1995 Liquid and Gaseous Fuels From Biotechnology - Challenge and Opportunities

ELSEVIER FEMS Microbiology Reviews 16 (1995) 111-142
MICROBIOLOGY
REVIEWS
Liquid and gaseous fuels from biotechnology: challenge and
opportunities
N. Kosar i c *, J. Vel i konj a
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
Co nt e nt s
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2. Fermentation ethanol for fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2.1. The semi-continuous (modified fed-batch) process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
2.2. Continuous process: internal yeast settling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3. Biotechnology in the production of 2,3-butanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
* Corresponding author. Tel.: (+ 1-519) 661 2131; Fax: (+ 1-519) 661 3498
Federation of European Microbiological Societies.
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
5. Biotechnology in methane and biogas production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ! 29
6. Biotechnology in diesel fuel and gasoline production from microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
7. Biotechnology in the production of other oxygenated alternative fuels and fuel extenders . . . . . . . . . . . . . . . . . 135
8. Biotechnology in direct energy conversion: microbial fuel cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
1. I n t r o d u c t i o n
The energy needs of the world today are esti-
mated at close to 1021 joules per year, which amounts
globally to about 20- 30 TW continuous power re-
quirement (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
t oday' s needs, would cover the basic energy require-
ments of the world population (apart from physiolog-
ical 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 re-
gions 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 sup-
plies (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 develop-
ing countries. The world population distribution
(around 3:1 for developing vs. industrialized coun-
tries) 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 super-
structure of the modern industrial society is built
upon oil which, at the present production rate, will
be exhausted in the next century with grave conse-
quences of global warming and environmental pollu-
tion. Apart from having the potential to affect and
upset the climatic, geological and biological equilib-
rium in nature, its production and use directly influ-
ence 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) 111-142 113
Tabl e l
Per capita commerci al energy usage (1988)
Count ry kV Count ry kW
Canada 9. 0 Japan 3.6
Uni t ed States 8.3 Italy 3. 2
Saudi Arabia 7.0 Spain 2.3
Former E. Ger many 6.8 Brazil ~ 1.4
Sweden 6.2 Mexi co a 1.2
Former W. Ger many 5.1 Chi na a 0.7
Former Soviet Uni on 4. 9 Ni geri a a 0.5
Uni t ed Ki ngdom 4.0 India a 0.3
Af t er Cole [1].
Count ri es wi t h hi gh consumpt i on of traditional fuels.
Another major peak of liquid fuel production is
expected around the year 2030, with a rapid decline
to zero by 2090.
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 qual-
ity 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 per i od- -
mostly in underdeveloped countries. The answer to
that depends primarily on three other issues:
(i) development and worldwide implementation
of viable technologies for industrial produc-
tion, heating, and transportation based on al-
ternative (renewable) fuels and feedstocks;
(ii) availability of sufficient quantities of renew-
able energy and feedstocks;
(iii) development and implementation of tech-
nologies for the reduction of environmental
pollution and emission of CO 2.
Tabl e 2
Ma xi mum power out put s of renewabl e sour ces
Source P o we r / W Comme nt s ( as s umed 100% l and coverage)
Solar phot ovol t ai c 1015
Solar phot osynt het i c bi omass 9 1012
Aeol i an 1 1015
Undul ar
Hydroelectric
Tidal
Geot hermal
Uncert ai n
< 6 1012
Uncert ai n
< 1012
< 7 1012
< 3 1013
7 - 1 0 % conversi on effi ci ency; REQUI RED, heavy dut y st orage syst em and hi gher
conversi on efficiency Land coverage difficulties; vi sual pollution
Land coverage and harvest i ng: social pr obl ems
REQUIRED: heavy dut y st orage syst em Land coverage: technical and social
probl ems, vi sual pol l ut i on
Usef ul near t he sea; heaviest, most expensi ve engi neeri ng
Restricted in global application
Restricted to tidal regi ons
Restricted to specific areas Present potential < 3 109 W Mi d-ocean ridges: in
ver y di st ant perspect i ve
Adapt ed from Cole [1].
Tabl e 3
Worl dwi de reserves of fossil fuel s (vari ous sources)
Resour ces Tot al reserves proven + undi scovered
Vol ume equ. (109 bbl oil) Energy a (1018 j ) %
Pr oven/ r ecover abl e reserves
Vol ume equ. (109 bbl oil) Energy a (1018 j ) %
Oil 1 177 7 203 15.4
Heavy oil ~ 543 3323 7.1
Shale oil ~ 1066 6 524 13.9
Bi t umen b 345 2 111 4.5
Coal c 3 175 19430 41.5
Natural gas J 1 335 8 172 17.5
Total 7 641 46 763 99. 9
703 4 303 11.1
450 2 752 7.1
1 066 6 524 16.8
345 2 111 5. 4
3 175 19430 50.1
593 3 629 9.4
6 332 38 749 99. 9
Based on a heat i ng val ue of oil of 6. 12 GJ / b b l (1 bbl = 42 US gal = 159 1).
b Heavy oil, shal e oil and bi t umen were gi ven t he same heat i ng val ue as oil.
c Based on a met ri c-t on-of-coal equi val ent of 7 109 c a l / t (29.3 GJ / t ) .
d Based on an heat i ng val ue of 37.3 MJ / m 3 natural gas.
~ Consurnption
< ~ r ' - I Domestic supply
~ . o ~ ~ _~
e 3 ~
c "-. 2 ~ C ._
11 ".iT,
u ~ 1.C ]
, J , < , ,~
Fig. 1. World's principal consumers of oil and their domestic
supply of oil products in 1985 (source [46]).
Th e o n l y a l t e r na t i ve t o f ul f i l l t he a b o v e g o a l s
r e l i e s u p o n r e n e wa b l e e n e r g y r e s our c e s . Th e c h o i c e s
a r e e v e n mo r e l i mi t e d wh e n l i qui d a nd g a s e o u s f ue l s
f or c o mb u s t i o n a nd t r a ns por t a t i on a r e i n que s t i on:
b i o ma s s e n e r g y a nd wa t e r ( f or h y d r o g e n ge ne r a t i on) .
A mo n g t he v a r i o u s t e c h n o l o g i e s a v a i l a b l e or d e v e l -
o p e d f o r e n e r g y p r o d u c t i o n f r o m t he a b o v e t wo
s our c e s , b i o t e c h n o l o g y o f f e r e d a nd c o n t i n u e s t o o f f e r
i nt e r e s t i ng a nd p r o mi s i n g a l t e r na t i ve s . Ma n y o f t h e m
a r e not c o mp e t i t i v e wi t h t he c h e a p f os s i l f ue l s t o d a y
- - a f act t hat wi l l , h o we v e r , dr a s t i c a l l y c h a n g e in t he
f ut ur e.
Bi o t e c h n o l o g y - d e r i v e d f ue l s f or he a t g e n e r a t i o n
2 0 -
0
oT7
2 0
3 0
5 0 -
Produced. 1859 - 1985
Remaining Reserves.
End of 1985
0
Fig. 2. Total discovered oil (source [46]).
7
l /
, / /
".N
!
u

<
150
1 0 0
5O
5 0
i O 0
~00
N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142 115
Hy d r o e l e c t r i c
6. 7 */0
~ i i ~ . : . . ~ L - ~ ~___.____N u c l e a r
/ - ' ~ . : : . . ' . : - . . . . . % , 4G 0/,
Coal
30. 7 *
Na t u r a l
Gas
20.1 */0
Oil
37.9 */ .
Fig. 3. Worl d consumpt i on of commercially provi ded energy
(source [46]).
and for i nt ernal -combust i on engi nes (ICEs) whi ch
wer e bei ng st udi ed so far as economi cal l y and tech-
nol ogi cal l y promi si ng are:
Li qui d fuel s:
(i) ethanol;
(ii) ot her al cohol s (e.g. but anol , 2, 3-but anedi ol );
(iii) short-chain aliphatic aci ds as precursors (e.g.
acet i c to valeric);
1o

.2
K
30(3 Cool
2OO
Nat ur al gas
Nucleor
10(: New SOurCes
Pet rol eum
Hydro elect r i
Non commerciQI
energies
19~0 1980 2000 2020 2040 2060
Yeor
Fig. 4. Evolution of world energy supplies (From [47]).
