Lignocellulosic biomass includes various agricultural residues (straws, hulls, stems,
stalks), deciduous and coniferous woods, municipal solid wastes (MSW, paper,

This chapter is not subject to U. S. copyright. Published 1997 American Chemical Society

cardboard, yard trash, wood products), waste from pulp and paper industry and herbaceous energy crops. The compositions of these materials vary. The major component is cellulose (35-50%), followed by hemicellulose (20-35%) and lignin (10­25%). Proteins, oils and ash make up the remaining fraction of lignocellulosic biomass

(2) . The structure of these materials is very complex and native biomass is resistant to an enzymatic hydrolysis. In the current model of the structure of lignocellulose, cellulose fibers are embedded in a lignin-polysaccharide matrix. Xylan may play a significant role in the structural integrity of cell walls by both covalent and non-covalent associations

(3) . The pretreatment of lignocellulosic biomass is crucial before enzymatic hydrolysis. Various pre-treatment options are available now to fractionate, solubilize, hydrolyze and separate cellulose, hemicellulose and lignin components (4-7). These include steam explosion, dilute acid treatment, concentrated acid treatment, alkaline treatment, treatment with S02, treatment with hydrogen peroxide, ammonia fiber explosion, and organic solvent treatments. In each option, the biomass is treated to reduce its size and open its structure. Pretreatment usually hydrolyzes hemicellulose to its sugars (xylose, L — arabinose, and other sugars) that are water soluble (4). The residue contains cellulose and lignin. The lignin can be extracted with solvents such as ethanol, butanol or formic acid. Alternatively, hydrolysis of cellulose with lignin present makes water — soluble sugars and the residues are lignin plus unreacted materials. The use of S02 as a catalyst during steam pretreatment resulted in the enzymatic accessibility of cellulose and enhanced recovery of the hemicellulose derived sugars (8). Steam pretreatment at 200- 210°C with the addition of 1% SO 2 (w/w) was superior to other forms of pretreatment of willow (9). A glucose yield of 95%, based on the glycan available in the raw material, was achieved. A summary of various pretreatment options is given in Table I.

Recently, supercritical carbon dioxide explosion was found to be very effective for pretreatment of cellulosic materials before enzymatic hydrolysis (10). The sequential steps for production of fuels and chemicals from lignocellulosic biomass involve feedstock preparation, pretreatment, fractionation, enzyme production, hydrolysis, fermentation, product recovery, and waste treatment. The pretreatment of lignocellulosic biomass is an expensive procedure with respect to cost and energy.

Computer-Mediated Addition of Fresh
Medium in Continuous Culture of Zymomonas
mobilis by Monitoring Weight Changes

H. D. Zakpaa1, Ayaaki Ishizaki1, and K. Shimizu2

department of Food Science and Technology, Faculty of Agriculture,
Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan
department of Biochemical Engineering and Science,

Faculty of Computer Science and Systems Engineering,

Kyushu Institute of Technology, Iizuka 820, Japan

A major problem encountered in pH-stat continuous culture of Zymomonas mobilis which employed growth-dependent pH changes to control the rate of addition of fresh medium into the culture was that of low dilution rate. Glucose concentration in the culture broth also became very low after the initiation of continuous fermentation.

A new control system for delivering fresh substrate into the fermentor is discussed that solves the problems of low dilution rate and low residual glucose. This new system involves linking the feeding of fresh substrate into the fermentor with the outflow of alkali (NaOH) from the alkali reservoir by monitoring weight changes of the two solutions. Dilution rates of about 0.33 h_1 and productivity values of about 11.00 g. Hlr1 could be achieved with this method, while maintaining the concentration of ethanol in the fermentor at about 33.00 gH when the ratio (a) of weight of glucose fed into the fermentor to the unit weight of alkali used in neutralizing acid produced in the broth was 25.00.

Zymomonas mobilis (/), a bacterium that occurs as motile short rods (2), has attracted considerable attention as a promising microorganism for large scale production of ethanol because of its unusual physiological and biochemical properties (/), and more recently because of its high efficiency in ethanol production.

Three main approaches namely; physiological (3, 4-9), genetic (10-16), and engineering (17-23) are being pursued with an aim towards improving the productivity of ethanol fermentation. With the engineering approach to fermentation process improvement, fermentors are operated in continuous mode instead of the more conventional batch mode, resulting in an increase in productivity. The genetic approach aims at increasing process productivity through the improvement of the metabolic characteristics of the organism employed, by attempting to correct a recognized weakness or deficiency, such as

© 1997 American Chemical Society

broadening the range of substrates which the organism can metabolize as carbon source. The physiological approach attempts to control parameters that affect process productivity, by varying environmental factors such as the chemical composition of the fermentation medium, concentration of essential nutrients or inhibitory substances, as well as pH and temperature (4-9, 24).

Computer technology has had significant impact on fermentation in recent times. The advent of inexpensive, computer-coupled fermentation systems has facilitated close monitoring and direct-digital control (DDC) of environmental variables such as temperature and dissolved oxygen concentration. These systems are now commonly used for on-line monitoring of several fermentation variables such as oxygen uptake rate, carbon dioxide evolution rate, and respiratory quotient. Such variables have been successfully used for identifying the physiological state of microorganisms in many fermentation processes (25, 26). The use of computer-coupled fermentation systems to control pH in DDC mode also allows for on-line measurements of the amount of acid produced and the acid — production rate during fermentation. These variables can prove significant in on­line characterization of an anaerobic fermentation process, like ethanol fermentation by Z mobilis.

