Biofuels Review

ELSEVIER FEMS Microbiology Reviews 16 (1995) 111-142

MICROBIOLOGY REVIEWS

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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. 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 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 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 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 pollution. 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

Table l Per capita commercial energy usage (1988)

Country kV Country kW

Canada 9.0 Japan 3.6 United States 8.3 Italy 3.2 Saudi Arabia 7.0 Spain 2.3 Former E. Germany 6.8 Brazil ~ 1.4 Sweden 6.2 Mexico a 1.2 Former W. Germany 5.1 China a 0.7 Former Soviet Union 4.9 Nigeria a 0.5 United Kingdom 4.0 India a 0.3

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.

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.

Table 2 Maximum power outputs of renewable sources

Source Power /W Comments (assumed 100% land coverage)

Solar photovoltaic 1015

Solar photosynthetic biomass 9 × 1012 Aeolian 1 × 1015

Undular

Hydroelectric

Tidal Geothermal

Uncertain < 6 × 1012

Uncertain < 1012 < 7 × 1012 < 3 × 1013

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

Adapted from Cole [1].

Table 3 Worldwide reserves of fossil fuels (various sources)

Resources Total reserves proven + undiscovered

Volume equ. (109 bbl oil) Energy a (1018 j) %

Proven/recoverable reserves

Volume 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 Bitumen 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 heating value of oil of 6.12 GJ /bb 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 ca l / t (29.3 GJ/t) . d Based on an heating value of 37.3 M J / 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]).

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

2 0 -

0

oT7 20

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

50

iO0

~00

N. Kosaric, J. Velikonja / FEMS Microbiology Reviews 16 (1995) 111-142 115

H y d r o e l e c t r i c 6.7 */0

~ i i ~ . : . • . ~ L - ~ ~___.____N uc lear / - ' ~ . : : . . ' . : - . . . . . % , 4G 0/,

Coal 30.7 *

Natura l Gas

20.1 */0

Oil 37.9 */.

Fig. 3. World consumption of commercially provided energy (source [46]).

and for internal-combustion engines (ICEs) which were being studied so far as economically and tech- nologically 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);

1o

£ .2 K

30(3 Cool

2OO

Natural gas

Nucleor 10(: New SOurCes

Petroleum Hydro elect r i¢

Non commerciQI energies

19~0 1980 2000 2020 2040 2060

Yeor

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

(iv) other oxygenated solvents (e.g. ketones); (v) 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 gener-

ating 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 Exploration Extraction, production, pro- Transmission Use and disposal cessing

Atmosphere Emissions of hydro- Refinery emissions of SO 2, - Emissions of SO2, CO 2, carbons as a result of HzS, CO2, NOx, and hydro- and hydrocarbons a blowout carbons

Hydrosphere Blowouts and spills Blowouts and spills from exploratory Brine and drilling chemicals wells 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, leading to oil contamination

Groundwater contamination by leaking tanks

From Runnalls and Mackay [53].

2. Fermentation ethanol for fuel

E O O r )

- - tO 1 3 m

t : t -

O

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, 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

[ ] South 6.2e ~_271

5 . 4 4

4 . 5 9

4

2 . 7 3

9 7 1.10 6 0 74 • "

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}).

300 o °

u

200 .~

E

E

8 ~oo -

c c <

Concerning yeasts, S a c c h a r o m y c e s cerev i s iae 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

d ias ta t icus , for fuel alcohol production, as investi-

Q

u :3

" o o 1,. o.

1960

• ~ 2 Ill

%

I I I I I |

1980 2000 2020 2040 2010 2080

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

Calendar years

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 EmissionsofgasandH2S 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 accidents 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 - Emissions of CO2, NOx, COz, from processing to gas or liquid and particulates fuel Coal dust dispersal

Hydrosphere - Leaching of acids and metals - Thermal effects Organic compounds formed with 'synfuels' Siltation

Lithosphere - Disruption from strip mining - Fly ash disposal and subsidence Slag heaps

Human impact - Lung disease Mine safety - 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 of spent fuel and tion lines waste

Human impact - Accidents and mine-plant ex- Accidents during Exposure to wastes Terror- plosive mining hazards fuel transport ism



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].

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. 'Biosti l l ' developed by Alfa Laval).

