Bioprocess Economics

Introduction

Economic considerations play a continuing role at nearly every stage of a

plant design. The steps involves are
• Inception
• Preliminary evaluation of economics and market
• Development of data necessary for final design
• Final economic analysis
• Procurement
• Construction
• Start-up and trial runs
• Production
 Bioproduct regulation
• Involvement of government in bioproduct regulation stems from legislated
agency responsibility for workers health and safety , consumer safety,
environmental statues applicable to biotechnology , and specific regulations
concerning newer aspects of biotechnology (recombinant DNA in particular).
• The three most important U.S. agencies in regulation are the Food and Drug
Administration (FDA), the U.S. Department of Agriculture (USDA) and
Environmental Protection Agency (EPA)

In Nepal Department of Drug Administration

 Bioproduct regulation
• FDA
• Office of New Drug Evaluation ( all new drugs require animal and human
testing ; any r DNA drug products are all new)
• Office of Biologics : clinical trials required , followed by licensing of both
product and producer
• National Center for Devices and Radiologic Health: regulates medical
devices and medical diagnostic (including monoclonal antibody diagnostics,
except those related to blood bank operation). Monoclonal antibody (MAb)
system currently must demonstrate performance equivalent to prior product,
rather than safety and efficacy
 General Fermentation Process Economics

Laboratory inoculum Primary seed Secondary seed

Liquid storage
Sterilization Production fermentor
Dry raw materials

steam
Media preparation Harvest vessel

water

Byproducts Recovery steps Recovery steps

Fig: Generalized
Packaging and storage Product
process flow sheet
 General Fermentation Process Economics

• Process consist of fermentation (bioreactor) section and a product recovery

section.
• Upstream is a section for receiving and storing raw materials , and
downstream is final product preparation ( formulation), packaging and
shipping.
• Cost analyses are typically performed on three bases: CAPITAL cost
( bioreactor and product recovery equipment), PRODUCTION costs
(salaries, supplies , maintenance, taxes, depreciation etc), and MATERIALS
and UTILITIES cost (feedstock nutrients, additives, packaging agents and
materials, electrical gas, steam and water needs)
 Estimation of Manufacturing Costs

Direct Costs

• Raw Materials • Maintenance and Repairs

• Waste Treatment • Operating Supplies
• Utilities • Laboratory Charges
• Operating Labor • Patents and Royalties
• Supervisory and Clerical
Labor
Fixed Costs
• Do not vary with production rate but relate “directly” to production
function.
• Local Taxes and Insurance
• Plant Overhead Costs
General Expenses
• Administration Costs
• Distribution and Selling Costs
• Research and Development
 Fine chemicals
• Biotechnological application of Bio-production of fine chemicals
includes
• Vitamins
• Hormones
• Enzymes
• Antibiotics
• Monoclonal antibodies
• Further, bioconversion of high value starting materials ( antibiotics,
steroids , etc) to yet more value products .
 Enzyme
• The global enzymes market size was USD 8.18 billion in 2015 and is
expected to witness significant growth over the next eight years on
account of its increasing application in detergents, pharmaceuticals, and
food & beverages.

• Enzymes market is projected to show high gains in light of its extensive

usage in food processing, germination in breweries, pre-digestion of baby
food, fruit juice clarification, meat tenderization, cheese manufacturing,
and conversion of starch into glucose
Important Therapeutic enzyme
 Protein via Recombinant DNA
• The three earliest products available via recombinant E. coli in clinically significant
amounts are :
• Human insulin ( 1979)
• Human growth hormones(1981) and
• Human leukocyte interferon (1981)
The economic assessment of these various products must include
• Initial research and development costs: there products are among the first from a new
generation of biotechnology industries . Early products must pay for recovery of a
(considerable) initial investment in laboratory, personnel and production facility
• Conventional bioreactor and product recovery cost of the actual production process
• Recovery of cost for clinical testing and other procedures required by US Food and Drug
Administratoin, U.S. Department of Agriculutre or other corresponding agency.
Protein via Recombinant DNA