Low dilution rate and low residual glucose concentration in the fermentor broth were encountered in the previous set-up of computer-mediated, pH — dependent addition of fresh substrate in continuous culture of Z. mobilis (27). In this paper an alternative but similar method of supplying fresh substrate to the culture is designed and tested. This approach involved linking the feeding of fresh substrate into the fermentor with the outflow of alkali (NaOH) from the alkali reservoir into the fermentor, achieved through the monitoring of weight changes. The rationale behind this method is that by monitoring the NaOH addition resulting from decrease in the pH of the culture broth, better control of glucose concentration in the fermentor can be accomplished This contrasts with previous methods (27), where fresh substrate was only fed when the pH in the fermenting culture rose above the set pH limit on the control unit as a result of excretion of NH4+ ions into the medium. In anaerobic culture employing Z. mobilis, this pH rise is delayed. By the time it occurred the cells had been starved of glucose and most had lost viability.

Glycerol can be converted to 1,3-PD by a number of bacteria including strains of the genera Klebsiella, Citrobacter, and Clostridium under anaerobic conditions. Among these, Klebsiella pneumoniae and Clostridium butyricum have been most intensively studied. The pathway of anaerobic dissimilation in these strains is depicted in Fig. l.

Fig. l Pathway of anaerobic glycerol dissimilation in К pneumoniae and C. butyri­cum.

Glycerol is fermented by a dismutation process involving two parallel pathways. Through the oxidative pathway, glycerol is dehydrogenated by an NADMinked enzy­me, glycerol dehydrogenase, to dihydroxyacetone (DHA) which is then further metabo­lized to pyruvate. Through the parallel reductive pathway, glycerol is dehydrated by a В ^-dependent glycerol dehydratase to form 3-hydroxypropionaldehyde which is then reduced to 1,3-PD by an NADH-linked oxidoreductase, 1,3-PD dehydrogenase. The physiological role of the 1,3-propanediol pathway is to regenerate the reducing equiva­lents (NADH2) which are released from the formation of DHA and during the further oxidation of dihydroxyacetonephosphate (DHAP) as well as from biosynthesis. The enzymes leading to the formation of 1,3-PD and DHAP have been studied by many re­searchers with strains of Enterobacteriaceae (9,19,20,25,29). Enzymes active in the gly­cerol metabolism of C butyricum were measured very recently (1,2).

The further metabolism of DHAP is essential to provide ATP for cell growth and for the necessary phosphorylation of dihydroxyacetone. In addition, it provides the reducing equivalents for the 1,3-PD pathway, leading to the formation of 1,3-PD. De­spite its importance for an optimum production of 1,3-PD the metabolism of DHAP and its subsequent metabolites has received little attention in the past. In fact, the pathways of DHAP (and pyruvate) oxidation have been taken from glucose metabolism without experimental evidence. There is few work concerning the enzymes which catalyze the metabolism of pyruvate in pure glycerol fermentation. Zeng et al. (33,38) applied the pathway stoichiometry to analyze the fermentation of glycerol in both K. pneumoniae and C. butyricum, with particular emphasis on the regulation of pyruvate metabolism and its influences on the 1,3-PD yield and selectivity. Fig. 2 summarizes the pathways of pyruvate metabolism in K. pneumoniae and C. butyricum grown anaerobically on glycerol, respectively.

(1) Pyruvate formate-lyase

(2) Pyruvate dehydrogenase

(3) Pyruvaterferredoxin oxidoreductase

Fig. 2. Pyruvate metabolism during anaerobic fermentation of glycerol.

The cleavage of pyruvate to acetyl-CoA and C02 is assumed to be carried out by the enzyme pyruvate: ferredoxin oxidoreductase in C. butyricum and by the enzyme pyruvate-formate lyase in K. pneumoniae in the literature. Acetic acid and butyric acid are the main fermentation products of pyruvate in C. butyricum. K. pneumoniae produ­ces no butyric acid, but ethanol as one of the main products. Minor products include 2,3-butanediol, lactic and formic acid. At low pH value a significant amount of 2,3-bu — tanediol is formed in the glycerol fermentation of K. pneumoniae (7).

Analysis of continuous culture data of C. butyricum revealed that the reduced ferredoxin (Fdred) formed during oxidation of pyruvate to acetyl-CoA is not completely cleaved into hydrogen and oxidized ferredoxin under conditions of glycerol excess (38). Instead, part of the reducing power from Fdred is transferred to NAD+ under the forma­tion of NADH2. The enzymes catalysing this reaction had been previously described for C acetobutylicum (21) and recently for C. butyricum (2). In K. pneumoniae reducing equivalents released from pyruvate cleavage by the pyruvate formate lyase are trapped in formate and cannot be transferred to NAD. It was therefore surprising to find sub­stantial deviations of the ratio of 1,3-PD to hydrogen from the calculated one based on the action of pyruvate formate lyase in this species. We could recently demonstrate by enzyme assays that pyruvate dehydrogenase, which is normally the enzyme complex for an aerobic pyruvate decarboxylation in Enterobacteriaceae, is simultaneously involved in this anaerobic fermentation process (Menzel et al., GBF, unpublished data). Factors that affect the pyruvate dehydrogenase activity in K. pneumoniae are being studied in continuous culture with the goal of further increasing the activity of this enzyme for a high yield and flux of 1,3-PD. Although the activity of this enzyme is desirable its si­multaneous involvement in addition to pyruvate formate lyase in the K. pneumoniae culture gives rise to unfavorable dynamic behavior of the pathways such as oscillation and hysteresis under a variety of conditions (37).

