Influence of different chemical pretreatments of elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis>, Schum.) used as a substrate for cellulase and xylanase production in submerged cultivation

This study evaluated the potential use of elephant grass biomass, a highly productive species, for cellulase and xylanase production by the cellulolytic mutant Penicillium echinulatum 9A02S1 in submerged cultivation, using untreated biomass, biomass pretreated with different concentrations of NaOH, H₂SO₄ or NH₄OH, or biomass pretreated with H₂O at 121 °C. For filter paper activity, all cultivation carried out with pretreated elephant grass under the evaluated conditions showed superior activity when compared with the control (untreated elephant grass). The activities of endoglucanases and β-glucosidases were higher in the cultivation prepared from pretreated samples than the control made with cellulose (Celuflok^®). Without pretreatment, elephant grass can be used for xylanase production, enabling similar activities to those obtained in the cultivation with cellulose, reducing the enzyme production cost. These results indicate that the pretreatment of elephant grass, especially when pretreated with H₂SO₄, may be used as a partial or total replacement for cellulose to cellulase production, and untreated elephant grass may be used for xylanase production.     

Bioconversion of wheat straw to ethanol: Chemical modification,enzymatic hydrolysis,and fermentation

Native wheat straw (WS) was pretreated with various concentrations of H₂SO₄ and NaOH followed by secondary treatments with ethylene diamine (EDA) and NH₄OH prior to enzymatic saccharification. Conversion of the cellulosic component to sugar varied with the chemical modification steps. Treatment solely with alkali yield 51–75% conversion, depending on temperature. Acid treatment at elevated tempeatures showed a substantial decrease in the hemicellulose component, whereas EDA-treated WS (acid pretreated) showed a 69–75% decrease in the lignin component. Acid-pretreated EDA-treated straw yielded a 98% conversion rate, followed by 83% for alkali–NH₄OH treated straws. In other experiments, WS was pretreated with varying concentration of H₂SO₄ or NaOh followed by NH₄OH treatment prior to enzymatic hydrolysis. Pretreatment of straw with 2% NaOH for 4 h coupled to enzymatic hydrolysis yield a 76% conversion of the cellulosic component. Acid–base combination pretreatment yielded only 43% conversions. A reactor column was subsequently used to measure modification–saccharification–fermentation for wheat straw conversion on a larger scale. Thirty percent conversions of wheat straw cellulosics to sugar were observed with subsequent fermentation to alcohol. The crude cellulase preparation yielded considerable quantities of xylose in addition to the glucose. Saccharified materials were fermented directly with actively proliferating proliferating yeast cells without concentration of the sugars.     

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6 % H₂SO₄ (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1 % NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/g_substrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/g_substrate and β-glucosidase 13.3 CBU/g_substrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06 %).     

Pretreatment and enzymic saccharification of water hyacinth cellulose

Water hyacinth was pretreated, under variable conditions, with NaOH, alkaline H₂O₂, peracetic acid and sodium chlorite. Combined pretreatments included sodium chlorite with each of NaOH, alkaline H₂O₂ and peracetic acid. Combined pretreatment with 0.1% NaClO₂ for 1 h at 100 °C and peracetic acid at 100 °C for 15 min afforded the most promising sample. The recovered lignin, cellulose and hemicellulose of this sample was 2.56%, 96.69%, and 81.38%, respectively. The same sample, by cellulase hydrolysis showed the highest cellulose conversion (80.8%) and 90% saccharification using 200 FPU/g substrate. Some ambient factors affecting saccharification of pretreated water hyacinth were investigated. Enzymic saccharification after 6 h was about 50% of that at 48 h, indicating a slow hydrolysis rate by time. Addition of 8% glucose at the beginning of the enzymic hydrolysis decreased the saccharification to about its half while addition of 8% ethanol brought about complete inhibition of the enzyme. Addition of cellobiase to the reaction mixture increased cellulose conversion and saccharification by 10%.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

稀酸和稀碱预处理对四种不同生物质资源制备还原糖的影响

本论文探讨了不同浓度的稀H_2SO_4和稀NaOH预处理对大豆秸秆、水稻秸秆、象草和狼尾草四种不同生物质酶解制备还原糖的影响。结果表明,大豆秸秆、水稻秸秆、象草和狼尾草具有较高的纤维素和半纤维素含量,是制备还原糖的理想原料。与稀H_2SO_4预处理相比,经稀NaOH预处理后的样品表现出较好的酶解性能。通过使用4%的NaOH对大豆秸秆和狼尾草进行预处理,还原糖产量分别为145.8 mg/mL和319.2 mg/mL。此外,以1%NaOH预处理后的水稻秸秆和象草为原料,可以分别获得385.2 mg/mL和231.6 mg/mL还原糖产量。     