(i v) ot her oxygenat ed sol vent s (e.g. ket ones);
(v) lipids (fat t y-aci d t ri gl yceri des, ot her fat t y-aci d
esters).
Gas eous fuels:
(i) methane;
( i i ) medi um- heat cont ent bi ogas;
(iii) hydrogen.
Bi ot echnol ogy al so of f er s the possi bi l i t y of gener-
ating electricity by direct conversi on of fuel ener gy
in mi crobi al fuel cells.
Maj or alternative rout es are present ed bel ow.
Table 4
Environmental impacts of oil
Energy activity
Envi ronment Exploration Extraction, production, pro- Transmission Use and disposal
cessing
At mosphere Emi ssi ons of hydro- Refinery emi ssi ons of SO 2, - Emissions of SO2, CO 2,
carbons as a result of HzS, CO2, NOx, and hydro- and hydrocarbons
a blowout carbons
Hydrosphere Bl owout s a nd spi l l s Bl owout s a nd spi l l s
from expl orat ory Brine and drilling chemi cal s
wel l s at sea, leading disposal Refinery effluents
to oil contamination
Lithosphere Blowouts and spills Blowouts and spills Sludge Pipeline construction and Used oil disposal
on land disposal spills Damage due to per-
mafrost
Human im- Disruption of life Interference with fisheries Interference with fish- Hydrocarbons and poly
pact style eries or land use Disrup- nuclear aromatic hydro-
tions of life style during carbons from combustion
construction
Tanker accidents, lead-
ing to oil contamination
Groundwater contamina-
tion by leaking tanks
From Runnalls and Mackay [53].
2. Ferment at i on et hanol for fuel
E O
Or )
- - tO
13m
t :
t -
O
Fermentation of sugars by yeasts is one of the
oldest practised biotechnology processes. Major em-
phasis in the past was to produce potable alcohol in
the form of beer and wine. More recently, particu-
larly 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 ' Melle-
Boinot' 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].
e-
~ J N o r t h 7.11
[ ] Sout h 6. 2e ~_271
5 . 4 4
4 . 5 9
4
2 . 7 3
9 7 1.10
6 0 7 4 "
1960 1980 2 0 0 0 2 0 2 0 2 0 4 0 2 0 6 0
Year
Fig. 5. Levels of energy consumption (From [47}).
3 0 0 o
u
200 . ~
E
E
8
~oo -
c
c
<
Concerning yeasts, S a c c h a r o my 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 my c e s
d i a s t a t i c u s , for fuel alcohol production, as investi-
Q
u
:3
" o
o
1,.
o.
1960
~ 2
I l l
%
I I I I I |
1980 2000 2020 2040 2010 2080
l 16 N. Kosaric, J. Velikonja / FEMS Microbiology Reuiews 16 (1995) 111 - 142
Cal endar year s
Fig. 6. Projected rate of world production of fossil fuels (From [48]).
2100
N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142
Table 5
Environmental impacts of natural gas
117
Energy activity
Environment Exploration Extraction, production, Transmission Use and disposal
processing
Atmosphere Emi ssi onsofgasandH2S Gas plant emissions of - Emissions of CO 2,
during an accidental H2S, SO 2, and hydrocar- NO~
blowout bons
Hydrosphere Blowouts Blowouts and drilling - -
Disposal of chemicals
Lithosphere - - Construction of pipeline -
Damage due to per-
mafrost
LNG accidents Disrup-
tions of life style during
construction
Human impact - LNG acci dent s H 2 S
emissions
From RunnaUs and Mackay [53].
Table 6
Environmental impacts of coal
Energy activity
Environment Exploration Extraction, production, processing Transmission Use and disposal
Atmosphere - Emissions of SO 2 and PNAs - Emi s s i ons o f CO2, NOx, COz,
from processing to gas or liquid and part i cul at es
fuel Coal dust dispersal
Hydrosphere - Leachi ng o f aci ds and metals - Thermal effects
Organic compounds formed with
' synfuels' Siltation
Lithosphere - Di s rupt i on f rom st ri p mi ni ng - Fly ash disposal
and s ubs i dence Slag heaps
Human impact - Lung di s eas e Mi ne saf et y - Exposure to emissions from
combustion and coke ovens
Table 7
Environmental impacts of nuclear power
Energy activity
Environment Exploration Extraction, production, process- Transmission Use and disposal
ing
Atmosphere - Accidents Radon emissions from - -
mine railings
Hydrosphere - Accidents Leachate from mine - Thermal effects
tailings
Lithosphere - Accidents Tailings contamina- Transmission Disposal o f s pent f uel and
tion lines was t e
Human impact - Ac c i de nt s and mi ne- pl ant ex- Accidents during Exposure to wastes Terror-
pl osi ve mi ni ng haz ar ds fuel transport i sm
Table 8
Productivities of alternative batch and continuous fermentation
systems utilizing yeast
System C 2 H5 OH productivity
(g1-1 h - 1)
Continuous, vacuum recycle 80
Continuous, recycle 40
Batch, recycle 15
Continuous, multi-stage 12
Continuous 5
Batch 2
Adapted from Vergara [54].
s e r i e s o f p h o t o g r a p h s wh i c h we r e t a ke n in 10- s
i nt e r va l s a f t e r t he mi x i n g wa s s t o p p e d ( Fi g. 8). Th i s
c l e a r l y i l l us t r at es a s pe c i a l c a p a b i l i t y o f t hi s s y s t e m
as c o mp a r e d t o p r o c e s s e s wh e r e y e a s t ha s t o be
c o n c e n t r a t e d b y c os t l y c e n t r i f u g e s f o r its r et ur n t o
t he f e r me n t a t i o n br ot h.
Ba s e d on t hi s p r o p e r t y , t wo p r o c e s s e s we r e d e v e l -
ope d: a s e mi c o n t i n u o u s ( mo d i f i e d f e d - b a t c h ) p r o c e s s
a nd a c o n t i n u o u s p r o c e s s wi t h i nt er nal cel l r e c yc l e ,
wi t h o u t t he ne e d f or us e o f a n y me c h a n i c a l s et t l i ng
de vi c e s , as i s t he c a s e in o t h e r c o n t i n u o u s p r o c e s s e s
( e. g. ' Bi o s t i l l ' d e v e l o p e d b y Al f a La va l ) .
g a t e d i n o u r l a b o r a t o r y . Th i s y e a s t h a s a hi gh e f f i -
c i e n c y i n c o n v e r t i n g s u g a r s t o a l c ohol . An o t h e r a d-
v a n t a g e i s i n i t s hi gh f l o c c u l a t i n g c a pa bi l i t y. Th e
y e a s t p r o d u c e s v e r y s t a bl e f l oc s d u r i n g g r o wt h wh i c h
c a n r a p i d l y set t l e i f ne e de d, but c a n a l s o be e f f i -
c i e nt l y ma i n t a i n e d i n s u s p e n s i o n wh e n s uf f i c i e nt
mi x i n g i s a ppl i e d. Th e s et t l i ng i s i l l us t r at ed i n t he
2.1. The s emi - cont i nuous ( modi f i ed f ed- bat ch) pr o-
cess
Th e s c h e ma t i c o f t he ope r a t i on o f t hi s p r o c e s s i s
p r e s e n t e d i n Fi g. 9. A c o n v e n t i o n a l b i o r e a c t o r ( s t i r r ed
t a nk r e a c t or ) i s f i l l ed wi t h t he me d i u m, i n o c u l a t e d
HzSO~ _ !
Molasses
o r c a n e j u i c e
[ !