Microorganisms. Saccharomyces yeasts 1400 and genetically engineered 1400(pLNH33) were used in the experimental work. The yeast 1400 (pLNH33) was obtained from Dr. Nancy Ho at LORRE.

The yeast strain 1400 (77) is a protoplast fusion product of Saccharomyces diastaticus and Saccharomyces uvarum.

Culture Conditions. The recombinant yeast 1400 (pLNH33) was maintained on YEPX seed cultures. The composition of the seed culture media per liter of distilled water is as follows: D-xylose 20g, yeast extract lOg, Bactopeptone 20g. The yeast was grown to an OD of 400-450 Klett Unit (measured by a Klett-Summerson colorimeter) and then maintained at 4°C. The medium for the preparation of the inoculum was the YEPX medium described above. 1 ml of the seed culture was added to a sterilized 250 ml Erlenmeyer flask with silicone sponge closure, containing 50 ml of medium. The inoculum was incubated at 30°C in a floor shaker at 150-200 rpm for 18-20 hours (when the cells were in the late exponential phase) before being used to inoculate the fementation medium.

The parent yeast strain 1400 was maintained on YEPD agar plates. The composition of the plate media per liter of distilled water is as follows: glucose 20g, yeast extract lOg, Bactopeptone 20g and agar 20g. The yeast was inoculated on the plate medium at 30°C for 48 hours and then maintained at 4°C. For the preparation of the inoculum, yeast extract-Bactopeptone medium with 20 g/L glucose was used. A loopful of yeast cells were transferred from the agar plate into 50 ml sterilized medium in a 250 ml Erlenmeyer flask.

Fermentation Conditions. The fermentation was performed in 250 ml Erlenmeyer flasks with silicone sponge closures, containing 100 ml sterilized medium. The fermentation medium consisted of 20 g/L Bactopeptone, 10 g/L yeast extract and appropriate concentrations of glucose and/or xylose. The inoculum sizes used were in the range of 0.1 g/L-2.5 g/L. The fermentation conditions were same as those indicated earlier for the inoculum preparation.

Pretreatment and Hydrolysis of Corn Fiber. Com fiber (com hull from A. E. Staley, Lafayette, IN) was pretreated with 0.5% dilute hydrochloric acid at 120°C for 45 minutes. Enzymatic hydrolysis of the pretreated com fiber was performed at 45 °С using Iogen cellulase having an activity of 154 FPU/ml. The pretreated com fiber was thoroughly washed, following which cellulase was added to the glucose free medium.

Simultaneous Saccharification and Fermentation (SSF) of Corn Fiber. Dry

pretreated com fiber (25% w/v) was added into a 250 ml side-arm Erlenmeyer flask. Yeast extract (10 g/L) and Bactopeptone (20 g/L) were also added and the pH was adjusted to 5. The yeast was inoculated from the seed culture to give an initial cell concentration of 0.5 g/L. Iogen cellulase was added to the medium to give an activity of about 5-10 FPU per gram of cellulose from com fiber. This fermentation medium was diluted using deionized water to give the desired solid fraction. The SSF of pretreated com fiber was conducted at 30°C in a shaker at 150-200 rpm.

Dilution is an additional possible solution to the viscosity problem of vegetable oils as discussed above. Results with this technology have been mixed and engine problems similar to those found with neat vegetable oils as fuels were observed here also. A model on vegetable oil atomization showed that blends of DF2 with vegetable oil should

contain from 0 to 34% vegetable oil if proper atomization was to be achieved (69).

A 75:25(vol-%) petrodiesel / sunflower oil blend had a viscosity of 4.88 mm2/s at 40°С, exceeding the ASTM maximum value of 4.0. The blend was not recommended for long-term use in the DI diesel engine (64). A 75:25 (vol-%) petrodiesel / high-oleic safflower oil blend with a viscosity of 4.92 mm2/s passed the 200 hr EMA (Engine Manufacturers Association) test. The different results were attributed to the degree of unsaturation of the respective vegetable oil (32). The more unsaturated oil (sunflower) that accumulates in the crankcase and hot engine parts tends to oxidize and polymerize due to its reactivity. Accumulation of such products in the lube oil could lead to lubricant thickening. A lube oil change is called for by the EMA test after 100 hr and at that time the viscosity of the lube oils had not varied greatly in either test.

Other reports include successfully using a 70:30 winter rapeseed oil / DF1 mixture (47) or blends of £ 15% rapeseed oil with DF2 (77), and an 80:20 DF2 / safflower oil blend with reduced CO and hydrocarbon emissions (72). A 75:25 DF / crude sunflower oil blend produced greatest solids contamination in the lubricating oil (49) similar to the results mentioned above, while another report mentions satisfactory performance of a 75:25 DF / sunflower oil blend (67). In early studies on sunflower oil, 80:20 DF / sunflower oil blends (31) were run for prolonged periods of time before exhaust smoke increased due to carbon build-up or power loss ensued. Another engine, due to inadequate atomization, showed more of the engine problems associated with neat vegetable oils.

