Use of co-cultures in the production of ethanol by the fermentation of biomass

Production of ethanol and enzymes by fermentation of biomass with co-cultures of C. thermocellum and C. thermohydrosulfuricum.

This invention relates to the direct fermentation of plant cellulose and 
hemi-cellulose to ethanol and commercial enzymes by co-cultures of 
thermophilic anaerobic bacteria. 
The cost and lesser availability of petroleum and natural gas has generated 
research interest in bio-conversion processes that makes use of renewable 
biomass resources for the production of fuels and chemical feed stocks. 
Anaerobic fermentations have formed the basis for such microbial chemical 
and fuel production. By catabolizing organic matter in the absence of 
oxygen, a variety of reduced organic compounds such as ethanol, methane, 
acetic acid, lactic acid and the like have been produced in lieu of 
complete aerobic combustion of such organic matters to CO.sub.2 and 
H.sub.2 O. 
Several factors account for the technological interest in thermophilic 
bacterial fermentation as opposed to mesophilic yeast and/or bacteria. 
Bacterial fermentation enables direct conversion of the cellulosic and 
hemi-cellulosic components of preferred delignified biomass to chemicals 
or fuels, without pretreatment to depolymerize the substrates. As a 
consequence of growth at high temperatures and unique macromolecular 
properties, obligately thermophilic bacteria can possess high metabolic 
rates, physically and chemically stable enzymes, and a higher end product 
to cell ratio than in metabolically similar mesophilic species. 
Thermophilic processes are more stable, rapid, and facilitate reactant 
activity and product recovery. Fermentation of biomass at temperatures 
above 60.degree. C. is limited to thermophilic bacteria. 
These features of thermophilic bacterial systems are important for the 
development of viable biotechnology. Important is the fact that 
thermophilic fermentations may lower the energy requirements for recovery 
of volatile products, such as enabling reduced pressure distillation from 
a fermenter operating under vacuum conditions. Ethanol producing 
thermophiles, with rapid metabolic rates, are especially desirable from 
the standpoint of self heating and novel methods for product recovery. 
It is an object of this invention to provide a method for the production of 
ethanol in high yield and purity by fermentation of plant cellulosic and 
hemi-cellulosic materials with thermophilic and anaerobic bacteria. 
Relatively little is known about the entire spectrum of thermophilic 
anaerobic bacteria as applied to bio-fuel and bio-chemical production. A 
fundamental approach for development of the desired bio-conversion 
technology has been adopted by way of examination of the microorganisms in 
pure and in co-cultures to define their metabolism, especially in terms of 
defining key rate limiting and regulatory factors of catabolism. 
These investigations have led to the discovery that ethanol production in 
high yield can be achieved with strains that utilize the .alpha. and 
hemi-cellulose components in plant cellulose or biomass and by the use of 
strains that produce ethanol in high yields. It has been found that such 
high conversion ratio of cellulose to ethanol can be achieved in a single 
stage fermentation when use is made of co-cultures of Clostridium 
thermocellum and Clostridium thermohydrosulfuricum in the fermentation 
process.

These two thermophilic anaerobic bacteria differ one from the other in many 
respects. Comparison of their growth properties are tabulated in the 
following Table 1, using C. thermocellum LQRI and C. thermohydrosulfuricum 
39E as representative. From the table it will be apparent that the growth 
properties of these two species differed significantly. The optimum 
temperature for growth of C. thermocellum and C. thermohydrosulfuricum 
were 62.degree. C. and 65.degree. C. respectively. C. thermocellum 
fermented cellulose, cellodextrins and glucose as energy sources, whereas 
C. thermohydrosulfuricum fermented a variety of hexoses and pentoses but 
not cellulose. Significant is the fact that C. thermocellum LQRI did not 
ferment pentoses or hemi-cellulose. C. thermohydrosulfuricum displayed a 
faster growth rate on glucose than did C. thermocellum and the growth of 
the latter was almost three times more rapid on cellobiose than on 
cellulose. 
TABLE 1 
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Growth Characteristics of C. thermocellum LQRI 
and C. thermohydrosulfuricum 39E 
Conditions LQRI 39E 
______________________________________ 
Temperature range 
for growth: 
Min. -40.degree. C. 
40.degree. C. 
Opt. 62.degree. C. 
65.degree. C. 
Max. &lt;70.degree. C. 
75.degree. C. 