W e i g h i n g a n d 1 B r i x
s t e r i l i z i n g I ~ a d j u s t m e n t
. l t . t o 22"1o v/v
P r e p a r a h o n
of yeast
l
W o r t = [
I - I
Decanted w i n e
R e c u p e r a t i o n o f yeast
F e r m e n t a h o n
1 Wine [ D e c a n t a t i o n J
a n d
J ~1 centrifu~ation
I
t
Rechhed
Alcohol 20/. v / v a l c o h o l
L , Raw alcohol
P h l e g F u s e l
_ ~ S t i l l a ~ o i l
D I S T I L L A T I O N RECTIFICATION
Benzene
, ~ r ' '
DEHYDRATION
D e c a n t a t i o n
1
H~O
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
Table 9
Yields in production of ethanol from sugar cane
119
Alcohol, indirectly Alcohol, directly
from molasses from sugar cane
63 63
8.32 8.32
7.0
2.21 66.2
1 ~32 8.73
675 4 460
11.5 75
730 4 800
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].
wi t h a b o u t 10% o f an i n o c u l u m ( p r e v i o u s l y p r e p a r e d
i n s h a k e f l a s k s ) a nd t he f e r me n t a t i o n i s r un t o c o m-
pl e t e ut i l i z a t i on o f t he s ubs t r a t e s uga r s . At t hi s t i me
t he mi x e r i s s t o p p e d a nd t he y e a s t i s a l l o we d t o
r a p i d l y s et t l e, l e a v i n g a s u p e r n a t a n t d e v o i d o f y e a s t
f l ocs . S o me n o n - f l o c c u l a t i n g mi c r o o r g a n i s ms ma y
st i l l b e i n t he u p p e r z o n e at t hi s t i me . As s o o n as t he
s e t t l i ng i s c o mp l e t e ( t o t he de s i r e d l e ve l ) , t he u p p e r
c l e a r s u p e r n a t a n t i s wi t h d r a wn a nd s ent t o di s t i l l a-
t i on, n e w me d i u m i s p u mp e d i nt o t he s a me v e s s e l
c o n t a i n i n g t he s e t t l e d y e a s t a nd t he n e x t f e r me n t a t i o n
s t a r t e d u n d e r f ul l mi x i n g . As c a n b e s e e n i n Fi g. 10,
as s oon as t he hi gh b i o ma s s c o n c e n t r a t i o n i n t he
b i o r e a c t o r i s a c h i e v e d , t he s u b s e q u e n t f e r me n t a t i o n
t i me s ar e c o n s i d e r a b l y r e d u c e d d o wn t o a b o u t 3 h
f r o m t he i ni t i al 20 + h, as o b t a i n e d i n s i mp l e b a t c h
e x p e r i me n t s .
T h e s e f e r me n t a t i o n c y c l e s h a v e b e e n s e p a r a t e l y
r un i n a d o z e n o t h e r f e r me n t a t i o n s , f o r u p t o 10
c o n s e c u t i v e r uns wi t h o u t a v i s i b l e l os s i n e t ha nol
p r o d u c t i v i t y ( Fi g. 10).
Th e r e a r e a n u mb e r o f a d v a n t a g e s o f t hi s p r o c e s s
as c o mp a r e d t o s i mp l e b a t c h f e r me n t a t i o n s a nd t he s e
c a n b e s u mma r i z e d as f o l l o ws :
( i ) Hi g h p r o d u c t i v i t i e s ar e a c hi e ve d.
( i i ) Hi g h b i o ma s s c o n c e n t r a t i o n i n t he r e a c t o r i s
ma i n t a i n e d i n t he o r d e r o f u p t o 50 g / l . T h e
b i o ma s s r e p r e s e n t s an e x c e l l e n t y e a s t b y -
p r o d u c t wh i c h c a n b e e a s i l y d e wa t e r e d ( i n-
c l u d i n g a s i mp l e s e t t l i ng o p e r a t i o n ) wh i c h
wo u l d c o n s i d e r a b l y r e d u c e i t s r e c o v e r y cos t .
Th i s b i o ma s s c o u l d b e a r i c h s o u r c e o f p r o -
t ei n a nd o t h e r nut r i e nt s f o r e i t he r h u ma n
nut r i t i on or a n i ma l f e e d.
( i i i ) Th e o p e r a t i o n i s s i mpl e , r e qui r i ng o n l y a
s t i r r ed t a n k a nd s t o r a g e v e s s e l s a nd p u mp s .
( i v) T h e y e a s t i s b e i n g c o n c e n t r a t e d wi t h o u t t he
us e o f a n y c e n t r i f u g e s or o t h e r me c h a n i c a l
c o n c e n t r a t i o n de vi c e s .
( v ) Wh i l e t he f e e d b r o t h mu s t b e s t e r i l i z e d ( l i ke
f o r a ny o t h e r f e r me n t a t i o n pr oc e s s ) , t he a c -
t ual f e r me n t a t i o n r un c a n b e done u n d e r
non- s t e r i l e c ondi t i ons . Th e f e r me n t a t i o n t i me
i s s hor t f o r a ny i nt e r f e r i ng d e v e l o p me n t o f
c o n t a mi n a t i n g mi c r o o r g a n i s ms ( b a c t e r i a a nd
wi l d ye a s t ) , a nd i f t he s e d o d e v e l o p , t he y
wi l l p r o b a b l y not f l o c c u l a t e a nd wi l l t hus b e
wi t h d r a wn f r o m t he b i o r e a c t o r at t he e nd o f
e a c h c y c l e i n t he s p e n t br ot h.
( vi ) Co n s i d e r a b l e r e duc t i on i n t he f e r me n t a t i o n
t i me t o a b o u t 3 h f r o m c o n v e n t i o n a l 20 h i n
s i mp l e b a t c h ope r a t i on. Du e t o t hi s f a c t ,
mu c h s ma l l e r r e a c t o r s a nd e q u i p me n t ar e
n e e d e d , wh i c h r e d u c e s t he o v e r a l l c a pi t a l
a nd i n v e s t me n t cos t s .
( vi i ) T h e e n e r g y r e qui r e d t o r un t he f e r me n t a t i o n
p r o c e s s i s o n l y r e qui r e d f o r mi x i n g and
p u mp i n g o f t he l i qui d.
At t he p r e s e n t t i me , t he r e i s no s uc h p r o c e s s i n
c o mme r c i a l o p e r a t i o n . Al l r uns we r e ma d e in o u r
pi l ot p l a n t 10-1 f e r me n t e r s .
2.2. Cont i nuous pr oces s : i nt ernal y e as t set t l i ng
T h e s a me f l o c c u l a t i n g y e a s t , S. di ast at i cus, al -
l o we d t he d e v e l o p me n t o f t hi s c o n t i n u o u s pr oc e s s .
I t s c a p a b i l i t y t o r a p i d l y s et t l e a g a i n s t an u p f l o w o f
f l ui d a l l o we d a d e v e l o p me n t wi t h i n t he r e a c t or o f a
1 2 0 N. Kosaric, J . Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142
M l x e m ' s t o p p e d , T i m e : (I s e c
A f t e r I 0 s e c A f t e r 2 0 s e c
~ L ~ " ~ ~
A f t e r 3(I s e c
A f t e r 4 ( I s e c
A f t e r 5 0 s e c A f t . e r 6 0 s e c
F i g . 8. S e t t l i n g o f Saccharomyces diastaticus ( F r o m [ 4 9 ] ) .
OPERATE ST~L DP~W FILL
Fig. 9. Schematic diagram of the semi-continuous (modified fed-
batch) 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 arti-
choke juices by this yeast showed that a high con-
centration 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 h-
is 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 con-
centration within the bioreactor.
(vi) Improved economics and energetics of the
system.
(vii) Less capital investment due to elimination of
external cell recycling and concentration sys-
tem.
No such system is at present operating commer-
cially. Further developments are under way to scale-
up the process, modify and optimize the bi or eact or
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 main-
tained 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 utiliz-
ing 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 al cohol / 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 compari-
son 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. Bi otechnol ogy in the producti on of 2,3-
butanedi ol
Butanediol has a heating value of 114 MJ / k g [3],
as compared to ethanol (122 MJ / kg) , and an
equimolar mixture of the two (116 MJ/ kg) . How-
ever, its price is not competitive with that of fermen-
tation 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
r . ~
I =~ 0. . j
v l w
, j
- ~ R ~ ~ , h '
= , 0 , , , 9 N
v l N
~ n m
. i i / . n j ,~-
- ~ j
: ,,l~
( 1 _ 1 5 ) ' [ O U e q ; 3
, j
( K . 1 6 ) ' J ~ 6 n s
o
L 6
: =
L~
a= ',O
o
o
F
o
X
o
S
0
D
o
0
- 0
CLEAR Z O N E
+
M I X I N G ZONE
! t
- e l - - - V M - - - - ~
t
" - F r
e
X
e
S
e
P
e
F = F
o e
V " VOLUME OF SETTLING ZONE
V M VOLUI4E OF MI XI NG ZONE
= + V M
V V s
F
D : ~ D I L U T I O N R A T E
Fig. ] I. S c h e m a t i c d i a g r a m o f the c o n t i n u o u s f e r m e n t a t i o n p r o c e s s w i t h o u t external r e c y c l e ( F r o m [49]).
boi l i ng point. Al t ernat i vel y, condensat i on wi t h MEK
and subsequent hydr ogenat i on yi el d oct ane i somers
f or hi gh-qual i t y avi at i on fuels.
