Selective solvent delignification for fermentation enhancement

Cellulose and hemicellulose in renewable biomass resources such as cornstover and wheat straw have been examined as substrates for the production of ethanol. A mixed culture of selected strains of Clostridium thermocellum and Clostridium thermosaccharolyticum are used to accomplish both the hydrolysis and fermentation of these carbohydrates in a single step. However, lignin and related phenolic materials are shown to diminish the rate, extent, and yield at which these carbohydrates can be utilized for ethanol production. In order to overcome this problem, a selective solvent pretreatment with alkaline-ethanol-water mixtures was examined for the delignification of cellulosic biomass under conditions where very little loss of fermentable carbohyrates results. Under optimal conditions, up to 67% of the initial lignin in cornstover can be extracted while 95% of the alpha-cellulose and pentosan carbohydrates remain insoluble. Subsequent mixed culture fermentation of the treated material has shown a 400% increase in the rate of degradation and greater than 85% utilization of the substrate. The effects of various extraction parameters on delignification kinetics and subsequent fermentation performance are discussed.     

Influence of moisture content and cultivation duration on Clostridium thermocellum 27405 end-product formation in solid substrate cultivation on Avicel

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.     

Electrically enhanced ethanol fermentation by <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethanol production by Clostridium thermocellum ATCC 35609 and Saccharomyces cerevisiae ATCC 26603 was improved in an electrochemical bioreactor system. It was increased by 61% with Cl. thermocellum and 12% with S. cerevisiae in the presence of -1.5 V of electric potential. These increases were attributed to high production rates due to regeneration and availability of increased reduced equivalents in the presence of electric potential. The electric current caused considerable shift in the metabolite concentrations on a molar basis in Cl. thermocellum fermentation but less in S. cerevisiae fermentation. Increasing electric potential in Cl. thermocellum fermentation resulted in less acetate and more lactate production. Acetate production was also reduced with increased electric potential in S. cerevisiae fermentation. The high electric potential of -5 V adversely affected the Cl. thermocellum fermentation, but not the S. cerevisiae fermentation even at a high electric potential of -10 V.     

Immobilized purple bacteria for light-driven H2 production from starch and potato fermentation effluents

The goal of the study was to show that immobilized purple bacteria could photoproduce H(2) using dark fermentation effluent (FE) as substrate. Simple pretreatment of an inexpensive glass-fiber matrix accelerated the immobilization process. Photobioreactors (PhBR) containing immobilized Rhodobacter sphaeroides GL produced 0.128 L H(2) h(-1) L(-1) of PhBR volume (0.570 L h(-1) L(-1) of matrix) for up to 3 months when continuously fed artificial media with volatile fatty acids (VFAs) or FE from potato and starch fermentations. Hydrogen production was insensitive to NH(4)(+) up to 1 mM and saturated at 8 mM lactate or 1.5% potato FE (diluted in water and supplemented with critical micronutrients). The efficiency of VFA transformation to H(2) was 50-70% of theoretical. At nonlimiting substrate concentrations in artificial media or FE, acetate was utilized before butyrate. High volumetric rates of continuous H(2) photoproduction and stability of the process are advantages of using immobilized cultures. Use of H(2) photoproduction as a polishing step in the treatment of FEs from dark fermentations increased the total amount of H(2) produced from 0.9 to 4.7 mol mol(-1) glucose equivalent in the original potato homogenate.     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

A consolidated bio-processing of ethanol from cassava pulp accompanied by hydrogen production

A biphasic fermentation approach was undertaken for the production of ethanol and hydrogen from cassava pulp. The glucose generated by co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense was 13.65±0.45 g L(-1), which was 1.75 and 1.17-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. The accumulated glucose could be utilised rapidly by subsequently inoculated Saccharomyces cerevisiae. An inoculum ratio of 1:1, a thermophilic fermentation of 84 h, and a pulp concentration of 4% proved optimal for ethanol production, fermentation efficiency, and productivity. With these conditions, the ethanol level reached 8.83±0.31 g L(-1) with a fermentation efficiency of 64.95±2.71%. Hydrogen production of 4.06 mmol by the co-culture system was 1.54 and 2.09-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. This sequential co-culture approach provided a consolidated bio-processing means to produce ethanol and hydrogen from cassava pulp.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

Continuous production of ammonium lactate by Streptococccus cremoris in a three-stage reactor

Batch and continuous fermentation studies were performed to optimize the production of ammonium lactate from whey to optimize the production of ammonium lactate from whey permeate. The product known as fermented ammoniated condensed whey permeate (FACWP) is a very promising animal feed. After an initial screening of four strains which produce predominantly L(+)- lactic acid, the desired isomer [D(-)-lactic acid is toxic], Streptococcus cremoris 2487 was chosen for further study. In batch mode, pH between 6.0 and 6.5 and 35 degrees C provided optimum incubation conditions. To stimulate a plug flow reactor, three CSTRs (continuous stirred tank reactors) were connected in tandem. For a 7.5-h retention time, 1.6-fold and 1.3-fold higher productivities were obtained for three-stage than for the single- and two-stage reactors, respectively. Various retentions times were examined (5, 7.5, and 10 h; 5g/L yeast extract). Although maximum lactate productivity occurred at a 5-h residence time (5.38 g/L H. 75% lactose utilization), lactose utilization was more complete at 7.5 h (4.38 g/L h productivity, 91% lactose utilization and a productivity, 91% lactose utilization). Retention time was increased to 15 h to obtain 95.9% lactose utilization and a productivity of 2.42g/L h for 2g/L yeast extract. Based on this lower yeast extract concentration, it was determined that ammonium lactate production and subsequent concentration by 11-fold would yield a product (FACWP) 17% more than soybean meal (crude protein contents are equivalent, 44%) at current market prices.     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

