. (2007) Modeling Neisseria meningitidis metabolism: from genome previous to metabolic fluxes. Genome Biology. 8:36. experience 2. Bosma, R., Zessen, E. van, Reith, J.H., Tramper, J. and Wijffels, R.H. (2007) related to Prediction of volumetric productivity of an outdoor photobioreactor. Biotechnology the task and Bioengineering. 97: 1108-1120. 3. Cuaresma, M., Janssen, M.G.J., Vilchez, C. and Wijffels, R.H. (2009). Productivity of Chlorella sorokiniana in a short light-path (SLP) panel photobioreactor under high irradiance. Biotechnology and Bioengineering. 104: 352-359. 4. Martens, D.E. (2006). Metabolic Flux Analysis of Mammalian Cells. In M. Al-Rubeai and M. Fussenegger (Eds.), Systems Biology (Cell engineering, 5) (pp. 275-300). 2. Marteijn, R.C.L., Jurrius, O., Dhont, J., de Gooijer, C.D., Tramper, J., Martens, D.E. (2003) Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm. Biotechnology and Bioengineering. 81: 269-278. 3. Zijffers, J.F., Janssen, M.G.J., Tramper, J. and Wijffels, R.H. (2008) Design process of an area-efficient photobioreactor. Marine Biotechnology. 10: 404-415. 4. Wijffels, R.H. , Barbosa, M., Janssen, M., Wessels, H.S., Tramper, J., Vilchez, C., Leon, R. and Akkerman, I. (2003) Marine biotechnology: basics and applications. Biomolecular Engineering. 20: IX-X. P16 Universitaet Bielefeld, for Biotechnology, CeBiTec (UNIBI) General The research planned within this project is backed by the outstanding facilities of the description Center of Biotechnology (CeBiTec). The CeBiTec is a central scientific institution at Bielefeld University dedicated to interdisciplinary research in life sciences. It is a place to encourage and support the creation of innovative projects that cross discipline boundaries. For this purpose, it consolidates research activities of high international reputation in the fields of biotechnology, molecular biology, genome research, systems biology, biochemistry, nano- and biophysics, as well as bioinformatics and computer sciences. Role WP2 biotechnological engineering, metabolomics and transcriptomics in microalgae, Key persons Prof. Dr. Olaf Kruse, leader Algae Biotechnology & Bioenergy, >10 years experience Prof. Dr. Alfred Phler, leader Genome Research of Industrial Microorganisms, >10 years experience Prof. Dr. Karsten Niehaus, leader Proteome and Metabolome Research, >10 years experience Dr. Jrn Kalinowski, leader Technology Platform Genomics, >10 years experience Dr. Alex Goesmann, leader Bioinformatics Resource Facility, >10 years experience Selected 1. Mussgnug, J.H., Klassen, V., Schlter, A., Kruse, O. (2010) Microalgae as substrates publications/ for fermentative biogas production in a combined biorefinery concept. Journal of previous Biotechnology. 150: 51-56. experience 2. Stephens, E., Ross, I.L., King, Z., Mussgnug, J.H., Kruse, O., Posten, C., Borowitzka, related to M.A., Hankamer, B. (2010) An economical and technical evaluation of microalgal the task biofuels. Nature Biotechnology. 28:126-128. 3. Wobbe, L., Blifernez, O., Schwarz, C., Mussgnug, J.H., Nickelsen, J., Kruse, O. (2009) Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas. PNAS. 106:1329013295. 4. Nguyen, A.V., Thomas-Hall, S.R., Malno, A., Timmins, M., Mussgnug, J.H., Rupprecht, J., Kruse, O., Hankamer, B., Schenk, P.M. (2008) Transcriptome for photobiological hydrogen production induced by sulphur deprivation in the green alga Chlamydomonas reinhardtii. Eucaryotic Cell. 7: 1965-1979
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311956 SPLASH 5. Schneiker-Bekel, S., Perlova, O., Kaiser, O., Gerth, K., Alici, A., Altmeyer, M.O., Bartels, D., Bekel, T., Beyer, S., Bode, E., Bode, H.B., Bolten, C.J., Choudhuri, J.V., Doss, S., Elnakady, Y.A., Frank, B., Gaigalat, L., Goesmann, A., Groeger, C., Gross, F., Jelsbak, L., Jelsbak, L., Kalinowski, J., Kegler, C., Knauber, T., Konietzny, S., Kopp, M., Krause, L., Krug, D., Linke, B., Mahmud, T., Martinez-Arias, R., McHardy, A.C., Merai, M., Meyer, F., Mormann, S., Munoz-Dorado, J., Perez, J., Pradella, S., Rachid, S., Raddatz, G., Rosenau, F., Rckert, C., Sasse, F., Scharfe, M., Schuster, S.C., Suen, G., Treuner-Lange, A., Velicer, G.J., Vorhlter, F.J., Weissman, K.J., Welch, R.D., Wenzel, S.C., Whitworth, D.E., Wilhelm, S., Wittmann, C., Blocker, H., Phler, A., Mller, R. (2007) Complete genome sequence of the myxobacterium Sorangium cellulosum. Nature Biotechnology. 25: 1281-1289 P17 Westfaelische Wilhelms-Universitaet Muenster (UMUE) General The research of the laboratory of Prof Michael Hippler is focusing on proteomics, description functional analysis and engineering of bioenergetic pathways. The laboratory has a very broad experience in molecular biology of algae, proteomic analysis and in related bioinformatic tools. The principal scientist Prof. Michael Hippler is chair of Plant Biochemistry and Biotechnology and managing director of the Institute of Plant Biochemistry and Biotechnology at the Department of Biology of the University of Mnster. He is author or co-author of more than 40 peer-reviewed publications. The research group is momentarily compromised of four post-docs, six PhD students, and three staff members: a chemical engineer responsible for mass spectrometric and liquid chromatography systems, a computer scientist and a technician. In the Hipplers laboratory, a nano-liquid chromatography (LC) system (Ultimate 3000, Dionex) and a hybrid linear ion-trap mass spectrometer (MS) are available (LTQ-Orbitrap, Thermo Fisher Scientific). The LTQ-Orbitrap mass spectrometer was granted in 2007 via an instrument grant from the Deutsche Forschungsgemeinschaft (INST 211/409-1 FUGG; About 20 % of the time budget of the LC-MS set-up will be available for the consortium. Role WP2 Genome reconstruction, proteomics and metabolic engineering Key persons Prof. Michael Hippler, chair of Plant Biochemistry and Biotechnology and managing director of the Institute of Plant Biochemistry and Biotechnology at the Department of Biology of the University of Mnster A Chemical engineer, a software engineer, technician and two PhD students will partially work on the project. Selected 1. Peers, G., Truong, T.B., Ostendorf, E., Busch, A., Elrad, D., Grossman, A.R., Hippler, publications/ M. and Niyogi, K.K. (2009) An ancient light-harvesting protein is critical for the previous regulation of algal photosynthesis. Nature. 462, 518-521. experience 2. Petroutsos, D., Terauchi, A.M., Busch, A., Hirschmann, I., Merchant, S.S., Finazzi, G. related to and Hippler, M. (2009) PGRL1 participates in iron-induced remodeling of the the task photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii. J. Biol. Chem. 284, 32770-32781. 3. Stauber, E.J., Busch, A., Naumann, B., Svatos, A. and Hippler, M. (2009) Proteotypic profiling of LhydrocarbonsI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex. Proteomics. 9, 398-408. 4. Naumann, B., Busch, A., Allmer, J., Ostendorf, E., Zeller, M., Kirchhoff, H., Hippler, M. (2007). Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii. Proteomics 7. 3964-3979. 5. Merchant, S.S.,.Hippler, M.,and Grossman, A.R. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. 318: 245250.
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311956 SPLASH P18 Ege University (EGE) General Ege University, Department of Bioengineering was established in 2000 as the first and description only Bioengineering Department in Turkey. At the present it has 26 staff of various multidisciplinary backgrounds. 161 undergraduate students and 42 post-graduate students are studying in the Department. The Department has 11 Research Groups; namely, Animal Cell and Tissue Engineering Group, Bioprocess Group and Pilot Plant, Cancer Molecular Biology Group, Bioenergy and Environmental Biotechnology Group, Industrial Microbiology Group, Microalgal Technology Group, Molecular Genetics Group, Natural Product Chemistry Group, Plant Cell and Tissue Culture Group, Plasma Based Technologies Group, Supercritical Fluid Technologies Group. Equipment, facilities, research infrastructure Thar SFE 100, supercritical fluid equipment, a wide range of bioreactors and photobioreactors in different scales from 500 ml to 100 L, incubators, rotary shakers, autoclaves, filter press, tubular centrifuge, APV Homogenizer, refrigerated centrifuge, ultrafiltration, pervaporation and lyophylisation equipment, FLPC, UPLC, glucose analyzer, spectrophotometers (UV visible), cell disintegration, cross flow membrane filtration constitute the main infrastructure. Role WP3 Extraction with supercritical CO2 Key persons Ass. Prof. Dr. Ozlem Yesil Celiktas is head of the Supercritical Fluid Technologies Group and research interests are concentrated on optimization of process parameters for the separation of value-added compounds from plant materials, fermentation broths and biomass. Furthermore, isolation of bioactive compounds from renewable resources for sustainable development is a major focus as well. A variety of matrices have been investigated including Stevia rebaudiana, Rosmarinus officinalis, various Pinus species, sea phanerogams (Posidonia ocenica, Zostera marina) for the recovery of high-value components such as glycosides, carnosic acid, taxifolin, catechin derivatives, ferulic acid and zosteric acid and conversion of biomass to biofuels with the full contribution of Muge Pilavtepe. Encapsulation of drug molecules and proteins is another major area in which our resources are allocated with the aim of developing micro- and nano particles with improved properties. Prof. Dr. Fazilet Vardar-Sukan is head of the department and leading the Bioprocess Group which focuses on enzyme production and optimisation (Xylanase, Laccase, Lipase, Protease, Dipeptidil Peptidase IV, Pectinase, L-asparaginase, X-L-fucasidase), microalgal production in photobioreactors (Spirulina & Haematococcus,Chlorella), microbial production techniques (submerged culture, solid-state, co-culture; termophiles; Lactic acid, Chitin, Chitosan, okrotoxin-A, biopolymers) and bioenergy (Biogas, Bioethanol, Biodiezel, Hydrojen) Dr. Suphi ONCEL, chemical engineer 1. Yesil-Celiktas, O., Otto, F., Gruener, S., Parlar, H. (2009) Determination of Selected extractability of pine bark using supercritical CO2 extraction and different solvents publications/ optimization and prediction. Journal of Agricultural and Food Chemistry, 57, 341-347. previous 2. Yesil-Celiktas, O., Ganzera, M., Akgun, I.H., Sevimli, C., Korkmaz, K.S., Bedir, E. experience (2009) Determination of polyphenolic constituents and biological activities of bark related to extracts from different Pinus species. Journal of the Science of Food and Agriculture, the task 89, 1339-1345. 3. Erkucuk, A., Akgun, I.H., Yesil-Celiktas, O. (2009) Supercritical CO2 extraction of glycosides from Stevia rebaudiana leaves: identification and optimization. Journal of Supercritical Fluids, 51, 29-35. 5. Oncel, S., Vardar-Sukan F. (2009) Photo-bioproduction of hydrogen by Chlamydomonas reinhardtii using a semi-continuous process regime, International Journal of Hydrogen Energy, 34: 7592-7602. 6. Oncel, S., Vardar-Sukan, F. (2008) Comparison of two different pneumatically mixed column photobioreactors for the cultivation of Artrospira platensis (Spirulina platensis), Bioresource Technology, 99: 4755-4760. ANNEX 1 SPLASH 43 | P a g e