2, 3-But anedi ol is anot her chemi cal whos e pr oduc-
tion and process devel opment wer e st i mul at ed by
war. Dur i ng Wor l d War II it was needed f or conver -
S o = 97.0 GL"
Sugar in fermentor
= 40- 50 GL"
B i o ma s s Concentration in
f e r me n t o r
Ypts= 0 . 4 9 GG "~
For al l d i l u t i o n rates
o ~ GL"
S u g a r c o n c e n t r a t i o n i n
f e r m e n t o r
o~ GL"
E t h a n o l c o n c e n t r a t i o n in
f e r me n t o r
PR GL" HR "I
V o l u me t r i c e t h a n o l
product i v i t y
aS. U.
/o of sugar u t i l i z e d
o~ op ePRAS.U
10C 5 0 - 5 C 10C
80- 4C-4C-BC
60. 30-3C .6(]
:/
4 0 . 2 0 - 2C ' 2C
2 0 . 10- 1C , 2 0
~0 0 ~ O- 0 - ' - - " - ' " ~"
o~2 o14 0.'6 o18
Fig. 12. Continuous fermentation of fodder beet juice (From [49]).
i
1.0 D. ~R "I
124 N. Kos ar i c, J. Vel i konj a / F E MS Mi c r o b i o l o g y Re v i e ws 16 (1995) 1 1 1 - 1 4 2
Table 10
Continuous fermentation of fodder beet j ui ce with S a c c h a r o my c e s di as t at i c us
First ferment or Second fermentor
F o ] ) D S O S 1 P , Yp/~ Qp
( ml h - (h - l ) ( g l - l ) ( g 1 - 1 ) ( g l 1) (g g - 1) (g I i h - ')
$2 P2 Yp/s Qp
(g 1- ] ) (g 1-1) ( g g 1 ) ( g l - I
h ~)
31.0 0,258 111.8 4.0 53.8 0.50 13.88
62.0 0.517 111.8 5.0 53.8 0.50 27.81
93.0 0.779 111.8 5,0 54.0 0.51 42.07
108.5 0.904 111.8 10.0 51.5 0.51 46.56
124.0 1.033 111.8 14.2 49.5 0.51 51.13
2.0 54.0 - -
2.0 43.0 - -
5.1 54.0 2.26 0.51
3.2 54.8 5.48 0.48
From Kosaric [49].
Table 11
Continuous fermentation of Jerusalem artichoke j ui ce with S a c c h a r o my c e s di as t at i c us
First ferment or
F o D S o S 1 Pl Yp/ s Qp
( ml h I) ( h - I ) ( g l - I ) ( g l - l ) ( g l 1) ( g g - l ) ( g l - l h - J )
Second fermentor
$ 2 P 2 Y p / s i) QP
( g l 1) ( g l J) ( g g - ( g1- 1 h l )
15.5 0.129 161.0 36 61 0.49 7.9
31.0 - 0.258 161.0 51 54 0.49 13.9
46.5 0.387 161.0 47 56 0.49 21.7
62.0 0.517 161.0 52 55 0.49 28.4
108.5 0.904 164.0 64 49 0.49 44.3
132.5 1.162 164 72 38 0.41 44.1
28 65 0,50 0.5
27 66 0.50 3.1
39 60 0.46 1.5
39 60 0.49 2.6
44 59 0.50 9.0
46 51 0.50 15.1
From Kosaric [49].
s i on i nt o 1, 3- but adi ene, o ne o f the bui l di ng bl o c ks o f
s ynt het i c rubber. It can oc c ur in the f orm o f t wo
enant i omers : D - ( - ) , or l e v o , and L - ( + ) , or dext ro,
as we l l as an opt i c a l l y i na c t i ve meso-form. Al l ar e
ba c t e r i a l pr oduc t s : pur e D - ( - ) - 2 , 3 - b u t a n e d i o l i s pr o-
d u c e d b y Bacillus polymyxa, wh e r e a s meso-2,3-
So: 1G1.0 GL"
Tot al s u g a r in j ui c e
: 5 0 - 7 0 GL"
B i o mo s s c o n c e n t r a t i o n i n
f e r me n t o r
Ypts: 0. 49 GG "1
F o r al l d i l u t i o n r a t e s
a g G L "
S u g a r c o n c e n t r a t i o n in
f e r m e n t o r
o P G L "
E t h a n o l c o n c e n t r a t i o n i n
f e r m e n t o r
P R G L "I H R "I
V o l u me t r i c e t h a n o l
p r o d u c t i v i t y
AS. U
*t , 01 s u g a r u t i l i z e d
aS op ePR~S.U
12(
Bt3 4 8( A
4 . L , 1 / ~ / ~ , , , , .
4 0
2 C
0. 2 0. 4 0. 6 0. 8 1.O 1.2 - D. HR "1
Fig. 13. Continuous fermentation of Jerusalem artichoke j ui ce (not hydrolysed) (From [49]).
N. Kosaric, J. Velikonja / FEMS Microbiology Reciews 16 (1995) 111-142 125
16,
15
14
13
12
11
8
7
6
5
4
3
2
1
0
i - . . I . . . . . . % . . . . - i - . . . . . r . . . - . n . . . . . - / - . - . - . r . - . - . 1
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . , . . . . . . . . . , . . . , . . . . . . - . . . . . - . - . . . . . . . - . . o . .
i i i i l i i i i i i i i i i i i i i i i i i i i i i : i
i i i i i i i i i i i i i i ! i i i i i i ! i i i i ! i ! i i i i i i ! i i ! i ! i ! i 1 i ! i i i i i i ! i i i i i i i i i ! i i ! ! i ! i ! i i i i i i i ! i ! i t i i i
s t i l
I I I I I 1 I
1 2 3 4 5 G 7
F I N
| |
8 9 10
Fig. 14. Ratios of stillage and ethanol volumes obtained at differ-
ent F/ N conditions for conventional and biostill fermentations
(F, fermentable components; N, nonfermentable components)
(From [49]).
Table 12
Comparison of the biostill with a conventional plant at Sao Luiz
(Brazil) using the same substrate (Source: Alpha Laval)
Parameter Biostill Conventional
Yield (% of theoretical) 94.5 87
Stillage (1/1 alcohol) 0.8 11
Manpower 3 7
Space requirement (m 2) 350 1350
but anedi ol is a pr oduct of Klebsiella pneumoni ae
( Aerobact er aerogenes), whi ch al so pr oduces s ome
of t he L - ( + ) i somer . Ther e ar e al so ot her bact er i a
whi ch s ynt hes i ze mi xt ur es of di f f er ent f or ms (e. g.
Bacillus subtilis, Serratia marcescens, Aeromonas
(Pseudomonas) hydrophila), and al so s ever al yeast s,
but t hey ar e cons i der ed e c onomi c a l l y uni mpor t ant .
The use of var i ous or gani s ms and subst r at es as
f eeds t ocks is s hown in Tabl es 13, 14, and 15. Yi el ds
are not i mpr es s i ve, mos t l y under 1 g l - ~ h - =, wi t h
8. 2 g 1 = h - 1 (i n t he pr esence of 4.5 g / 1 acet at e) as
t he hi ghest ever r ecor ded [4]. One of t he i nt r i nsi c
di f f i cul t i es wi t h pr oduct r ecover y is its hi gh boi l i ng
poi nt of about 180C and hi gh wat er af f i ni t y, for
whi ch al t er nat i ves to di s t i l l at i on wer e be i ng devel -
oped, such as sol vent ext r act i on ( wi t h et hyl acet at e,
Conc . Feed
h , . .