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

2.1. The semi-continuous (modified fed-batch) process

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

HzSO~ _ ! Molasses or cane juice

[ ! Weighing and 1 Brix sterilizing I ~ adjustment

.lt. to 22"1o v/v

Preparahon of yeast

l Wort = [

I -I Decanted wine

Recuperation of yeast

Fermentahon 1 Wine [ Decantation 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

Phleg Fusel _ ~ Stilla ~ oil

DISTILLATION RECTIFICATION

Benzene , ~ r ' '

DEHYDRATION

Decantation 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].

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 by- product which can be easily dewatered (in- cluding 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.

(iii) 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

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


Mlxem' stopped, Time : (I sec

After I0 sec After 20 sec

~ L ~" ~ ~

After 3(I sec After 4(I sec

After 50 sec Aft.er 60 sec

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


OPERATE S£T~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 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 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 system.

No such system is at present operating commercially. Further developments are under way to scale- up 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 in the production of 2,3- butanediol

Butanediol has a heating value of 114 MJ/kg [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 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

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.16) 'J~6ns

o L6

:= L~

a= ',O

o

o


F o

X o

S 0

D o

• 0

-0 CLEAR ZONE +

MIXING ZONE

! t -el--- VM ----~

t "- F r

e

X e

S e

P e

F = F o e

V " VOLUME OF SETTLING ZONE

V M • VOLUI4E OF MIXING ZONE

= + V M V V s

F D : ~ DILUTION RATE

Fig. ] I. Schematic diagram of the continuous fermentation process without external recycle (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-

S o = 97.0 GL" Sugar in fermentor

= 4 0 - 5 0 GL" B i o m a s s Concentration in f e r m e n t o r

Ypts= 0 . 4 9 GG "~

For all d i lu t ion rates

o ~ GL"

S u g a r c o n c e n t r a t i o n in fermentor

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 m e n t o r

• PR GL" HR "I

V o l u m e 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 zed

o~ op ePRAS.U

10C • 50 -5C 10C

80- 4C-4C-BC

60. 30-3C .6(]

: / 4 0 . 20- 2C ' 2C

20 . 10- 1C ,20

~ 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


Table 10 Continuous fermentation of fodder beet juice with Saccharomyces diastaticus

First fermentor Second fermentor

F o ] ) D S O S 1 P, Yp/~ Qp ( m l h - (h - l ) (g l - l ) (g1-1) (g l 1) (g g - 1) (g I i h - ')

$2 P2 Yp/s Qp (g 1-]) (g 1-1) (gg 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 juice with Saccharomyces diastaticus

First fermentor

F o D S o S 1 Pl Yp/s Qp (mlh I) ( h - I ) ( g l - I ) ( g l - l ) (g l 1) ( g g - l ) ( g l - l h - J )

Second fermentor

$2 P2 Yp/s i) QP (gl 1) (gl 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].

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,

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-

So: 1G1.0 GL" Total suga r in ju ice

: 5 0 - 7 0 GL" B i o m o s s c o n c e n t r a t i o n in f e r m e n t o r

Ypts: 0.49 GG "1 For all d i l u t i o n r a t e s

a g GL" Sugar concentration in fermentor

oP GL" E t h a n o l c o n c e n t r a t i o n in fermentor

• PR GL "I HR "I V o l u m e 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 sugar u t i l i zed

aS op ePR~S.U

12(

Bt3 4£ 8( A

4£ . L , 1 ° / ~ / ~ , , , , .

40 2C

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 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 . .

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

s t i l

I I I I I 1 I

1 2 3 4 5 G 7

FIN

| |

8 9 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]).

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

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. 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 180°C and high water affinity, for which alternatives to distillation were being developed, such as solvent extraction (with ethyl acetate,

Conc. Feed

h, . . ! ........ n

llllllIlllll ~c

I~""""U I FERMENTER|

OOLER 4k T Yeast C ream, I

® AIR 8 LO~,'ER

I,, CENTRIFUGE

,,~.r-i I

REGENERAT IVE HEAT EXCHANGER

d

AIcoho l

(q0-S0 t vvl

- - l-SECTION ~--" 1BEER ST ILL

Stillage

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

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

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].

ether, or n-butanol), membrane technologies, adsorp- tion, and chemical recovery. None of these have as yet yielded commercial ly applied solutions.