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P19 Lankhorst Yarns Portugal (LEP) General Lankhorst Yarns is the trade name of the registered company Lankhorst Indutech Cerfil description SA, which is one of the six divisions within the Royal Lankhorst Euronete Group bv. we develop innovative solutions for specific applications. The majority of products, branded under the name Elite, is based on a wide range of polyethylene and/or polypropylene monofilaments, tapes and yarns, in several tenacities and elongations. These products are used in many industries, such as the weaving industry, the rope-maker industry, the filter industry and the packing industry. Besides these materials, also polyamide, polyester, aramide, ultra high modulus polyethylene, are used for a specific range of specialties. The main production facilities are located in Portugal (Maia). Role WP4: product development & testing WP5: process demonstration at pilot scale Lankhorst will test the biobased polyesters on a pilot extrusion scale. They will prepare tapes and yarns and perform multiple types of tests on the prepared tapes. Lankhorst has considerable research experience in the developing and production of new tapes and yarns and rope constructions for many years. Innovation is a leading strategy for Lankhorst. They have the availability of 2 R&D extrusion lines and a production park for extrusion and twisting, therefore the level of knowledge and experience with these types of conversions is high. Key persons Fernando Eblagon, senior researcher materials (>10 years experience) Nuno Lopes Rui Barros, senior researcher mechanical engineering(>10 years experience) Rui Bastos - researcher materials (5 years experience) Selected Lankhorst has several patents pending on the use of biobased polymers in tapes and publications/ yarns. Lankhorst is also a participant in the FP7 HAWE project. previous experience related to the task P20 Rhodia Operations (FR) General Rhodia (14.000 employees), headquartered in France and is part of the Solvay Group. description Rhodia is a world leader in the development and production of specialty chemicals, servicing sectors like automotive, electronics, flavors and fragrances, health, personal and home care, consumer goods and industrial. Rhodia has a very strong R&D track record with 700 employees in Europe. The participating R&D centre in Aubervilliers hosts 200 experts recognized for their key competencies in design, formulation, processes and characterization of fine inorganic products, specialty polymers, complex fluids and materials. Role WP4: product development & testing WP5: process demonstration at pilot scale Rhodia will concentrate on chemical analysis, evaluation of the bio-adipic acid vs requirements of final materials such as Polyamide and chemical analysis of the diacid and final polymers. pilot quantities will be tested and produced. Rhodia will also do a chemical evaluation of polysaccharides properties vs. application criteria and modification to make them suitable to the applications Key persons Dr. Franck Touraud is a polymer expert in Rhodia. He has over 40 patents and publications in polymer and material sciences. His experience & centres of interest are polycondensation polymers, from engineering plastics to high performance polymers, including biodegradable polymers for pharmaceutical applications. His main expertise is in the design of any (co)polymers, playing with various levers like composition, architecture (statistical or block copolymers, branched, hyperbranched, networks), functionalization, supramolecular chemistry to meet the required properties for a given
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311956 SPLASH application and market. Dr. Philippe Marion is Laboratories Manager for Organic Synthesis Dr. Grard Mignani is a scientific expert at the Rhodia Operations R&D centre in Lyon. Selected Rhodia has been investing strongly in research in polysaccharides. It is one of their core publications/ competences. Similarly the Lyon research facility boasts state-of-the-art pilot facilities for previous type of research needed in SPLASH. Rhodia already has many patents related to the experience topic at hand. The company is an experienced project partner, currently participating in related to three FP7 projects. 1. F. Touraud, T. Loughman, R. Russell, Kinerton Ltd, Dublin, Ireland, WO199740085, the task 30 Oct. 1997, Acidic polylactic polymers 2. F. Touraud, N. Bryson, Flamel Technologies, Vnissieux, Fr., WO200137809, 31 May 2001, Colloidal suspension of submicronic particles as vector for active principles and method for preparing same 3. J.P. Lagrange, C. Paulo, J.-F. Sassi, F. Touraud, C. Vidil, Rhodianyl, Fr., WO2003002668, 9 Jan. 2003, Thermoplastic polymers, use thereof in polyamide compositions with improved hydrophily and anti-staticity 4. F. Touraud, S. Jol, Rhodia Oprations, WO2010034803, 1 Apr. 2010, Reinforced Polyamide Composition
B2.3 Consortium as a whole The aim of the present project is develop technology for the industrial production of biopolymers with a market volume of potentially over 100.000 ton/yr, and a market value of .1-3/kg, using algae as a raw material. Large-scale Industrial Biotechnology (IB) with algae is a non-existing industry and compared to IB with microbes, the algal biotechnology tools, needed to build a new IB, are missing or under development. Synergies between established IB and developing processes will fasten this process. For this reason, in SPLASH partners with expertise in microalgae and other microorganisms, such as partners DLO-FBR, WU and LG are present. One of the required enabling tools needed to establish IB with algae is a stable, robust, rapid growing production organism (called production platform in the project), that is liable to metabolic engineering. UCAM will be responsible for this. For this, partners UMUE, UNIBI, DLO-PRI and LG will develop enabling knowledge and technology for successful metabolic pathway engineering and improvement of wild strain leading to (1) availability of a well annotated genome of the industrial production platform (a suitable algal strain), (2) quantitative tools or genome-scale metabolic models that permit manipulation of industrial algae, (3) the possibility of expressing algal genes in a production platform. In addition to pathway engineering, fermentation science, innovative downstream processing (partners PDX, DLO-FBR and EGE), process (BIT and AVT), products development (Partners FRAUNHOFER, RHO, AVT, DLO-FBR) and functional testing (LAN) are key enabling technologies needed to establish IB with microalgae as a raw material for the production of biopolymers . The consortium consists of a powerful mix of established research organisations (UCAM, UNIBI, UMUE, WU, DLO, CERTH, UHU, EGE), small and medium enterprises (LG, PDX, OWS, VFT, AVT, PAQ, BIT, NOVA, PNO) large scale industry (FRAUNHOFER, RHO and LAN). All partners have substantial experience in working on complex projects in international environments and have participated in EUfunded projects before. They have strong complementary knowledge and skills required for the successful implementation of the SPLASH project aims, combining high quality research capacity and substantial industrial participation. This will ensure that the research is focussed on the implementation of a new industrial platform for the sustainable production of biopolymers. Excellence of the SPLASH Consortium - The right players at the right time SPLASH has been assembled with the aim to combine the different complementary expertises that are needed to successfully address the topics of the call in a well-balanced consortium. The participants that comprise SPLASH are leaders in their respective fields of expertise and cover the broad range of competences and resources required to achieve the ambitious, but realistic goals set in this project. Overall, SPLASH is a balanced consortium comprising 9 SMEs, 6 Universities, 2 Research organisations and 3 large companies. The entire production chain, from the development of enabling technologies by
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311956 SPLASH partners UMUE, UNIBI, DLO-PRI, LG and from metabolic pathways achieved by WU, UCAM until product development and testing by AVT, FRAUNHOFER, RHO, DLO-FBR and LEP is well covered by the consortium. The presence of industrial parties and the management of exploitable results by an experienced dissemination and exploitation team will ensure that the knowledge tools and technologies developed within SPLASH will result in the establishment of a new sustainable bio-based industry. Comprehensive and complementary assembly of expertises and technologies SPLASH combines forces in all areas of expertise and technologies that are needed to fulfil the proposed tasks and deliverables. These include the genomics, metabolomics, proteomics, modelling, and process engineering. In addition to the summaries given below, information on specific aspects of individual expertises is provided in the individual partner descriptions. The complementarity of expertises held by the individual partners within SPLASH, and the role of participants throughout the project, is presented in figure 14. This overview illustrates the limited redundancy across the consortium, further illustrating the adequateness of the partner acquisition strategy. Modification, Metabolic Engineering andomics One of the main project objectives is to develop an industrial production platform, which harbours the unique hydrocarbon producing ability and can be genetically engineered. In Europe, only a limited number (about 10) of scientific parties are recognised as experts in genetic engineering of algae. Two of them, UCAM and UNIBI are part of the SPLASH consortium. Mutation analysis, another essential biotechnology tool which is presently not developed for algae in Europe, will be covered by DLO-FBR, which has ample experience in mutation analysis of microbes. Of all fields of expertise relevant to SPLASH, the omics (genomics, transcriptomics, metabolomics) is currently the most revolutionary field, which has a big impact on other disciplines. Genomics of algae is only marginally developed in Europe. The parties invited for this task all have ample experience in proteomics, metabolomics, transcriptomics, genome sequencing and bioinformatics of plants (PRI), fungi and bacteria (LG).
Expertise Area Role in CASH
Downstream Processing
Bioprocess Engineering
Metabolic Engineering
Polymer Technology
Systems Biology
Coordinator
WP Leader
Chemistry
WP1
WP2
WP3
WP4
WP5
WP6
M M S M
Nr. Short name Country Department Involved