!
........ n
llllllIlllll
~ c
I ~" " " " U I
F E R M E N T E R |
OOLER 4 k
T
Yeast
C ream, I

A I R 8 LO~,'ER
I , ,
C E N T R I F U G E
,,~.r-i I
RE GE NE RAT I VE
H E A T EXCHANGER
d
A I c o h o l
( q 0 - S 0 t v v l
- - l - S E C T I O N
~--" 1 B E E R ST I L L
St i l l a g e
Fig. 15. Schematic of the biostill process (From [49]).
Table 13
Batch fermentation of 2,3-butanediol: summary of data for various bacterial strains and substrates
Substrate Organism Overall butanediol Overall butanediol
productivity (g 1-l h- l) yield ( g/ g substrate)
Glucose Aerobacter aerogenes NRRL B199 2.02 0.45
Glucose Klebsiella pneumoniae NRRL B199 0.36
Xylose Klebsiella pneumoniae NRRL B199 0.27
Xylose Klebsiella oxytoca ATCC 8724 1.35 0.36
Xylose Bacillus polymyxa NRCC 9035 0.1 0.24
Mannose Klebsiella pneumoniae AU- l-d3 0.64 0.30
Lactose Klebsiella pneumoniae NCIB 8017 0.06 0.24
Whey permeate Klebsiella pneumoniae 0.08 0.46
Hydrolysed whey permeate KlebsieUa pneumoniae 0.14 0.39
Whey Klebsiella pneumoniae ATCC 13882 0.38
Whey Bacillus polymyxa ATCC 1232 0.02 0.16
Starch Aeromonas hydrophila NCIB 9240 0.17 0.2
Citrus waste Aerobacter aerogenes 1.1
Xylan Bacillus polymyxa NRCC 9035 0.02
Wood hemicellulose hydrolysate Klebsiella pneumoniae ATCC 8724 0.45
Agricultural residues Klebsiella pneumoniae ATCC 8724
Jerusalem artichoke Bacillus polymyxa ATCC 12321 0.79 0.4
From Maddox [56].
et her, or n- but anol ) , me mbr a ne t echnol ogi es , adsor p-
t i on, and c he mi c a l r ecover y. None of t hese have as
yet yi e l de d c omme r c i a l l y appl i ed sol ut i ons.
In si t u conver s i on t o MEK ( boi l i ng poi nt 79. 6C)
by aci d cat al ys i s woul d make r ecover y much easi er
and mor e ef f i ci ent , but decr eas ed conver s i on r at es in
t he c ompl e x br ot h awai t f ur t her devel opment .
The cost of 2, 3- but anedi ol does not c ompa r e fa-
vor a bl y wi t h ot her f er ment at i on pr oduct s ( Tabl e 16),
al t hough s ever al f act or s, l i ke t he choi ce of r aw mat e-
r i al s, coul d i mpr ove pr oces s economi cs . Li ke wi t h
ot her f er ment at i ons f or f uel pr oduct i on, t he c ombi na -
t i on wi t h was t e ut i l i zat i on or wi t h t he el i mi nat i on o f
wast e mat er i al s whi ch pr es ent an envi r onment al nui -
sance, yi el d i mpr ove d over al l per f or mance. Thus,
del i gni f i ed wat er hyaci nt h was r ecent l y st udi ed as a
pot ent i al subst r at e for 2, 3- but anedi ol f er ment at i on
[5], wi t h s i mi l ar r esul t s as t hose obt ai ned f or ot her
subst r at es. Mor e encour agi ng r esul t s wi t h t hi s we e d
( 1. 4 g i - 1 h- 1 at 4 h hydr aul i c r et ent i on t i me) wer e
obt ai ned in cont i nuous oper at i on wi t h anaer obi c
f i xe d- f i l m and UAS BR r eact or s f ed wi t h al kal i ne
hydr ol ys at e (19 g / l r educi ng sugars).
I n concl usi on, i t can be st at ed t hat f er ment at i ons
f or oxyge na t e d f uel s or fuel pr ecur s or s ot her t han
et hanol wi l l be i nevi t abl y out compet ed by pet r o-
Table 14
Some intensified fermentation technologies for 2,3-butanediol: summary of data
Method Substrate Organism Butanediol productivity
(g i -1 h - l )
Continuous flow Sucrose
Continuous flow Glucose
Metabolistat Lactose
Continuous flow/carrageenan-immobi- Glucose
lized cells
Continuous flow/alginate-immobilized Whey permeate
cells
Continuous flow/cell recycle Whey permeate
Aerobacter aerogenes NCIB 8017 4.6
Klebsiella pneumoniae NRRL B199 4.25
Enterobacter cloacae 0.03
Enterobacter aerogenes IAM 1133
0.75
Klebsiella pneumoniae NCIB 8017
2.3
Bacillus polymyxa 1.04
From Maddox [56].
c h e mi c a l s as l o n g as oi l c o n t i n u e s t o b e t he c h e mi c a l
a n d e n e r g y f o u n d a t i o n o f t h e i n d u s t r i a l c i v i l i z a t i o n .
I n al l l i k e l i h o o d t hi s s i t u a t i o n wi l l c h a n g e d r a ma t i -
c a l l y i n t he n e x t c e n t u r y f or we l l - k n o wn r e a s o n s , Bu t
e v e n t h e n t he 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 -
g i e s i s u n c l e a r , s i n c e t he s e a r c h f or a l t e r n a t i v e f u e l s
g o e s ma n y d i f f e r e n t wa y s a n d ma y u l t i ma t e l y pr o-
v i d e mu c h b e t t e r s o l u t i o n s i n t he f o r m o f c l e a n e r a n d
mo r e e f f i c i e n t e n e r g y c a r r i e r s , s u c h as h y d r o g e n .
4. Bi otechnol ogy in the product i on of l i qui d and
gaseous fuels f rom coal
I n t e r ms o f e n e r g y , c o a l r e p r e s e n t s 7 1 . 4 % ( 1 6 1 0 0 0
EJ ) o f wo r l d ' s f os s i l f ue l r e s e r v e s , as c o mp a r e d t o
7 5 0 0 EJ i n c r u d e oi l ( 3 . 3 %) . Th e r e c o v e r a b l e c oa l
c o n t a i n s 9 1 . 1 % o f e n e r g y a n d 9 3 . 9 % c a r b o n c o n -
t a i n e d i n oi l , g a s a n d c oa l c o mb i n e d [6]. A t r ul y
r e ma r k a b l e r e s o u r c e !
Table 15
Summary of 2,3-butanediol production from potential substrates
Substrate a Initial Fermentation Yields ( g/ l ) Butanediol
monosaccharide time (h)
g / l % used Diol EtOH HAc g / g used g / g available g 1- J h i
Waste sulfite liquor 38.0 69.7 72 9.0 3.3 - 0.34 0.24 0.13
Citrus press juice 215.0 91.5 56 51.0 - - 0.26 0.24 0.91
Sugar beet molasses 56.6 71.0 24 20.1 - - 0.50 0.36 0.84
Sugar beet pulp 11.0 78.0 11 2.4 1.6 - 0.28 0.21 0.21
Wood hydrolysate ( l a) 100.0 55.0 46 16.5 - - 0.30 0.17 0.34
Wood hydrolysate ( l b) 100.0 93.0 3.1 35.5 - - 0.38 0.36 1.04
Wood hydrolysate (2) 12.1 95.0 NR c 6.0 3.8 2.1 0.52 d 0.50 o _
Wood hydrolysate (3) 9.7 100.0 24 0.2 0.5 3.8 0.02 d 0.02 o 0.01
Wood hydrolysate (4) 40.0 100.0 46 20.0 5.9 0.1 0.50 d 0.50 d 171.42
Adapted from Magee and Kosaric [57].
a Wood hydrolysate: ( l a) southern red oak hydrolysate (Scholler process); ( l b) same as l a, 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 Selling price (1985 US$)
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)
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 Mt / a production; lower value estimated in Eur. Chem. News, June 4, 1984
for 180 Mt / a.