Substrates cellulose pyruvate 
supporting cellodextrins 
glucose 
growth cellobiose cellobiose 
glucose xylose 
mannose 
fructose 
starch 
Substrates not fructose cellodextrins 
supporting xylose cellulose 
growth mannose xylan 
starch mannan 
pyruvate 
Growth rate on: 
glucose 0.44 hr.sup.-1 
0.78 hr.sup.-1 
cellobiose 0.5 hr.sup.-1 
0.67 hr.sup.-1 
cellulose 0.15 hr.sup.-1 
-- 
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Comparison between the end products of glucose and cellobiose fermentation 
of C. thermohydrosulfuricum and C. thermocellum give further indication of 
the vast differences between the two microorganisms. As shown in Table 2, 
both species produced the same fermentation products which include 
H.sub.2, CO.sub.2, acetic acid, ethanol, and lactic acid, but in 
drastically different proportions. Most significantly, the ethanol to 
acetate ratio of C. thermohydrosulfuricum was 18 as compared to 1.2 for C. 
thermocellum. Similar ethanol to acetate ratios were observed when these 
two strains grew on substrates other than cellobiose or glucose. The yield 
of ethanol per mole of glucose equiv. fermented by C. 
thermohydrosulfuricum (1.84) approximated the maximum yield for ethanol 
fermentation; whereas, a value of 0.8 was observed for C. thermocellum. 
Also worth noting, C. thermohydrosulfuricum produced lower amounts of 
H.sub.2 than C. thermocellum. C. thermohydrosulfuricum strain 39E produced 
the highest yield of ethanol of any other described thermophilic 
saccharolytic anaerobe. 
TABLE 2 
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Fermentation Products of C. thermocellum LQRI 
and C. thermohydrosulfuricum 39E 
LQRI 39E 
______________________________________ 
Substrate 131 .mu.moles 
298 .mu.moles 
consumed cellobiose glucose 
Products formed Total .mu.moles 
H.sub.2 275 31 
CO.sub.2 340 580 
Ethanol 207 549 
Acetic 170 31 
Lactic 46 50 
Ethanol/glucose 
equivalent 
(mole/mole) 0.79 1.84 
Ethanol/acetate 
(mole/mole) 1.22 17.71 
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Since such vast differences have been found to exist in the growth and 
fermentation characteristics of C. thermocellum and C. 
thermohydrosulfuricum, it was indeed unexpected to find that co-cultures 
formed of these two species gave conversion ratios of plant cellulose 
(i.e. .alpha. and hemi-cellulose) to ethanol which far exceeded the ratios 
obtainable from either species alone. A synergism is clearly indicated by 
the co-culture, especially when employed in the conversion of plant 
cellulose to ethanol. 
This has been demonstrated by the fermentation of several substrates by 
mono- and co-cultures of C. thermocellum in the absence or presence of C. 
thermohydrosulfuricum. The experimental conditions under which the 
comparisons were carried out comprised the use of anaerobic tubes filled 
with 10 ml of GS medium and 0.7% by weight substrate. Fermentation was 
carried out at 62.degree. C. Amounts of substrate degraded after 120 hours 
were 100% (MN300 cellulose) and 80% (Solka Floc cellulose). Delignified 
wood was also fermented to ethanol. The delignified wood was aspen wood 
chemically delignified with SO.sub.2 or treated by steam exploding poplar 
wood. The results are set forth in Table 3 and Table 4. Ethanol and acetic 
acid production and CMCase activity were examined in cultures growing on 
MN300, Solka Floc, chemically delignified wood or untreated wood. Under 
these conditions, mono- and co-cultures degraded (&gt;80%) Solka Floc and 
MN300, degraded 50% of delignified wood but did not significantly 
metabolize the untreated wood even after 168 hours of incubation. The 
ethanol to acetate ratio in the co-culture was greater than three times 
higher than in the mono-culture. Similarly, the yield of ethanol per mole 
of glucose equiv. metabolized was 100% higher in co-culture as opposed to 
mono-culture. Reducing sugars were in much lower amounts in the co-culture 
at the termination of the fermentation. 
TABLE 3 
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Comparison of cellulose fermentation in a mono-culture 
of C. thermocellum LQRI and a co-culture of C. thermocellum 
LQRI and C. thermohydrosulfuricum 39E 
Products formed 
Ethanol/ 
Ethanol/ 
Ethanol 
Acetate 
Acetate 
Glu. Equiv. 