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 DLO-FBR DLO-PRI CERTH OWS PAQUES BIT VFT AVT LG PDX Nova NL NL GR BE NL DK BE NL PT UK DE Bi ofuel s Pl a nt Sci ences Al ga l Bi otechnol ogy Bi oproces s Engi neeri ng Centre for Bi otechnol ogy Ins titute of pl a nt bi ochemi s try a nd bi otechnol ogy Supercri tica l Fl ui d Technol ogi es Group Orga ni c Innova tion La bora tory x x x x x x x M M M x x WP2 M M M Supercri tica l Fl ui d Technol ogi es Group x x WP5 WP7 M M x x x x x WP6 WP4 M M M M M M M S M Bus i nes s Uni t Bi oba s ed product Bi os ci ence Bus i nes s Uni t Chemi ca l Proces s Engi neeri ng Res ea rch Ins titute x x x x x x x x WP3 M M S M M M S
FRAUNHOFER FI UCAM ES PNO UHU WU UNIBI UMUE EGE LEP RHO UK ES NL DE DE TR PT FR
x x
x x
M x
x
M M M M M
Figure 14: SPLASH consortium: areas of expertise and role in the project. Symbols Indicate M = Main Work package, participant playing an important role for at least one of the tasks; S = Supporting Work package participant providing technology or knowledge needed to perform the work
Systems models With the large amounts of data generated by the omics techniques, it is essential to take a systems approach and build appropriate models, particularly of metabolic flux. The SME LG has an in-depth and
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311956 SPLASH in-breadth experience in systems biology, WU has been at the forefront of metabolic flux engineering of animal cells, and has recently extended its expertise to algae. Complementary modelling expertise on statistical models is brought in by PRI. This combination of expertises is ideal for the development of systems models. Bioprocess Engineering and downstream processing Fermentation, process design and knowledge of scaling up are further essential tools. The leading parties in Europe in these expertise fields (both for algae and microbes) are WU and DLO-FBR. The infrastructure required for scale-up for algae production and product extraction will be provided by DLOFBR (see next section AlgaePARC) and an innovative reactor concept will be provided by PAQ. In addition, the company BIT, which has a long experience in the cultivation of microalgae, will contribute to the optimisation and scale-up of microalgae cultivation systems, while producing biomass throughout the duration of the project, for the partners developing downstream processes. DLO-FBR together with the industrial partner PDX, an SME specialised in supersonic flow fluid processing will develop an in situ continuous extraction process. Integration with the cultivation system and scale-up of the integrated system will require an excellent coordination of expertises from the SMEs PAQ, PDX and research institute DLO-FBR. RHO will perform lab evaluations and run a pilot on the transformation of monomers to polymers using the produced polysaccharides. Product development and functional testing Three industry partners (FRAUNHOFER, RHO and LEP), two SMEs (AVT, LEP) and one research organizations (DLO-FBR) are involved in product development. The industrial partners represent the producers of intermediates and different end users for the main algal products (FRAUNHOFER, RHO and LEP); AVT is a worldwide recognised leader (Cleantech Top 100 Company) in catalytic biomass conversion into furanic building blocks and high throughput technology; DLO-FBR has a large expertise in the production and industrial application of a variety of chemicals, has insight on the determination of physico-chemical properties, chemo-enzymatic methods and product blending for the development of new chemicals. The role of SMEs in SPLASH The small- and medium-sized enterprise (SME) sector is considered crucial to European competitive development. It employs the majority of the European labour force and commands two thirds of sales volume in the non-primary sector. Most of the expansion of employment in Europe over the last decade has been in very small firms. The sector's importance is reflected in a consistent long-term European SME policy, with the major emphasis on creating a favourable competitive business environment. SPLASH involves eight SMEs representing different industrial sectors. Collaboration with SMEs is extremely important as they are the drivers behind development of new markets, playing therefore a key role in the development of bio-refineries using microalgae as many new application still need to be developed. All SMEs in SPLASH have different roles; PAQ will provide a demo pilot plant and will be closely involved in improvement and development of new microalgae cultivation techniques. Their involvement of both companies will improve the acceleration of microalgae cultivation in Europe. LG will play a role in the commercialisation of microalgae strain development using a systems biology approach, PDX will play a role in downstream processing, an essential step to be developed before commercialisation of bulk microalgae products. Avantium will test suitability of polysaccharides for the new field of furanic chemicals (one of de US DOE appointed top 12 biomass chemicals of the future). Being a new field in development, it is important that it is proven as an added value to the European economy and environment. For this reason, SMEs specialised in LCA (OWS), market analyses (VFT and PNO) and communication (NOVA, PNO) are on board of SPLASH. The consortium shows both strength in depth of expertise of individual members, effective collaboration mechanisms, and the excellent integration between them to realize the objectives of the project. This integration will be further facilitated by dissemination / communication activities of WP7 and input of the Scientific Board and Dissemination, Exploitation and Intellectual Property Advisory board.
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311956 SPLASH Synergies with other projects (with partner involved) Recently, two large projects were funded, which are expected to develop strong links with the SPLASH Project. The first project is AlgaePARC, an 8 million pilot facility for the industrial production (open and closed cultivation) with algae. The facility is co-funded by the Province of Gelderland, the Dutch Ministry of Agriculture and 14 European Industry partners active in different sectors (oil, chemical, food and technology development). The pilot plant will allow fast implementation of the SPLASH findings and developments accomplished at lab scale. Moreover, the pilot plant will provide practical experience, real productivities and shorten the time steps for full commercial exploitation of the processes developed within SPLASH. The second project is the Center for Photosynthesis Research, Towards BioSolar Cells (TBSC)40, an initiative of four Dutch Universities (Wageningen, Delft, Groningen and Amsterdam) and a total budget of 45 million. The research focus of TBSC is on improving the photosynthetic and metabolic efficiency of plants and algae. Botryococcus serves as model for algal photosynthesis in TBSC. The genomics and metabolic engineering work planned in SPLASH will certainly benefit from the interaction with work planned in TBSC. Additionally, AquaFUELS Algae and aquatic biomass for a sustainable production of 2nd generation biofuels is an EU coordination and support action. Project objectives are; (1) provide complete set of surveys identifying actual status quo of algae for biofuels; (2) provide complete set of assessments (technological, economic, environmental and social); (3) identifying future research needs (4); concretely contribute to structuring the field by means of networking with more than 30 ongoing projects (over 64 million) covering all AquaFUELS issues which are creating a European Algae Biomass Association (EABA) and building consensus among major stakeholders (science, industry, policy, standardization , society at large). AquaFUELS can be used as a platform for dissemination and exploitation of PolyAlgae. Furthermore, SUNBIOPATH Towards a better sunlight to biomass conversion efficiency in microalgae is an EU Small or medium Collaborative project. SUNBIOPATH is aimed at improving biomass yields and valorisation of biomass of Chlamydomonas reinhardtii and Dunaliella salina through the production of antigens in the chloroplast and/or biomethane production. Synergy with Polyalgae will be on genetic engineering of Chlamydomonas and biosafety, more specifically scale up of processes for engineered algae.
Synergies with EU projects FP6 EPOBIO (POLICIES1.2) FP7 - AquaFUELS (ENERGY.2009.3.2.1) FP7 SUNBIOPATH (KBBE2009-3-2-02) FP7 - BIOREF-INTEG (ENERGY-2007-3.3-03) FP7 - SOLARH2 (ENERGY2007-3.5-01) Germany: Ecological innovation politics Research into more resource efficiency and climate protection through sustainable industrial material use of biomass Germany: The development of instruments to support the material use of renewable raw materials in Germany Netherlands: AlgaePARC. International research program aimed at comparing different microalgae bioreactors at pilot scale Netherlands: Algicoat. Development of a whole crop biorefinery concept for large-scale algal cultures, co-producing poly-unsaturated fatty acids and polyols as raw materials for coating components, oil for fuels and heat / electricity Netherlands: "Towards BioSolar Cells programme. Cat-Fur organosolv pulping of lignocellulose for furanics production and techno-economic analysis including LCA of the process. Netherlands: Algae and genetic modification Present an overview on the technical developments on genetic modification of algae for food and non-food applications with focus on changes in fitness and environmental risks FP7 BioAlgaeSorb (SME-2) Synergies with EU national projects Belgium: Alchemis (IWT)
FP7 - SYSINBIO (KBBE-20073-2-05) FP7 BIOCORE (KBBE2009-3-7-01)
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http://documents.plant.wur.nl/pri/biosolar-safe.pdf
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Finally, DLO (PRI) and WU recently established a Research Agreement with the University of Tsukuba (Japan). This University is one of the very few in the world having expertise in growing Botryococcus and is presently working on engineering Botryococcus. The University of Tsubuka is not a partner in SPLASH, but we agreed to exchange non-confidential information between SPLASH and the Tsukuba project (CREST). Also, a workshop will be planned on Botryococcus between SPLASH and CREST members. The members of the CREST project are University of Tsukuba, National Institute for Environmental Studies (NIES), Tokyo Institute of Technology, Institute of Optics, DENSO Co. LtD, Mitsubishi Chemical Holdings Co. LtD, Nippon Steel Co. LtD and TEPCO, Idemitsu. B2.4 Resources to be committed The overall financial plan of the SPLASH project is summarised in the tables below. The project will take 48 month to achieve its objectives. The overall project cost is 12.368.381 and the overall EC funding requested is 8.942.933. The total costs are split over the following categories: Requested PERSONNEL costs: 5.868.709 These costs represent the person-months in WP1-WP7. The bulk of the project budget concerns RTD. The costs are based on an accurate estimation of person-months necessary to carry out the various tasks that form the work plan. They have been calculated through average monthly rates cost of personnel involved by each one of the partners. Requested OTHER costs (consumables, travel, parts, dissemination): 2.178.460 Other Costs in this project are substantial in size. The costs have been carefully calculated, taking into account existing infrastructure and facilities. Most of the cost will go into consumables. For instance, microalgae cultivation will need a large amount of production raw materials (consumables and usage of a 5 m3 photo-bioreactor in order to produce the required amount of biomass and the delivery of standardised quality biomass of reactors; BIT). WP2 also has substantial Other Costs related specifically to sequencing of the Botryococcus genome and ESTs. WP3 and WP4 have high Other Costs specifically related to transcriptomics, proteomics and metabolomics. In WP3 substantial costs have been reserved for PDX to cover the transfer of one of the supersonic fluid pilot reactors (PDX) to Wageningen to operate in continuous production. WP5 costs relate to equipment and consumables for the demonstration setup, like the pilot from AVT for production of 2,5 FDCA and PEF thin films. FRAUNHOFER and RHO will have costs related to chemical analysis (consumables) and product development.WP6 and WP7 have limited Other Costs related to dissemination activities described in section 3.2.3. DLO-FBR will use the infrastructure of AlgaePARC. PAQUES will use the infrastructure of its workshop to construct the pilot scale photo-bioreactors. FRAUNHOFER, AVT, PDX will use their (pilot) research facilities for new applications and product development. LEP will use their pilot line to perform the required tests. RHO will use its pilot facilities in Lyon for transforming polysaccharide-originated monomers to polymers. A new outdoor pilot plant flat panel photobioreactor will be built at Wageningen (DLO-FBR, PAQ) and in Spain (UHU and BIT). Travel costs for work package meetings, (annual) consortium meetings and dissemination have been calculated on the basis 500 for travel costs per person and 150 for hotel and subsistence costs per person. The no. of meetings has been minimized and are planned to coincide with official Consortium Meetings. Each partner will attend the annual Consortium meeting, as well as the initial kick-off meeting, the interim report meeting and the final report meeting. The project coordinator will claim an amount of 6.000 for travel expenses of the two external Advisory Boards which will meet with the coordinator during the consortium meetings. Any additional meetings will in principle - be held electronically. Travel costs have also been allocated to dissemination activities which are listed in section 3.2.3. No costs have been calculated for travel outside Europe! Requested INDIRECT costs: 3.926.274 49 | P a g e
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311956 SPLASH Indirect costs have been calculated through the cost model and the overheads provided by the partners. The industrial partners LEP and PDX use the 20% flat rate method. Nine partners work on the basis of Real Indirect Costs method. The others use the 60% transitional rate. Requested SUBCONTRACTING costs: 131.500 This project will involve subcontracting of certain aspects of work. OTHER Website development: The total expected subcontracting costs for developing, hosting and maintaining the website are: 15.000 (part of WP7, partner Nova). 15.000 is also reserved to cover costs related to printing of dissemination materials and presentation materials (part of WP7, partner Nova). MNG Partners claim a total of 5.000 per audit certificate each to cover the costs of financial guarantees and audit certificates for each 375 000 subsidy-, with a total of 90 000 (DLO- 25.000; PAQ, BIC, AVT, Nova, UCAM, UNIBI, UMUE, 5.000 each; LG and PDX, 10.000 each) RTD . PAQ - Euro 5 000. This budget is meant to cover analytical costs at laboratories for chemical analysis that cannot be done in house VFT - Euro 5 000 to buy market studies to support the economic analysis LEP - Euro 1 500 for performing flame retardancy en UV tests from the produced yarns. These are important properties for these kind of yarns. Mechanical tests can be done at LEP but these specific properties need to be done by an external lab.