Table 17
Some coal-solubilizing microorganisms
Fungi (Basidiomycetes)
Coriolus versicolor
Phanerochaete chrysosporium
Poria placenta
Fungi (Hyphomycetes)
Acremonium sp.
Aspergillus spp.
Cunninghamella sp.
Mucor spp.
Paecilomyces spp.
Penicillium spp.
Sporothrix sp.
Yeast-like fungi
Candida sp.
Actinomycetes
Streptomyces badius
Streptomyces setonff
Streptomyces viridosporus
Eubaeteria
Bacillus sp.
Pseudomonas sp.
From Faison [6].
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
MIXED CULTURE No. 2
(PRIMARILY BACTERIA)
PRODUCTION
OF L)OUIO FUELS
FUNGI OR ACTINOMYCETES
OAEREAcToR~ C O A L SOLUBILIZATION
ROBI C~)
t t
METHANOl. CH4
ETHANOL (METHANE)
(PRIMARILY BACTERIA)
BI OGASI FI CATI ON
Fig. 16. Schematic of a two-stage process for the production of fuel chemicals from coal (From [6]).
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 sev-
eral microorganisms, from different taxa, for which a
coal solubilization capability was demonstrated (Ta-
ble 17). Various kinds of oxidative (hydrogen perox-
ide, ozone, 8 M nitric aci d/ 48 h, a i r / 7 days/ 150C),
as well as non-oxidative pretreatments with surfac-
tants (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 solubi-
lizates 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 solubiliza-
tion.
Subbituminous, nitric-acid pretreated coal was
partly (approx. 10%) solubilized by Paecilomyces
TLi [10]. The medium, containing 2.5 g / l unfrac-
tionated, solubilized coal as the sole C-source, sub-
jected to methanogenic fermentation by an accli-
mated 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 exhaus-
tion of components which were metabolized into
methane. Cultures supplemented with CI. aceto-
butylicum 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 concentra-
tions 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 bacte-
rial 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 sub-
strates (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 so-
lutions 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 consider-
able 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
aerobi c/ anaerobi c process of solubilization and fuel
production would be most advantageous.
5. Biotechnology in methane and biogas produc-
tion
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 kJ / mol (at 25C and 101.3
kPa). Enormous quantities are constantly being re-
leased into the atmosphere from geological sources
(natural gas vents and coal deposits), decaying or-
ganic 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 / FEMS Microbiology Reviews 16 (1995) 111-142
Can run 2 horsepow er
engine fo r one hour
II !
Can ru n 3 0 0 l i t r e
r e f r i g e r a t o r f o r 3 h o u rs
Can illu m in a te m a n t l e la m p
] e q u i v a l e n t to 6 0 w a l t
, ~ f o r a b o u t 7 h o u rs
l
% N ~ Y Can co o k 3 meals
fo r f a m i l y o f t, persons
One m 3
o f b io g as
(;a n g e n e ra te 1 . 2 5 kw
e le c t r ic it y
Fig. 17. Possible applications of biogas (From [18]).
wi de yi el ds about 50 E J / a , whe r e a s s ome 30 E J / a
c ome f r om ge ol ogi c a l sour ces. Bes i des car bon di ox-
i de, a t mos phe r i c met hane i s a ma j or gr eenhous e gas.
Pos s i bl e appl i cat i ons of bi ogas ar e pr es ent ed in Fi g.
17.
Bi ogas i s t he f i nal gas eous pr oduct o f anaer obi c
degr adat i on. Al mo s t al l of it i s met hane ( 5 4 - 8 0 %)
and car bon di oxi de ( 2 0 - 4 5 %) , in a t ypi cal vol ume
r at i o of 3 / 2 [13]. Ot her , us ual l y mi nor , const i t uent s
are hydr ogen, mol e c ul a r ni t r ogen, oxygen, hydr oge n
sul f i de, and car bon monoxi de. The el ement al c ompo-
si t i on of degr adabl e bi oma s s di r ect l y i nf l uences t he
me t h a n e / c a r b o n di oxi de r at i o, a fact r ef l ect ed in t he
Bus we l l - Mue l l e r s t oi chi omet r i c equat i on [14]:
( a b )
C. H~O b + n 4 2 H2 0
----.> - - _ _ q . - CO 2 + - - + - - _ _ CH 4
8 2 8 4
As wi t h any ot her ki nd of fuel , met hane pr oduc-
t i on r equi r es cheap r aw mat er i al s. Al mos t al l
bi oma s s - ba s e d f uel s mus t neces s ar i l y come f r om t he
bi oconver s i on of l i gnocel l ul os i cs , i. e. t he pr oduct s of
Table 18
Raw materials for biogas production
Origin Type of waste
Agricultural wastes
.Human wastes
Animal wastes
Agriculture-based
Forestry wastes
Aquatic wastes
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
From Aziz [18].
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 consi der ed to have no
methanogenic potential, because its decomposition
rate is far too low. Useful waste materials are sum-
marized 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 poten-
tial for fuel production, though they have a better
potential for alcohol fuels, as represented in Table
20.
Primary production of biomass through photosyn-
thesis is estimated at 172 billion tons per year (ap-
prox. 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 produc-
tion is estimated at 3.21 x 1021 J / a [16], assuming a
heating value of 18.6 GJ / 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 worl d' s fuel,
equivalent to 20 million barrels of oil per day, which
Tabl e 19
Composi t i on and met hane potential of muni ci pal refuse
Chemi cal const i t uent Dry wei ght (%) Met hane potential (%)
Cel l ul ose 51. 2 73.4
Hemi cel l ul ose 11.9 17.1
Protein 4.2 8.3
Li gni n 15.2 0
Starch 0.5 O. 7
Pectin < 3. 0 -
Soluble sugar s 0.35 0.5
From Barlaz [58].
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 hemi-
sphere have been converting most of their agricul-
tural waste into fuel ethanol rather than into biogas,
although dedicated fuel crops prevail as raw materi-
als. These nations traditionally continue to generate
Tabl e 20
Worl d bi omass potential for et hanol production
Source Mass( l O 9 t / a ) Et hanol equi val ent s Oil equi val ent s Energy (EJ)
(109 1) (106 bbl)
Cane and beet mol as s es 38 11 69 0.23
Cane and beet j ui ce - 5 31 0.11
Bagasse sur pl us to fuel 24 7.5 47 0. 16
Grain, dedicated 23 8 50 0.17
Grain, low grade 80 27 170 0.57
B starch 116 52 327 1.10
Straw, chaff, st over 3 300 1000 6 290 21.20
Cassava, cull 2 1 6 0. 02
Cassava, t ops 45 14.4 90 0.31
Potato, cull 12 3.8 24 0.08
Jerusal em artichoke, t ops 3 1 6 0. 02
Forest l oggi ng resi dues + 360 125 786 2.65
non- commer ci al harvest
Plantation forest s 60 24 152 0.51
Muni ci pal wast e 250 37 232 0.78
Total 4 313 1316. 7 8 280 27.91
From Wa yma n and Parekh [59].
(1 1 C2H5OH approx. 21. 2 MJ).
Table 21
Composition of municipal solid waste (1986)
Component Percent by wet weight
Paper, paperboard 41.0
Yard refuse 17.9
Food 7.9
Subtotal 66.8
Metals, ceramics 8.7
Glass 8.2
Textile, rubber, leather, wood 8.1
Plastics 6.5
Inorganic (ash, rock), etc. 1.6
Subtotal 33.1
From Lewis [60].
wast e (Tabl e 21), wi t h the highest met hane potential
for cel l ul ose, f ol l owed by hemi cel l ul ose and prot ei ns
(Tabl e 19).
Pot ent i al l y, landfilled refuse coul d generat e 0.13
m 3 met hane per kg dry wast e [20], but pract i cal l y the
product i on has ranged f r om 1% to 52% of that val ue
[ 2 1 ] .