Effect of physicochemical pre-treatment of rice straw on its digestibility by <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis>

Rice straw (RS) may serve as a low-cost biomass for the production of biofuels and biochemicals, but its native structure is resistant to enzymatic and microbial deconstruction. Therefore, an efficient pre-treatment method is required to modify crystalline cellulose to a more reactive amorphous form. This work investigated pre-treatments of rice straw involving size reduction (S) followed by either sodium hydroxide (NaOH) or diluted sulfuric acid (H₂SO₄) and liquid hot water (LHW). The shrinkage of the vascular bundles in the rice straw structure pre-treated with NaOH–LHW–S was higher than that with LHW–S and H₂SO₄–LHW–S pre-treatments. The highest levels of total fermentative products and residual sugars were obtained at the concentrations of 7.8 ± 0.2 and 2.1 ± 0.3 g/L, respectively, after fermentation by Clostridium cellulolyticum for NaOH–LHW–S pre-treated rice straw at 121 °C for 120 min. Overall, the combined physicochemical pre-treatment of RS led to improved microbial hydrolysis during cellulose degradation at the percentage of 85.5 ± 0.5.     

Simultaneous saccharification and fermentation (SSF) of <Emphasis Type="Italic">Jatropha curcas</Emphasis> shells: utilization of co-products from the biodiesel production process

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H₂SO₄ and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H₂SO₄- and NaOH-pretreated biomasses, respectively.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

EFFICIENT PRODUCTION OF BIOETHANOL FROM CORN STOVER BY PRETREATMENT WITH A COMBINATION OF SULFURIC ACID AND SODIUM HYDROXIDE

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid–alkali combination (1% H₂SO₄, 150°C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35°C for 36 hr after presaccharification at 50°C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.     

Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies

In order to investigate changes in substrate chemical and physical features after pretreatment, several characterizations were performed on untreated (UT) corn stover and poplar and their solids resulting pretreatments by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough, lime, and SO₂ technologies. In addition to measuring the chemical compositions including acetyl content, physical attributes determined were biomass crystallinity, cellulose degree of polymerization, cellulase adsorption capacity of pretreated solids and enzymatically extracted lignin, copper number, FT-IR responses, scanning electron microscopy (SEM) visualizations, and surface atomic composition by electron spectroscopy of chemical analysis (ESCA). Lime pretreatment removed the most acetyl groups from both corn stover and poplar, while AFEX removed the least. Low pH pretreatments depolymerized cellulose and enhanced biomass crystallinity much more than higher pH approaches. Lime pretreated corn stover solids and flowthrough pretreated poplar solids had the highest cellulase adsorption capacity, while dilute acid pretreated corn stover solids and controlled pH pretreated poplar solids had the least. Furthermore, enzymatically extracted AFEX lignin preparations for both corn stover and poplar had the lowest cellulase adsorption capacity. ESCA results showed that SO₂ pretreated solids had the highest surface O/C ratio for poplar, but for corn stover, the highest value was observed for dilute acid pretreatment with a Parr reactor. Although dependent on pretreatment and substrate, FT-IR data showed that along with changes in cross linking and chemical changes, pretreatments may also decrystallize cellulose and change the ratio of crystalline cellulose polymorphs (Iα/Iβ).     

Alkaline peroxide assisted wet air oxidation pretreatment approach to enhance enzymatic convertibility of rice husk

Pretreatment of rice husk by alkaline peroxide assisted wet air oxidation (APAWAO) approach was investigated with the aim to enhance the enzymatic convertibility of cellulose in pretreated rice husk. Rice husk was presoaked overnight in 1% (w/v) H₂O₂ solution (pH adjusted to 11.5 using NaOH) (equivalent to 16.67 g H₂O₂ and 3.63 g NaOH per 100 g dry, untreated rice husk) at room temperature, followed by wet air oxidation (WAO). APAWAO pretreatment resulted in solubilization of 67 wt % of hemicellulose and 88 wt % of lignin initially present in raw rice husk. Some amount of oligomeric glucose (?8.3 g/L) was also observed in the APAWAO liquid fraction. APAWAO pretreatment resulted in 13‐fold increase in the amount of glucose that could be obtained from otherwise untreated rice husk. Up to 86 wt % of cellulose in the pretreated rice husk (solid fraction) could be converted into glucose within 24 hours, yielding over 21 g glucose per 100 g original rice husk. Scanning electron microscopy was performed to visualize changes in biomass structure following the APAWAO pretreatment. Enzymatic cellulose convertibility of the pretreated slurry at high dry matter loadings was also investigated. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