Requested PROJECT MANAGEMENT costs: 438.302 (including 90.000 for audit certificates) EU funded Management costs amount to 4.9% of total EC requested funding. The management costs reflect the complexity of the project.
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B3. Impact B3.1 Contribution of this project to the expected impacts listed in the work programme The SPLASH project addresses KBBE.2012.3.4-02 Biotechnology for novel biopolymers and complies with the technical content and scope of the expected project results as stated in the Work Programme: 1. SPLASH will develop a new pathway for industrial production of hydrocarbons and polysaccharides to create biopolymers from gene-transfer in micro-algae. This will lead to a factor-5 increase of hydrocarbon and polysaccharide productivity and the establishment of a new industrial platform. 2. SPLASH will establish and test an industrial production platform for hydrocarbons and polysaccharides by optimizing growth conditions and strain improvement. In total 60kilograms of polysaccharides will be produced to demonstrate its conversion to the final product (PEF for packaging and polyesters and polyamides for yarns). Ten kilograms of green naphtha will be produced and cracked to allow comparison with fossil based naphtha. 3. SPLASH will research recycling options for novel biopolymers through energy recovery, mechanical recycling, composting, anaerobic digestion and chemical recycling 4. The close research collaboration between SPLASH partners will include standardisation as an integral part of its R&D work packages. This will facilitate transfer of knowledge and establish a credible roadmap for production, processing and product-optimisation of bio-based hydrocarbon and carbohydrate production. SPLASH will significantly contribute toward long-term and sustained improvement of its current trade deficit on polymers with the US and Japan, and help ensure that it will not be overtaken by Brazil, China and the Middle East. As evidenced by the partner list, SPLASH is rooted in a strong industry drive to reduce dependence on hydrocarbons from fossil sources. There are several clear reasons why industry is looking toward more economically and environmentally sustainable alternatives to maintain global longterm competitiveness: high volatility in fossil energy prices, which negatively influence competitiveness and production continuity. expected demand-growth of 5% per year for propylene and ethylene41. How can this demand be satisfied with diminishing availability of current raw materials? predictions that by 2020 bio-based products will account for 20% of sales within the chemical industry42. Once oil and natural gas reserves become more depleted, the chemical sector needs to be able to switch to other primary raw. Increased demand for chemical products like propylene and ethylene will also put further environmental pressure (emission rights, waste disposal etc.) on the industry, making old-style production less and less economically attractive. By being able to deduct bio-based carbon from a companys total footprint (see below for details), its competitive position may improve instead of decline further. Environmental considerations Substantial emission reductions from energy consumption and chemicals production have already been achieved over the past 20 years, but from an environmental and economic perspective, more needs to be done to maintain long term competitiveness. SPLASH will have the following environmental benefits:
41 42
EPTQ.com (the refining, gas and petrochemicals processing website). Quote taken from EPQT quarterly journal, Q3 2010, p. 87. Accelerating the development of the market for bio-based products in Europe. Report of the Taskforce on Bio-based products. Composed in preparation of the Communication A Lead Market Initiative for Europe. {COM(2007) 860 final}. http://ec.europa.eu/enterprise/policies/innovation/files/lead-market-initiative/bio_based_products_taksforce_report_en.pdf
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311956 SPLASH Reduction in energy usage and GHG emission at petroleum refining stage as a result of not having to pump crude oil in order to produce naphtha at a specified quality level, avoiding e.g. the need for sulphur removal. SPLASH will investigate whether hydrocarbons can be tailored such that the energy cost to crack hydrocarbon chains are less, due to higher purity of algal hydrocarbons (ie. no need to filter out unwanted byproducts like mercury and sulphur from natural gas condensate). This could result in significant emission and energy consumption reduction. Research43 shows an expected annual growth of nearly 5% for fermentation products. By producing biopolymers (polysaccharides) from Chlamydomonas reinhardtii through oxidation into adipic acid: o no need for petroleum as base resource for producing cyclohexanone (as reaction of cyclohexane with air; cyclohexane is produced by reacting oil-derived benzene with hydrogen) o no need for production of benzene, which is highly toxic the production of hydrocarbons and polysaccharides from algae does not compete with land use or plant crops needed for food production. Algae production facilities can be constructed on industrial or brownfield sites, whereby the best location (local at chemical site or centralised production sites) will be investigated as part of WP6. the algae production systems are expected to be built at locations with higher solar irradiance since this will lead to higher productivities and the need for less ground area. Preferably this should be done at the plant site. For an average plant site with a capacity of 940000 tonnes/year of polyethylene and 620000 tonnes/year of polypropylene an estimated algae installation of 30000 ha will be required to supply this quantities. Estimated total fixed capital costs per year (depreciation in 10 years) and operational costs are 3000 M and 6000 M, respectively, per year. These values will be validated in WP5 and used in WP6 for the sustainability assessment.
Potential market, conditions and actions needed toward commercial application The European chemical sector produces 24 per cent of the worlds chemicals, employs 1.2 million workers and contributes 449 billion to the EU economy. According to Eurostat and Cefic Chemdata analysis 2011, the EU chemical trade surplus was at a record level in 2010. The long-term target of the European chemistry policy is to derive at least 25% of the bulk chemicals from biomass in 202544. The petrochemical industry currently relies on only a few so-called platform chemicals, generated by steam cracking of naphtha: ethylene, propylene, butadiene, and the aromatic compounds benzene, toluene and xylenes (BTX). In 2009, Western European production of platform chemicals amounted 46 million tonnes.45 In petrochemical conversions, functionality, in the form of oxygen, is added. The production of chemicals from biomass usually takes the reverse approach: oxygen-rich biomass such as a carbohydrate is subjected to treatments which remove a significant share of oxygen. Current petrochemical infrastructure is in most cases not suited for such de-oxygenations, or cannot handle oxygen-rich biomass very well. From a cost perspective the chemical industry would strongly prefer a biological feedstock of renewable hydrocarbons that can be processed with existing infrastructure. Botryococcus braunii has been chosen as the carrier in SPLASH precisely because the hydrocarbons can be used directly as a feedstock in existing petrochemical processes whilst the isolated carbohydrates (ie. polysaccharides) can be converted to an existing polymer building block (ie. adipic acid). In the table below, current and expected production volumes can be found of the chemicals covered by SPLASH:
43
World Market for Fermentation Ingredients, Study GA-103R by Business Communications Company Inc.,Norwalk, - March 2005 & Fermentation Chemicals, Industry Study 1921 by The Freedonia Group Inc., Cleveland, May 2005. 44 A European Technology Platform for sustainable chemistry. SusChem (June 2004). 45 http://www.petrochemistry.net/facts-and-figures-statistics.html (January 2011).
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j SPLASH products Platform b chemicals Adipic acid Furanics
a b
European yearly production 6 a (10 tons) 46 1 47 46
European predictions 6 for 2015 (10 tons) for biobased chemicals and polymers 4 0.1 4
European 25% 6 target in 2025 (10 tons) for biobased chemicals and polymers 9.2 0.2 10
Expected contribution c of SPLASH in 2025
8% 13% 13%
In Western Europe in 2009 [http://www.petrochemistry.net/facts-and-figures-statistics.html (Jan 2011] Includes ethylene (18.8 Mt), propylene (14.4 Mt), butadiene (1.8 Mt), benzene (7.2 Mt), toluene (1.6 Mt), and xylenes (2.2 Mt). Assumptions: 50% of biomass dry weight are hydrocarbons and 30% are polysaccharides. Location of production facility is Southern Europe, 4% photosynthetic efficiency is assumed leading to a productivity of around 100 ton DW biomass /ha/year. Northern Europe 4 % PE, c.a. 50 ton DW /ha/year an average of both is used. Total arable irrigated land available 48 in Europe is 25892340 ha (10% of total land). We assumed that only 1% of this land will be available for microalgae production in 2025 as this industry is still in development.
The above table is the expected contribution of SPLASH in 2025; the last column represents the expected contribution-percentage to an average chemical plant foreseen as end-user (ie. like Solvay etc.). The uptake and volumes as presented in the table are only possible if marine biotechnology is able to bring the cost of algal down from currently 4.00 per kg49 to around 0.40 per kg. Fragmented research and (SME) piloting capacity Commercial application of microalgae has thus far primarily concentrated on high-value compounds (>10/kg). Little research has been carried out with respect to industrial bulk production, where a much lower cost price is crucial. However, this is exactly what is needed in order to maintain and improve competitiveness of the European chemical sector. Companies and national governments recognise the potential of micro-algae, but are uncertain about the technical and economic viability as well as consistency of the end-product quality of large-scale production facilities. SPLASH has a strategic approach in which research, innovation and capitalisation are combined. The high industry-participation in SPLASH, particularly from SMEs (40%), underlines the link between research output and industrial impact. Biotechnology, and the cultivation of microalgae more particular, has potential to give European SMEs a very strong international competitive position. Latest data of the Marine Board of the European Science Foundation (ESF)50 show a market value of 2.8 million in 2010 and a potential to grow 12% each year. This might not seem much by todays standards, but marine biotechnology is typically a domain in which highly innovative SMEs drive technological innovation with a steep turnover curve. However, individually these SMEs do not have the time and cannot take on the costs of researching the best feedstock and building larger-scale test facilities needed to convince their clients (chemical industries) of consistency in output and quality. This inability hampers their bio-based product and services development. In this sense, the SPLASH consortium is a response to the Commissions analysis that the EU has very good research and development capacities in some key enabling technology areas, but is not as successful in commercialising research results through manufactured goods and services51
46 47
http://www.icis.com/Articles/2011/02/28/9439026/european-chemical-profile-adipic-acid.html No. current production. Estimated global production volume of terephthalic acid is about 40 million tonne,, which can be replaced by furanic chemicals. 48 http://faostat.fao.org/site/377/DesktopDefault.aspx?PageID=377#ancor 49 Norsker NH, Barbosa MJ, Vermu MH, Wijffels RH (2011). Microalgal production a close look at the economics. Biotechnol Adv 29:24-27(November 2007). 50 Europe can be marine biotech leader. ESF report (January 2011). 51 European Commission: Preparing for our future: Developing a common strategy for key enabling technologies in the EU", COM(2009) 512/3, p. 2.
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The SPLASH project consortium consists of almost 50% SMEs. They will have an important role in microalgae cultivation, downstream processing and chemistry. Successful integration of the microalgae biotechnology sector with the existing chemical industrial sectors, as is the case in the SPLASH project, will improve the market for many SME microalgae producers and also stimulate the agricultural sector. Downstream processing and chemistry are already established markets, but expanding business and new innovation will strengthen these companies economic position globally. Necessary steps toward achieving industrial production The R&D intensity of the European chemical industry is rather limited (1.5% of sales in 2008), not only compared to Japan (4.1%) and the US (2.1%), but also compared to other industry sectors, where annual R&D investment can amount to between 15%-30% (telecommunications) 52. This percentage is, however, not unusual for a processing industry, whereby continuity and consistency is paramount. Microalgae biotechnology research is still in an early stage, even though industry clearly recognises its longer term industrial potential. There are two main hurdles to take in order to push production of biopolymers from microalgae to the next level: 1. speeding up hydrocarbon and polysaccharide metabolism to a level at which it becomes possible to set up larger-scale demonstration facilities which can be used to test and validate quantity and quality of the produced hydrocarbon 2. establishing and validating technical and economic data on the cultivation and processing of microalgae in an industrial setting. There is no doubt that the market demand for hydrocarbons is there, but it is unclear at which exact point in time microalgae could reach price parity with petroleum and natural gas. The information gathered will provide the chemical industry with credible data on which to base further R&D and production investment into microalgae. Once the principal hurdles are overcome, industry will then be in a position to jointly work with the science community on further tailoring the hydrocarbons toward desired molecule lengths that reduce energy consumption (and thus reduce energy cost) during cracking. Ramping up production to full production level to achieve volumes stated earlier in this chapter should be possible within the next 10-15 year. Transnational need for collaboration The concept of biorefineries, needed to cultivate and harvest hydrocarbons from microalgae, requires a true multidisciplinary and transnational approach with participation of many companies active in agriculture, biotechnology and chemistry. Scientific knowledge is spread over small research communities across Europe and applications exist only are at a small scale. Furthermore part of the research must also include finding optimal locations for cultivation of microalgae, as sunny areas (Southern Europe) are very well suited to produce microalgae due to their high production rate and locations close to chemical industries (mostly located in northern Europe), present advantages in terms of logistics and accessibility to nutrients. The technological development is at a too early stage for the existing sector to reach critical research and development mass on its own, as costs are simply too high and technical risks too many. It is therefore important that the European Commission has taken the initiative to set a clear innovation agenda which connects industry needs with national policies towards sustained international competitiveness for Europes bio-chemical businesses. Support from the FP7 programme is vital to bring the available expertise together to get to the stage at which a real demonstration project can deliver data on economic, technical and environmental viability and produce a credible market analysis. European Commission Initiatives & Communications In 200253 the European Commission identified biotechnology as the next wave (after ICT) of the knowledge-based economy, creating new opportunities for our societies and economies. SPLASH will deliver on several of the EUs economic (competitiveness), societal (prosperity, knowledge based society) and environmental policies and priorities, as explained below:
