Liquid and Gaseous Fuels From Biotechnology: Challenge and Opportunities

MICROBIOLOGY REVIEWS
ELSEVIER
FEMS MicrobiologyReviews 16 (1995) 111-142
Liquid and gaseous fuels from biotechnology: challenge and opportunities

N. Kosaric *, J. Velikonja
Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ont. N6A 5B9, Canada
Abstract This paper presents challenging opportunities for production of liquid and gaseous fuels by biotechnology. From the liquid fuels, ethyl alcohol production has been widely researched and implemented. The major obstacle for large scale production of ethanol for fuel is the cost, whereby the substrate represents one of the major cost components. Various scenarios will be presented for a critical assessment of cost distribution for production of ethanol from various substrates by conventional and high rate processes. The paper also focuses on recent advances in the research and application of biotechnological processes and methods for the production of liquid transportation fuels other than ethanol (other oxygenates; diesel fuel extenders and substitutes), as well as gaseous fuels (biogas, methane, reformed syngas). Potential uses of these biofuels are described, along with environmental concerns which accompany them. Emphasis is also put on microalgal lipids as diesel substitute and biogas/methane as a renewable alternative to natural gas. The capturing and use of landfill gases is also mentioned, as well as microbial coal liquefaction. Described is also the construction and performance of microbial fuel cells for the direct high-efficiency conversion of chemical fuel energy to electricity. Bacterial carbon dioxide recovery is briefly dealt with as an environmental issue associated with the use of fossil energy.
Keywords: Fuels; Liquid fuels; Gaseous fuels; Biotechnology; Ethanol; Biogas; Energy; Fermentation
Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Fermentation ethanol for fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The semi-continuous(modified fed-batch) process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Continuous process: internal yeast settling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Biotechnology in the production of 2,3-butanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 112 116 118 119 121
* Correspondingauthor. Tel.: (+ 1-519) 661 2131; Fax: (+ 1-519) 661 3498 Federation of European MicrobiologicalSocieties. SSDI 0168-6445(94)00049-2
112
N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142
4. Biotechnology in the production of liquid and gaseous fuels from coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Biotechnology in methane and biogas production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Biotechnology in diesel fuel and gasoline production from microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Biotechnology in the production of other oxygenated alternative fuels and fuel extenders . . . . . . . . . . . . . . . . . 8. Biotechnology in direct energy conversion: microbial fuel cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127 ! 29 132 135 137 141
1. Introduction The energy needs of the world today are estimated at close to 1021 joules per year, which amounts globally to about 20-30 TW continuous power requirement (perhaps rising to 100 TW) [1]. This equals a continuous average per capita consumption of about 4 - 6 kW, which is very unevenly distributed among individual countries (Table 1). An estimated 2.5 1012 W, or only 20% from today's needs, would cover the basic energy requirements of the world population (apart from physiological energy supplied by food), whereas the remaining 80% are spent on the ever growing technological activities and increasing life quality. The obtainable energy on earth is available in different renewable and non-renewable resources. An idealized representation of power outputs from the renewable world resources and some associated problems are given in Table 2. Most of the energy used today comes from fossil fuels (Table 3). There is a great discrepancy in production and consumption of energy worldwide as shown in Fig. 1. The remaining reserves of oil, estimated for the end of 1985, are presented in Fig. 2. One should point out, however, that the reserves of the former USSR and China may not be accurate and that much larger reserves are probably available in these regions which have not been so far fully evaluated. The world consumption of commercially provided energy is shown in Fig. 3, from which it is evident that coal, oil and natural gas (non-renewable fossil fuels) represent almost 90% of the world energy
consumption. Comparing these different energy supplies (Fig. 4), it is predicted that by the year 2060 the use of coal, natural gas, nuclear, hydroelectric, and new sources of energy will predominate, while petroleum supply will gradually diminish. Another breakdown of these trends is shown in Fig. 6. Another growing problem in terms of energy consumption represents also the great imbalance of annual energy consumption between north and south (Fig. 5) and between the industrialized and developing countries. The world population distribution (around 3:1 for developing vs. industrialized countries) aggravates the problem even more. Both population and pollution problems are also associated with energy use and distribution. The environmental impacts of these tremendous amounts of various energy forms are given in Tables 4,5, 6, and 7 for oil natural gas coal, and nuclear power, respectively. The non-renewable energy resources (fossil fuels) are by far the most exploited forms of energy today, as exemplified in Table 3. In fact, the whole superstructure of the modern industrial society is built upon oil which, at the present production rate, will be exhausted in the next century with grave consequences of global warming and environmental pollution. Apart from having the potential to affect and upset the climatic, geological and biological equilibrium in nature, its production and use directly influence all aspects of humanity. As shown in Fig. 6, the production of liquid fuels from oil is expected to peak around 2015, with approximately the same amount produced by coal liquefaction at that time.
N. Kosaric, J. Velikonja / FEMS Microbiology Reciews 16 (1995) 111-142

Table l Per capita commercial energy usage (1988) Country Canada United States Saudi Arabia Former E. Germany Sweden Former W. Germany Former Soviet Union United Kingdom kV 9.0 8.3 7.0 6.8 6.2 5.1 4.9 4.0 Country Japan Italy Spain Brazil ~ Mexico a China a Nigeria a India a kW 3.6 3.2 2.3 1.4 1.2 0.7 0.5 0.3
113
After Cole [1]. Countries with high consumption of traditional fuels.
Another major peak of liquid fuel production is expected around the year 2030, with a rapid decline to zero by 2090.
Table 2 Maximum power outputs of renewable sources Source Solar photovoltaic Solar photosynthetic biomass Aeolian Undular Hydroelectric Tidal Geothermal Adapted from Cole [1]. Power/W 1015 9 1012 1 1015 Uncertain < 6 1012 Uncertain < 1012 < 7 1012 < 3 1013
This projection into the not-so-far future does not reflect on the odds of maintaining a steady growth of technology and economy, keeping a reasonable quality of life for those who would not want to lose it, and possibly extending it to a larger part of the world population, which will double in the same period-mostly in underdeveloped countries. The answer to that depends primarily on three other issues: (i) development and worldwide implementation of viable technologies for industrial production, heating, and transportation based on alternative (renewable) fuels and feedstocks; (ii) availability of sufficient quantities of renewable energy and feedstocks; (iii) development and implementation of technologies for the reduction of environmental pollution and emission of CO 2.
Comments (assumed 100% land coverage) 7-10% conversion efficiency; REQUIRED, heavy duty storage system and higher conversion efficiency Land coverage difficulties; visual pollution Land coverage and harvesting: social problems REQUIRED: heavy duty storage system Land coverage: technical and social problems, visual pollution Useful near the sea; heaviest, most expensive engineering Restricted in global application Restricted to tidal regions Restricted to specific areas Present potential < 3 109 W Mid-ocean ridges: in very distant perspective
Table 3 Worldwide reserves of fossil fuels (various sources) Resources Total reserves proven + undiscovered Volume equ. (109 bbl oil) Oil Heavy oil ~ Shale oil ~ Bitumen b Coal c Natural gas J Total 1 177 543 1066 345 3 175 1 335 7 641 Energy a (1018 j) 7 203 3323 6 524 2 111 19430 8 172 46 763 % 15.4 7.1 13.9 4.5 41.5 17.5 99.9 Proven/recoverable reserves Volume equ. (109 bbl oil) 703 450 1 066 345 3 175 593 6 332 Energy a (1018 j) 4 303 2 752 6 524 2 111 19430 3 629 38 749 % 11.1 7.1 16.8 5.4 50.1 9.4 99.9
Based on a heating value of oil of 6.12 G J / b b l (1 bbl = 42 US gal = 159 1). b Heavy oil, shale oil and bitumen were given the same heating value as oil. c Based on a metric-ton-of-coal equivalent of 7 109 c a l / t (29.3 GJ/t). d Based on an heating value of 37.3 M J / m 3 natural gas.
114
<
~.o~
c "-. 2
~
e ~
~ C
~ Consurnption r'-I Domestic supply
._
~ _~
11 u~ 1.C
".iT,
]
,J,<,
,~
Fig. 1. World's principal consumers of oil and their domestic supply of oil products in 1985 (source [46]).
The only alternative to fulfill the above goals relies upon renewable energy resources. The choices are even more limited when liquid and gaseous fuels for combustion and transportation are in question: biomass energy and water (for hydrogen generation). Among the various technologies available or developed for energy production from the above two sources, biotechnology offered and continues to offer interesting and promising alternatives. Many of them are not competitive with the cheap fossil fuels today - - a fact that will, however, drastically change in the future. Biotechnology-derived fuels for heat generation
20-
150
100
7
l/ ,//
5O
oT7
20
30
!
".N u
50
<
Produced. 1859 - 1985 Remaining Reserves. End of 1985
iO0
50~00
Fig. 2. Total discovered oil (source [46]).

Hydroelectric 6.7 */0
115
/-'~.::..'.:-.....%,
Coal 30.7 *
~ii~.:.
. ~L - ~
~___.____Nu c l e a r
30(3
Cool
4G 0/,
1o
Natural Gas 20.1 */0
.2
2OO
N a t u r a l gas
K
Nucleor
10(: New SOurCes Petroleum Hydro elect r i Non commerciQI
energies
19~0
1980
2000
2020
2040
2060
Yeor
Oil 37.9 */. Fig. 3. World consumption of commercially provided energy (source [46]).
Fig. 4. Evolution of world energy supplies (From [47]).
(iv) (v)
and for internal-combustion engines (ICEs) which were being studied so far as economically and technologically promising are: Liquid fuels: (i) ethanol; (ii) other alcohols (e.g. butanol, 2,3-butanediol); (iii) short-chain aliphatic acids as precursors (e.g. acetic to valeric);
other oxygenated solvents (e.g. ketones); lipids (fatty-acid triglycerides, other fatty-acid esters). Gaseous fuels: (i) methane; (ii) medium-heat content biogas; (iii) hydrogen. Biotechnology also offers the possibility of generating electricity by direct conversion of fuel energy in microbial fuel cells. Major alternative routes are presented below.
Table 4 Environmental impacts of oil Energy activity Environment Atmosphere Exploration Emissions of hydrocarbons as a result of a blowout Blowouts and spills from exploratory wells at sea, leading to oil contamination Blowouts and spills on land Disruption style of life Extraction, production, processing Refinery emissions of SO 2, HzS, CO2, NOx, and hydrocarbons Blowouts and spills Brine and drilling chemicals disposal Refinery effluents Blowouts and spills Sludge disposal Interference with fisheries Transmission Use and disposal Emissions of SO2, CO 2, and hydrocarbons Groundwater contamination by leaking tanks
Hydrosphere
Tanker accidents, leading to oil contamination
Lithosphere
Human pact
im-
Pipeline construction and spills Damage due to permafrost Interference with fisheries or land use Disruptions of life style during construction
Used oil disposal
Hydrocarbons and poly nuclear aromatic hydrocarbons from combustion
From Runnalls and Mackay [53].
l 16
N. Kosaric, J. Velikonja / FEMS Microbiology Reuiews 16 (1995) 111 - 142
2. Fermentation ethanol for fuel