The CP of a 50:50 DF2/ high-oleic safflower oil was -13 °С and the PP was -15 °С, and similar blends with high-linoleic safflower oil had CP -13 °С and PP -15 °С or winter rapeseed oil had CP -11 °С and PP -18°C (55).

A 50:50 blend of Stoddard solvent (a dry-cleaning fluid, viscosity 0.95 mm2/s, estimated CN 50, heat of combustion 46,800 kJ/kg, CP < -16°C, PP < -35 °С, flash point 42.2°C) with soybean oil gave low CP (-18.9°C) and PP (-31.7°C) but performed less well in a diesel engine than DF2 (73).

Air-lift fermentors have often been used instead of the traditional stirred-tank fermentor for production of penicillin(21), a-amylase(22), xanthan(23) and single cell protein(24). As the air-lift fermentor does not require mechanical agitation, the energy consumption is lower than that of a stirred-tank fermentor. The effect of the change in medium viscosity by adding carboxymethylcellulose (CMC) in an air-lift type fermenter on gas-hold up, bubble formation, flow pattern and mass transfer of oxygen has been reported by many researchers(25-27). We investigated the application of an air-lift type fermentor and the effect of change in medium viscosity by addition of CMC. Figure 5 shows the air-lift type fermentor used in this study, which was assembled as described by Okabe et al(28). To obtain high mass transfer achieved through the formation of small bubbles, a sintered stainless steel sparger (pore size, 10 mm; diameter, 12 mm; length, 20 mm) was installed at the bottom of the reactor. The feeding rate of the substrate gas mixture in the air-lift fermentor was 2 dm3 •min-1, which is equivalent to a superficial gas velocity of 2.62 cmes_1. Figure 6 shows the changes in medium viscosity and gas hold-up at various concentrations of CMC in the air-lift fermentor. Gas hold-up increased in proportion to the increase in CMC concentration up to 0.1% (w/v) but the gas hold-up decreased above 0.1% (w/v) of CMC. Deducing from this result, addition of CMC up to 0.1% (w/v) was expected to increase oxygen transfer rate with resultant increase in P(3HB) productivity. Figure 7 shows the time courses of autotrophic culture of A. eutrophus in the air-lift fermentor with addition of various concentrations of CMC. The productivity of P(3HB) in the culture with addition of 0.05 %(w/v) CMC (shown in Fig.7b) was increased to twice as that of the control culture with no addition of CMC(shown in Fig.7a). In the culture with addition of 0.1 % CMC, P(3HB) productivity was about 1.5 times higher than that of the control culture(Fig.7c). However, there was no apparent effect of the addition of CMC on the productivity of P(3HB) in the cultivation using the stirred-tank fermentor. A comparison was made for the effect of CMC addition on the mass transfer of oxygen in the air-lift and stirred-tank fermenters. When measurements were done by the static method, maximum KLa value for the air-lift fermenter was obtained at 0.05% of CMC concentration. The values of KLa for the air-lift fermentor measured by the sulfite oxidation method was observed to decrease with an increase in CMC concentration. For stirred-tank fermentor on the other hand, there was no increase in KLa values by addition of CMC into the culture medium. In the measurement by the sulfite oxidation method, the KLa of the stirred-tank fermenter was lowest at 0.1 % CMC. It is generally known that some kinds of surfactants, such as CMC, affect the sulfite-oxidation reaction. P(3HB) production rate observed in the fermentation experiments using the air-lift fermentor, correlated to the KLa measured by the static method but did not correlated to the KLa measured by the sulfite oxidation method. The KLa value measured by sulfite oxidation method was larger than those measured by the static method. These results mean that the static method is more reliable for the measurement of KLa than the sulfite oxidation method in autotrophic culture for P(3HB) production using air-lift fermentor. However, the relationship between the

The effect of CMC concentration on viscosity and gas-hold up of culture medium.

Cultivation time (h) Cultivation time (h) Cultivation time (h) Figure 7 Time course of autotrophic cultivation of A. eutrophus in air-lift type fermenter under various concentrations of CMC. (a): no addition of CMC, (b): 0.05 %(w/v) CMC, (c): 0.1 % (w/v) CMC.

KLa measured by the static method and the gas hold-up was not close, especially for the case where 0.1% CMC was used. This cannot be easily explained at present.

As mentioned in the section ’’Influence of the raw material” it is important for the economic viability of the process to reach high yields and to utilise the raw material efficiently. In the following example, the effect of recycling liquid streams on the various unit operations was investigated through a combination of simulations and experiments in the bench-scale unit in order to reduce the use of fresh water in the process. In this case, it is also important to examine the build-up of inhibitory substances in the conversion steps, such as hydrolysis and fermentation, as they may affect the yield and productivity in these steps. One of the simulated process alternatives was then experimentally verified in the bench-scale process development unit. A base case was established (Figure 4) which serves as a reference case. The base case simulates a plant where fresh water is added if needed to modify the concentration of dry matter in a step, and also for washing steps. Obviously, this is not realistic for an industrial process, but it reflects the normal procedure used in most lab-scale experiments. A number of different process configurations were simulated to establish the concentration levels of soluble components at various locations in the process, but in the following, two examples are reviewed in more detail. The flowsheets shown have been simplified to speed up calculations and to make the results easier to interpret.