52 53
Cefic Chemdata, 2011 European Commission: Communication on Life Sciences and Biotechnology; COM(2002) 27 final, p. 3
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311956 SPLASH Developing a common strategy for Key Enabling Technologies in the EU: SPLASH is a prime example of a KET54 because it provides the chemical industry with new sustainable tools to meet the increasing demand for goods that can be used in all types of new applications that may help meet future Societal Challenges on topics like climate change, energy transport and storage or even medical applications. SPLASH is a knowledge intensive project with a high R&D requirement which will produce rapid innovation cycles during the development of a new high metabolism green algae. EU-Lead market Initiative: SPLASH will create the conditions (ie. development of bio-polymers and green naphtha) that will help remove uncertainty about product properties.55 According to the Commissions policy recommendations, industry will benefit from the SPLASH project as biobased carbon in products will be deducted from their total carbon footprint56. In doing so, SPLASH will give a major boost to the European chemical industry, which, according to the SusChem European Technology Platform57, is currently declining. Environmental Technology Action Plan (ETAP) and the 20-20-20 Plan: SPLASH is focused on demonstration activities which will help build experience of the different stakeholders and reduce uncertainty about the technologys economic viability58. The SPLASH project will also provide a serious contribution toward Europes 20-20-20 targets on CO2 reduction. SPLASH is a logical next step in the chemical industrys ongoing drive59 to reduce GHG emissions. With state of the art closed photo-bioreactor systems, a photosynthetic efficiency on solar energy of 3% can be reached with the current technology. This can be translated to a productivity of circa 70 ton dry algal biomass/ha/yr under solar conditions found in South Europe (Seville was used for calculations). Future technology will likely allow production levels of up to 150 ton/ha/yr (7% photosynthetic efficiency at the same location, maximum theoretical efficiency is 9%). For each hectare of photo-bioreactor system (at a productivity of 70 ton/ha/yr algal biomass) 130 ton CO2 will be captured per year. With only 1% of agricultural land used for microalgae cultivation already 33.6 * 106 ton CO2 per year could be captured (2% of the total emission)60. By further using industrial CO2, some additional revenues may be provided, estimated at 50/ton algal biomass (at carbon capture and storage costs of 25/ton).61 Tackling challenges in commodity markets and on raw materials: Microalgae will deliver the means for establishing locally available (as opposed to outside the EU) raw materials production (hydrocarbons and polysaccharides) which is environmentally sustainable and thus offers long-term security of raw material supply to the European chemical sector. In doing so, SPLASH demonstrates the potential of green algae to be part of the progressive replacement of non-renewable materials currently used in various industries with renewable resources62 Innovation Union: The Commissions Flagship on Innovation Union wants to establish well integrated projects which deliver rapid R&D breakthrough with the express aim to transfer the gained knowledge into concrete innovative and competitive (marketable) products and services for both large and small companies. The SPLASH project has a strong focus on results, outcomes and impacts related to the efficient and sustainable production of micro-algae based products as non-energy raw materials along the entire chemical value chain. SPLASH can build upon ongoing nationally supported research, like in the Netherlands with the Wageningen AlgaePARC (which was partfinanced by the Dutch national government). This will make SPLASH a truly European Innovation Partnership and an example of a successful European Research Area. Resource-efficient Europe: The SPLASH project responds to all 4 framework aims63:
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European Commission, Preparing for our future: Developing a common strategy for key enabling technologies in the EU, COM(2009) 512/3, p. 3 55 COM(2007) 860 final, pp. 4-6. 56 See also: European Commission: "A resource-efficient Europe: flagship initiative under the Europe 2020 strategy, COM(2011) 21, p.18 57 Innovating for a better future. Sustainable chemistry: a catalyst for innovation and growth in Europe. SusChem report (February 2006). 58 http://ec.europa.eu/environment/etap/index_en.htm (January 2011). 59 www.icca-chem.org 60 http://europa.eu/rapid/pressReleasesAction.do?reference=IP/08/787&format=HTML&aged=0&language=EN&guiLanguage=en. Retrieved 61 Sustainable growth - for a resource efficient, greener and more competitive economy. (January 2011) http://ec.europa.eu/europe2020/priorities/sustainable-growth/index_en.htm 62 European Commission: Preparing for our future: Developing a common strategy for key enabling technologies in the EU", COM(20 09) 512/3, p. 3. 63 European Commission: A resource-efficient Europe Flagship initiative under the Europe 2020 Strategy, COM(2011) 21, p. 3
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311956 SPLASH 1. it will help boost economic performance while reducing resource use by providing the chemical sector with locally abundant available raw materials which have the potential to replace fossil fuels as primary carrier for base chemicals. If hydrocarbons and polysaccharides can be produced on an industrial scale independent of scarcity (and therefore price volatility) and geographic distance to the source, European companies are in a better position to compete worldwide 2. SPLASH will significantly change the way many chemistry-products are produced, whilst also making the process of industrialized harvesting of modified green algae a highly marketable product in itself (particularly for the SME companies) 3. If hydrocarbons and polysaccharides can be harvested locally in Europe, this leads to a significant increase of security of supply of essential resources for European chemical companies, compared to the use of for example - petroleum (in case of naphtha) or natural gas condensate. 4. If hydrocarbons can be produced sustainably and in sufficient quantities from green algae, less petroleum and natural gas condensate needs to be converted (as a by-product) at existing refineries. Industrial policy for the globalization era: Standardisation of the engineering process and in particular downstream processing of the green algae biomass toward a consistent quality of produced hydrocarbons and polysaccharides will be key in facilitating European and worldwide market penetration of bio-produced base chemicals, fucose, rhamnose and galactose. The SPLASH consortium will make the definition of standards in this field an integral part of all R&D WPs and will take into account standardization issues in the work package on market analysis and economic viability. Agenda for new skills & jobs: By supporting large-scale projects like SPLASH, the EC and the consortium will promote biotechnologys research and entrepreneurial potential, making it attractive for young researchers to join the small (currently estimated at less than 500 researchers in Europe) research community. The projects combination of strong academic research partners, specialized SMEs and large corporate end-users will allow researchers to build-up knowledge in EU marine biotechnology from several angles and to take advantage of cross-discipline research career possibilities. At present, there is no large-scale microalgae production in Europe. The successful outcome of SPLASH would have significant impact on future jobs ranging from agricultural to middle/senior-level scientists within the chemical industry. The consortium estimates the sector could grow to as large as 10.000 full-time jobs in Europe. ERA framework: SPLASH contributes to the ERA vision64 by: o mitigating the fragmentation of resources, know-how and technology, across the ERA and thereby fostering effective cross-border and cross-sectoral innovation. SPLASH is clearly aimed at bringing together all relevant stakeholders around microalgae biotechnology and biorefinery in Europe and creating a common vision and standards for the future. o establishing a research-friendly ecology needed to allow actors and institutions to work together in productive innovation networks. This proposal is a major step in creating such an integrated innovation network, in which basic research and industrial research are combined to establish market applicability and viability of a whole value chain.
Assumptions, external factors that may influence achievement of expected impact This project works on the assumption that scarcity of resources combined with a need to mitigate climate change leads companies to search for sustainable alternatives which provide longterm supply security and provide a new competitive edge. From a technical perspective the project assumes that it will be possible to isolate genes and transfer/engineer them to an extend that a significant production of relevant products can be achieved through a harvesting mechanism which still needs to be developed. It is also assumed that through this project new ways will be discovered that bring the process fairly quickly to some form of a price-parity with fossil resources, and similar to first generation biomass derived raw materials.
64
http://ec.europa.eu/research/era/pdf/eg7-era-rationales-final-report_en.pdf.
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311956 SPLASH The success of SPLASH is thus influenced by the ability of the project team to tackle several multidisciplinary technological, economic and societal challenges. The SPLASH consortium is strongly aware of these risks and hurdles. As far as technical risks from assumptions and external factor are concerned, these have been dealt with in chapter 1.3, where each work package description is followed by a risk mitigation plan. There are several short term and long term factors influencing wider implementation of microalgae for bulk chemical production. Short term factors are likely to be political issues and public perception of the use of gene-tailored algae. Another important factor is the fluctuation of oil prices and its effect on an uncertain global economic market. This short term thinking may in turn have significant impact on long term necessary changes in the economy and the environment. Public perception on climate change also influences the investment level. A EC study showed that respondents who feel that climate change is a serious global problem declined from 62% in spring 2008 to 47% in autumn 200965. Budget cuts as a result of the economic decline may (further) reduce national governments programmes on climate change and sustainability. This may in turn lower private investment confidence in new biotechnologies. Other national or international research activities Several EU and national projects are investigating the use of micro-algae for fuel or industrial applications. As shown in section 2.3.3 the consortium has identified 7 FP7 projects, 1 FP6 project and 4 EU national projects which each address very specific needs and challenges. The projects and their results have been closely analysed and taken into account whilst writing this proposal. The SPLASH project is different in that it is the first large-scale project which attempts to engineer hydrocarbonmetabolism by transferring genes to a different type of algae. It is the only project which encompasses the full value chain from bio-tailoring of hydrocarbon production to product development, optimisation and testing for the chemical industry. It is also the only European project which bridges academic, large company and SME interests in the field of biotechnology at this time. The SPLASH project team also reviewed other global projects mainly focused in the US. NREL is presently applying for a larger program in the framework of sustainability programs initiated under the administration of president Obama. Companies like Sapphire ($150 million funding via the Bill Gates foundation) and Solix biofuels, a spin off from Colorado State University are also actively researching this topic. The company Seambiotic was founded in Israel to produce biodiesel from microalgae.
B3.2 Dissemination and exploitation of project results, management of intellectual property There is a lack of European patents in the field of biorefinery compared to North America. SPLASH is expected to generate significant Intellectual Property (IP). Therefore, much attention will be placed on the analysis of patentable issues and awareness building of on Intellectual Property Right Issues (IPRissues). Intellectual property protection The Consortium will follow European Commission guidelines for Intellectual Property (IP) management66 with recommendations on IP management67. Further points will be covered in the Consortium Agreement (the FP7 Consortium Agreement of DESCA will be used as a model for the CA). The consortium recognises the following main categories of IP: 1) Background IP (BIP), 2) Foreground IP (FIP) and 3) Sideground IP (SIB). Management of intellectual property IPR-issues will mainly be the responsibility of the Dissemination and Exploitation Officer (DEO). The DEO has overall responsibility for the earliest possible identification of protectable FIB. The DEO will be assisted by the Dissemination, Exploitation and Intellectual Property Advisory Board (DEIPAB).