• Only a small number of hosts are currently used or under serious consideration for
industrial application ( E. coli, Sachhromyces cerevisiae, several mammalian cell lines,
bacillus substilis and several Pseudomonas species)
• For E. coli, for example , the simple genetics, good characterization , and high growth rate
are advantages.
• Bioreaction and recovery problems associated with E. coli hosts are:
• E.coli contain at least eight soluble protease: these may have played a role in observed
degradation of the expressed recombinant proteins somatostatin and human growth
hormones. Their influence is counter during product recovery by use of the protease
inhibitor but not metalloenzymes. Minimum iron levels diminish metalloenzyme
protease activity, but induce a metal complexation agent which may hinder product
purification.
• Cell envelope lipopolysaccharides is a very powerful pyrogen, which provides fever
response even at 0.5 ng per kg body weirth (one half part per million).
 Protein via Recombinant DNA

• Other high value products undergoing rDNA research and development include
• Regulatory proteins ( interferons, human and animal growth hormones,
neuroactive peptides and lymphokines)
• Blood factors ( human serum albumin, antihemophilic factor, thrombolytic
and fibrinolytic enzymes)
• Vaccines (viral and bacterial)
• Antibiotics
 Antibiotics
• Secondary metabolism products which inhibit growth of other microbial
species , even at low level are called antibiotics
• Uses for antibiotics are found in antimicrobial ( human diseases),
antitumor agents, fungicides and pesticides for plant protection , animal
diseases and animal growth products and research.
• More than 6000 antibiotics are known, of which nearly 100 are produced
commercially via fermentation
• Antibiotics classification are based on breadth of antimicrobial action (
broad vs narrow range) , basis of activity ( mechanism), sources (producer
strains), biosynthetic pathway, and molecular structure
 Penicillin
• All penicillin have the same general 6 – aminopenicillanic acid (6-APA)
structure, consisting of a 5- member thiazolidine ring , and associated 4-
member beta- lactum ring , and an acyl (R-CO-) side chain on the lactum
ring
• The natural penicillin are produced microbially from a useful medium
formulation , Penicillin G is a major example
• Addition of side chain precursor to the medium formulation allow change
in the dominant acyl group substituted on the 6- APA structure; penicillin
V and O are some of the consequent biosynthetic penicillins.
• See Table 12.13 from book for details structure
History of antibiotics discovery
Platensimycin 2006 S. platensis
Daptomycin 2003 S. roseosporus
2000
Linezolid 2000 Synthetic
1970 Mupirocin 1985 Pseudomonas fluorescens
Ribostamycin 1970 S. ribosidificus
Fosfomycin 1969 S. fradiae
1960
Trimethoprim 1968 Synthetic
Gentamicin 1963 Micromonospora purpurea
Fusidic acid 1963 Fusidium coccineum
Nalidixic acid 1962 Synthetic
Tinidazole 1959 Synthetic
Kanamycin 1957 S. kanamyceticus
Rifamycin 1957 Amycolatopsis mediterranei
Noviobiocin 1956 S. niveus
1950
Vancomycin 1956 S. orientalis
Cycloserine 1955 S. garyphalus
Lincomycin 1952 S. lincolnensis
Erithromycin 1952 Saccharopolyspora erythraea
Virginiamycin 1952 in S. pristinaespiralis S. virginiae
Isoniazid 1951 Synthetic
Isoniazid 1951 Synthetic
Viomycin 1951 S. vinaceus e S. capreolus
Nystatin 1950 S. noursei
Tetracycline 1950 S. aureofaciens
Neomycin 1949 S. fradiae
Chloramphenicol 1949 S. venezuelae
Polymyxin 1947 Bacillus polymyxa
Nitrofurantoin 1947 Synthetic
Cephalosporins 1945 S. clavuligerus
Bacitracin 1945 Bacillus licheniformis
Cephalosporins 1945 S. clavuligerus
Streptomycin 1944 S. griseus
Penicillin 1941 Penicillium chrysogenum
1940
Rudi et al., 2012
Ref: Antibiotics in the clinical pipeline in 2013 journal of antibiotics 2013 66, 571-591
9 th jan
• 9, 5,26,2128
 Vitamins
• In addition to protein (enzymes) and antibiotics, many other complex
metabolites are produced by microbes
• Of the microbially synthesized Vitamins [B12, riboflavin(B2), thiamine, folic
acid, pantothenic acid, pyridoxal], only B12 and B2 are produced microbially
• Vitamin B12 , also called cyanocobalamin, is synthesized exclusively by
microbes
• Microbial flora of the large intestine can synthesize B12, its assimilation by
human does not occur, hence it must by obtained from food.
• Early 1980s, world production was roughly ten tons, of which
pharmaceutical industries consumed about two-thirds ( as cyanocobalamin,
hydroxocobalamin, coenzyme B12, and methaylocobalamin)
 Vitamin B12 production
• Propionibacteria ( freudenreichii and shermanii) are used primarily in
batch process employing two phases:
• a 2-4 day anerobic operation followed by a 3-4 days aerobic stage.
• The anaerobic products (5'- deoxyadenosylcobinamid guanosine
phosphate) is coupled with the aerobic product (5,6-dimethyl
benzimidazole) to give, ultimately a desired vitamin B12.
• Pseudomonas denitrificans also produces B12 in a one stage process,
cobalt and 5,6-dimethyl benzimidazole are also added.
 Folic acid (B9)