The simulations are based on a feed capacity of 20,000 kg/h wood chips with a moisture content of 50%. This gives the minimum amount of water that enters the system and which must be disposed of. The dry matter is assumed to consist of 36% cellulose, 24% hemicellulose, 21% lignin, and 19% solubles, which is the composition of willow (55). It is assumed that the conversion in the hydrolysis stage is 90%, in the fermentation step 95%, and the recovery in the distillation step is 99%.

The outputs from the process include ethanol as the major product, lignin as the major by-product, resulting from the filtering of the hydrolysis residue, and the stillage waste stream. In the process configuration for the base case, large volumes of fresh water are used. This will result in a very dilute distillation feed, containing around 2.5% (wt/wt) ethanol. Distillation of such a dilute feed makes the operation cost-sensitive for changes in the feed concentration for nearly all distillation technologies (56, 57). The base case yields a liquid waste stream of about 38 tonnes for every tonne of ethanol produced. This results in high costs for fresh water and for waste treatment. The main and probably the only advantage of this process alternative is the low concentration of inhibitors in all the reaction steps, due to the dilution with fresh water. One reason to recycle liquid process streams is to decrease the fresh water demand and to considerably reduce the amount of waste water. Other reasons for recycling are the opportunities of increasing the concentrations of glucose in the fermenter and ethanol in the distillation column.

Recycling of Distillation Stillage. Several recycling options are possible. The simulations were performed to investigate how various components were distributed

Figure 4. The base case flowsheet. 1: Pretreatment; 2: Filtration; 3: Enzyme production; 4: Hydrolysis; 5: Fermentation; 6: Distillation.

in the process assuming that they would not alter the yields in the various reaction steps. A number of components were chosen to represent very volatile (furfural), moderately volatile (acetic acid) and non-volatile (’’soluble”) components. Figure 5 shows a configuration where the stillage resulting from the distillation column is recycled to the washing steps. This reduces the amount of fresh water required in the washing steps to virtually zero. In this case, the stillage stream is reduced to about 7 tonnes/h but the concentration of by-products is much higher. The total amount of by­products is, of course, unaltered. Through this change in the original configuration, it is possible to increase the ethanol concentration to approximately 4.5% (wt/wt), which reduces the energy consumption in the distillation step with about 40%. However, the concentration of by-products is greatly increased and in the hydrolysis step is up to 18 times higher than that obtained in the base case for acetic acid, furfural, and non-volatile components (Figure 6). It can also be seen in the same figure that there is a 9-10 times. higher concentration of acetic acid in the fermentation step compared with the base case, about 1-1.5% (wt/wt). This may cause a negative effect on the yield and on the productivity in the fermentation.

The results obtained from the simulation were examined experimentally in the bench-scale unit using willow as raw material (36). The experiments comprised pretreatment, enzymatic hydrolysis, fermentation, and distillation. The overall ethanol yield in this experiment was only 65% of the theoretical, which was lower than previously obtained in lab-scale investigations (14, 58). But enhanced yields are expected when, for example, hydrolysis is performed in fed-batch mode and fermentation is run continuously. To evaluate the effect of recycling, the stillage stream was fractionated into several volatile fractions and one non-volatile fraction by evaporation, thus simulating a multi-effect evaporation unit. The inhibitory effects of the various fractions were assessed by fermentation using S. cerevisisae after the addition of glucose. The non-volatile residue of the stillage was found to be inhibitory to fermentation already at a concentration five times higher than in the original stillage. The ethanol yield decreased from 0.37 g/g in a pure sugar reference to 0.31 g/g in the residue and the average fermentation rate decreased from 6.3 g/(L h) to 2.7 g/(L h). The acetic acid concentration in the residue was 9.2 g/L, a concentration previously found not to inhibit S. cerevisiae significantly (36), but it is more likely that lignin-degradation products are responsible for the inhibitory action. The evaporation condensates, containing the volatile components, showed no negative effects on fermentation.

The COD and the BOD7 in the stillage stream, the volatile fractions, and the non-volatile residue were used to estimate the environmental impact of disposal. The most volatile part and the non-volatile residue exhibited a considerably higher COD and BOD7 than the intermediate fraction (Figure 7). This indicates that the stream most suited for disposal is the intermediate part of the stillage stream and the parts most suited for recycling are the more volatile fractions. These results show that although the simulation indicates that a high degree of recirculation is an attractive option, in practice it is not possible to achieve an optimised process without the removal of non-volatile compounds. Incorporation of an evaporation unit is one way of removing the non-volatile residue from the process. Since this residue contains high amounts of organic compounds it is well suited to be used for steam generation.

3: Enzyme production; 4: Hydrolysis; 5: Fermentation; 6: Distillation.

Furfural HAc Glycerol Ethanol Solubles Figure 6. Concentration of ethanol and by-products in the hydrolysis. 1: Base case; 2: Recycling of the stillage; 3: Recycling of the distillation feed.