Sanitary landfills are s l ow but, due to their large
di mensi ons, qui te producti ve bi ogas reactors. It was
estimated that more than 8 2 5 0 0 0 t onnes o f coal
equi val ents per year were gl obal l y saved by their
methane producti on, wi t h a t endency to increase
further [22].
most o f the bi ogas from wastewater treatment and
sol i d wastes. The mai n reason for this l i es in the fact
that amount s o f domest i c refuse and s ewage present
formi dabl e wast e management and envi ronmental
probl ems, whi c h can be al l evi ated by fuel produc-
tion. An esti mated average o f 1. 64 kg MSW per
person per day was generated i n the US i n 1986
( Lewi s, 1989). About 95% o f the MS W i n the US
was l andfi l l ed i n 1984 [19].
Man-generated refuse represents an excel l ent
f eedst ock for me t ha ne / bi o g a s generati on by:
( i ) l andfi l l gas col l ect i on;
( i i ) anaerobi c di gest i on o f MSW i n reactors.
It i s estimated that more than two-thi rds o f MSW
( on a wet basi s) consi st o f easi l y fermentable organi c
6. Bi o t e c h n o l o g y i n di es el f uel a nd gas ol i ne pr o-
duc t i o n f r om mi c r oal gae
Among the vari ous possi bl e fuel s from bi ot ech-
nol ogy research and devel opment , there is consi der-
abl e interest to tap the hi gh-densi t y energy st ored in
lipids (35. 6 MJ / I for veget abl e oil) as possi bl e
alternatives for di esel (39.1 MJ / 1 ) engines. Vehi cl es
(e.g. publ i c transportation buses) are al ready testing
veget abl e oils, but there is still s ome cont r over sy
over the envi ronment al benefi t s vs. risks. However ,
it wi l l be a lasting endeavour of the const ruct ors of
ICEs (internal combust i on engi nes) to decrease pol -
lutant emi ssi on l evel s and i mprove fuel effi ci ency,
envi ronment al safet y and overal l market abi l i t y of
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
Maximal lipid Species Maximal lipid
content (% w/ w) content (% w/w)
72 Peridinium cincture 36
53- 70 Neochloris oleabundans 35- 54
50 Oocystis polymorpha 35
49 Chrysochromulina spp. 33- 48
48 Scenedesmus acutus 26
47 Scenedesmus spp. 26
Chlorella minutissima 23
45 Prymnesium parvum 22-38
43 Navicula pelliculosa 22- 32
40 Scenedesmus dimorphus 16-40
40 Scotiella sp. 16-35
40 Ch lorella spp. 15-26
39-71 Euglena gracilis 14-20
36 (72) Porphyridium cruentum 14 (22)
From Ratledge [36].
diesel engines, which currently use 17% of energy
for transportation purposes [23].
This chapter discusses the substrates, microorgan-
isms and biotechnological processes useful for fuel
lipid production. A relatively limited number of mi-
croorganisms can accumulate large amounts of stor-
age 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 pho-
toautotrophically 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 di-
rectly for ICEs, but have to be either transesterified
into low-viscosity and low-melting point esters (e.g.
methyl esters), or catalytically converted into hydro-
carbons as gasoline substitutes. The potential advan-
tages 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 Mono-
raphidium minutum show similarities with the trans-
esterification 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 sol-
vent [28]. It was earlier postulated that transesterifi-
cation has the same economical potential as catalytic
conversion, if the by-product glycerol can be mar-
keted [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. sub-
salsum, 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 transesterifi-
cation 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. Rest-
ing green cells of this alga produce traces of hydro-
carbons. 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 hy-
drocarbons, 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 immo-
bilization), 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).
"o ~ r- i n medi um~,
, , , , / | / C , p i d 1% biomass
/
0 10 20 30 40 50 60 70
Ti me (h)
Fig. 19. Idealized pat t ern of lipid accumul at i on in an ol eagi nous
mi cr oor gani sm gr own in bat ch cul t ure (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 competi-
tive 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 photo-
synthesis of Nannochloropsis sp. and Phaeodacty-
lum 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 illu-
mination and insufficient mixing. Although no at-
tempt was made to determine the lipid content of the
algae, such an approach might be a promising alter-
native 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.
Tabl e 23
Changes of bi omass and lipid yi el ds of Hantzschia DI-60 and Cyclotella cryptica under ni t rogen or silica deficiency at 20 and 30C
Or gani sm Ni t rogen suffi ci ency Ni t rogen defi ci ency Silicon suffi ci ency Silicon defi ci ency
AFDW a Tot al lipids Change Change AFDW a Total lipids Change Change
( g / I ) ( g / l ) AFDF a (%) lipids (%) ( g / l ) ( g / l ) AFDF a (%) lipids (%)
Hantzschia 20C 1.314 0. 319 - 36 + 15 1.185 0.271 - 17 + 4 8
DI-60
30C 1.582 0. 468 - 29 + 28 1.264 0. 334 - 16 + 41
Cyclotella 20C 1.114 0.181 - 16 + 120 1.161 0. 222 - 16 + 3 8
cryptica
30C 1.321 0. 224 - 26 + 88 1.219 0. 257 - 27 + 46
Adapt ed f r om Sri haran et al. [32,33].
a AFDW, ash-free dry wei ght ( measur ed after 5 - 7 days of cultivation).
7. Bi otechnol ogy in the product i on of other oxy-
genated al ternati ve fuel s 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 poten-
tial to produce it biotechnologically.
In 1915 Chaim Weizmann patented his process
for the production of acetone and butanol by fermen-
tation of carbohydrates by Clostridium aceto-
butylicum. 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 explo-
sive cordite. Scientific research, although mostly of a
serendipitous nature, can have far-reaching conse-
E~
teck
A
E
- t A
ow
E2
.o .~_
~ - ~ B i o m a s s
" ~ ~ Nitrogen
. . . . .
I ipi(! 1% I , i - m ; L ' ; s ) ~ N ~ l
/
n I I J ~ l
0-1 0-2
Dilution rate (h -I)
CO=
Exhaust Gas COs
II p o . , P , n , . i ' ,
Algal Growth Pond
Water Recy'elo
Seawater. Nutrients
Nutrients Recycle
I
i . l o-.,oo
Fig. 20. ( A) Idealized pattern of l i pi d accumulation in an oleaginous yeast grown in continuous culture (From [36]). (B) Conceptual system
of algae-based bioprocess for CO 2 removal (From [51]).
N A D + N A D H + H + ~ l
' CHz-CHOH-COOH I < ~ z j cH3_co_coo H
Lactate Pyruvete
J
CHs-COOi.I e . - . ~ t ,
Acott, C.,-CO-CH -CO-CoA
Acetyl-P CoA Acetyl-CoA CoA Acetoaeetyl- CoA
ATP ADP -i
Co.A ~ ~ t r r r ] - c , a
CHs - CH0 CH3-CO-CHz-C00H
Acetaldehyde Acetoecetate
Ethanol Acetone
FdHs e ) F ~
Reactions in Clostri-
di um acet obut yl i cum leading to
the formation of organic acids
and solvents. 1, reactions of the
Embden-Meyerhof-Pamas path-
way: 2, lactate dehydrosenase:
3, p.vruvate: ferredoxin oxidore-
ductase 4, ferredoxin: NAD *
oxidoreductase; 5, ferredoxin
NADP" oxidoreductase; 6, hy-
drogenase; 7, phosphotransace-
tylase: 8, acetate kinase: 9,
acetaldehyde dehydrogenase;
10, alcohol dehydrogenase: 11,
B-ketothiolase; 12, acetoacetyl-
CoA: butyrate/acetate CoA
transferase; 13, acetoacetate de-
carboxylase: 14, p-hydroxybuty-
ryl-CoA dehydrogenase: 15,
crotonase; 16, butyryI-CoA de-
hydrogenase; 17, phosphotrans-
butyry, lase; 18, butyrate kinase;
19, butyraldehyde dehydrogen-
ase; 20, butanol dehydrogenase
l CH3-CHz-CH2-CH20H
Butanol
CH3-CHz-CHz-COOH
Butyl'ate I
Fig. 21. Biochemistry of the acetone-butanol-ethanol fermentation (From [52]).
CH 3- CH z- CH z-CH0
Bu*.yraldehyde
t
2 ~ NADPH + H
NADP +
CHs-CHOH-CHz-CO-CoA
- Hydroxybutyryl - CoA
t ~Hz O
CHs-CH=CH-C0-CoA
Crotonyl- CoA
t e ~- - NADH + H +
NAD
CHs-CHz-CHz-C0-CoA
Butyryl - CoA
N A D ~ CoA
CH~-~-Iz-CHz-CO- 0
Butyryl-P
, e ~ ADP
ATP
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 pro-
cess. 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. beijer-
inckii, 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 tox-
icity (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, fermen-
tation butanol is not likely to outcompete petrochem-
ical butanol in the near future.