Vitamin B2 - riboflavin

Vitamin B12
 Alkaloids
• Alkaloids are naturally occurring chemical compounds containing
basic nitrogen atoms . The name derives from the word alkaline and
was used to describe any nitrogen - containing base.
• Alkaloids are produced by a large variety of organisms, including
bacteria, fungi, plants, and animals and are part of the group of natural
products (also called secondary metabolites )
• They often have pharmacological effects and are used as medications,
as recreational drugs, or in entheogenic rituals.
Alkaloids contd…..

• Ergot is the dried sclerotium of a fungus, Claviceps purpurea (Fam.

Hypocreacea) that arise on the ovaries of the rye plant (Secale cereale, Fam.
Gramineae).
• Ergot alkaloids are N-monosubstituted amide derivatives of both lysergic
acid and its isomer isolysergic acid that differ only in configuration at C-8.
• On treatment with ammonia lysergic and isolysergic acids give the
corresponding amides ergine and isoergine respectively.
• Members related to lysergic acid (e.g. ergotamine and ergometrine) are
levorotatory, more active and designated by suffix “ine”.
• Members related to isolysergic acid (e.g. ergotaminine and
ergometrinine), are dextrorotatory, less active and designated by suffix
“inine”.
 Nucleosides
• Nucleosides and nucleotides are used as flavor enhancer in food
• The most effective substances are the purine 5'-monophosphates: guanylic
acid (5'-GMP), inosinic acid (5'-IMP) and xanthylic acid (5'-XMP).
• Sodium glutamate has a synergistic flavor-enhancing action with these 5'-
monophosphates
• Approximately 300 tones per year of 5'-IMP (66%) and 5'-GMP (34%) are
produced annually in Japan.
• Commercial products are ultimately produced either microbially or by
hydrolysis of cellular RNA ( either in vitro using yeast RNA and microbial
enzymes or in vivo using endogenous enzymes followed by mnonucleotides
excretion)
• All steroid hormones
are:
• Derived from
cholesterol &
differ only in the
ring structure &
side chains
attached to it.
• Lipid soluble & thus
are freely permeable
to membranes so are
not stored in cells
 Steroids
OH
O H3C
H3C
H3C H
H3C H