In situ conversion to MEK (boiling point 79.6°C) 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 fa- vorably 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 combina- tion with waste utilization or with the elimination of

waste materials which present an environmental nui- sance, 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 f ixed-fi lm and UASBR 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 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 m i c a l s as l ong as oi l c o n t i n u e s to be the c h e m i c a l

and ene rgy f o u n d a t i o n of the indus t r ia l c iv i l iza t ion .

In all l i ke l ihood this s i tua t ion wi l l c h a n g e d ramat i -

ca l ly in the next cen tury for w e l l - k n o w n reasons , B u t

e v e n then the fu ture o f such p roduc t s and t echno lo -

g ies is unc lear , s ince the sea rch for a l t e rna t ive fue ls

goes m a n y d i f fe ren t w a y s and m a y u l t ima te ly pro-

v ide m u c h be t t e r so lu t ions in the fo rm of c l eane r and

more ef f ic ien t ene rgy carr iers , such as hyd rogen .

4. Biotechnology in the production of liquid and gaseous fuels from coal

In t e rms o f energy , coal r ep resen t s 7 1 . 4 % (161 000

EJ ) o f w o r l d ' s fossi l fuel reserves , as c o m p a r e d to

7500 EJ in c rude oil (3 .3%) . The r ecove rab l e coal

con t a in s 9 1 . 1 % of ene rgy and 9 3 . 9 % c a r b o n con-

t a ined in oil, gas and 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 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 (la) 100.0 55.0 46 16.5 - - 0.30 0.17 0.34 Wood hydrolysate (lb) 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: (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 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) 1,4-Butanediol (tank) 2,3-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.

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

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~ COAL SOLUBILIZATION ROBIC~)

t t METHANOl. CH4 ETHANOL (METHANE)

(PRIMARILY BACTERIA)

BIOGASIF ICAT ION

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 several 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 acid/48 h, a i r /7 days/150°C), 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 solubilization.

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 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 100°C 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 kJ /mol (at 25°C and 101.3 kPa). Enormous quantities are constantly being re- leased 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 / FEMS Microbiology Reviews 16 (1995) 111-142

Can run 2 horsepower engine for one hour

II !

Can run 300 litre refrigerator for 3 hours

Can illuminate mantle lamp ] equivalent to 60 walt

, ~ f o r about 7 hours

l ° ° % N ~ Y Can cook 3 meals

for family of t, persons

One m 3 of biogas

(;an generate 1.25 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 of anaerobic degradation. Almost all of it is methane (54 -80%) and carbon dioxide (20-45%) , 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

me thane /ca rbon dioxide ratio, a fact reflected in the Buswell-Mueller stoichiometric equation [14]:

( a b ) C.H~O b + n 4 2 H 2 0

----.> - - _ _ q . - CO 2 + -- + -- _ _ CH 4 8 2 8 4

As with any other kind of fuel, methane production requires cheap raw materials. Almost all biomass-based fuels must necessarily come from the bioconversion of l ignocellulosics, i.e. the products 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 (18-30% 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 photosyn- thesis 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 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 world's fuel, equivalent to 20 million barrels of oil per day, which

Table 19 Composition and methane potential of municipal refuse

Chemical constituent Dry weight (%) Methane potential (%)

Cellulose 51.2 73.4 Hemicellulose 11.9 17.1 Protein 4.2 8.3 Lignin 15.2 0 Starch 0.5 O. 7 Pectin < 3.0 - Soluble sugars 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 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 Mass(lO 9 t / a ) Ethanol equivalents Oil equivalents Energy (EJ) (109 1) (106 bbl)

Cane and beet molasses 38 11 69 0.23 Cane and beet juice - 5 31 0.11 Bagasse surplus 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, stover 3 300 1000 6 290 21.20 Cassava, cull 2 1 6 0.02 Cassava, tops 45 14.4 90 0.31 Potato, cull 12 3.8 24 0.08 Jerusalem artichoke, tops 3 1 6 0.02 Forest logging residues + 360 125 786 2.65 non-commercial harvest Plantation forests 60 24 152 0.51 Municipal waste 250 37 232 0.78 Total 4 313 1316.7 8 280 27.91

From Wayman 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].