Influence of xylan on the enzymatic hydrolysis of steam‐pretreated corn stover and hybrid poplar

The focus of this study was to alter the xylan content of corn stover and poplar using SO₂‐catalyzed steam pretreatment to determine the effect on subsequent hydrolysis by commercial cellulase preparations supplemented with or without xylanases. Steam pretreated solids with xylan contents ranging from ～1 to 19% (w/w) were produced. Higher xylan contents and improved hemicellulose recoveries were obtained with solids pretreated at lower severities or without SO₂‐addition prior to pretreatment. The pretreated solids with low xylan content (<4% (w/w)) were characterized by fast and complete cellulose to glucose conversion when utilizing cellulases. Commercial cellulases required xylanase supplementation for effective hydrolysis of pretreated substrates containing higher amounts of xylan. It was apparent that the xylan content influenced both the enzyme requirements for hydrolysis and the recovery of sugars during the pretreatment process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Evaluation of Processing Technology for Triarrhena sacchariflora (Maxim.) Nakai for Ethanol Production

The effects of dilute H₂SO₄ concentration, forage:sulfuric acid ratio, digestion time, and digestion temperature were evaluated to determine effects on ethanol yield of Triarrhena sacchariflora (Maxim.) Nakai. Twenty single factor experiments were conducted to evaluate H₂SO₄ concentration (0.5, 1.0, 1.5, 2.0, and 2.5%, w/w), forage:sulfuric acid ratio (1∶6, 1∶8, 1∶10, 1∶12, and 1∶14, g/ml), digestion time (15, 30, 45, 60, and 90, min), digestion temperature (80, 100, 110, 120, and 125 °C) for 3 replicates of the 5 levels of each factor. Based on results of the single factor experiments, an incomplete factorial was designed to evaluate ethanol yield from the best combinations of single factors. Finally, the best combination was tested by enzymatic hydrolysis and fermentation experiment in selected combinations according to pretreatment results. Percentage cellulose, hemicellulose, and lignin contents of forage residue after pretreatment, and glucose and xylose concentrations of the filtrate were analyzed prior to enzymatic hydrolysis, and percentage crystallinity was observed in untreated grass and pretreated residue. In addition, the solid residues were then hydrolysed and fermented by cellulase and yeast, the concentrations of glucose and ethanol being monitored for 96 h. Results showed that the order of the effect of main effect factors was as follows: digestion temperature > dilute H₂SO₄ concentration > digestion time > forage:sulfuric acid ratio. The best process parameters evaluated were sulfuric acid concentration of 1.5%, forage:sulfuric acid ratio of 1∶6, digestion time of 15 min, and digestion temperature of 120°C. With this combination of factors, 80% of the cellulose was hydrolysed in 96 h, and 78% converted to ethanol. The findings identified that hemicelluloses were the key deconstruction barrier for pretreatment of Triarrhena sacchariflora (Maxim.) Nakai for ethanol production. The results of this research provide evidence of appropriate combinations of processing factors for production of ethanol from Triarrhena sacchariflora (Maxim.) Nakai.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

Production of H2 from cellulose by rumen microorganisms: effects of inocula pre-treatment and enzymatic hydrolysis

H₂ production from cellulose, using rumen fluid as the inoculum, has been investigated in batch experiments. Methanogenic archaea were inhibited by acid pre-treatment, which also inhibited cellulolytic microorganisms, and in consequence, the conversion of cellulose to H₂. Positive results were observed only with the addition of cellulase. H₂ yields were 18.5 and 9.6 mmol H₂ g cellulose^?1 for reactors with 2 and 4 g cellulose l^?1 and cellulase, respectively. H₂ was primarily generated by the butyric acid pathway and this was followed by formation of acetic acid, ethanol and n-butanol. In reactors using 4 g cellulose l^?1 and cellulase, the accumulation of alcohols negatively affected the H₂ yield, which changed the fermentation pathways to solventogenesis. PCR–DGGE analysis showed changes in the microbial communities. The phylogenetic affiliations of the bands of DGGE were 99 % similar to Clostridium sp.     

Visible light-induced H2 production from cellulose using photosensitization of Mg chlorophyll a

A photoinduced-H₂ production system, coupling cellulose degradation by cellulase and glucose dehydrogenase (GDH) and H₂ production with colloidal Pt as a catalyst using the visible light-induced photosensitization of Mg chlorophyll a, has been developed. When the sample solution containing methylcellulose, cellulase, GDH, NAD⁺, Mg chlorophyll a, Methyl viologen and colloidal Pt was irradiated, continuous H₂ production was observed. The amount of H₂ production was about 12 mol after 4 h irradiation.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.