It is also possible to recycle part of the distillation feed back to the washing stage of the pretreated material. This configuration is shown in Figure 8. This alternative gives the same distribution of by-products as the former example (see Figure 6). But the ethanol concentration of the distillation feed could be increased to about 7% (wt/wt) (59), which is comparable to the concentration obtained when ethanol is produced from corn. This reduces the energy demand in the distillation by approximately 30%. However, in this particular case, the ethanol concentration in the hydrolysis reactor is increased to roughly 4% (wt/wt), which could yield a negative effect on the enzymatic hydrolysis. This configuration is presently being investigated using the bench-scale unit.

There are several other ways of increasing the ethanol concentration in the feed to the distillation unit (59). One of these is to recycle part of the stream from the hydrolysis tank. This will increase the ethanol concentration in the distillation feed, but it will also increase the risk of infection, since the recycling of sugar-containing liquids is involved.

In this laboratory, our current research has focused on improving the energy efficiency of photosynthetic hydrogen production. Using mutants of Chlamydomonas that lack PSI but contain PSII, we have demonstrated a new type of photosynthesis: that is, photoevolution of 02 and H2 and photoassimilation of C02 by PSII light reaction alone (J5, 36). This work builds on the original demonstration by Biochenko et al. (53) of hydrogen and oxygen transients in PSI-deficient mutants of Chlamydomonas. Based on studies of the electron transport pathway (Lee and Greenbaum, 1996, unpublished), the newly discovered water-splitting reaction for H2 and 02 production (reaction 1) or for

C02 fixation and 02 evolution (reaction 2) may require only half the number of photons of conventional Z-scheme photosynthetic reactions 3 and 4.

Newly Discovered PSII Photosynthesis:

H20 + 2 hv——— > H2 + 1/2 02 AG* = — 115 kJ/mol (1)

Energy efficiency = 67.4% (X = 680 nm)

C02 + H20 + 4 hv———— > 1/6(C6HI206) + 02 AGe = — 224 kJ/mol (2)

Energy efficiency = 68.2% (X = 680 nm)

Conventional Z-scheme Photosynthesis:

H20 + 4 hv——— > H2 + 1/2 02 AGe = — 446 kJ/mol (3)

Energy efficiency = 33.7% (X = 680 nm)

C02 + H20 + 8 hv———— > 1/6(C6H1206) + 02 AG° = — 927 kJ/mol (4)

Energy efficiency = 34.1% (X = 680 nm)

Both reactions 1 and 2 have a significantly large negative value of AG*. They should be able to occur spontaneously. Therefore, although the discovery is surprising and novel, it still obeys the laws of thermodynamics. Since reactions 1 and 2 require half the number of photons of reactions 3 and 4, the discovery can potentially lead to H2 production and/or C02 fixation technology with twice the energy conversion efficiency of conventional Z-scheme photosynthesis.

The demonstration of photosynthesis by a single light reaction proved that a single light reaction can span the potential difference between water oxidation and proton reduction for sustained evolution of H2 and 02, which was previously thought to be difficult to achieve (54). Therefore, we can now propose a new reactor system containing biometallocatalysts that requires only a single type of photochemical reaction center (Figure 6) but is able to perform the same function as the reactor system in Figure 3. As illustrated in Figure 6, when water is split to 02 and protons by PSII, electrons from the reducing side of PSII should, neglecting resistive loss, be able to reduce protons on a platinum catalyst surface to evolve H2 in a separate compartment without PSI.

At low light intensity and under ideal laboratory conditions, the maximum sunlight to H2 energy conversion efficiency for Z-scheme photosynthesis has been measured to be about 10% (55). From a practical point of view, application of PSII photosynthesis can potentially double the sunlight conversion efficiency from 10 to 20% (35). This potentially higher efficiency can put photosynthetic H2 production in a much more competitive position vis-a-vis other solar technologies. Moreover, since PSII photosynthesis can also photoassimilate C02, it should also be able to improve the energy efficiency of photosynthesis (C02 fixation) in general. Therefore, the discovery also provides a new opportunity to improve energy efficiency for production of H2 by the photosynthesis/fermentation combined system of Miura et al. (34).

Figure 6. A proposed biometallocatalytic reactor system for production of H2 and 02 in separate compartments by a single light reaction (PSII).

Acknowledgments

This research was supported by the U. S. Department of Energy, the Pittsburgh Energy Technology Center, and the ORNL Laboratory Director’s R&D Fund. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp., for the U. S. Department of Energy under contract DE-AC05-96OR22464.

Cellulose is a linear polymer of 8,000-12,000 D-glucose units linked by 1,4-B-D — glucosidic bonds. The enzyme system for the conversion of cellulose to glucose comprises endo-1,4-B-glucanase (ЕС 3.2.1.4), exo-1, 4-B-glucanase (EC 3.2.1.91) and B-glucosidase (EC 3.2.1.21). Cellulolytic enzymes with B-glucosidase act sequentially and cooperatively to degrade crystalline cellulose to glucose. Endoglucanase acts in a random fashion on the regions of low crystallinity of the cellulosic fiber whereas exoglucanase removes cellobiose (B-l, 4 glucose dimer) units from the non-reducing ends of cellulose chains. Synergism between these two enzymes is attributed to the endo — exo form of cooperativity and has been studied extensively between cellulases in the degradation of cellulose in Trichoderma reesei (11). B-Glucosidase hydrolyzes cellobiose and in some cases cellooligosaccharides to glucose. The enzyme is generally responsible for the regulation of the whole cellulolytic process and is a rate limiting factor during enzymatic hydrolysis of cellulose as both endoglucanase and

cellobiohydrolase activities are often inhibited by cellobiose (12-14). Thus, P — glucosidase not only produces glucose from cellobiose but also reduces cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently. However, like B-glucanases, most 6-glucosidases are subject to end-product (glucose) inhibition. The kinetics of the enzymatic hydrolysis of cellulose including adsorption, inactivation and inhibition of enzymes have been studied extensively (75). For a complete hydrolysis of cellulose to glucose, the enzyme system must contain the three enzymes in right proportions.