Carboxymethyl 
Substrate 
Culture 
(.mu.mol) 
(.mu.mol) 
(mole/mole) 
(mole/mole) 
Cellulase (IU/mg) 
__________________________________________________________________________ 
MN300 Mono 316 264 1.2 0.81 3.30 
Cellulose 
Co 688 45 15.4 1.77 1.31 
Solka Floc 
Mono 317 225 1.4 0.81 2.66 
Co 703 37 19.3 1.81 1.02 
Delignified 
Mono 171 156 1.1 0.88 -- 
Aspen Wood 
Co 323 144 2.3 1.66 -- 
Aspen Wood 
Mono 6 27 0.2 -- -- 
Control 
Co 26 38 0.7 -- -- 
__________________________________________________________________________ 
Experimental conditions: Anaerobic tubes contained 10 ml of modified GS 
medium and 0.7% substrate. Amounts of substrate degraded were 90% (after 
48-96 hrs), 50% (after 168 hrs) and &lt;1% (after 168 hrs) respectively for 
cellulosic substrates, delignified wood and wood. Aspen wood was 
chemically delignified by SO.sub.2 treatment. 
TABLE 4 
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Comparison of fermentations of cellulosics by a mono-culture 
of C. thermocellum LQRI and a co-culture of C. thermocellum and C. 
thermohydrosulfuricum.sup.a 
Reducing 
Substrate 
Products Formed (mM) 
CMCase 
Sugar 
Consumed 
Ethanol/Acetate 
Substrate 
Condition 
Ethanol 
Acetate 
(U/ml) 
(mg/ml) 
(%) Ratio (mol/mol) 
__________________________________________________________________________ 
MN300 Mono- 31.2 22.5 2.66 1.28 60 1.39 
Co- 88.9 4.2 0.79 0.08 100 21.1 
Solka Floc 
Mono- 30.8 27.3 4.61 2.89 50 1.13 
Co- 98.7 11.3 1.54 .15 80 8.73 
SO.sub.2 --Treated 
Mono- 16.9 13.6 2.14 1.55 -- 1.24 
Wood Co- 54.3 11.2 1.0 .64 -- 4.85 
Steam Exploded 
Mono- 14.9 15.2 2.97 1.58 -- 0.98 
Wood Co- 63.9 6.6 0.72 0.12 -- 9.68 
Untreated Wood 
Mono- 2.2 6.4 -- -- -- 0.34 
(control) 
Co- 8.4 9.3 -- -- -- 0.90 
__________________________________________________________________________ 
.sup.a Experiments were performed in anaerobic culture tubes that 
contained 10 ml of GS medium and 1.0% substrate except for MN300 cellulos 
(0.8%). Tubes were incubated without shaking at 62.degree. C. for 120 h. 
FIG. 1 shows the kinetics of product formation during Solka Floc cellulose 
fermentation in C. thermocellum mono-culture and in co-cultures with C. 
thermohydrosulfuricans. Solka floc is a wood cellulose that contains both 
cellulose and hemicellulose. Ethanol, acetate, cellulase and reducing 
sugars were formed by the co-culture but at drastically different rates 
than that observed in mono-culture. Reducing sugar accumulation in the 
co-culture was 10 fold lower than in the mono-culture. At 1% Solka floc 
the accumulated sugars consisted mainly of xylobiose, lower amounts of 
glucose and xylose, and only traces of cellobiose. Carboxymethycellulase 
(CMCase) was produced during the fermentation but the rate of production 
was three times lower in the co-culture than the mono-culture. Despite the 
lower production of CMCase, the co-culture fermented cellulose (as 
measured by residual cellulose) at similar rates to that of C. 
thermocellum alone. Most importantly the rate of ethanol production in 
co-culture increased three fold; whereas acetate production ceased early 
in the fermentation (.about.30 h) and the final acetate concentration was 
&gt;2 times less than that of the mono-culture. The co-culture was very 
stable at 62.degree. , repeatably transferable, and contained 
approximately equal numbers of each species. Essentially the same high 
ethanol production rates and yields were obtained in C. 
thermohydrosulfuricum mono-culture fermentation of Solka Floc cellulose 
that contained cellulase from C. thermocellum. Several physiological and 
biochemical properties of C. thermocellum and C. thermohydrosulfuricum 
help explain the basis for enhanced fermentation of Solka floc cellulose 
to ethanol in co-culture. These features include: the ability of C. 
thermocellum cellulase to degrade .beta.(1,4)-xylan, or glycans; the 
ability of C. thermohydrosulfuricum to ferment xylose and xylobiose and to 
incorporate cellobiose and glucose faster than C. thermocellum; and a 
lower proton concentration associated with fermentation of equivalent 
amounts of cellulose by the co-culture. 
Several thermostable enzymes were obtained from the cellulose fermenting 
co-culture that displayed high rates of catalysis. 