~J North 7.11
Fermentation of sugars by yeasts is one of the oldest practised biotechnology processes. Major emphasis in the past was to produce potable alcohol in the form of beer and wine. More recently, particularly in countries which lack petroleum but have abundant sugar crops (e.g. Brazil with sugar cane), a large scale fermentation industry for production of fuel alcohol has been developed. Productivities of alternative batch and continuous systems are shown in Table 8. A typical process for production of ethanol in a batch mode, as applied in Brazil, is the 'MelleBoinot' process, presented in Fig. 7. Two approaches are being practised by use of molasses or sugar cane juice and the industrial yields are shown in Table 9. Other raw materials for fuel alcohol production have also been investigated, such as sugar beets, Jerusalem artichokes, cassava, wood hydrolysates, starches, sweet sorghum, etc. A comprehensive review on alcohol production, recovery, and biotechnology has been presented by Kosaric and Duvnjak [2].
[ ] South
E O Or)
6.2e
5.44
~_271
300
4.59 4
200 .~
E
- - tO 13m
2.73
~oo
t: tO
8
-
97
1.10
c c <
60
74
"
1960 1980 2 0 0 0
2020
2040
2060
Year
Fig. 5. Levelsof energyconsumption(From[47}). Concerning yeasts, S a c c h a r o m y c e s c e r e v i s i a e has been mainly used. Of particular interest is also the use of flocculating yeasts, such as S a c c h a r o m y c e s diastaticus, for fuel alcohol production, as investi-
eQ u :3
"o ~
Ill
o.
1,.
1960
1980
2000
2020
2040
2010
2080
2100
C a l e n d a r years
Fig. 6. Projectedrate of world productionof fossil fuels(From [48]).

Table 5 Environmental impacts of natural gas Energy activity Environment Atmosphere Exploration EmissionsofgasandH2S during an accidental blowout Blowouts Extraction, processing production, Transmission Use and disposal Emissions of NO~ CO 2,
117
Hydrosphere Lithosphere
Gas plant emissions of H2S, SO 2, and hydrocarbons Blowouts and drilling Disposal of chemicals -
Construction of pipeline Damage due to permafrost LNG accidents Disruptions of life style during construction
Human impact
LNG accidents emissions
H 2S
From RunnaUs and Mackay [53].
Table 6 Environmental impacts of coal Energy activity Environment Atmosphere Exploration Extraction, production, processing Emissions of SO 2 and PNAs from processing to gas or liquid fuel Coal dust dispersal Leaching of acids and metals Organic compounds formed with 'synfuels' Siltation Transmission Use and disposal
Emissions of CO2, NOx, COz, and particulates

Thermal effects
Hydrosphere
Lithosphere Human impact
Disruption from strip mining and subsidence Slag heaps Lung disease Mine safety
Fly ash disposal Exposure to emissions from combustion and coke ovens
Table 7 Environmental impacts of nuclear power Energy activity Environment Atmosphere Hydrosphere Lithosphere Human impact Exploration Extraction, production, processing Accidents Radon emissions from mine railings Accidents Leachate from mine tailings Accidents Tailings contamination Accidents and mine-plant exTransmission Transmission lines Accidents during fuel transport Use and disposal Thermal effects Disposal of spent fuel and
waste
Exposure to wastes Terror-
plosive mining hazards

ism
118
N. Kosaric, J. Velikonja/ FEMS Microbiology Reviews 16 (1995) 111-142
Table 8 Productivities of alternative batch and continuous fermentation systems utilizing yeast System C 2H5OH productivity (g1-1 h -1) Continuous, vacuum recycle Continuous, recycle Batch, recycle Continuous, multi-stage Continuous Batch Adapted from Vergara [54]. 80 40 15 12 5 2
series of photographs which were taken in 10-s intervals after the mixing was stopped (Fig. 8). This clearly illustrates a special capability of this system as compared to processes where yeast has to be concentrated by costly centrifuges for its return to the fermentation broth. Based on this property, two processes were developed: a semicontinuous (modified fed-batch) process and a continuous process with internal cell recycle, without the need for use of any mechanical settling devices, as is the case in other continuous processes (e.g. 'Biostill' developed by Alfa Laval).
2.1. The semi-continuous (modified fed-batch) process
gated in our laboratory. This yeast has a high efficiency in converting sugars to alcohol. Another advantage is in its high flocculating capability. The yeast produces very stable flocs during growth which can rapidly settle if needed, but can also be efficiently maintained in suspension when sufficient mixing is applied. The settling is illustrated in the
HzSO~ _!
The schematic of the operation of this process is presented in Fig. 9. A conventional bioreactor (stirred tank reactor) is filled with the medium, inoculated
Molasses
or cane juice
of yeast
~
Preparahon
Recuperation of
yeast
Weighing and 1 sterilizing I
Brix adjustment .lt. to 22"1ov/v
Wort
=[
Decanted wine Benzene

,~r ' '
-I
Fermentahon
Wine
Decantation
and
~1
centrifu~ation
Decantation
Alcohol20/. v/v
Rechhed
alcohol
, Raw alcohol
H~O
~ Stilla _ DISTILLATION
~ Phleg RECTIFICATION
Fusel oil
DEHYDRATION
Recycle RECUPERATION OF BENZENE
Fig. 7. Typical process for the production of ethanol from sugar cane (From [49]).
N. Kosaric, J. Velikonja/ FEMS Microbiology Reciews 16 (1995) 111-142
119
Table 9 Yields in production of ethanol from sugar cane Alcohol, indirectly from molasses Sugar cane yield in 1.5-2-year cycle (south central region), t/ha Average sucrose yield (13.2 wt %), t/ha Crystal sugar production, t/ha Final molasses or cane juice production, t/ha Fermentable sugar, molasses, or juice, t/ha Alcohol yield at 100% global efficiency, kg/ha Alcohol yield at reasonable 85% global efficiency, 1/ton of cane or in l/ha From Lindeman and Rocchicciolo [55]. 63 8.32 7.0 2.21 1~32 675
11.5
Alcohol, directly from sugar cane 63 8.32 66.2 8.73 4 460 75 4 800
730
with about 10% of an inoculum (previously prepared in shake flasks) and the fermentation is run to complete utilization of the substrate sugars. At this time the mixer is stopped and the yeast is allowed to rapidly settle, leaving a supernatant devoid of yeast flocs. Some non-flocculating microorganisms may still be in the upper zone at this time. As soon as the settling is complete (to the desired level), the upper clear supernatant is withdrawn and sent to distillation, new medium is pumped into the same vessel containing the settled yeast and the next fermentation started under full mixing. As can be seen in Fig. 10, as soon as the high biomass concentration in the bioreactor is achieved, the subsequent fermentation times are considerably reduced down to about 3 h from the initial 20 + h, as obtained in simple batch experiments. These fermentation cycles have been separately run in a dozen other fermentations, for up to 10 consecutive runs without a visible loss in ethanol productivity (Fig. 10). There are a number of advantages of this process as compared to simple batch fermentations and these can be summarized as follows: (i) High productivities are achieved. (ii) High biomass concentration in the reactor is maintained in the order of up to 50 g / l . The biomass represents an excellent yeast byproduct which can be easily dewatered (including a simple settling operation) which would considerably reduce its recovery cost. This biomass could be a rich source of protein and other nutrients for either human nutrition or animal feed.
The operation is simple, requiring only a stirred tank and storage vessels and pumps. (iv) The yeast is being concentrated without the use of any centrifuges or other mechanical concentration devices. (v) While the feed broth must be sterilized (like for any other fermentation process), the actual fermentation run can be done under non-sterile conditions. The fermentation time is short for any interfering development of contaminating microorganisms (bacteria and wild yeast), and if these do develop, they will probably not flocculate and will thus be withdrawn from the bioreactor at the end of each cycle in the spent broth. (vi) Considerable reduction in the fermentation time to about 3 h from conventional 20 h in simple batch operation. Due to this fact, much smaller reactors and equipment are needed, which reduces the overall capital and investment costs. (vii) The energy required to run the fermentation process is only required for mixing and pumping of the liquid. At the present time, there is no such process in commercial operation. All runs were made in our pilot plant 10-1 fermenters.
2.2. Continuous process: internal yeast settling
(iii)
The same flocculating yeast, S. diastaticus, allowed the development of this continuous process. Its capability to rapidly settle against an upflow of fluid allowed a development within the reactor of a
120
Mlxem' s t o p p e d ,
Time
(I s e c
After
I0 s e c
After
20
sec
~ L
~" ~~
After
3(I s e c
After
4(I s e c
After
50
sec
Aft.er
60
sec
Fig. 8. Settling of Saccharomyces diastaticus (From [49]).
121
OPERATE
ST~L
DP~W
FILL
Fig. 9. Schematicdiagram of the semi-continuous(modifiedfedbatch) process(From [49]).
well mixed turbulent yeast zone and of a clear liquid zone at the top. Depending on the mixing applied, the height of the top clear zone can be regulated and maintained during the operation of the bioreactor. The process is depicted schematically in Fig. 11. Fermentation of fodder beet and Jerusalem artichoke juices by this yeast showed that a high concentration of yeast (40-70 g / l ) can be kept in the bioreactor at high dilution rates, and therefore a high volumetric ethanol productivity of 40-50 g L - i his achieved. Figs. 12 and 13, as well as Tables 10 and 11, show the results of this system with the following advantages: (i) High ethanol productivities at high dilution rates. (ii) High ethanol yield at 96% of theoretical. (iii) No need for external yeast recycling and for cell concentration within the bioreactor. (iv) Simple bioreactor configuration. (v) Easy maintenance of desired biomass concentration within the bioreactor. (vi) Improved economics and energetics of the system. (vii) Less capital investment due to elimination of external cell recycling and concentration system. No such system is at present operating commercially. Further developments are under way to scaleup the process, modify and optimize the bioreactor for this fermentation and demonstrate the process on a large production facility. The key to this process is the flocculating ability of the selected yeast. This ability was never lost in our tests and the flocculating stability must be maintained in a large scale installation. These tests are now under way in our laboratories.
Two continuous processes, which are at the pilot plant demonstration stage, can be compared to our process. These are the Hoechst-Uhde process utilizing another flocculating yeast which apparently is sometimes washed out from the system (personal communication) and which achieves a volumetric ethanol productivity of about 16 g 1-~ h J (as compared to 40-50 g 1-~ h-~ in our system). The Hoechst-Uhde process incorporates an internal cell setling and recycle system, which is also not required in our process. Another process for comparison is the Biostill continuous process installed by Alfa-Laval in Brazil as a pilot plant demonstration project producing 150000 I alcohol/day (Alfa-Laval Report, 1983). The Biostill process claims: (i) High alcohol yield because of low by-product formation (mainly glycerol). (ii) Low stillage flow because of low dilution water requirements (see Fig. 14). (iii) Low manning cost because of continuous processing. (iv) Compact plant containing just one fermenter. A comparison of the Biostill with a conventional alcohol plant in Sao Luiz (Brazil) using the same substrate is shown in Table 12. A schematic representation of the Biostill process is given in Fig. 15. As can be seen from the figure, the centrifuge represents an integral part for cell concentration and recycling to the fermenter. Even though at the present time we do not possess the values for all parameters required for a comparison with the pilot plant Biostill data, our process appears to be at least as efficient, but is definitely cheaper as no external cell recycle is required.
3. Biotechnology butanediol
in the production of 2,3-
Butanediol has a heating value of 114 M J / k g [3], as compared to ethanol (122 MJ/kg), and an equimolar mixture of the two (116 MJ/kg). However, its price is not competitive with that of fermentation or synthetic ethanol. Thus its main prospect in the fuels industry is in the dehydration to MEK (methyl ethyl ketone or 2-butanone), which is much more suited as a fuel because of its much lower
122
N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111 142

