Selection of a mixed culture of cellulosolytic thermophilic anaerobes from various natural sources

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60 degrees C, pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g ethanol per g substrate utilized at the ethanol/acetate ratio 4.7:1.     

Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL‐H₂/g‐sugars (14.2 ± 0.2 mmol‐H₂/g‐sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 ± 10.1 mL‐H₂/g‐sugars (7.9 ± 0.4 mmol H₂/g‐sugars) and 184.0 ± 10.7 mL‐H₂/day L_reactor (8.2 ± 0.5 mmol‐H₂/day L_reactor), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum. Biotechnol. Bioeng. 2010;105: 899–908. © 2009 Wiley Periodicals, Inc.     

Feasibility of acrylic acid production by fermentation

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via -alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.     

Effect of initial bacteria concentration on hydrogen gas production from cheese whey powder solution by thermophilic dark fermentation

Dark fermentative hydrogen gas production from cheese whey powder solution was realized at 55°C. Experiments were performed at different initial biomass concentrations varying between 0.48 and 2.86 g L^?1 with a constant initial substrate concentration of 26 ± 2 g total sugar (TS) per liter. The highest cumulative hydrogen evolution (633 mL, 30°C), hydrogen yield (1.56 mol H₂ mol^?1 glucose), and H₂ formation rate (3.45 mL h^?1) were obtained with 1.92 g L^?1 biomass concentration. The specific H₂ production rate decreased with increasing biomasss concentration from the highest value (47.7 mL g^?1 h^?1) at 0.48 g L^?1 biomass concentration. Total volatile fatty acid concentration varied beetween 10 and 14 g L^?1 with the highest level of 14.2 g L^?1 at biomass concentration of 0.48 g L^?1 and initial TS content of 28.4 g L^?1. The experimental data were correlated with the Gompertz equation and the constants were determined. The most suitable initial biomass to substrate ratio yielding the highest H₂ yield and formation rate was 0.082 g biomass per gram of TS. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 931–936, 2012     

A continuous thermophilic cellulose fermentation in an upflow reactor by aClostridium thermocellum-containing mixed culture

Summary A continuous thermophilic cellulose fermentation by aCl. thermocellum-containing mixed culture was carried out in an upflow reactor for a period of 100 days. The cellulose conversion rate was finally 0.35 g.1^–1.h^–1. Evidence that the fermentation process was influenced by both pH and dilution rate was given by the changes of concentration of the main fermentation products, acetic acid and ethanol. The role of cellodextrins and glucose as reactive intermediates in the process of cellulose breakdown was established.     

Cellulase production by mixed fungi in solid-substrate fermentation of bagasse

Ammonia-treated bagasse with 80%(w/w) moisture content was subjected to mixed-culture solid-substrate fermentation (SSF) with Trichoderma reesei LM-UC4 and Aspergillus phoenicis QM 329, in flask or pot fermenters, for cellulase production. Significantly higher activities of all the enzymes of the cellulase complex were achieved in 4 days of mixed-culture SSF than in single-culture (T. reesei) SSF. The highest filter-paper-cellulase and -glucosidase activities seen in mixed-culture SSF were 18.7 and 38.6 IU/g dry wt, respectively, representing approx. 3- and 6-fold increases over the activities attained in single-culture SSF. The mixed-culture SSF process also converted about 46% of the cellulose and hemicellulose to reducing sugars and enriched the product with 13% fungal protein. The biomass productivity, 0.29 gl^-1.h, and enzyme productivity, 28.0 IU I^-1.h, were about twice as high in the mixed-culture than in the single-culture.R. Dueñas is with the Departamento de Biologia, Universidad Nacional San Antonio Abad, Cusco, Peru. R. Tengerdy is with the Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. M. Gutierrez-Correa is with the Laboratorio de Micologia y Biotecnologia, Universidad Nacional Agraria La Molina, Apdo. Postal 456, Lima 1. Peru;     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Feasibility of hydrogen production from ripened fruits by a combined two-stage (dark/dark) fermentation system

Anaerobic fermentation for hydrogen (H₂) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H₂ production (4463.7 mL-H₂ L⁻¹-culture) with a maximum H₂ yield (2.2 mol H₂ mol⁻¹ glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H₂ L⁻¹-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H₂ conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.     

Hydrogen production from glucose by anaerobes

Various anaerobes were cultivated in media containing glucose. When 100 mL of thioglycollate medium containing 2.0% (w/v) glucose was used, Clostridium butyricum ATCC 859, NBRC 3315, and NBRC 13949 evolved 227-243 mL of biogas containing about 180 mL of hydrogen in 1 day. Although some strains had some resistance against oxygen, C. butyricum ATCC 859 and 860 did not have it. C. butyricum NBRC 3315 and Enterobacter aerogenes NBRC 13534 produced hydrogen in the presence of glucose or pyruvic acid, and E. aerogenes NBRC 13534 produced hydrogen by not only glucose and pyruvic acid but also dextrin, sucrose, maltose, galactose, fructose, mannose, and mannitol. When a medium containing 0.5% (w/v) yeast extract and 2.0% (w/v) glucose was used, E. aerogenes NBRC 13534 evolved more biogas and hydrogen than C. butyricum NBRC 3315 in the absence of reducing agent.     