Product inhibition, thermal inactivation, substrate inhibition, low product yield and high cost of cellulase are some barriers to commercial development of the enzymatic hydrolysis of cellulose. Many microorganisms are cellulolytic. However, only two microorganisms (Trichoderma and Aspergillus) have been studied extensively for cellulase. There is an increasing demand for the development of thermostable, environmentally compatible, product and substrate tolerant cellulase with increased specificity and activity for application in the conversion of cellulose to glucose in the fuel ethanol industry. Thermostable cellulases offer certain advantages such as higher reaction rate, increased product formation, less microbial contamination, longer shelf — life, easier purification and better yield.

In our work, forty-eight yeast strains belonging to the genera Candida, Debaryomyces, Kluyveromyces and Pichia (obtained from the ARS Culture Collection, Peoria, IL) were screened for production of extracellular glucose tolerant and thermophilic B-glucosidase activity using p-nitrophenyl-6-D-glucoside as substrate (16). Enzymes from 15 yeast strains showed very high glucose tolerance (< 50% inhibition at 30%, w/v glucose). The optimal temperatures and pH for these B-glucosidase activities varied from 30 to 65°C and pH 4.5 to 6.5. The B-glucosidase from D. yamadae Y — 11714 showed highest optimal temperature at 65°C followed by C. chilensis Y — 17141(60°C) and K. marxianus Y-l 195 (60°C). The optimal pH of these three enzyme preparations were 6.5, 6.0 and 6.5, respectively. The temperature and pH profiles of p-glucosidases from C. chilensis Y-17141, D. yamadae Y-11714 and K. marxianus Y-l 195 are shown in Figure 1. The p-glucosidases from all these yeast strains hydrolyzed cellobiose. Novel glucose tolerance and thermoactivity found in the enzyme preparations from D. yamadae, K. marxianus and C. chilensis are desired

Table П. Biochemical characteristics of thermostable 0-glucosidase from Aureobasidium pullulans NRRL Y-12974 (77)

attributes of a P-glucosidase suitable for industrial application for enzymatic hydrolysis of cellulose to glucose. We have purified and characterized a highly thermophilic P — glucosidase from a color variant strain of Aureobasidium pullulans (17). Some properties of this enzyme are summarized in Table II.

The cellulose hydrolysis step is a significant component of the total production cost of ethanol from wood (18). Achieving a high glucose yield is necessary (>85%

theoretical) at high substrate loading (>10% w/v) over short residence times (< 4 days). It was shown that simultaneous saccharification (hydrolysis) of cellulose to glucose and fermentation of glucose to ethanol (SSF) improve the kinetics and economics of biomass conversion by reducing accumulation of hydrolysis products that are inhibitory to cellulase and P-glucosidase, reducing the contamination risk because of the presence of ethanol, and reducing the capital equipment requirements {19). An important drawback of SSF is that the reaction has to operate at a compromised temperature of around 30°C instead of enzyme optimum temperature of45-50°C. Enzyme recycling, by ultrafiltration of the hydrolyzate, can reduce the net enzyme requirement and thus lower costs {20). Hinman et al. {21) reported that a preliminary estimate of the cost of ethanol production for SSF technology based on wood-to-ethanol process is $ 1.22/gal of which the wood cost is $ 0. 459/gal. Wright et al. (22) evaluated a separate fungal enzyme hydrolysis and fermentation process for converting lignocellulose to ethanol. The cellulase enzyme was produced by the fungal mutant Trichoderma Rut C-30 (the first mutant with greatly increased P-glucosidase activity) in a fed batch production system that is the single most expensive operation in the process. The conversion of lignocellulosic biomass to fermentable sugars requires the addition of complex enzyme mixtures tailored for the process and parallel reuse and recycle the enzymes until the cost of enzymes comes down. Enzyme recycling can increase the rates and yields of hydrolysis, reduce the net enzyme requirements and thus lower costs (23). The first step in cellulose hydrolysis is considered as the adsorption of cellulase onto cellulosic substrate. As the cellulose hydrolysis proceeds, the adsorbed enzymes (endo — and exo-glucanase components) are gradually released in the reaction mixture. The P-glucosidase does not adsorb onto the substrate. These enzymes can be recovered and reused by contacting the hydrolyzate with the fresh substrate. However, the amount of enzyme recovered is limited because some enzymes remain attached to the residual substrate and some enzymes are thermally inactivated during hydrolysis. It has been shown that several substrates containing a high proportion of lignin result in the poor recovery of cellulase {24).