-
~r r,l (,j
o
L6
r.~
I=~ 0..j
:= L~
vl
,j a= ',O
=,0,,,9
vl N
~R~~,
h'
N
o
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ii/.n j
,~-
-~
: ,,l~
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F X S
0
123
o o 0
"r
F X S P
e e e e
-0
CLEAR ZONE
!t
-el--- VM ----~
MIXING ZONE
=F
V " VOLUMEOF SETTLING ZONE

VM VOLUI4E OF MIXING ZONE
Vs
VM
DILUTION RATE
F D: ~
Fig. ] I. Schematic diagram of the continuous fermentationprocess without externalrecycle(From [49]).
boiling point. Alternatively, condensation with MEK and subsequent hydrogenation yield octane isomers for high-quality aviation fuels.
2,3-Butanediol is another chemical whose production and process development were stimulated by war. During World War II it was needed for conver-
So = 97.0 GL" Sugar in fermentor = 40-50 GL"

B i o m a s s Concentration in fermentor Ypts= 0 . 4 9 o ~ GL" Sugar concentration in GG "~
o~
op ePRAS.U
10C 5 0 - 5 C 10C
For all d i l u t i o n rates
80- 4C-4C-BC
fermentor
o ~ GL"
Ethanol concentration fermentor
PR GL" HR"I
60. 30-3C .6(]

in 4 0 . 2 0 - 2C ' 2C
Volumetric ethanol product i v i t y aS.U. /o of sugar u t i l i z e d
2 0 . 10- 1C , 2 0
:/
~0 0 ~ O- 0 -'--"-'"~"
o~2
o14
0.'6
o18
1.0
D.~R "I
Fig. 12. Continuousfermentationof fodder beetjuice (From [49]).
124
Table 10 Continuous fermentation of fodder beet juice with Saccharomyces diastaticus First fermentor Second fermentor 1)
Fo ] ) (mlh31.0 62.0 93.0 108.5 124.0
D (h - l )
0,258 0.517 0.779 0.904 1.033
SO (gl - l )
111.8 111.8 111.8 111.8 111.8
S1 (g1-1)
4.0 5.0 5,0 10.0 14.2
P,
(gl 53.8 53.8 54.0 51.5 49.5
Yp/~ (g g - 1)
0.50 0.50 0.51 0.51 0.51
Qp (g I i h - ')
13.88 27.81 42.07 46.56 51.13
$2 (g 1-])
2.0 2.0 5.1 3.2
P2 (g 1-1)
54.0 43.0 54.0 54.8
Yp/s (gg
2.26 5.48
1)
Qp ( g l - I h ~)
0.51 0.48
From Kosaric [49]. Table 11 Continuous fermentation of Jerusalem artichoke juice with Saccharomyces diastaticus First fermentor Second fermentor 1)
Fo (mlh
15.5 31.0 46.5 62.0 108.5 132.5
I)
D (h-I)
0.129 - 0.258 0.387 0.517 0.904 1.162
So (gl-I)
161.0 161.0 161.0 161.0 164.0 164
S1 (gl-l)
36 51 47 52 64 72
Pl (gl
61 54 56 55 49 38
Yp/s (gg-l)
0.49 0.49 0.49 0.49 0.49 0.41
Qp (gl-lh-J)
7.9 13.9 21.7 28.4 44.3 44.1
$2
28 27 39 39 44 46
(gl
1)
(gl 65 66 60 60 59 51
P2
J)
(gg0,50 0.50 0.46 0.49 0.50 0.50
Yp/s
i)
QP (g1-1 h l) 0.5 3.1 1.5 2.6 9.0 15.1
From Kosaric [49].
sion into 1,3-butadiene, one of the building blocks of synthetic rubber. It can occur in the form of two enantiomers: D - ( - ) , or levo, and L - ( + ) , or dextro, So:
1G1.0 GL" Total s u g a r in juice
as well as an optically inactive meso-form. All are bacterial products: pure D - ( - )-2,3-butanediol is produced by Bacillus polymyxa, whereas meso-2,3-
aS op ePR~S.U
: 50-70 GL" Biomoss concentration fermentor
in
12(
Ypts: 0.49
For
GG "1 all d i l u t i o n
rates
a g GL"
Sugar concentration in fermentor oP GL"
Ethanol concentration in
Bt3
4 8(
fermentor
PR GL "IHR "I
Volumetric ethanol productivity AS.U *t, 01 s u g a r u t i l i z e d
40
2C
4 ,
/.~ /L~ 1
, .
0.2
0.4
0.6
0.8
1.O
1.2 -D. HR "1
Fig. 13. Continuous fermentation of Jerusalem artichoke juice (not hydrolysed) (From [49]).
N. Kosaric,J. Velikonja/ FEMS MicrobiologyReciews 16 (1995) 111-142

16,
15 14 13 12 11
. . . . . . . . . . . . . . . . . . . . . . . . ..,......... ,...,......-.....-.-.......-..o.. ... i -.. I......%....-i-.....r...-.n.....-/-.-.-.r.-.-.1
125
Table 12 Comparison of the biostill with a conventional plant at Sao Luiz (Brazil) using the same substrate (Source: Alpha Laval) Parameter Yield (% of theoretical) Stillage (1/1 alcohol) Manpower Space requirement (m2) Biostill 94.5 0.8 3 350 Conventional 87 11 7 1350
iiiiiiiiiiiii iiiii!i iiiiiiii!ii! iiiiii!iiii!i!i!i!i i! !i1i!iiiiii!i !i!i!iiiiiii!i!itiii i

8
iiiiliiiiiiii i iiiiiiiiiiiii: i
butanediol is a product of Klebsiella pneumoniae (Aerobacter aerogenes), which also produces some of the L - ( + ) isomer. There are also other bacteria which synthesize mixtures of different forms (e.g.
6
5 4 3 2 1
I I I I I 1
Bacillus subtilis, Serratia marcescens, Aeromonas (Pseudomonas) hydrophila), and also several yeasts,
but they are considered economically unimportant. The use of various organisms and substrates as feedstocks is shown in Tables 13, 14, and 15. Yields are not impressive, mostly under 1 g l-~ h -=, with 8.2 g 1 = h - 1 (in the presence of 4.5 g / 1 acetate) as the highest ever recorded [4]. One of the intrinsic difficulties with product recovery is its high boiling point of about 180C and high water affinity, for which alternatives to distillation were being developed, such as solvent extraction (with ethyl acetate,
AIcohol
stil
I | |
5 FIN
10
Fig. 14. Ratios of stillage and ethanol volumes obtained at different F / N conditions for conventional and biostill fermentations (F, fermentable components; N, nonfermentable components) (From [49]).
(q0-S0 t vvl
Conc. Feed
!
~c
h,..
,,~.r-i
........ n
--
llllllIlllll
FERMENTER| OOLER 4k
REGENERAT IVE HEAT EXCHANGER
l-SECTION ~--"1BEER ST ILL
I~""""U
Yeast C ream,I
AIR 8 LO~,'ER
I,,
CENTRIFUGE
d
Stillage
Fig. 15. Schematic of the biostill process (From [49]).
126
N. Kosaric, J. Velikonja / F E M S Microbiology Reviews 16 (1995) 111-142
Table 13 Batch fermentation of 2,3-butanediol: summary of data for various bacterial strains and substrates Substrate Glucose Glucose Xylose Xylose Xylose Mannose Lactose Whey permeate Hydrolysed whey permeate Whey Whey Starch Citrus waste Xylan Wood hemicellulose hydrolysate Agricultural residues Jerusalem artichoke From Maddox [56]. Organism
Aerobacter aerogenes NRRL B199 Klebsiella pneumoniae NRRL B199 Klebsiella pneumoniae NRRL B199 Klebsiella oxytoca ATCC 8724 Bacillus polymyxa NRCC 9035 Klebsiella pneumoniae AU- l-d3 Klebsiella pneumoniae NCIB 8017 Klebsiella pneumoniae KlebsieUa pneumoniae Klebsiella pneumoniae ATCC 13882 Bacillus polymyxa ATCC 1232 Aeromonas hydrophila NCIB 9240 Aerobacter aerogenes Bacillus polymyxa NRCC 9035 Klebsiella pneumoniae ATCC 8724 Klebsiella pneumoniae ATCC 8724 Bacillus polymyxa ATCC 12321
Overall butanediol productivity (g 1-l h- l) 2.02
Overall butanediol yield (g/g substrate) 0.45 0.36 0.27 0.36 0.24 0.30 0.24 0.46 0.39 0.16 0.2
1.35 0.1 0.64 0.06 0.08 0.14 0.38 0.02 0.17