The following Table 5 compares the properties of the enzymes that were 
obtained. 
TABLE 5 
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Activity and Stability of Enzymes Recovered 
from the Cellulose Fermentating Co-culture 
Specific Activity 
Thermal (.mu.moles/min/mg 
stability 
Q.sub.10 protein at 60 C. 
______________________________________ 
Extracellular 
70 C. 1.57 0.42.sup.a 
cellulase (30.degree. C.-70.degree. C.) 
Cell-bound 
86 C. 1.9 1.6 
Reversible (&gt;50.degree. C.) 
NADP linked 2.9 
Alcohol (&lt;50.degree. C.) 
dehydrogenase 
Cell-bound 
65 C. 2.1 6.70 
Malic (22.degree. C.-60.degree. C.) 
Enzyme 
______________________________________ 
.sup.a Specific activity is expressed in .mu.moles of carbohydrate/min/mg 
protein, and determined by the continuous spectrophotometric assay for th 
quantification of cellulase solubilizing activity. 
The cellulase was easily recovered from the culture supernatant by a 
variety of common precipitation procedures. The cellulase was stable, 
active at 70.degree. C. and devoid of proteolytic activity. The high 
cellulose solubilizing activity of the crude enzyme was not affected by 
oxygen. Enzymes were easily extracted from the cell pellet including the 
reversible NADP linked alcohol dehydrogenase of C. thermohydrosulfuricum 
and the malic enzyme of C. thermocellum. Both of these enzymes displayed 
high thermostability and activity, and were present in catabolic amounts. 
These data indicate that the direct fermentation of cellulose to ethanol 
and commercial enzymes by co-cultures of C. thermocellum and C. 
thermohydrosulfuricum has utility as a biological system for biomass 
conversion. The ethanol yield during cellulose hydrolysis by C. 
thermocellum was increased 100% in co-culture with C. 
thermohydrosulfuricum which rapidly metabolizes mono- and disaccharides of 
hexose and pentose. Active, thermal stable enzymes produced by the 
co-culture that are of potential industrial and/or analytical value 
included: supernatant cellulase and cell-bound alcohol dehydrogenases and 
malic enzyme. 
It will be apparent from the foregoing that co-cultures of different 
species provide for efficient conversion of plant biomass to ethanol. 
Higher ratios and yields of ethanol are obtainable from these co-cultures 
as compared to mono-cultures and co-culture fermentations have been found 
to be more stable from the corresponding mono-cultures. From regulation 
studies in thermophilic saccharolytic bacteria, it appears that for higher 
ethanol ratios and yield, the presence of exogenous electron acceptors 
should be avoided in the fermentation. Hydrogen pressure should be kept 
sufficiently high to enhance ethanol formation and to inhibit hydrogen and 
acetate formation. For increased ethanol production, it is desirable to 
make use of co-cultures with high saccharide to ethanol conversion rates 
that use both hemi-cellulose and cellulose, i.e. hexose and pentose 
derivatives, are important in biomass fermentation. 
The co-culture data suggests that the rate and yield of ethanol production 
from cellulose was increased as a result of more effective uptake of 
cellulase hydrolytic products than by increasing the specific activities 
of cellulase. The rate of cellulolysis as a rate-limiting step can be 
improved for process development, as well as the rate of uptake of 
hydrolysis products by C. thermocellum. While this may be achieved by 
genetic manipulation of C. thermocellum, it is preferably achieved by the 
use of the described co-culture technique. 
It has been found that the inhibition of acetaldehyde reductase by low NAD 
concentration gives a higher level of acetate production in C. 
thermocellum as opposed to C. thermohydrosulfuricum. High alcohol 
concentration (e.g.&gt;1%) also decreased the rate of ethanol production by 
the alcohol dehydrogenases of both C. thermocellum and C. 
thermohydrosulfuricum. Thus, mutants of both species can be used to 
increase the rate and yield of ethanol production from cellulose as a 
result of derepressing alcohol dehydrogenase activity by genetic 
manipulations. 
Thermophilic ethanol fermentations are of interest to industrial alcohol 
production because both the pentosic and hexosic fraction of biomass can 
be directly fermented in high yield (i.e. mol ethanol/mol substrate 
consumed), and because of potential novel process features associated with 
high temperature operation. As a net result, the co-culture cellulose 
fermentations described here have the potential to convert more substrate 
to alcohol than other bioconversion systems as shown in FIG. 2. 
Therefore, having described the invention process and product it is 
understood that mutants of both species can be used without departing from 
the invention as set forth in the following claims.