Two-stage system for hydrogen production by immobilized cyanobacterium Gloeocapsa alpicola CALU 743

Previous studies showed that cell suspensions of unicellular nondiazotrophic cyanobacterium G. alpicola grown under nitrate-limiting conditions intensively produces H2 via fermentation of endogenous glycogen with hydrogen yield more then 90% of theoretical maximum (3.8 mol H2 per mol glucose). H2 production is realized by a Hox hydrogenase on the stages of NAD(P)H generation. Exploiting this property, the two-stage cyclic system for sustained hydrogen production was developed using a photobioreactor (PhBR) with G. alpicola immobilized on glass fiber TR-0.3. Immobilization of the cells on the matrix occurred during growth directly in PhBR operated in continuous mode; the density of culture immobilized achieved 37 g Chl alpha cm(-2). The first stage of the cycle was the photosynthetic incubations of G. alpicola in the flow of the culture medium, which contained limiting concentrations of nitrate for efficient glycogen accumulation and activation of hydrogenase. The second stage was the fermentation of glycogen, with H2 production realized in darkness with continuous Ar sparging and without medium flow. Standardization of optimal parameters for both stages provided a stable cyclic regime of the system: photosynthesis (24 hours)-fermentation (24 hours). The total amount of H2 evolved in one cycle was 957.6 mL L(-1)(matrix), and the overage rate of H2 production during the cycle (48 hours) was about 20 mL h(-1) L(-1)(matrix). Ten consequent cycles was carried out in this regime with reproducible H2 production, although PhBR with the same sample of immobilized culture was operated over a period of more then three months.     

Effect of Yeast Extract and Vitamin B(12) on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B(12), biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B(12) alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Effect of nutrient limitation on product formation during continuous fermentation of xylose with <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 Fe(7)

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

A study of producing ethanol from cellulose using Clostridium thermocellum

The feasibility of producing ethanol in a continuous system from cellulose using Clostridirrrn thermocellum was investigated. This anaerobic and therniophilic bacterium was able to degrade cellulose directly into ethanol with acetic acid, hydrogen. and carbon dioxide as by-products of this fermentation. The fermentation was first carried out in a batch mode to study the effects of buffers, temperature, and agitation on microbial growth and ethanol production. From the compounds used to control pH. sodium bicarbonate had the most preferred effects on generation time and ethanol production. As expected, there was a positive relationship between temperature and growth rate. On the other hand, agitation did not benefit from ethanol production or microbial growth. The possibility of noncompetitive inhibition within such a system was deduced from the calculation of the kinetic constants K(m) and V(max). Continuous fermentations were carried out at 60 degrees C and pH 7.0 using 1.5 and 3% pure cellulose as a limiting substrate. The maximum ethanol concentration reached during the 1.5% cellulose fermentation was 0.3%. and 0.9% during the 3% cellulose fermentation. The yield of ethanol was about 0.3 grams per gram of consumed cellulose. The overall yield in both schemes was around 0.45 and 0.75 grams per gram of cellulose degraded. It was concluded that cellulose could be degraded continuously in a system with C. thermocellum for production of ethanol. While the continuous system like the batch method is feasible, it may not be promising as yet because of the slow generation time of this microorganism.     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.     

甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。     

Influence of process conditions on end product formation from Clostridium thermocellum 27405 in solid substrate cultivation on paper pulp sludge

Solid substrate cultivation of thermophilic, anaerobic bacteria offers an alternative production method for many bio-based chemicals; however the process must be optimized for each substrate-organism fermentation. The effects of initial substrate moisture content (SMC, 30%, 50% and 70% wet-basis), supplemental nutrient concentration (SNC, 12%, 50% and 100%) and duration of cultivation time (6, 10 and 14 days), on product formation (lactate, ethanol and acetate) by Clostridium thermocellum 27405 were examined during growth on paper pulp sludge. Water activities at moisture contents above 30% wet-basis were essentially identical ( approximately 0.99), yet the water contents differed significantly, and affected the metabolic activity of C. thermocellum. Increases in initial substrate moisture content from 50% to 70% for cultures supplemented with 50% or 100% nutrients resulted in a 75-145 mM increase in total end products. At 70% SMC, the addition of 100% SNC generated a 56% increase in product formation above the addition of 50% nutrient supplementation. Increases in the quantity of free water present in the solid substrate cultivation system up to the water holding capacity of the paper pulp sludge led to improved performance of this anaerobic bacterium. While nutrient supplementation is common in the form of salts for many aerobic microorganisms, efficient metabolism for anaerobic C. thermocellum grown in SSC was highly dependent on added salts, vitamins and reducing agents. Further studies are needed to determine if this is a general effect for other anaerobes grown in solid substrate cultures.     

Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW

Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H?) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose·L?1, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO?), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H? production was 14.2 mmol·(L culture)?1 and the total production of ethanol was 0.4 mmol·(L culture)?1. The maximum yield of H? was 1.3 mol·(mol glucose equivalent)?1 at a carbon recovery of 94%. The specific production rates of H?, CO?, and ethanol were 0.45, 0.13, and 0.003 mol·h?1·(g dry cell mass)-1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate-synthesizing clostridia.