1.1
0.02 0.45 0.79 0.4
ether, or n-butanol), membrane technologies, adsorption, and chemical recovery. None of these have as yet yielded commercially applied solutions. In situ conversion to M E K (boiling point 79.6C) by acid catalysis would make recovery much easier and more efficient, but decreased conversion rates in the complex broth await further development. The cost of 2,3-butanediol does not compare favorably with other fermentation products (Table 16), although several factors, like the choice of raw materials, could improve process economics. Like with other fermentations for fuel production, the combination with waste utilization or with the elimination o f
waste materials which present an environmental nuisance, yield improved overall performance. Thus, delignified water hyacinth was recently studied as a potential substrate for 2,3-butanediol fermentation [5], with similar results as those obtained for other substrates. More encouraging results with this weed (1.4 g i-1 h-1 at 4 h hydraulic retention time) were obtained in continuous operation with anaerobic fixed-film and U A S B R reactors fed with alkaline hydrolysate (19 g / l reducing sugars). In conclusion, it can be stated that fermentations for oxygenated fuels or fuel precursors other than ethanol will be inevitably outcompeted by petro-
Table 14 Some intensified fermentation technologies for 2,3-butanediol: summary of data Method Continuous flow Continuous flow Metabolistat Continuous flow/carrageenan-immobilized cells Continuous flow/alginate-immobilized cells Continuous flow/cell recycle From Maddox [56]. Substrate Sucrose Glucose Lactose Glucose Whey permeate Whey permeate Organism Butanediol productivity (g i -1 h - l )
Aerobacter aerogenes NCIB 8017 4.6 Klebsiella pneumoniae NRRL B199 4.25 Enterobacter cloacae 0.03 Enterobacter aerogenes IAM 1133
0.75 2.3
1.04
Klebsiella pneumoniae NCIB 8017 Bacillus polymyxa

c h e m i c a l s as l o n g as oil c o n t i n u e s to b e the c h e m i c a l and energy foundation of the industrial civilization. In all l i k e l i h o o d this s i t u a t i o n will c h a n g e d r a m a t i cally in the n e x t c e n t u r y for w e l l - k n o w n r e a s o n s , B u t e v e n t h e n the f u t u r e o f s u c h p r o d u c t s a n d t e c h n o l o gies is u n c l e a r , s i n c e the s e a r c h for a l t e r n a t i v e f u e l s g o e s m a n y d i f f e r e n t w a y s a n d m a y u l t i m a t e l y prov i d e m u c h b e t t e r s o l u t i o n s in the f o r m o f c l e a n e r a n d m o r e e f f i c i e n t e n e r g y carriers, s u c h as h y d r o g e n .
127
4. Biotechnology in the production of liquid and gaseous fuels from coal

In t e r m s o f e n e r g y , coal r e p r e s e n t s 7 1 . 4 % (161 0 0 0 E J ) o f w o r l d ' s fossil fuel r e s e r v e s , as c o m p a r e d to 7 5 0 0 EJ in c r u d e oil ( 3 . 3 % ) . T h e r e c o v e r a b l e coal contains 91.1% of energy and 93.9% carbon cont a i n e d in oil, g a s a n d coal c o m b i n e d [6]. A truly r e m a r k a b l e resource!
Table 15 Summary of 2,3-butanediol production from potential substrates Substrate a Initial monosaccharide g/l Waste sulfite liquor Citrus press juice Sugar beet molasses Sugar beet pulp Wood hydrolysate (la) Wood hydrolysate (lb) Wood hydrolysate (2) Wood hydrolysate (3) Wood hydrolysate (4) 38.0 215.0 56.6 11.0 100.0 100.0 12.1 9.7 40.0 % used 69.7 91.5 71.0 78.0 55.0 93.0 95.0 100.0 100.0 72 56 24 11 46 3.1 NR c 24 46 Fermentation time (h) Yields (g/l) Diol 9.0 51.0 20.1 2.4 16.5 35.5 6.0 0.2 20.0 EtOH 3.3 1.6 3.8 0.5 5.9 HAc 2.1 3.8 0.1 Butanediol g / g used 0.34 0.26 0.50 0.28 0.30 0.38 0.52 d 0.02 d 0.50 d g / g available 0.24 0.24 0.36 0.21 0.17 0.36 0.50 o 0.02 o 0.50 d g 1- J h i 0.13 0.91 0.84 0.21 0.34 1.04 _ 0.01 171.42
Adapted from Magee and Kosaric [57]. a Wood hydrolysate: (la) southern red oak hydrolysate (Scholler process); (lb) same as la, except for acclimatized culture; (2) steam-exploded aspen, hemicellulose fraction, acid hydrolysis; (3) steam-exploded aspen, hemicellulose fraction, enzyme hydrolysis; (4) steam-exploded aspen, cellulose fraction, acid hydrolysis. b Average value. c Not reported. o Conversion of HAc and uronic acids not considered.
Table 16 Selling prices for selected solvents Solvent Methanol (synthesis, tank) Ethanol (fermentation, tank) Ethanol (synthesis, 190 proof, tank) Ethanol (absolute, 200 proof, tank) iso-Propanol (anhydrous, 99%, tank) n-Butanol (synthesis, fermentation, tank) Acetone (tank) 2-Butanone (MEK) (tank) 1,3-Butanediol (tank) 1,4-Butanediol (tank) 2,3-Butanediol (tank) Selling price (1985 US$) 0.14-0.19/1 0.39-0.45/1 0.48-0.50/1 0.51-0.53/1 0.53/1 0.79/1 0.51/kg 0.79/kg 1.59/kg 1.76/kg 3.02/kg
Adapted from Magee and Kosaric [57]. Higher value estimated by Magee and Kosaric (1987) for a 1.2 M t / a production; lower value estimated in Eur. Chem. News, June 4, 1984 for 180 Mt/a.
128
N. Kosaric, J. Velikonja /FEMS Microbiology Reviews 16 (1995) 111-142
Table 17 Some coal-solubilizing microorganisms
Fungi (Basidiomycetes)
Coriolus versicolor Phanerochaete chrysosporium Poria placenta
Yeast-like fungi
Candida sp.
Actinomycetes
Streptomyces badius Streptomyces setonff Streptomyces viridosporus
Fungi (Hyphomycetes)
Acremonium sp. Aspergillus spp. Cunninghamella sp. Mucor spp. Paecilomyces spp. Penicillium spp. Sporothrix sp.
From Faison [6].
Eubaeteria
Bacillus sp. Pseudomonas sp.
Under the term coal are classified many different products of carbonization of ancient organic matter. The younger ones are low-rank coals (lignites and subbituminous coals), whereas the older ones are high-rank coals (bituminous coals and anthracite). Coal constituents are organic compounds, inorganics (clays, quartz, calcite, iron sulfides, etc.) and water. All of these vary considerably in their amounts in coals. The organic part consists of aromatic and aliphatic compounds, the latter being considerably more abundant in lower rank coals. Oxygen is more abundant in younger coals (60% in ether bonds and hydroxylic groups, 40% in esters, carboxylic and carbonyl groups).
COAL FUNGI OR ACTINOMYCETES

OAEREAcToR~
ROBIC~)
COAL SOLUBILIZATION
MIXED CULTURE No. 2 (PRIMARILY BACTERIA) PRODUCTION OF L)OUIO FUELS METHANOl. ETHANOL
(PRIMARILY BACTERIA)
BIOGASIFICATION
CH4 (METHANE)
Fig. 16. Schematic of a two-stage process for the production of fuel chemicals from coal (From [6]).
129
Some coals (mostly low-rank and in some cases bituminous coals) have the potential to be solubilized by the action of microorganisms [7]. There are several microorganisms, from different taxa, for which a coal solubilization capability was demonstrated (Table 17). Various kinds of oxidative (hydrogen peroxide, ozone, 8 M nitric acid/48 h, air/7 days/150C), as well as non-oxidative pretreatments with surfactants (SDS), buffers (Tris, Gly-Gly, phosphate) at alkaline pH, and acid extraction (HCI) have shown to enhance microbial solubilization. It appears that ligninolytic organisms more readily attack lignite (chemically related to lignin) than non-ligninolytic organisms [8]. The product thus obtained is a dark, acidic, polar, water-soluble liquid, consisting of polycondensated, oxidized aromatics, some of them with molecular masses between 30 and 300 kDa [9]. Coal solubilizates of fungal and bacterial origin do not differ markedly from one another. Solubilized and non-solubilized lignite can be a potential substrate for the microbial production of methane, alcohol and fatty acids. It is unlikely that any organism will ever be able to use all or most of the complex organic structure of coal. But a two-stage process, similar to the one in Fig. 16, could be a promising future technology. Some examples cited below illustrate studies on laboratory coal solubilization. Subbituminous, nitric-acid pretreated coal was partly (approx. 10%) solubilized by Paecilomyces TLi [10]. The medium, containing 2.5 g / l unfractionated, solubilized coal as the sole C-source, subjected to methanogenic fermentation by an acclimated culture, yielded 56% from predicted volume of biogas (approx. 25% methane) after 100 days of incubation and a lag of 25 days. After 60 days, only carbon dioxide was produced, because of the exhaustion of components which were metabolized into methane. Cultures supplemented with CI. acetobutylicum ATCC 824 (to break down aromatics into low molecular mass oxygenates) caused an increase in production. Sheep rumen, sewage sludge and soil isolates were tested for alcohol and acetate production [11]. After 10 days incubation of 1 ml solubilized lignite with sewage sludge isolates, the concentration of ethanol was 0.072 g / l , and that of acetic acid 0.83
g/1. With 10 ml solubilized lignite, the concentrations were 0.386 g / ! and 0.642 g / l , respectively. Two soil isolates gave very little ethanol with 1% lignite, gradually disappearing at 48 h of incubation, whereas acetate showed a peak of 1.16 g/1 at 48 h. Another mixed culture from soil gave within 48 h an increase in ethanol concentration of 0.35 g / l and an acetate concentration of 0.40 g/I. The above study, however, has shown that bacterial isolates from coal environments were able to solubilize > 30% untreated lignite in 28 h. Another very interesting finding was that small quantities of cells (bacterial isolate LSC), grown on cheap substrates (e.g. crushed barley hulls) and subsequently added to pretreated lignite in water, brought about a 45% solubilization at 100C in only 10 min (90 g solubilized coal per liter). Subbituminous coal solubilization with various oxidoreductases in organic solvents and aqueous solutions under both aerobic and anaerobic conditions was also studied [12], however without convincing results. From the above it may be concluded that research on coal solubilization and subsequent fuel production is still in its beginnings, although there is considerable interest to convert cheap, low-grade lignite into more valuable products like liquid fuels or biogas. Accumulated data seem to indicate that a two-stage aerobic/anaerobic process of solubilization and fuel production would be most advantageous.
5. Biotechnology in methane and biogas production