8. Biotechnology in direct energy conversion: mi-
crobial 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.
Oxidation A
product s "
Fuel ---'-
-7
f
\
F
. ~ L o a d ~ .
e e
A n o d e lC o th o d .
e
Reduced
~ oxi dant
Medi at or
red)
e e
J
Medi at or
( o x )
H + p
I
Oxldont
V
Ion-exchange
membrane
Fig. 22. Schematic diagram of a microbial fuel cell (From [44]).
Ener gy- r i ch chemi cal bonds of a fuel can be
br oken by t he act i on of oxi dant s. Ther eby an overal l
redi st ri but i on o f el ect ri c char ge bet ween the partici-
pat i ng mol ecul es o f fuel and oxi dant t akes place. The
fuel is bei ng reduced, l osi ng some of its internal
ener gy in the f or m o f electrons. Si mul t aneousl y, the
oxi dant i ncreases its internal ener gy cont ent by gai n-
i ng the same el ect rons. The free ener gy of the reac-
R,Nka ~ . ~ ~ J t t t ~ ~ . = i . ~ , M ptmr,wia, (~). ,,,.=i,,,.~ am~m,= .e.,w,lh (A.J
C a,.,
$1t~Ktutal formul,i Redox Incdillor (V) (lun)
C. # { , CHz - - I ~ ~ / N- - CH: C, H, gena# viololcn -0.359 YEI
Cl
%_._ ~
O==~: y ~=N- - ~__~OH Z.e.~icmorot,l~'uotlnUot~e,,oI +0.217
cI
N~ PIIENAZINES
~nazi fl tho~ulphale +0.065
I
CzH,
CH~ N.~ CH~
Hz N i NH: Sairanine-O - 0 289 512
CH, )
S O~ - PHENOTHI AZI NES
( CHt ) 2 N/ ~ : ~ O Alizm' in Brillian, Blue -0.173 641
OH
SOl ~ SOs-
CH, HN
N.N-dimet hyl-di~ulphonat cd iheonine +0+~"0 620
C: HsHN S 1~/ C: H,
Mclhylene Blue +0.011 f ~l
New Methylene Blue -0.021 590
~ O ~.o~.++z,~,~ 0 13o s.o
Fig. 23. Redox mediators and their mid-point potentials (ETm) and maximum absorbance wavelengths (Area x) (From [44]).
llzN NI4.,
f C H ~ ) 2 N ..
C~H
( C I l O z N ~ 0
OH
H O , , ~ N ~ O I~.soeurm
+0.064
Toluidlne BhJe-O +0.034
0.04"/
PHENOXAZINES
BliUilnl Cfesyl Blue
G:tll~'yanin +0.021
- o . ~ t
Fig. 23 (continued).
F/2
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 chan-
nel 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 con-
tact 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. Theoreti-
cally, any organic or inorganic compound or a mix-
ture 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 tem-
peratures a nd/ or under extremes of pH if the neces-
sary 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 oxidore-
ductases, 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 elec-
tron 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 de-
scribed [45] in which carbohydrates (simple sugars,
starch) or hydrocarbons (crude oil) were fed to a
mixed culture of Prot eus vulgaris, Escherichia coli,
CO2 H20
~ / SO4 "
' ~ bacteria ~ H* ' ~
S -o
l y O z
02
CxHyOz (fuel)
+ trace elements
Fig. 24. Biochemical fuel cell with sulfate reduction (From [45]).
Ps eudomonas aerugi nosa a nd Desul f ovi bri o desul f u-
ri cans i n 0 . 1 - 0 . 5 % s o d i u m s ul f a t e s ol ut i on, s ol i di -
f i e d as a b u l k y mi c r o b i a l a n o d e wi t h c l a y or s l at e
di s pe r s i ons . He r e t he e n e r g y o f t he o r g a n i c mo l e c u l e s
i s c o u p l e d t o s ul f a t e r e duc t i on. Th e g e n e r a t e d sul -
f i de, a t e r t i a r y f uel , s e r v e d as t he a n o d i c r e d o x
me d i a t o r :
Bi o l o g i c a l r e a c t i o n s ( ( C H 2 0 ) i s s i mp l i f i e d c a r b o -
h y d r a t e f uel ) :
2 < C H 2 0 > + 2 H 2 0 ~ 2 CO 2 + 8 H + + 8 e -
SO42- + 8 H + + 8 e - ~ $ 2 - + 4 H 2 0
An o d e r e a c t i on:
S 2 - + 4 H 2 0 ~ SO42- + 8 H + 8 e -
( a n d 8 / 3 S 2 - + 4 H 2 0 ~ 4 / 3 S 2 0 3 2 - + 8 H + + 8 e -
Ca t h o d e r e a c t i on:
2 0 2 + 8 H + + 8 e - ~ 4 H 2 0
T h e p r i n c i p l e o f o p e r a t i o n i s s h o wn i n Fi g. 24.
Th i s b i o c h e mi c a l f ue l cel l wa s s h o wn t o o f f e r a
p r o b l e m- a nd ma i n t e n a n c e - f r e e o p e r a t i o n f o r 5 ye a r s .
P u r ifie d w a te r
/ C a t h o d e ~ k
Anode Waste-water Slate materials
Fig. 25. Construction of a wastewater fuel cell with slate materials
(From [45]).
I t ha d a s t o r a g e c a p a c i t y o f up t o 0. 2 A c m - 2 , a nd
c o u l d p r o v i d e a c ur r e nt o f 6 A / k g cel l we i g h t (1 h
c o n t i n u o u s l oad) , or 15 A / k g ( 10 mi n c ont i nuous l y) .
Th e s a me a ut hor s p a t e n t e d a l s o a s i mi l a r l y bui l t
wa s t e wa t e r f ue l cel l , s h o wn on Fi g. 25. Th e wa s t e
e l i mi n a t i o n e f f i c i e n c y a c h i e v e d i n s h o r t - p e r i o d i nc u-
ba t i on, da i l y f o r 6 mo n t h s , i s r e p r e s e n t e d i n Ta b l e
24.
T h e b a s i c d i s a d v a n t a g e s o f mi c r o b i a l f ue l c e l l s
ar e t he i r g e n e r a l l y l ow c o u l o mb i c yi e l ds a nd l ow
p o we r out put s , at t r i but ed t o p H v a r i a t i o n dur i ng
o p e r a t i o n a nd l ow f ue l s t o r a g e c a pa c i t i e s . Ma n y
mo r e i mp r o v e me n t s , e s p e c i a l l y i mp r o v e me n t s o f t he
e l e c t r ode ma t e r i a l s a nd c o n s t r u c t i o n wi l l b e ne c e s -
s a r y, b e f o r e b i o l o g i c a l f ue l cel l p r o d u c t i o n a nd us e
c a n b e c o mme r c i a l i z e d .
Ho we v e r , mi n i a t u r i z e d v e r s i o n s o f s uc h s y s t e ms ,
not me a n t as p o we r s o u r c e s t h o u g h i n a n a d v a n c e d
s t at e o f d e v e l o p me n t a nd ma r k e t e d wo r l d wi d e , c on-
s t i t ut e a l a r ge s u b g r o u p o f bi os e ns or s .
Table 24
Wastewater purification efficiency of a fuel cell
Type of wastewater TOC (mg/1) COD ( mg/ I ) Incubation time (h) De g r a d a t i o n (%)
Sewage works effluent 50 140 0.5 35
(fulvic acids)
Effluent from landfill 2 000 6 000 2 65-75
From Habermann and Pommer [45].
TOC, total organic carbon; COD, chemical oxygen demand.
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1995 Liquid and Gaseous Fuels From Biotechnology - Challenge and Opportunities

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1995 Liquid and Gaseous Fuels From Biotechnology - Challenge and Opportunities

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ELSEVIER FEMS Microbiology Reviews 16 (1995) 111-142

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