H H
H H O
O
Progesterone Testosterone
 Bioconversion
• Bioconversion is the conversion of organic materials, such as plant or animal waste, into
usable products or energy sources by biological processes or agents, such as certain
microorganisms or enzymes. In other words it is defined as transformation of a chemical
added to the medium into a commercially valuable compound.
• Steroid biotransformation is a multimillion dollar industry and pharmaceutical uses of
steroids are numerous
• Applications for natural and derivatized steroids and sterols includes therapeutic uses
( estrogens, progesterons and androgens), contraceptives (derivatized estrogen and
progesterone), sedatives, antitumor therpay, veterenary products, anti-inflammatory
for skin diseases and arthritis(cortisone), and medication for controlling sodium
retention potassium excretion.
• Specific microbial transformation of new steroids and their evaluation as drugs and
hormones
• Biotraformation have provided adequate tools for the large scale production of natural or
modified steroids analog.
Enzyme and Microbial Technology 32(2003) 688-705
 Monoclonal Antibodies (MAb)
• When a foreign molecule (antigen) is injected into an animal such as a
mouse , the animals immune system will often recognize the foreign
molecule and produce specific antibodies which complex very selectively
with the antigen, there by activating it and or tagging it so that other body
function will recognize the complex and remove it from circulation.
• Typically, antibodies are produced in specialized cells known as B
lymphocytes (spleen, blood, lymph); a characterstics immune response will
produce a number of antibodies (i.e. in mixture) to a given antigen.
• Each B lymphocyte produces one kind of antibody.
• These cells ( e.g. spleen) can be removed and cultured external to the
mouse, to produce antibody.
 MAb
• MAb technical application have appeared rapidly in commercial use in the 1980s ,
primarily because the earliest major products, in vitro medical diagnostic kits, did not
require the level of testing needed for in vivo application.
• The driving forces for MAbs – based diagnostic are several:
• Time savings: a number of diseases were previously diagnosed by time-demanding sample
cultures: MAbs kits provide rapid determinations . Example:
• Human diseases such as ( gonorrhea, chlamydia and herps simplex virus) , and several
common bacterial infections found in hospitalized patients ( e.g. pseudomonas
aeruginosa and B streptococci.
• Potentially , cancer detections are also possible provided a tumor-specific marker is
identifiable; a market example is an acid phosphatase released by a cancerous prostate
glands.
• Increased sensitivity: one MAb hepatitis B detection system is claimed to be 100 times
more sensitive than a polyclonal antibodies determination.
 Monoclonal Antibody Production
Bioreactor fluid
with cells

Sterile filtration
Cells
Continuous
centrifugation
IgG product

Formulation
100,000 MWCO
membrane

IgG eluted
Affinity Membrane
chromatography concentration

Unbound material
IgG Economics – Commercial Plant

Direct Fixed Cost $ 15.3 million

Total capital investment $ 16.3 million
Plant throughput 6.2 kg of IgG per year
Manufacturing cost $ 5.64 million / year
Unit production cost $ 908 / g of IgG
Selling price $ 2,500 / g of IgG
Revenues $ 15.5 million / year
Gross profit $ 9.9 million / year
Taxes (40 %) $ 4.0 million / year
Net profit $ 7.4 million / year
Internal Rate of Return 47.4 %
Net present value ( 7 % ) $ 32.5 million

Ref:genetech
 Brewing and Wine Making
• Beer is defined as a fermented, alcoholic beverage made from barley,
wheat , rice etc and flavoured with hops.
• Ingredient of Beer:
• Barely,
• Hops,
• Yeast,
• Water,
• Sugar
 Yeast
• Discovered by Anton Van Loeuwenhoeck in 1685
• In 1875 Louise Passter explained the function of yeast in details
• Types:
• Saccharomyces Cerevaciae: Top fermentating yeast work at 15 to 19 degree
and settle at top of wort after fermentation. Hence top-fermenting beer is
drung at 15 to 19 oc
• Saccharomyces Carlsbergeneces - Bottom fermenting , settle in bottom of
wort after fermentation , work at 2- 6 degree celcius hence these beer ar
taken at 4-5 oC
• Emil Hansen developed this yeast in 1883 by isolating the first single cell of
yeast in Carlsberg brewery of Denmark.
Yeast powder
Process…..