Methane, the main constituent of natural gas and the principal combustible component in biogas, has the highest molar heat of combustion of all organic compounds: -890.31 k J / m o l (at 25C and 101.3 kPa). Enormous quantities are constantly being released into the atmosphere from geological sources (natural gas vents and coal deposits), decaying organic matter (lake and river sediments, peat bogs, marshes), agricultural areas (paddy rice fields), waste processing facilities (sewage treatment plants and landfills), and from the digestive tracts of mammals (most notably ruminants) and some insects. It is thought that microbially produced methane world-
130
N. Kosarie,J. Velikonja/ FEMSMicrobiologyReviews16 (1995)111-142
,~for
Can illuminatemantle lamp equivalentto 60 walt about 7 hours
Can run 2 horsepower engine for one hour %N~
l
One m3 of biogas
Can cook 3 meals for family of t, persons
II
refrigerator for 3 hours
Can run 300 litre
(;an generate1.25 kw electricity
Fig. 17. Possible applications of biogas (From [18]).
wide yields about 50 E J / a , whereas some 30 E J / a come from geological sources. Besides carbon dioxide, atmospheric methane is a major greenhouse gas. Possible applications of biogas are presented in Fig. 17. Biogas is the final gaseous product o f anaerobic degradation. Almost all of it is methane ( 5 4 - 8 0 % ) and carbon dioxide ( 2 0 - 4 5 % ) , in a typical volume ratio of 3 / 2 [13]. Other, usually minor, constituents are hydrogen, molecular nitrogen, oxygen, hydrogen sulfide, and carbon monoxide. The elemental composition of degradable biomass directly influences the
m e t h a n e / c a r b o n dioxide ratio, a fact reflected in the Buswell-Mueller stoichiometric equation [14]:
( a b ) C.H~O b +
----.> n 4 2 H20
--
__
q.-
CO 2 +
-- + -- _ _ CH 4 2 8 4
A s with any other kind of fuel, methane production requires cheap raw materials. Almost all biomass-based fuels must necessarily come from the bioconversion of lignocellulosics, i.e. the products of
Table 18 Raw materials for biogas production Origin Agricultural wastes .Human wastes Animal wastes Agriculture-based Forestry wastes Aquatic wastes From Aziz [18]. Type of waste Crop-related stubble, straw, spoiled fodders, weeds Excrements, sewage sludge, refuse Cattle dung, pig, sheep, goat manure, poultry litter; slaughterhouse, tannery, fishery, wood wastes Wastes from: palm oil and rubber mills, sugar cane bagasse, tobacco manufacture, breweries, distilleries, food, fruit and vegetable processing, sugar and tapioca mills, tea and coffee plantations, textile and jute mills, rice brans Twigs, barks, branches, leaves, dead trees, plants Algae, weeds, water hyacinth, other aquatic plants
N. Kosaric, J. Velikonja / FEMS Microbiology Reuiews 16 (I 995) 1 l 1-142
131
hydrolysis of its primary components: cellulose, hemicellulose and, to a lesser extent, pectin. These are abundantly found in the form of lignoceUulosic waste from silviculture, agriculture and industry, as well as in municipal sewage sludge and municipal solid waste. Lignin, a major constituent of wood ( 1 8 - 3 0 % d.s.), is considered to have no methanogenic potential, because its decomposition rate is far too low. Useful waste materials are summarized in Table 18. Methanogenic potentials of various classes of precursor compounds are given in Table 19, as represented in municipal solid waste. Dedicated fuel crops present an enormous potential for fuel production, though they have a better potential for alcohol fuels, as represented in Table 20. Primary production of biomass through photosynthesis is estimated at 172 billion tons per year (approx. 2 / 3 terrestrial and 1 / 3 aquatic) [15], which is roughly one-tenth of the standing biomass present on earth. In terms of energy, the annual biomass production is estimated at 3.21 x 1021 J / a [16], assuming a heating value of 18.6 G J / t dry biomass. This is very close to the actual world energy demand of today (see above). A decade ago it was noted that biomass supplied about one-seventh of the world's fuel, equivalent to 20 million barrels of oil per day, which
Table 19 Composition and methane potential of municipal refuse Chemical constituent Cellulose Hemicellulose Protein Lignin Starch Pectin Soluble sugars From Barlaz [58]. Dry weight (%) 51.2 11.9 4.2 15.2 0.5 < 3.0 0.35 Methane potential (%) 73.4 17.1 8.3 0 O.7 0.5
was twice the Saudi Arabian oil production and equal to the daily oil use in the USA [17]. Many developing countries, most notably China (9 million digesters, serving some 35 million people in rural ares) and India (over 70000 biogas plants producing annually an estimated 152 million tonnes of biogas, i.e. about 130-140 billion m 3, or some 3 EJ of energy) [18], have been successfully applying anaerobic digestion of domestic and agricultural waste in small- to medium-scale biogas generation. Industrialized countries of the Western hemisphere have been converting most of their agricultural waste into fuel ethanol rather than into biogas, although dedicated fuel crops prevail as raw materials. These nations traditionally continue to generate
Table 20 World biomass potential for ethanol production Source Cane and beet molasses Cane and beet juice Bagasse surplus to fuel Grain, dedicated Grain, low grade B starch Straw, chaff, stover Cassava, cull Cassava, tops Potato, cull Jerusalem artichoke, tops Forest logging residues + non-commercial harvest Plantation forests Municipal waste Total From Wayman and Parekh [59]. (1 1 C2H5OH approx. 21.2 MJ). Mass(lO 9 t / a ) 38 24 23 80 116 3 300 2 45 12 3 360 60 250 4 313 Ethanol equivalents (109 1) 11 5 7.5 8 27 52 1000 1 14.4 3.8 1 125 24 37 1316.7 Oil equivalents (106 bbl) 69 31 47 50 170 327 6 290 6 90 24 6 786 152 232 8 280 Energy (EJ) 0.23 0.11 0.16 0.17 0.57 1.10 21.20 0.02 0.31 0.08 0.02 2.65 0.51 0.78 27.91
132
Table 21 Composition of municipal solid waste (1986) Component Paper, paperboard Yard refuse Food Subtotal Metals, ceramics Glass Textile, rubber, leather, wood Plastics Inorganic (ash, rock), etc. Subtotal From Lewis [60]. Percent by wet weight 41.0 17.9 7.9 66.8 8.7 8.2 8.1 6.5 1.6 33.1
waste (Table 21), with the highest methane potential for cellulose, followed by hemicellulose and proteins (Table 19). Potentially, landfilled refuse could generate 0.13 m 3 methane per kg dry waste [20], but practically the production has ranged from 1% to 52% of that value
[21].
Sanitary landfills are slow but, due to their large
dimensions, quite productive biogas reactors. It was estimated that more than 825000 tonnes of coal equivalents per year were globally saved by their methane production, with a tendency to increase further [22].
most of the biogas from wastewater treatment and solid wastes. The main reason for this lies in the fact that amounts of domestic refuse and sewage present formidable waste management and environmental problems, which can be alleviated by fuel production. An estimated average of 1.64 kg MSW per person per day was generated in the US in 1986 (Lewis, 1989). About 95% of the MSW in the US was landfilled in 1984 [19]. Man-generated refuse represents an excellent feedstock for methane/biogas generation by: (i) landfill gas collection; (ii) anaerobic digestion of MSW in reactors. It is estimated that more than two-thirds of MSW (on a wet basis) consist of easily fermentable organic
Table 22 Lipid contents of selected microalgae Species Monalanthus salina Botryococcus braunii Outirococcus sp. Scenedesmus obliquus Nannochloris sp. Dunaliella bardawil ( ~ D. salina) Navicula pelliculosa Radiosphaera negevensis Biddulphia aurita Chlorella vulgaris Nitzschia palea Ochromonas dannica Chlorella pyrenoidosa From Ratledge [36]. Maximal lipid content (% w / w ) 72 53-70 50 49 48 47 45 43 40 40 40 39-71 36 (72)
6. Biotechnology in diesel fuel and gasoline production from microalgae
Among the various possible fuels from biotechnology research and development, there is considerable interest to tap the high-density energy stored in lipids (35.6 M J / I for vegetable oil) as possible alternatives for diesel (39.1 MJ/1) engines. Vehicles (e.g. public transportation buses) are already testing vegetable oils, but there is still some controversy over the environmental benefits vs. risks. However, it will be a lasting endeavour of the constructors of ICEs (internal combustion engines) to decrease pollutant emission levels and improve fuel efficiency, environmental safety and overall marketability of
Species Peridinium cincture Neochloris oleabundans Oocystis polymorpha Chrysochromulina spp. Scenedesmus acutus Scenedesmus spp. Chlorella minutissima Prymnesium parvum Navicula pelliculosa Scenedesmus dimorphus Scotiella sp. Ch lorella spp. Euglena gracilis Porphyridium cruentum
Maximal lipid content (% w/w) 36 35-54 35 33-48 26 26 23 22-38 22-32 16-40 16-35 15-26 14-20 14 (22)
133
diesel engines, which currently use 17% of energy for transportation purposes [23]. This chapter discusses the substrates, microorganisms and biotechnological processes useful for fuel lipid production. A relatively limited number of microorganisms can accumulate large amounts of storage lipids under specific growth conditions. Most productive among them, and industrially the only important ones, are some yeasts [24] and algae [25]. Yeasts, however, are less favorable because for lipid production they must grow aerobically, and that has a negative effect on substrate conversion yields. The ability of microalgae and cyanobacteria to grow photoautotrophically makes them far more interesting, since algae cultivation can be directly coupled with carbon dioxide elimination from power plant flue gas. Microalgae accumulate up to 60% or more lipids (based on dry biomass weight) intracellularly (Table 22), and these lipids, predominantly triglycerides, can be transformed into low-sulfur diesel substitutes [26]. The extracted triglycerides cannot be used directly for ICEs, but have to be either transesterified into low-viscosity and low-melting point esters (e.g. methyl esters), or catalytically converted into hydrocarbons as gasoline substitutes. The potential advantages and usefulness of such production seem to have escaped wider attention. Thus, in a review on microalgae biotechnology from 1987 [27], there was no mention of a fuel production potential of algae, although a price of (then) US$ 0.4-0.6 per kg of algae with an average of 30% lipid content was given for research-level production in unlined solar ponds and a projected market of at least US$ 100 million. Studies of extraction and transesterification of algal oils from Chaetoceros muelleri and Monoraphidium minutum show similarities with the transesterification of vegetable oils, with the difference that lipids from these algae had much higher free fatty acid contents (about 25% FFA) than vegetable oils, and thus an acid-catalysed reaction with strictly time-controlled duration was recommended, along with 1-butanol as the most efficient extraction solvent [28]. It was earlier postulated that transesterification has the same economical potential as catalytic conversion, if the by-product glycerol can be marketed [26].
Catalytic upgrading of pyrolysed microalgae lipids and whole cells over medium-pore, shape-selective zeolite (HZSM-5) to a high-octane, aromatic C-5 to C-10 gasoline was also studied recently [29]. The studied algae were Chaetoceros muelleri var. subsalsum, Monoraphidium minutum, Navicula saprophila, and Nannochloropsis sp. With the latter two algae at low partial pressures 50-65% alkenes and 15-25% aromatics were obtained, with almost no alkanes. Processing of whole algae cells, although highly desirable because of high extraction costs (5-6 cents per liter oil at 90% + extraction), gave ambiguous results due to high ash content (10-50%). Additional research is needed for both transesterification and the more promising catalytic upgrading. Outstanding among other algae is Botryococcus braunii strain B, a fresh-water green alga isolated from the so-called 'Boghead Coal' oil deposits. Resting green cells of this alga produce traces of hydrocarbons. Fast-growing green cells produce up to 17% of C-27, C-29, and C-31 dienes, whereas brown resting cells accumulate 70-90% of their dry weight as predominantly two polyunsaturated terpenoid hydrocarbons, botryococcene and isobotryococcene (Fig. 18). Under laboratory growth conditions the hydrocarbon content is lower (up to about 45%). Catalytic cracking of this hydrocarbon produced 67% gasoline, 15% aviation turbine fuel, 15% diesel fuel and 3% residual oil [30]. Due to low production rates of 0.12-0.15 g 1-1 d -1 (20-30% more after immobilization), that alga is still not promising as potential fuel producer. This emphasizes the need for genetic improvement of potential producer strains [31]. Microorganisms accumulate storage lipids when grown under nutrient limitation. Idealized curves for
Botryococcene
Fig. 18. Structures of botryococcene and isobotryococcene (From
[501).
134
"o ~
r- in medium~,
,,,
/|
/C, /
pid 1% biomass
10
20
30
40 50 Time (h)
60
70
Fig. 19. Idealized pattern of lipid accumulation in an oleaginous microorganism grown in batch culture (From [36]).
batch and continuous growth are given in Figs. 19 and 20A. The limiting nutrient is mostly nitrogen, but highly efficient diatoms had also considerably increased lipid yields under conditions of silicon deprivation, notwithstanding a concomitant decrease in biomass yield. Thus for two diatoms, Hantzschia DI-60 [32] and Cyclotella cryptica [33], the results shown in Table 23 have been obtained. The cultivation of algae for the production of cheap oils as precursors for economically competitive fuels, must use substrates and conditions other than those for food-grade and specialty chemical production. They can be grown in open ponds, in marine, brackish or waste water. Thus, algae from municipal sewage treatment plants were reported to yield up to 50 g dry biomass per m 2 per day [34]. It
has been estimated that a yield of 25 g m -2 d-~ is less than 10% of the theoretical maximum [35], and with oil contents of 25-50%, these algae would be much more efficient oil crops than plant seed, with yields from 12.5 to 25 t ha-1 a-1 [36]. A readily available source of carbon dioxide for algae cultivation is flue gases from power plants. An additional advantage is the environmentally highly desirable concomitant contribution to the elimination of waste carbon dioxide. A conceptual scheme for such a process [37] is represented on Fig. 20B. Recent tests in such a pilot facility in Japan indicate that an actual flue gas (10-12% carbon dioxide, 70-90 ppm sulfur and nitrogen oxides) directly blown into the mechanically mixed raceway-type pond did not adversely affect the growth and photosynthesis of Nannochloropsis sp. and Phaeodactylum sp. in seawater. Results were comparable to those obtained with pure carbon dioxide and with desulfurized flue gas: approximately 10 g m -z d -~ at 400 L y / d or half the value from laboratory tests. These lower yields were attributed to changing illumination and insufficient mixing. Although no attempt was made to determine the lipid content of the algae, such an approach might be a promising alternative to algal lipid production. In conclusion it can be said that in the long range algal lipid production has a promising potential for fuel, especially diesel fuel production since prices for such biodiesel have dropped from between approx. US$ 4.49/1 and US$ 4.76/1 in the early 1980s to about US$ 0.92/1 in 1992, with opportunities to lower them to US$ 0.26/1 [38]. Taxes for carbon dioxide release could make these fuels even more competitive.
Table 23 Changes of biomass and lipid yields of Hantzschia DI-60 and Cyclotella cryptica under nitrogen or silica deficiency at 20 and 30C Organism Nitrogen sufficiency AFDW (g/I)
a
Nitrogen deficiency Change AFDF a (%) - 36 - 29 - 16 - 26 Change lipids (%) + 15 + 28 + 120 + 88
Silicon sufficiency AFDW (g/l) 1.185 1.264 1.161 1.219