Gusakov et al. (25) found that cellolignin was completely converted to glucose by cellulase from I viride and A. foetidus. Cellolignin was an industrial residue obtained during the production of furfural from wood and com cobs when pretreated by dilute H2S04 at elevated temperature. The concentration of glucose in the hydrolyzate reached 4-5.5%, cellulose conversion being not less than 80%. Kinetic analysis of cellolignin hydrolysis, using a mathematical model of the process, has shown that, with product inhibition, nonspecific adsorption of cellulase onto lignin and substrate induced inactivation seem to affect negatively the hydrolysis efficiency. Borchert and Buchholz (26) investigated the enzymatic hydrolysis of different cellulosic materials (straw, potato pulp, sugar beet pulp) with respect to reactor design. The kinetics was studied including enzyme adsorption, inhibition, and inactivation. The results suggest the use of reactors with plug flow characteristics to achieve high substrate and product concentrations and to avoid back-mixing to limit the effect of product inhibition. For efficient use of cellulases, a reactor with semipermeable hollow fiber or an ultrafilter membrane was used and this allowed cellulases to escape end-product inhibition {27-30). A totally integrated biotechnology of rice straw conversion into ethanol was reported (37). It dealt with (i) ethanol refining of rice straw to segregate cellulose from pentose sugars and lignin, (ii) preparation of highly active mixed cellulase enzymes, (iii) a novel reactor system allowing rapid product formation involving enzymatic hydrolysis of cellulose to sugars followed by microbial conversion of the later into ethanol and its simultaneous flash separation employing a programmed recompression of ethanol vapors and condensation, and (iv) concentration of ethanol via alternative approaches.

In direct microbial conversion of lignocellulosic biomass into ethanol that could simplify the ethanol production process from these materials and reduce ethanol production costs, Clostridium thermocellum, a thermoanaerobe was used for enzyme production, hydrolysis and glucose fermentation (52). Cofermentation with C. thermosaccharolyticum simultaneously converted the hemicellulosic sugars to ethanol. However, the formations of by-products such as acetic acid and low ethanol tolerance are some drawbacks of the system. Several recent reviews have dealt with the molecular biology of cellulose degradation, cellulolytic enzyme systems, and the structure and function of various domains found in the enzymes involved (55-56).

Microorganism: Zymomonas mobilis NRRL-B14023 was used in this study. Stock culture was incubated in YM liquid medium (Difco laboratories, Michigan, USA) for 18 h at 30°C and stored for up to two weeks at 4°C in YM liquid medium.

Growth medium: The growth medium consisted of 100 g.1-1 glucose, 10 g/*1 yeast extract, 1 g. H KH2PO4, 1 g. tl (NH^SC^, and 0.5 %.tx MgS04.?H20. Composition of fresh medium feed was the same as the medium employed as growth medium. The substrate medium had the same composition as the growth medium.

Inoculation and Preculture: Z mobilis was first propagated at 30°C for 18 h without agitation by transferring 0.5 ml of culture from the stock culture to

In Fuels and Chemicals from Biomass; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

10 ml of YM liquid medium. The starter culture was then transferred to 40 ml of seed culture medium at pH 5.5 and incubated for 18 hours at 30°C. This seed culture was transferred into 450 ml of growth medium in the fermentor to make a final volume of 500 ml.

Continuous culture: The schematic diagram of the experimental set-up is shown in Figure 1. Continuous culture was performed in а 1-/jar fermentor with a working volume of 500 ml. The composition of the medium and culture conditions were the same as used for the batch and continuous culture experiments. Fresh substrate of the same composition and same initial pH of 5.5 as that in the fermentor was fed intermittently into the fermentor. The amount of fresh substrate fed into the fermentor depended on the amount of alkali (0.5 M NaOH) pumped into the fermentor by the pH control unit (Biott Co. Ltd.,Току о Japan, model KVS-15-B) to neutralize the acid produced by the metabolizing cells, and to maintain the pH of the fermenting broth at the set level of 5.5. pH was monitored using a glass Pt/KCl electrode (Toko Chemical Laboratories Co., Ltd. Tokyo Japan). Fresh medium and 0.5 M NaOH solution were contained in 1 / and 100 ml graduated cylinders, respectively. The fresh substrate and alkali reservoirs were placed on sensitive electronic balances (Shinko Denshi Co. Ltd. Tokyo Japan, models SK-6000H and SK-600H) with weight limits of 6000 g and 600 g, respectively. The weight signals (voltage) from the feed substrate and alkali balances were inputted into a computer (NEC, PC 9801 VM) through an analog-to-digital (A/D) converter. The digital signal was converted to the voltage signal by means of a digital-to-analog (D/A) converter. Using software written in C++ computer language loaded on the computer hard disk, any decrease in weight detected by the alkali reservoir balance, resulting from the pumping of alkali into the fermentor to neutralize any acid produced, led to the switching on of a peristaltic pump (Tokyo Rikikai Co. Ltd., Tokyo Japan, EYELA Microtube pump MP-3) connected to the D/A converter. The pump then delivered specific weight of fresh substrate into the fermentor. This was achieved by monitoring the decrease in weight recorded by the fresh medium pump when the pump was activated. The pump was switched off when the required amount of fresh substrate was delivered. The same pump was employed to draw off exactly the same volume of culture broth equal to the volume of fresh substrate fed into the fermentor. This was to ensure that culture volume was maintained at constant level in the fermentor. Each experiment was carried out until steady state was achieved, after about 30 to 50 hours.