Optimization of multienzyme production by two mixed strains in solid-state fermentation

F₃ and F₄ strains of Aspergillus niger were screened from five strains of fungi to produce multienzyme preparations (containing cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase) as dietary supplementation. Enzyme activities indicated that 1:4 (F₃ to F₄) was the optimum mixture proportion, and 0.3% (W/W) was the preferable pitching rate. In bran mash containing 54.5% (W/W) water, F₃ and F₄ could produce the supplementation better when cultured 30 to 36 h at 30 °C. Monofactorial and orthogonal experiments were performed to optimize media. Results of the variance and range analysis showed that the optimum medium contained 80 g of bran, 20 g of cottonseed powder, 1 g of (NH₄)₂SO₄, and 0.1 g of KH₂PO₄. When F₃ and F₄ strains were cultured in the optimum medium containing 54.5% (W/W) water, the activity of cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase reached 996; 15,863; 13,378; 7,621; and 5,583 U/g, respectively.     

Diversity and ecophysiological features of thermophilic carboxydotrophic anaerobes

Both natural and anthropogenic hot environments contain appreciable levels of carbon monoxide (CO). Anaerobic microbial communities play an important role in CO conversion in such environments. CO is involved in a number of redox reactions. It is biotransformed by thermophilic methanogens, acetogens, hydrogenogens, sulfate reducers, and ferric iron reducers. Most thermophilic CO-oxidizing anaerobes have diverse metabolic capacities, but two hydrogenogenic species are obligate carboxydotrophs. Among known thermophilic carboxydotrophic anaerobes, hydrogenogens are most numerous, and based on available data they are most important in CO biotransformation in hot environments.     

利用餐厨垃圾循环半连续厌氧发酵产氢研究

利用餐厨垃圾采用半连续厌氧发酵进行产氢的研究。实验结果表明以高温(100℃)预处理15 min的厌氧活性污泥为种泥,在温度37℃,pH 6.0左右,较宽的稀释率(1.0~4.0 d-1)范围内,均能较好的实现厌氧发酵产氢。在稀释率D=2.4 d-1下,流出液中乙醇、乙酸、丙酸、丁酸和戊酸的质量分数分别为5.6%、29.6%、5.4%、58.5%和0.9%,产氢过程属于典型的丁酸型发酵,最终氢气的体积分数可达60%,氢气的产生速率为5.49 m3/(m3.d)。将厌氧发酵液相产物作为稀释液返回到反应器中,反应器的产氢能力大幅度的提高,当回流比R=0.8时,最大产氢速率可达10.9 m3/(m3.d),最终氢气的含量可达65%,厌氧发酵反应器的产氢能力提高了约130%。     

Swine manure fermentation for hydrogen production

Biohydrogen fermentation using liquid swine manure as substrate supplemented with glucose was investigated in this project. Experiments were conducted using a semi-continuously-fed fermenter (8 L in total volume and 4 L in working volume) with varying pHs from 4.7 through 5.9 under controlled temperature (35 ± 1 °C). The hydraulic retention time (HRT) tested include 16, 20, and 24 h; however, in two pH conditions (5.0 and 5.3), an additional HRT of 12 h was also tried. The experimental design combining HRT and pH provided insight on the fermenter performance in terms of hydrogen generation. The results indicated that both HRT and pH had profound influences on fermentative hydrogen productivity. A rising HRT would lead to greater variation in hydrogen concentration in the offgas and the best HRT was found to be 16 h for the fermenter in this study. The best pH value in correspondence to the highest hydrogen generation was revealed to be 5.0 among all the pHs studied. There was no obvious inhibition on hydrogen production by methanogenesis when methane content in the offgas was lower than 2%. Otherwise, an inverse linear relationship between hydrogen and methane content was observed with a correlation coefficient of 0.9699. Therefore, to increase hydrogen content in the offgas, methane production has to be limited to below 2%.     

Amino acid fermentation and hydrogen transfer in mixed cultures

Abstract The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated.     

Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation

Summary Six thermophilic fungi were examined for their ability to produce cellulolytic enzymes in liquid (LF) and solid-state fermentation (SSF). The best cellulase activities were achieved by Thermoascus aurantiacus and Sporotrichum thermophile. Taking into consideration that solid-state medium obtained from 100 g of dry sugar-beet pulp occupies about 11 of fermentor volume equivalent to 11 of LF, it was confirmed that enzyme productivity per unit volume from both fungi was greater in SSF than in LF. The cellulase system obtained by SSF with T. aurantiacus contained 1.322 IU/1 of exo--d-glucanase, 53.269 IU/1 of endo--d-glucanase and 8.974 IU/1 of -d-glucosidase. The thermal and pH characteristics of cellulases from solid-state fermentation of T. aurantiacus and S. thermophile are described.     

Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions (approximately 60 degrees C) was studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of approximately 0.93 g g(-1). Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO2 and H2. The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO2 and H2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose was converted to ethanol by C. thermolacticum.     

Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production

Among different technologies of hydrogen production, bio-hydrogen production exhibits perhaps the greatest potential to replace fossil fuels. Based on recent research on dark fermentative hydrogen production, this article reviews the following aspects towards scaled-up application of this technology: bioreactor development and parameter optimization, process modeling and simulation, exploitation of cheaper raw materials and combining dark-fermentation with photo-fermentation. Bioreactors are necessary for dark-fermentation hydrogen production, so the design of reactor type and optimization of parameters are essential. Process modeling and simulation can help engineers design and optimize large-scale systems and operations. Use of cheaper raw materials will surely accelerate the pace of scaled-up production of biological hydrogen. And finally, combining dark-fermentation with photo-fermentation holds considerable promise, and has successfully achieved maximum overall hydrogen yield from a single substrate. Future development of bio-hydrogen production will also be discussed.