a
Silicon deficiency Change AFDF a (%) - 17 - 16 - 16 - 27 Change lipids (%) +48 + 41 +38 + 46
Total lipids (g/l) 0.319 0.468 0.181 0.224
Total lipids (g/l) 0.271 0.334 0.222 0.257
Hantzschia
DI-60
20C 30C 20C 30C
1.314 1.582 1.114 1.321
Cyclotella cryptica
Adapted from Sriharan et al. [32,33]. a AFDW, ash-free dry weight (measured after 5 - 7 days of cultivation).
135
7. Biotechnology in the production of other oxygenated alternative fuels and fuel extenders
Butanol has a boiling point of 118C, a heat of combustion of about 32 MJ/kg, and it is fully miscible with diesel fuel (a microemulsion), where it acts as a co-solvent. For that reason it may be interesting to know whether there is some real potential to produce it biotechnologically.
A
In 1915 Chaim Weizmann patented his process for the production of acetone and butanol by fermentation of carbohydrates by Clostridium acetobutylicum. The interest for this fermentation in the second year of World War I was enormous, since the british military industry needed large quantities of acetone as a solvent in the production of the explosive cordite. Scientific research, although mostly of a serendipitous nature, can have far-reaching conse-
-t ow A
~-~Biomass
" ~
Nitrogen
I ipi(! 1%I , i - m ; L ' ; s ) ~ N ~ l

. . . . .
E2 .o .~_
n I I J
/
~ l
0-1 0-2 Dilutionrate(h-I)

E~
teck
CO= Exhaust Gas COs
II po., P,n, .i',
Seawater. Nutrients Nutrients Recycle
i.l
Algal Growth Pond Water Recy'elo
o-.,oo
Fig. 20. (A) Idealized pattern of lipid accumulation in an oleaginous yeast grown in continuous culture (From [36]). (B) Conceptual system of algae-based bioprocess for CO 2 removal (From [51]).
136
NAD +
NADH
+ H+
~l
cH3_co_cooH
Pyruvete
' CHz-CHOH-COOH I <

Lactate
~ zj
FdHs
CHs-COOi.I Acott, e .-. ~ t,
) F~
C.,-CO-CH -CO-CoA
ATP ADP Acetyl-P CoA Acetyl-CoA -i
CoA
AcetoaeetylCoA
Co.A
~trrr]-c,a
CHs-CH0 Acetaldehyde
CH3-CO-CHz-C00H CHs-CHOH-CHz-CO-CoA - Hydroxybutyryl- CoA Acetoecetate
t~HzO
Ethanol
Reactions in Clostridium acetobutylicum leading to the formation of organic acids and solvents. 1, reactions of the Embden-Meyerhof-Pamas pathway: 2, lactate dehydrosenase: 3, p.vruvate: ferredoxin oxidoreductase 4, ferredoxin: NAD * oxidoreductase; 5, ferredoxin NADP" oxidoreductase; 6, hy-
Acetone
CHs-CH=CH-C0-CoA Crotonyl- CoA te~-- NADH + H +
NAD CHs-CHz-CHz-C0-CoA
Butyryl - CoA
N A D ~
CoA
drogenase;7, phosphotransacetylase: 8, acetate kinase: 9, CH~-~-Iz-CHz-CO- 0 CH3- CHz-CHz-CH0 acetaldehyde dehydrogenase; Butyryl-P Bu*.yraldehyde 10, alcohol dehydrogenase: 11, B-ketothiolase; 12, acetoacetyl2~ NADPH + H CoA: butyrate/acetate CoA , e ~ ADP transferase; 13, acetoacetate deATP NADP+ carboxylase: 14, p-hydroxybutyryl-CoA dehydrogenase: 15, crotonase; 16, butyryI-CoA deCH3-CHz-CHz-COOH hydrogenase; 17, phosphotrans- lCH3-CHz-CH2-CH20H Butanol Butyl'ate I butyry,lase; 18, butyrate kinase; 19, butyraldehyde dehydrogenase; 20, butanol dehydrogenase Fig. 21. Biochemistryof the acetone-butanol-ethanolfermentation(From [52]).
t
137
quences for human life, history, and even politics. In his 'War Memoirs' David Lloyd George, who was Chairman of the Munitions of the War Committee at the time, recounts that the Crown was so grateful to Weizmann for acetone from the ABE process, that this led to the famous Balfour Declaration, which enacted Palestine as the Jewish national home [39]. However, since much cheaper feedstocks and more efficient processes for acetone and butanol were later provided by petrochemistry, the ABE fermentation went into obsolescence. Today this well established technology is used in only a few places, most notably in the Republic of South Africa, where again politics, along with economic and climatic factors, have contributed to the survival of the process. The interest for this fermentation has never ceased and many improvements have been made since the gradual disappearance of ABE fermentation after World War II. The biochemistry of the process (Fig. 21) and its technological features are well characterized [40,41]. The main advantage of this technology lies in the fact that Clostridium acetobutylicum and C. beijerinckii, the two principal microorganisms, ferment not only hexoses but also pentoses from hemicellulose, and can utilize a vast array of adequately pretreated substrates.
The main drawbacks of ABE fermentations are: (i) low solvent yields (30-35% by weight of carbohydrate); (ii) low solvent concentration due to butanol toxicity (usually 20-25 g/l); (iii) difficult and costly recovery by distillation; (iv) phage sensitivity; (v) autolysin-induced culture autolysis by the end of the exponential phase; (vi) yield lowering due to ethanol production. Many of these problems have been partly solved by mutant selection and genetic engineering of the commercial strains. However, the economically most promising alternative to a cheaper butanol as a diesel fuel extender seems to be in the improvement of low-energy product recovery [42]. Anyhow, fermentation butanol is not likely to outcompete petrochemical butanol in the near future.
8. Biotechnology in direct energy conversion: microbial fuel cells
This chapter gives a brief description of specific and unique processes of energy generation from fuels, rather than processes of energy conservation in the form of fuels.
Anode Cothod. l
~
Reduced oxidant
.~
Load
~.
e
Oxidation products
A "
f
Mediator
red)
J
Mediator
Fuel ---'-
-7
(ox)
H+ p
Oxldont Ion-exchange membrane
Fig. 22. Schematic diagram of a microbial fuel cell (From [44]).
138
Energy-rich chemical bonds of a fuel can be broken by the action of oxidants. Thereby an overall redistribution of electric charge between the participating molecules of fuel and oxidant takes place. The
fuel is being reduced, losing some of its internal energy in the form of electrons. Simultaneously, the oxidant increases its internal energy content by gaining the same electrons. The free energy of the reac-
R,Nka ~
~Jttt~ ~ .
=i.~,M
ptmr,wia, ,,,.=i,,,.~am~m,=.e.,w,lh (A.J (~).