• Barley is used as a main source for beer production but can be produced from wheat ,
rice combination of grains
• To prepare for processing, it is incubated for 2 to 6 days so that germination occurs
• This malting step promotes the formation of active alpha and beta amylase as well as
protease enzyme.
• The resulting grains are then carefully dried
• Preparation of nutrient medium called beer wort for yeast growth requires mashing,
which is carefully controlled warming of an aqueous mixture of malt and starch
• Success of the mashing step requires artful exploitation of a complex mixture of
substrate and enzymes
• Proteins and carbohydrates in the malt starch mixture must be hydrolyzed, since the
yeast involved in the fermentation step can utilize only simple sugars and amino acid
as nutrients
Process…..
• On the other hand, total hydrolysis is not desirable because dextrins, peptides, and
peptones contribute flavor and body of the beer.
• Hydrolytic enzymes participate in mashing
• Elaborate cooking recipes with temperature varying with time over a range from
about 40 to 100 oC are employed to optimize mashing process
• The end product contain a suitable mixture of yeast nutrients and flavor
components.
• Next the beer wort is clarified and then boiled for about 2 h with periodic addition of hops
(ripened, dried cones of the hop vine), which contribute flavor, aroma , and color to the
beer and also exert an antibacterial action.
• The sterilized wort is then cooled and inoculated with a propriety strains of brewers yeast
(Sacchromyces cerevisiae).
• The yeast if first cultivated under aerobic conditions followed by a switch to an anaerobic
environment to cause ethanol and CO2 production.
• Finally the product is clarified , pasteurized , aged and packaged.
• Water
• The beer consist of 90% of water, the quality and mineral content affect the
character of brew
• Water content six main salts namely bicarbonate, sodium ,chloride, magnesium and
sulfate
• High level of carbonate will produce acidic mash, which will reduce the extraction
of sugar from malts
• Too much sulphur makes bitterness in brew
• Magnesium is a essential ingredients for yeast.
• Sugar
• To speed up the fermentation
• To reduce the bitterness
• To give colour in the form of caramel
• To cause secondary fermentation
 Major production steps
• Steeping • Brew Kettle
• Malting • Hop Back
• Germination • Hop Extraction
• Klining • Fermentation
• Sieving • Fining
• Grinding • Carbonation
• Extraction of • Bottling
• Sugar • Pasteurization
• Infusion
• Decoction
 The Brewing Process
Malt Mill

Cereal
Mash Tun
Cooker Brink Fermentation

Lauter Tun

Brew Hops
Kettle Aeration

Lagering
Hot Wort
Receiver

Wort
Cooler
Ref: Tom Pugh Miller Brewing Company
Cost
• The magnitude of brewing industry is impressive
• The U.S. industry had 1975 sales of 160 million barrels (31 gallons per
barrel).
• The approximate cost distribution , in million for this production was
• $610 malt
• $188 other grain
• $49 hops
• $860 direct salary
• $2500 packaging and containers
• $80 fuel, powder and water
• $360 transportation
• $600 equipment and improvement
• $230 advertising
Fuel Alcohol Production
• Inexpensive petroleum and new petrochemical processes developed
after 1945 provided a feedstock source which then displaced
fermentation derived products, including ethanol and virtually all other
simple oxychemicals ( acetone, butanol, acetic acid etc)
• Petroleum prices rises and demand also increases over the last decade,
which makes fuel production from alternative source i.e. biofuel more
attractive.
 Introduction : Biofuel
• Biofuel is a type of fuel whose energy is derived from biological carbon
fixation.
• Biofuels include fuels derived from biomass conversion, as well as solid
biomass, liquid fuels and various biogases.
• Unlike other renewable energy sources, biomass can be converted directly
into liquid fuels, called "biofuels," to help meet transportation fuel needs.
• The two most common types of biofuels in use today are ethanol and
biodiesel
• Ethanol is a volatile liquid fuel that may be used in place of refined
petroleum. It is manufactured by the action of microorganism and is therefore
a fermentation process.Conventional fermentation has sugars as feedstock
First generation biofuel:
• Biofuel made from sugar, starchy crops, vegetable oil or animal fat using
conventional technology.
• The starch from the basic feedstocks is fermented into bioethanol, or the
vegetable oil through chemical process to biodiesel.
• These feedstocks could instead enter the animal or human food chain.
• They don’t seem to be more environment friendly than the fossil fuels
Second Generation
• Second Generation Biofuels come from Woody crops and grasses species
(lignocellulosic)
The third generation
• The third generation biofuels come from algae, that are low-input, high-yield
feedstock to produce Biofuels
Ethanol from Cornstarch