65
Europeans attitude towards climate change special report Eurobarometer 313 (November 2009) http: ec.europa.eu/public_opinion/archives/ebs/ebs_313_en.pdf 66 Strategic guide to successful use and dissemination of research and development projects-McNErney O. et al www.useanddiffuse.eu (2009) 67 www://ipr-helpdesk.org
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311956 SPLASH The DEIPAB acts as a first point of contact to IP generating parties, and will assist in valorization activities of individual partners or the consortium as a whole. The role of the DEIPAB is to: implement policies to increase awareness of SPLASH researchers and to stimulate their contribution to valorisation activities guarantee that research results with commercial potential are proactively identified at the earliest possible stage during (or after) the project guarantee that project results are presented to their maximum effect to stakeholders inside and outside the consortium, without compromising commercial interests of any consortium member guarantee that SPLASH IP is fully (commercially) exploited, in first instance by the consortium parties, but if possible also by stakeholders outside the consortium identify gaps in the necessary knowledge. A clear protocol has been developed to ensure that appropriate and uniform procedures are followed (figure 15). The DEIPAB and EO will ensure that the procedure from submission of the Results Sheet until the License-to-publish-letter takes less than 100 days. Publication of results and protection of intellectual property will be on the agenda of all Project meetings.
Figure 15. Protocol for dissemination and exploitation of results.
Exploitation of project results The DEO is responsible for exploitation of project results including market exploration and screening for exploitation opportunities of SPLASH results. This will be done through desk-studies and market data analysis. The DEO will create a draft business plan half way during the project and a final plan at the end of the project. This business plan will contain the required steps for further exploitation of the developed concept and technology by the partners. The success of further exploitation beyond the project lifetime lies with the industrial partners who will translate the business plan into a concrete actions and developments. The business plan will be prepared by the DEO who will be assisted by the PC and the partner(s) involved with specific IP. The business plan will include 1) background information, 2) assessment of the commercial potential of an invention (a marketing plan), 3) risk assessment (technology, market, financial, business model, management), 4) exploitation operation plan, and 5) financial plan. The DEO will assist the partners with licensing issues, advice and coaching in order to maximise chances of successful market penetration. The consortium expects to create project FIP and develop exploitation and dissemination strategies in the following areas: novel micro-organisms, algae biomass cultivation, downstream processing and
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311956 SPLASH biorefinery, innovations in chemical routes, integrated processes and industrial designs. The expected outcomes of the project:
Topic Expected results Base chemicals via cracking of hydrocarbons Polymers Building blocks Adipic acid from polysaccharides Industry Benefits Environmental friendly production, reduction CO2 Environmental friendly production, reduction CO2, new material properties Environmental friendly production, reduction CO2, new material properties Higher production yields Environmental friendly production, reduction CO2 Environmental friendly production Environmental friendly production, Reduction in CO2 emissions Process engineering knowledge for implementations in other biotechnology processes Process engineering knowledge for implementations in other biotechnology processes Performance targets and bottleneck identification Development route or exploitation Development to pilot scale Patenting/licensing , development to pilot scale Patenting/licensing , development to pilot scale Development to pilot scale, licensing of strains Development to pilot scale, licensing of strains Development to pilot scale licensing of strains Development to pilot scale, licensing of procedures Development to pilot scale, licensing of procedures Development to pilot scale, licensing of procedures Development to pilot scale, licensing of procedures
Chemical
Chemical
Furanic chemicals from polysaccharides Fast growing Botryococcus strains Microorganisms Botryococcus strains producing enhanced amounts of hydrocarbons and polysaccharides C. Reinhardtii that is capable of producing hydrocarbons (and polysaccharides) Optimised cultivation conditions for Botryococcus (as part of the complete chain) Continuous steam extraction Process design Continuous solvent extraction
Chemical Agricultural, Biotechnology & Chemical Biotechnology & Chemical
Biotechnology & Chemical
Biotechnology & Agriculture
Biotechnology
Biotechnology
Process integration studies
Biotechnology , Chemical
Dissemination and publication of project results SPLASH is about researching and promoting a key enabling technology for the production of much needed chemicals. Market uptake and economies of scale can only be achieved if a wide audience of scientists, end-user industries, supply-chain service providers and policy makers have access to the projects scientific and market analysis, so they can make inf ormed decisions about investing in the technology. Communication plan The DEO will develop a detailed communication and dissemination plan. The DEO will be supported in this by the DEIPAB and EC guidelines68 will be taken into account. The main target audiences for
68
EC Research: Guide to successful communication: http://ec.europa.eu/reserach/science-society/science-communication/index_en.htm
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311956 SPLASH dissemination are: scientific communities, chemical industries, national policy makers, other involved stakeholders (specifically: SMEs service providers), the general public (general interest in chemistry and biotechnology + providing evidence that funding is well spent on solving a societal problem) The aim of the communication plan is to highlight a realistic pathway for decreasing dependency on fossil resources and developing alternative sustainable production chains for different sectors. The communication plan will detail the following (non-exclusive) approaches and actions: seminars and workshops for existing scientific and business networks, using existing networks from consortium partners conferences and workshops for European chemical industry, to be organised an led by the European Chemical Industry Technology Platform SusChem (who is a consortium member) to understand and evaluate the opportunities of digital manufacturing for their specific business scientific conference(s) and publications on research results. Project documentation will be prepared and made available on different aspects of the full supply and value chain perspective (also to be distributed electronically) scientific, industry-focused and general public websites on project focus and results, so that relevance for target groups is maximised. The main site will include project objectives, overview of project status and results thus far, other relevant news, contact opportunities and funding support confirmation from the European Commission. Focus per target audience will be on: o scientific community: research results on gene transfer, metabolism engineering, cultivation and harvesting technology o industrial partners: information on biobased chemicals, microalgae production for biobased chemicals, project results (specifically: quality validation), market analysis (ie. economic viability) data, how to become involved in biobased chemical production or/and in microalgae biotechnology, overview of events and publications at which SPLASH will be presented o General public: Clear and simple explanation of biorefinery and microalgae biotechnology. Basic knowledge transfer of how biomass can be used for industrial production. The Communication Plan will also consider using the website for educational purposes to increase schoolchildren (616 year olds) to become interested in biotechnology as a possible future career choice. A separate section will be reserved for project partners to exchange project related information and research data. Event attendance (incl. speaking) All project partners will be encouraged to attend thematic conferences, seminars and workshops. Project results are expected to be presented at selected conferences during the course of the project. Annual events of relevant to the project include:
European Bioplastics ANTEC (organised by the Society of plastic Engineers) - USA EABA (European Algae Biomass Association) Conference ABO (Algal biomass organisation), USA ISAP (International Society for Applied Phycology) World Polymer Conference
Publication of results The Consortium Agreement will describe guidelines for publishing project results, in particular when it relates to the use of existing background and new foreground IP. Partners wishing to publish about SPLASH research and results (scientific paper, conference abstract, press release, etc), will notify the PC and DEIPAB in advance and provide them with the final draft version at least two weeks before the intended publication or submission of the article. The PC and DEIPAB will read the draft publication and can take steps to support, amend or refuse publication by the partner in accordance with the Consortium Agreement guidelines. The project partners will publish project results under peer-review in high impact international scientific and industry journals. Relevant journals are:
Science ( Impact factor 31.36) Trends in Biotechnology (Impact factor 9.351) Biotechnology advances (Impact Factor: 8.250)
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Biotechnology and Bioengineering ( Impact Factor 3.7) PNAS (impact factor is 9.771) Metabolic Engineering (Impact Factor 4.725) 60 | P a g e
311956 SPLASH
Molecular Systems Biology (Impact Factor 9.667) Biopolymers (Impact Factor 2.572)
Training & education SPLASH is submitted for EC-funding under the KBBE-Cooperation programme. This programme does not allow for costs related to training of researchers coming into the field. As said earlier, the research community is still very small and should be increased to take advantage of the huge development potential of biorefinery and (microalgae) biotechnology. If successful, the SPLASH consortium will actively pursue a strategy for connecting this project to a comprehensive training programme with full academic and industrial participation under the FP7 PEOPLE programme. The training will include onsite research under expert supervision, workshops, summer schools and computer modeling around topics like: metabolic modeling and engineering, bioconversion, microalgae cultivation, biotechnological downstream processing, conversion of biomass (especially hydrocarbons and polysaccharides into chemicals), life cycle assessment and process modeling.
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311956 SPLASH B4. Ethics Issues Biosafety and genetically modified organisms The SPLASH project involves work with genetically engineered cell lines (WP2, WP3). All project partners are highly recognised and experienced in their respective fields and follow established and officially approved safety procedures for laboratory agents and genetically modified organisms. Staff working at dedicated facilities have received appropriate training and use standard operating procedures. Manipulation of genetically modified cell lines are performed in specific culture rooms in strict accordance with national an EU regulations. Permissions for constructing and handling genetically engineered microorganisms are obtained from the relevant national authorities, which require a full description of the work and the facilities where the work is to be performed, before permission is given. Inspection of the facilities by the Health and Safety Executive of each partner (and Ministry in some countries) guarantees that the procedures comply with the safety requirements. All partners are currently compliant with relevant institutional, national, European and international regulations concerning the work they perform and intend to perform so under this proposal. Attention will in particular be paid to EU directive 219 with respect to the containment of genetically modified organisms, and EU directive 90 addressing the rules of behaviour with regard to biosafety. This will be monitored by the appointed Biosafety officer (in WP3) together with the Health and Safety Executives of the involved partners using genetically modified organisms and recombinant nucleic acid techniques. This Biosafety officer should take appropriate actions where needed to safeguard the biosafety of the project. Environmental safety issues have been described in WP3.5 (chapter 1.3.3). Labour safety and laboratory procedures Activities in all work packages will be conducted in compliance with national health and safety regulations so that the proposed research has no negative impact on the environment or health & safety of staff involved in the project. Molecular biology procedures (and other procedures in this field) will be performed according to the guidelines laid out by each partners Health and Safety Committee and are conform to national and European directives 90/219, 90/220, and 98/81. All materials (chemicals, residues GMOs) will be disposed using national safety procedures. As already stated, all facilities are regularly inspected by national health and safety authorities. Local policies are in place and monitored by local committees to ensure compliance with health and safety legislation. Laboratory procedures shall respect the fundamental issues of good laboratory practices and of general practices for the handling of (biological) agents. Persons involved in the work activities will have been instructed in all necessary procedures and safety measures before starting work. The nature of possible health hazards are (or will be before starting any activity in WP2 or WP3) documented and appropriate safety procedures will be in place. The PC, together with the Biosafety officer, shall take all measures to assure that contractors accept and closely follow strict (bio)safety procedures. Data protection issues/Research on animals/Human embryonic stem cells The project does not involve the use or exploitation of personal data, research on animals or research on Human embryonic stem cells.
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Ethical Issues Table