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$1t~Ktutal formul,i
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gena# viololcn
-0.359
YEI
Cl %_._
~
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O==~:y~=N--~__~OH
cI
+0.217
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CzH,
PIIENAZINES
~nazifl tho~ulphale
+0.065
CH~
N.~
CH~
HzN
i CH,)
NH:
Sairanine-O
-0 289
512
SO~-
PHENOTHIAZINES
(CHt)2N/~:~O
OH
Alizm'in lian, ue Bri Bl
-0.173
641
SOl
SOsN.N-dimethyl-di~ulphonat iheonine cd +0+~"0 620
CH,HN
Mclhylene Blue
+0.011
f~l
New
Methylene Blue
-0.021
590
C:HsHN
1~/ C:H,
~o . + , , . ~+ z~~
13o
s.o
Fig. 23. Redox mediators and their mid-point potentials (ETm)and maximum absorbance wavelengths(Area (From [44]). x)
139
+0.064
llzN
NI4.,
Toluidlne
BhJe-O
+0.034
fCH~)2N
..
PHENOXAZINES BliUilnlCfesylBlue
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Fig. 23 (continued).
+0.021
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tion is negative, i.e. the reaction is spontaneous, and under normal circumstances of fuel combustion, this free energy performs volumetric work, causing the gaseous reaction products to expand. If the process is rapid enough, or, in other words, operating with a sufficient power output, we have the thermodynamic rationale of internal combustion engines. There are, however, other possible routes to channel and use the free energy liberated from fuels. This has been recognized as early as 1839 by Growe and 1884 by Ostwald. Systems in which the direct contact of fuel and oxidant molecules is prevented and the electric charge transfer from fuel to oxidant is intercepted by a galvanic element coupled to an external circuit are called fuel cells. They are direct converters of chemical into electric energy, operating much more efficiently than power plants. Theoretically, any organic or inorganic compound or a mixture can serve as a fuel, provided it is oxidized by the appropriate organism. E.g. for glucose:
C6H1206 q- 6H20 ~ 6CO 2 + 2 4 e - + 24H +

Conventional fuel cells must operate at high temperatures a n d / o r under extremes of pH if the necessary activation energy for fuel oxidation at the anode is to be reached. The same can be achieved much
more elegantly and at low temperatures by letting the redox machinery of living cells, or isolated oxidoreductases, do the activation job and mediate in the transfer of electrons from fuel to anode. This transfer can be direct, but for better coulombic yields and a higher rate of charge transfer (higher currents) it is necessary that the electron transfer be mediated by a reversible redox couple. Since the power output of fuel cells is directly proportional to the electromotive force of the cell, it is desirable that the reduction potentials of the mediator molecule be as low as possible. If live aerobic ceils are used, the mediator molecule, apart from having long-term stability and being water-soluble, has to be able to reversibly cross the cell membrane and in its oxidized state to interact with the lowest-potential points in the electron transport chain (or at more than one reaction site along it) [43]. The principle of operation of a microbial fuel cell is shown in Fig. 22, along with examples of mediator molecules (Fig. 23). Both are taken from Roller et al. [44]. An interesting microbial fuel cell design was described [45] in which carbohydrates (simple sugars, starch) or hydrocarbons (crude oil) were fed to a mixed culture of Proteus vulgaris, Escherichia coli,
140
Purifiedwater
CO2 ~ / SO4"
02
H20
/Cathode~k
'~
bacteria~
H* ' ~
S-o lyOz
CxHyOz (fuel) + traceelements

Fig. 24. Biochemical fuel cell with sulfate reduction (From [45]).
Anode
Pseudomonas aeruginosa and Desulfovibrio desulfuricans in 0.1-0.5% sodium sulfate solution, solidified as a bulky microbial anode with clay or slate dispersions. Here the energy of the organic molecules is coupled to sulfate reduction. The generated sulfide, a tertiary fuel, served as the anodic redox mediator: Biological reactions ( ( C H 2 0 ) is simplified carbohydrate fuel):
Waste-water
Slatematerials
Fig. 25. Construction of a wastewater fuel cell with slate materials (From [45]). It had a storage capacity of up to 0.2 A cm -2, and could provide a current of 6 A / k g cell weight (1 h continuous load), or 15 A / k g (10 min continuously). The same authors patented also a similarly built waste water fuel cell, shown on Fig. 25. The waste elimination efficiency achieved in short-period incubation, daily for 6 months, is represented in Table 24. The basic disadvantages of microbial fuel cells are their generally low coulombic yields and low power outputs, attributed to pH variation during operation and low fuel storage capacities. Many more improvements, especially improvements of the electrode materials and construction will be necessary, before biological fuel cell production and use can be commercialized. However, miniaturized versions of such systems, not meant as power sources though in an advanced state of development and marketed worldwide, constitute a large subgroup of biosensors.
2 < C H 2 0 > + 2 H 2 0 ~ 2CO 2 + 8 H + + 8 e SO42- + 8 H + + 8 e - ~ Anode reaction: S 2- + 4 H 2 0 ~ SO42- + 8H + 8 e (and 8 / 3 S 2 - + 4 H 2 0 ~ 4/3S2032- + 8 H + + 8 e Cathode reaction: 20 2 + 8H++ 8e-~ 4H20 $2-+ 4H20
The principle of operation is shown in Fig. 24. This biochemical fuel cell was shown to offer a problem- and maintenance-free operation for 5 years. Table 24 Wastewater purification efficiency of a fuel cell Type of wastewater TOC (mg/1) Sewage works effluent 50 (fulvic acids) Effluent from landfill 2 000
COD (mg/I) 140 6 000
Incubation time (h) 0.5 2
Degradation (%) 35 65-75
From Habermann and Pommer [45]. TOC, total organic carbon; COD, chemical oxygen demand.
141
References
[19] [1] Cole, G.H.A. (1992) Provision of the world's energy need. Energy World 199, 15-19. [2] Kosaric, N. and Duvnjak, Z. (1988) Ethanol. In: UUmann's Encyclopedia of Industrial Chemistry, Vol. A9 (Lederberg, J., Ed.), pp. 587-653. Academic Press, New York, NY. [3] Flickinger, M.C. (1980) Current biological research in conversion of cellulosic carbohydrates into liquid fuels: how far have we come? Biotechnol. Bioeng. Symp. 22 (Suppl. 1, Ferment.: Sci. Technol. Future), 27-48. [4] Shazer, W.H. and Speckman, P.A. (1984) J. Dairy Sci. 67 (Suppl. 1), 50. [5] Motwani, M., Seth, R., Daginawala, H.F. and Khanna, P. (1993) Microbial production of 2,3-butanediol from water hyacinth. Bioresource Technol. 44, 187-195. [6] Faison, B.D. (1991) Biological coal conversions. CRC Crit. Rev. Biotechnol. 11, 347-366. [7] Cohen, M.S. and Gabrielle, P.O. (1982) Degradation of coal by the fungi Polyporus oersicolor and Poria monticola. Appl. Environ. MicrobioL 44, 23-27. [8] Wondrack, L., Szanto, M. and Wood, W.A. (1989) Depolymerization of water soluble coal polymer from subbituminous coal and lignite by lignin peroxidase. Appl. Biochem. Biotechnol. 20/21,765-780. [9] Scott, C.D., Strandberg, G.W. and Thewis, S.N. (1986) Microbial solubilization of coal. Biotechnol. Prog. 2, 131139. [10] Davison, B.H., Nicklaus, D.M., Misra, A., Lewis, S.N. and Faison, B.D. (1990) Utilization of microbially solubilized coal: preliminary studies on anaerobic conversion. Appl. Biochem. Biotechnol. 24/25, 447-456. [11] Ackerson, M.D., Johnson, N.L., Le, M., Clausen, E.C. and Gaddy, J.L. (1990) Biosolubilization and liquid fuel production from coal. Appl. Biochem Biotechnol. 24/25, 913-928. [12] Scott, C.D., Woodward, C.A., Thompson, J.E. and Blankship, S.L. (1990) Coal solubilization by enhanced enzyme activity in organic solvents. Appl. Biochem. Biotechnol. 24/25, 799-814. [13] Wheatley, B.I. (1980) The gaseous products of anaerobic digestion - - biogas. In: Anaerobic Digestion (Stafford, D.A., Wheatley, B.I., Hughes, D.E., Eds.), pp. 415-428. Applied Science Publishers, London. [14] Buswell, A.M. and Mueller, H.F. (1952) Mechanisms of methane fermentation. Ind. Eng. Chem. 44, 550-552. [15] Szmant, H.H. (1986) Industrial Utilization of Renewable Resources, Technomic Publishing Co., Thancaster, Basel. [16] Whittaker, R.H. and Likens, G.E. (1975) The biosphere and man. In: Primary Productivity of the Biosphere (Lieth, H. and Whitaker, R.H., Eds.), pp. 305-328. Springer Verlag, New York, NY. [17] Zsuffa, L. (1982) The production of wood for energy. In: Energy from Forest Biomass (Smith, W.R., Ed.), pp. 5-17. Academic Press, New York, NY. [18] Aziz, M.A. (1991) Biogas: an assessment of potentials, technologies and utilization in Asia. In: Energy and Environmental Progress. 1. Volume B: Solar Energy Applications, Bio-
[20]
[21]
[22] [23]
[24]
[25]
[26]
[27] [28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
conversion and Synfuels (Veziroglu, T.N., Ed.), pp. 419-439. Nova Science Publishers, New York, NY. Carra, J.S. (1987) Design criteria for sanitary landfills: the American concept. Proc. ISWA Symp. Process Technol. Environ. Impact Sanit. Landfills, Cagliari, Sardinia, Italy, October 20-23. Stanford, CA. Halvadakis, C.P. (1983) Landfill methanogenesis: literature review and critique. Technical Report No. 271, Department of Civil Engineering, Stanford University. Ham, R.K., Hekimian, K.K., Katten, S.L., Lockman, W.J., Lofy, R.J., McFaddin, D.E. and Daley, E.J. (1979) Recovery, processing, and utilization of gas from sanitary landfills. EPA Publication No. 600/2-79-001, Municipal Environmental Research Laboratory, Cincinnati, OH. Richards, K. (1988) Gas from waste makes growing contribution to fuel supplies. Energy Man. 1, 25. American Solar Energy Society (1992) Economics of Solar Energy Technologies, American Solar Energy Society, Boulder, CO. Rattray, J.B.M. (1988) Yeasts. In: Microbial Lipids. Vol. 1 (Ratledge, C. and Wilkinson, S.G., Eds.), pp. 555-697. Academic Press, London. Wood, B.J.B. (1988) Lipids of algae and protozoa. In: Microbial Lipids Vol. 1 (Ratledge, C. and Wilkinson, S.G., Eds.), pp. 807-867. Academic Press, London. Neenan, B., Feinberg, D., Hill, A., Mclntosh, R. and Terry, K. (1986) Fuels from Microalgae: Technology Status, Potential, and Research Requirements, Solar Energy Research Institute, SERI/SP-231-2550, Golden, CO. Benemann, J.R., Tillet, D.M. and Weismann, J.C. (1987) Microalgae biotechnology. Trends Biotechnol. 5, 47-53. Nagle, N. and Lemke, P. (1990) Production of methyl esther fuel from microalgae. Appl. Biochem. Biotechnol. 24/25, 355-361. Milne, T.A., Evans, R.J. and Nagle, N. (1990) Catalytic conversion of microalgae and vegetable oils to premium gasoline with shape-selective zeolites. Biomass 21, 219-232. Hillen, L.W., Pollard, G., Wake, L.V. and White, N. (1982) Hydrocracking of the oils of Botryococcus braunii to transport fuels. Biotechnol. Bioeng. 24, 193-205. Dunahay, T.G., Jarvis, E.E., Zeiler, K.G., Roessler, P.G. and Brown, L.M. (1992) Genetic engineering of microalgae for fuel production. Appl. Biochem. Biotechnol. 34/35, 331339. Sriharan, S., Bagga, D. and Sriharan, T.P. (1990) Effects of nutrients and temperature on lipid and fatty acid production in the diatom Hantzschia DI-60. Appl. Biochem. Bioteehnol. 24/25, 309-316. Sriharan, S., Bagga, D. and Nawaz, M. (1991) The effects of nutrients and temperature on biomass, growth, lipid production and fatty acid composition of Cyclorella cryptica Reimann, Lewin, and Guillard. Appl. Biochem. Biotechnol. 28/29, 317-326. Pearson, H.W. (1987) Algae associated with sewage treatment. In: Microbial Technology in the Developing World (DaSilva, E.J., Dommergues, Y.R., Nyns, E.J. and Ratledge, C., Eds.), pp. 260-288. Oxford University Press, Oxford. Shifrin, N.S. and Chisholm, S.W. (1980) In: Algal Biomass
142