Ref: online sources

• See fig 12.6 for wine making flow chart
• And Table 12.16 & 12.17 for cost and the example of microbial
production of chemcials from book.
Organic and Amino Acid Manufacture
• Submerged fermentation process with high citric acid yields of 70 kg
acid from 100 kg sugar has fermentation capital costs which are 15-
25% less than surface fermentation, but requires 16-26% greater
operating costs.
• The economic aspects of amino acid production are now considered
• Of the twenty amino acid , only two ( Lysine and Glutamic acid) were
produced primarily by ferementation in 1980 , although more are
potentially produciable ( arginine, glutamine, histidine, leucine,
ornithine, phenylalanine, proline, threonine an valine , See table 12.23
from book)
Why production of amino acid
• Uses:
• Food industry: 65%
• Feed Additives: 30%
• Pharmaceutical: 5%
• Food industry
• Flavor enhancers, MSG, glycine, alanine. Tryptophan and histidine act as antioxidants to
preserve milk powder. For fruit juices cysteine is used as an antioxidant.
• Aspartame, dipeptide (aspartyl-phenylalanine-methyl ester) produced by combination of
asp and Phe is 200 times sweeter than sucrose. Used as low calorie artificial sweetener in
soft drink industry
• Essential amino acids are those deficient in plant based foods like lys, met, thr, Trp
improves nutritional quality of food and feed additives (animal). Bread: lysine, soy
products or soyabean meal (pigs/animals): methionine
 Single-cell protein(SCP)
• Single-cell protein or SCP refers to proteinaceous materials which are
dried cells of microorganism,
• Example species which have been cultivated for use in animals or human
foods include algae, actinomycetes, bacteria, yeast, molds and high fungi.
• Human consumption of microbial protein is ancient in origin and includes
yeast in
• leavened bread, lactic acid bacteria in fermented dairy products ( milk,
sausages, cheese) and algae( Spirulina) harvested from ponds and lakes.
 Single cell protein
• single cell protein is a protein extracted from cultured algae, yeasts, or bacteria
and used as a substitute for protein-rich foods, especially in animal feeds or as
dietary supplements.
• Many types of animal feeds contain single cell proteins.
• 60-80% dry cell weight; contains nucleic acids, fats, CHO, vitamins and
minerals
• Rich in essential amino acids (Lys-Met)
• Microbes can be used to ferment some of the vast amounts of waste materials,
such as straws; wood and wood processing wastes; food, cannery and food
processing wastes; and residues from alcohol production or from human and
animal excreta.
SCP
• More recent studies have established microbial growth in aerated
fermentors, using substrates such as gas oil, paraffins, natural gas,
molases and cellulosics.
• The process variability includes factors such as
• aseptic vs nonaseptic,
• open vs enclosed growth vesslus,
• full vs partial substrate utilization,
• optimum pH values from acidic to alkaline and
• product recovery by filtration vs centrifugation.
 Some SCPs…

Microbes employed include Bacteria

Rhodopseudomonas capsulata
Yeasts Typical yields of 43 to 56%, with
Saccharomyces cerevisiae, protein contents of 44% to 60%.
Pichia pastoris,
Candida utilis=Torulopsis and Algae
Geotrichum candidum (=Oidium lactis)), • Chlorella and
Other fungi • Spirulina
Aspergillus oryzae,
Fusarium venenatum,
Sclerotium rolfsii,
Polyporus and
Trichoderma),
• See figure 12.8 for process flow chart for SCP production
Anaerobic methane production
• Four main stages or path way of Anaerobic Digestion
• Hydrolysis
• Acidification/acedogenesis
• Acetogenesis and dehydrogenation
• Methanogenesis
Hydrolysis
• In anaerobic digestion, hydrolysis is the essential first step, as Biomass is
normally comprised of very large organic polymers, which are otherwise
unusable.
• Through hydrolysis, these large polymers, namely proteins, fats and
carbohydrates, are broken down into smaller molecules such as amino acids,
fatty acids, and simple sugars.
Acidogenesis contd…

• In general, acetogenesis is the creation of acetate, a derivative of acetic

acid, from carbon and energy sources by acetogens. These
microorganisms catabolize many of the products created in
acidogenesis into acetic acid, CO2 and H2.
• Acetogens break down the Biomass to a point to which Methanogens
can utilize much of the remaining material to create Methane as a
Biofuel
Methanogenesis
• Methanogenesis constitutes the final stage of anaerobic digestion in which
methanogens create methane from the final products of acetogenesis as well as
from some of the intermediate products from hydrolysis and acidogenesis
• 4H2 +CO2 = CH4 +2H2O Methanobacterium formicicium
• 2CH3(CH2)2OH +CO2 =4CH3COOH +CH4 Methanobravi bacteria
• 2CH3(CH2)2COOH +2H2O+CO2 =4CH3COOH +CH4 Ruminatium methneospirilum hungatei
• CH3COOH = CH4 + CO2
4 th feb 8, 11, 27,28, 18, 23