Research on Human Embryo/Foetus Does the proposed research involve human Embryos? Does the proposed research involve human Foetal Tissues/ Cells? Does the proposed research involve human Embryonic Stem Cells (hESCs)? Does the proposed research on Human Embryonic Stem Cells involve cells in culture? Does the proposed research on Human Embryonic Stem Cells involve the derivation of cells from Embryos? I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL Research on Humans Does the proposed research involve children? Does the proposed research involve patients? Does the proposed research involve persons not able to give consent? Does the proposed research involve adult healthy volunteers? Does the proposed research involve Human genetic material? Does the proposed research involve Human biological samples? Does the proposed research involve Human data collection? I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL Pr Privacy Does the proposed research involve processing of genetic information or personal data (e.g. health, sexual lifestyle, ethnicity, political opinion, religious or philosophical conviction)? Does the proposed research involve tracking the location or observation of people? I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL ES Page 17 Research on Animals Does the proposed research involve research on animals? Are those animals transgenic small laboratory animals? Are those animals transgenic farm animals? Are those animals non-human primates? Are those animals cloned farm animals? I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL Research on Animals YES Page 18 Research Involving ICP Countries Is the proposed research (or parts of it) going to take place in one or more of the ICP Countries? Is any material used in the research (e.g. personal data, animal and/or human tissue samples, genetic material, live animals, etc): a) Collected in any of the ICP countries? b) Exported to any other country (including ICPC and EU Member States)? I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL Dual Use Research having direct military use Research having the potential for terrorist abuse I CONFIRM THAT NONE OF THE ABOVE ISSUES APPLY TO MY PROPOSAL Page NO NO YES Page NO NO NO NO NO YES Page NO Page NO NO NO NO NO YES Page NO NO NO NO NO NO NO YES
NO NO YES Page NO NO YES
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311956 SPLASH B5. Consideration of gender aspects SPLASH wants to comply with EC communication 21784 (Women and Science Excellence and Innovation. As microalgae biotechnology is still a young science field with a small science community, there are ample opportunities to get it right from the start whilst being real istic about share of women in the overall beta-science population. The target for women participation in SPLASH research groups, panels and committees is 40%. Based on data during proposal development, it is estimated that total woman participation will be no less than 25% of the total personnel committed to the project (excluding personnel to be hired from the EU contribution). The project coordinator is also a woman. Even though we are encouraged by the proportion of (younger) female scientists in biotechnology as a whole, the consortium will attempt to promote additional women participation through a number of measures, so that over time the ratio of women scientists in senior management positions in the European scientific field is increased. These measure include: Recruitment: All participants in the consortium are committed to promoting equality of opportunity in recruitment of staff and students. Statements to this effect will be included in all advertisements for positions. In advertisements for research fellows or for PhD studentships for this programme, where local and national regulations allow, the respective consortium partner will include an additional statement that as women are underrepresented in this scientific field, it welcomes applications from women. As far as possible, women will be equally represented on interview panels for these promotions. Employment: Conditions of employment may discriminate against employees with responsibilities for child care (in some cases this counts for both man and woman). Where possible and without detriment to the progress of the scientific programme, some flexibility in working hours or work locations will be allowed. Monitoring: Gender outcomes of recruitment of staff and students will be monitored by the Project Coordinator.
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THEME [KBBE.2012.3.