(Shelef, G. and Soeder, C.J., Eds.), pp. 627-645. Elsevier/North Holland, Amsterdam. Ratledge, C. (1989) Biotechnology of oils and fats. In: Microbial Lipids, Vol. 2 (Ratledge, C. and Wilkinson, S.G., Eds.), pp. 567-668. Academic Press, London. Negoro, M., Shioji, N., Miyamoto, K. and Miura, Y. (1991) Growth of microalgae in high CO 2 gas and effects of SOx and NO x . Appl. Biochem. Bioteehnol. 28/29, 877-886. Wyman, C.E. and Goodman, B.J. (1993) Biotechnology for production of fuels, chemicals, and materials from biomass. Appl. Biochem. Biotechnol. 39/40, 41-59. Litvinoff, B. (1982) The Essential Chaim Weizmann. The Man, the Statesman, the Scientist. pp. 184-185. Weidenfeld and Nicolson, London. Volesky, B. and Szczesny, T. (1983) Bacterial conversion of pentose sugars to acetone and butanol. Adv. Biochem, Eng./Biotechnol. 27, 101-118. Lovitt, R.W., Kim, B.H., Shen, G.J, and Zeikus, J.G. (1988) Solvent production by microorganisms. CRC Crit. Rev. Biotechnol. 7, 106-186. Ennis, B.M., Gutierrez, N.A. and Maddox, I.S. (1986) The acetone-butanol-ethanol fermentation: a current assessment. Proc. Biochem. 21, 131-147. Allen, R.M. and Benetto, H.P. (1993) Microbial fuel cells: electricity production from carbohydrates. Appl. Biochem. Biotechnol. 39/40, 27-40. Roller, S.D., Benetto, H.P., Delaney, G.M., Mason, J.R., Stifling, J.L. and Thurston, C.F. (1984) Electron-transfer coupling in microbial fuel cells: 1. Comparison of redoxmediator reduction rates and respiratory rates of bacteria. J. Chem. Technol. Biotechnol. 34B, 3-12. Habermann, W. and Pommer, E.H. (1991) Biological fuel cells with sulphide storage capacity. Appl. Microbiol. Biotechnol. 35, 128-133. BP Statistical Review of World Energy (1986) British Petroleum Company p.i.c., London. Frisch, J.R. (1986) Energy abundance: myth or reality? Paper presented at the 13th Congress of the World Energy Conference, Cannes, France, October. Veziroglu, T.N. (1987) Int. J. Hydrogen Energy 12, 99-129. [49] Kosaric, N. (1990) Development of short-residence time ethanol fermentation processes. Proc. XIV Conf. Energy from Biomass and Wastes, Lake Buena Vista, FL, January 29-February 2. [50] Finnerty, W.R. (1989) Microbial lipid metabolism. In: Microbial Lipids. Vol. 2 (Ratledge, C. and Wilkinson, S.G., Eds.), Academic Press, London, pp. 525-566. [51] Negoro, M., Shioji, N., Ikuta, Y., Makita, T. and Uchiumi, M. (1992) Growth characteristics of microalgae in high-concentration CO 2 gas, effects of culture medium trace components and impurities thereof. Appl. Biochem. Biotechnol. 34/35, 681-692. [52] Bahl, H. and Gottschalk, G. (1988) Microbial production of butanol/acetone. In: Biotechnology. Vol. 6b (Rehm, H.J., Ed.), pp. 1-30. Verlag Chemic, Weinheim. [53] Runnalls, O.J.C. and Mackay, D. (1989) Energy growth. In: Environmental Science and Engineering (Henry, J.G. and Heinke, G.W. Eds.), pp. 46-85. Prentice Hall, Englewood Cliffs, NJ. [54] Vergara, W. (1980) Proe. IV Int. Symp. Alcohol Fuels Technol. Sao Paolo, Brazil, October, p. 143. [55] Lindeman, L.R. and Rocchicciolo, C. (1979) Ethanol in Brazil: brief summary of the state of industry in 1977. Biotechnol. Bioeng. 21, 1107-1119. [56] Maddox, I.S. (1988) Microbial production of 2,3-butanediol. In: Biotechnology. Vol. "19 (Rehm, H.J., Ed.), pp. 31-50. Verlag Chemie, Weinheim. [57] Magee, R.J. and Kosaric, N. (1987) The microbial production of 2,3-butanediol. Adv. Appl. Microbiol. 32, 89-161. [58] Barlaz, M.A., Ham, R.K. and Schaefer, D.M. (1990) Methane production from municipal refuse: a review of enhancement techniques and microbial dynamics. Crit. Rev. Environ. Control 19, 557-584. [59] Wayman, M. and Parekh, S.R. (1990) Crops that go on forever. In: Biotechnology of Biomass Conversion: Fuels and Chemicals from Renewable Resources. pp. 11-17. Prentice Hall, Englewood Cliffs, NJ. [60] Lewis, J. (1989) What's in the solid waste stream. EPA J. 15, 15.
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46] [47]
[48]

Liquid and Gaseous Fuels From Biotechnology: Challenge and Opportunities

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MICROBIOLOGY REVIEWS

FEMS MicrobiologyReviews 16 (1995) 111-142

Liquid and gaseous fuels from biotechnology: challenge and opportunities

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

127 ! 29 132 135 137 141

N. Kosaric, J. Velikonja / FEMS Microbiology Reciews 16 (1995) 111-142

After Cole [1]. Countries with high consumption of traditional fuels.

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

~ Consurnption r'-I Domestic supply

Produced. 1859 - 1985 Remaining Reserves. End of 1985

Fig. 2. Total discovered oil (source [46]).

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

Natural Gas 20.1 */0

Fig. 4. Evolution of world energy supplies (From [47]).

Tanker accidents, leading to oil contamination

Used oil disposal

Hydrocarbons and poly nuclear aromatic hydrocarbons from combustion

From Runnalls and Mackay [53].

N. Kosaric, J. Velikonja / FEMS Microbiology Reuiews 16 (1995) 111 - 142

2. Fermentation ethanol for fuel

Fig. 6. Projectedrate of world productionof fossil fuels(From [48]).

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

LNG accidents emissions

From RunnaUs and Mackay [53].

Emissions of CO2, NOx, COz, and particulates

Lithosphere Human impact

From Runnalls and Mackay [53].

plosive mining hazards

N. Kosaric, J. Velikonja/ FEMS Microbiology Reviews 16 (1995) 111-142

Weighing and 1 sterilizing I

Brix adjustment .lt. to 22"1ov/v

Decanted wine Benzene

Recycle RECUPERATION OF BENZENE

N. Kosaric, J. Velikonja/ FEMS Microbiology Reciews 16 (1995) 111-142

N. Kosaric, J. Velikonja/ FEMS Microbiology Reviews 16 (1995) 111-142

Fig. 8. Settling of Saccharomyces diastaticus (From [49]).

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

Fig. 9. Schematicdiagram of the semi-continuous(modifiedfedbatch) process(From [49]).

in the production of 2,3-

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111 142

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

V " VOLUMEOF SETTLING ZONE

Fig. ] I. Schematic diagram of the continuous fermentationprocess without externalrecycle(From [49]).

So = 97.0 GL" Sugar in fermentor = 40-50 GL"

For all d i l u t i o n rates

60. 30-3C .6(]

Volumetric ethanol product i v i t y aS.U. /o of sugar u t i l i z e d

Fig. 12. Continuousfermentationof fodder beetjuice (From [49]).

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142

Fo ] ) (mlh31.0 62.0 93.0 108.5 124.0

(gg0,50 0.50 0.46 0.49 0.50 0.50

QP (g1-1 h l) 0.5 3.1 1.5 2.6 9.0 15.1

From Kosaric [49].

: 50-70 GL" Biomoss concentration fermentor

1.2 -D. HR "1

N. Kosaric,J. Velikonja/ FEMS MicrobiologyReciews 16 (1995) 111-142

iiiiiiiiiiiii iiiii!i iiiiiiii!ii! iiiiii!iiii!i!i!i!i i! !i1i!iiiiii!i !i!i!iiiiiii!i!itiii i

REGENERAT IVE HEAT EXCHANGER

l-SECTION ~--"1BEER ST ILL

Fig. 15. Schematic of the biostill process (From [49]).

N. Kosaric, J. Velikonja / F E M S Microbiology Reviews 16 (1995) 111-142

Overall butanediol productivity (g 1-l h- l) 2.02