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

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 MJ/ 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 pol- lutant emission levels and improve fuel efficiency, environmental safety and overall marketability 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, 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 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 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 Mono- raphidium 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. 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 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. Rest- ing 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 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- in medium~,

, , , ® , / | / C , pid 1% biomass /

0 10 20 30 40 50 60 70 Time (h)

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 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 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 30°C

Organism Nitrogen sufficiency Nitrogen deficiency Silicon sufficiency Silicon deficiency

AFDW a Total lipids Change Change AFDW a Total lipids Change Change (g/ I ) (g / l ) AFDF a (%) lipids (%) (g / l ) (g / l ) AFDF a (%) lipids (%)

Hantzschia 20°C 1.314 0.319 - 36 + 15 1.185 0.271 - 17 +48 DI-60

30°C 1.582 0.468 - 29 + 28 1.264 0.334 - 16 + 41 Cyclotella 20°C 1.114 0.181 - 16 + 120 1.161 0.222 - 16 +38 cryptica

30°C 1.321 0.224 - 26 + 88 1.219 0.257 - 27 + 46

Adapted from Sriharan et al. [32,33]. a AFDW, ash-free dry weight (measured after 5 - 7 days of cultivation).


7. Biotechnology in the production of other oxygenated alternative fuels and fuel extenders

Butanol has a boiling point of 118°C, 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.

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 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 po., P,°n, . i ' ,

Algal Growth Pond Water Recy'elo

Seawater. Nutrients

Nutrients Recycle

I i . l 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]).


NAD + NADH + H + ~l ' CHz-CHOH-COOH I < ~ z j cH3_co_coo H

Lactate Pyruvete

J

CHs-COOi.I e . - . ~ t, Acot°t, 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- dium acetobutylicum 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 oxidoreductase • 4, ferredoxin: NAD * oxidoreductase; 5, ferredoxin NADP" oxidoreductase; 6, hydrogenase; 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-hydroxybutyryl-CoA dehydrogenase: 15, crotonase; 16, butyryI-CoA dehydrogenase; 17, phosphotrans- butyry, lase; 18, butyrate kinase; 19, butyraldehyde dehydrogenase; 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~HzO

CHs-CH=CH-C0-CoA Crotonyl- CoA

te~-- 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 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. 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, fermentation butanol is not likely to outcompete petrochem- ical 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.

Oxidation A products "

Fuel ---'- -7

f

\

F . ~ Load ~ .

e e

Anode® ®lCothod.

e

Reduced ~ oxidant Mediator

red)

e e

J Mediator (ox)

H + p

I

Oxldont

V Ion-exchange membrane

Fig. 22. Schematic diagram of a microbial fuel cell (From [44]).


Energy-rich chemical bonds of a fuel can be broken by the action of oxidants. Thereby an overall redistribution of electric charge between the partici- pating 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 gain- ing the same electrons. The free energy 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 . # { , C H z - - I ~ ~ / N - - C H : C , H , gena# viololcn -0.359 YEI

Cl %_._ ~

O==~:y~=N--~__~OH Z.e.~icmorot,l~'uotlnUot~e,,oI +0.217

cI N ~ PIIENAZINES

~nazifl¢ ¢tho~ulphale +0.065

I CzH,

CH~ N.~ CH~

Hz N i NH: Sairanine-O -0 289 512 CH,)

S O ~ - PHENOTHIAZINES

(CHt )2N /~ :~ 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.,

fCH~)2N ..

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 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 and/or 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,


CO2 H20

~ / SO4"

' ~ bacteria ~ H* ' ~ S -o lyOz

02

CxHyOz (fuel) + trace elements

Fig. 24. Biochemical fuel cell with sulfate reduction (From [45]).

Pseudomonas aeruginosa and Desulfovibrio desulfu- ricans in 0 .1-0 .5% sodium sulfate solution, solidi- fied 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):

2 < C H 2 0 > + 2 H 2 0 ~ 2CO 2 + 8 H + + 8e -

SO42- + 8 H + + 8 e - ~ $ 2 - + 4 H 2 0

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 + + 8e -

Cathode reaction:

2 0 2 + 8 H + + 8 e - ~ 4 H 2 0

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.

Purified water

/ 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]).

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, con- stitute a large subgroup of biosensors.

Table 24 Wastewater purification efficiency of a fuel cell

Type of wastewater TOC (mg/1) COD (mg/I) Incubation time (h) Degradation (%)

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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