4-02] [Biotechnology for novel biopolymers]

Annex I - "Description of Work"

One form per project General information Project title

Sustainable PoLymers from Algae Sugars and Hydrocarbons

Duration in months Call (part) identifier

Activity code(s) most 7 relevant to your topic

311956 SPLASH - Part A - - Page 3 of 6

311956 SPLASH - Part A - - Page 5 of 6

311956 SPLASH - Part A - - Page 6 of 6

SPLASHSustainable PoLymers from Algae Sugars and Hydrocarbons

List of work packages

LIST OF WORK PACKAGES (WP) WP Number

Type of 54 activity MGT RTD RTD RTD DEM RTD OTHER

Lead beneficiary 55 number 1 12 1 7 9 6 10 Total

311956 SPLASH - Workplan table - - Page 1 of 49

List of Deliverables - to be submitted for review to EC Deliverable Number

Estimated WP Lead benefiindicative number ciary number person53 months 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D1.1 D1.2 D1.3 D1.4 D1.5 D1.6 D1.7 D1.8 D1.9

0.50 O 1.00 R 2.00 R 2.00 R 4.80 O 2.00 O 1.00 O 2.00 R 0.50 R

311956 SPLASH - Workplan table - - Page 2 of 49

Estimated WP Lead benefiindicative number ciary number person53 months 1 1

311956 SPLASH - Workplan table - - Page 3 of 49

Estimated WP Lead benefiindicative number ciary number person53 months

311956 SPLASH - Workplan table - - Page 4 of 49

Estimated WP Lead benefiindicative number ciary number person53 months

311956 SPLASH - Workplan table - - Page 5 of 49

Deliverable Title Conversion of hydrocarbons and sugars into biopolymers

Estimated WP Lead benefiindicative number ciary number person53 months 5 20

311956 SPLASH - Workplan table - - Page 6 of 49

Estimated WP Lead benefiindicative number ciary number person53 months 7 10

D7.7 D7.8 D7.9

2.40 O 3.50 O 1.50 O

311956 SPLASH - Workplan table - - Page 7 of 49

Estimated WP Lead benefiindicative number ciary number person53 months

311956 SPLASH - Workplan table - - Page 8 of 49

Work package description

311956 SPLASH - Workplan table - - Page 9 of 49

Work package description

Participant short name 1 DLO

Person-months per participant 27.30 Total 27.30

List of deliverables Deliverable Number

Lead beneficiary number 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Estimated indicative personmonths

311956 SPLASH - Workplan table - - Page 10 of 49

Work package description

Deliverable Title Plan for the use and dissemination of foreground

Lead beneficiary number 1 Total

Estimated indicative personmonths

311956 SPLASH - Workplan table - - Page 11 of 49

Work package description

Milestone name Kick-off Meeting Robust project management

Contractual deliverables met on time and in full

311956 SPLASH - Workplan table - - Page 12 of 49

Work package description

From systems biology to strain engineering

311956 SPLASH - Workplan table - - Page 13 of 49

Work package description

311956 SPLASH - Workplan table - - Page 14 of 49

Work package description

311956 SPLASH - Workplan table - - Page 15 of 49

Work package description