Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas

Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.     

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     

联合生物制氢方法及发展趋势

为了在生物制氢过程中最大限度提高产氢量和产氢速率,增大底物的利用率以及更好地发挥菌种间的协同作用,联合生物制氢技术成为近年来人们关注的焦点。综述了目前国内外几种联合生物制氢方法的研究现状。并从产氢机理的角度对几种联合制氢技术进行了分析比较,重点强调光合发酵和暗发酵联合生物制氢技术具有广泛的发展前景,并指出其存在的问题和未来的发展趋势。     

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.     

A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste

Aims:  The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H₂) producers from digested household solid wastes.
Methods and Results:  A strict anaerobic extreme thermophilic H₂ producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70°C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80°C and an optimal pH 8·1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H₂ and carbon dioxide. Maximal H₂ production rate on glucose was 1·1 mmol l⁻¹ h⁻¹ with a maximum H₂ yield of 1·9 mole H₂ per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 ± 5% and 13 ± 5% for Bacillus and Clostridium , respectively.
Conclusions:  An extreme thermophilic, strict anaerobic, mixed microbial culture with H₂-producing potential was enriched from digested household wastes.
Significance and Impact of the Study:  This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H₂ production from complex organic wastes.     

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.     

Keratinase of an anaerobic thermophilic bacterium <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp. Strain 1004-09 isolated from a hot spring in the Baikal rift zone

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and α- and β-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60°C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.     

Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora

For the transition to the hydrogen economy, hydrogen must be produced sustainably, e.g., by the fermentation of agricultural material. Continuous fermentative production of hydrogen from an insoluble substrate in nonsterile conditions is yet to be reported. In this study hydrogen production using mixed microflora from heat-treated digested sewage sludge in nonsterile conditions from a particulate co-product of the wheat flour industry (7.5 g L(-1) total hexose) at 18- and 12-hour hydraulic retention times, pH 4.5 and 5.2, 30 degrees C and 35 degrees C was examined. In continuous operation, hydrogen yields of approximately 1.3 moles hydrogen/mole hexose consumed were obtained, but decreased if acetate or propionate levels rose, indicating metabolism shifted towards hydrogen consumption by homoacetogenesis or propionate producers. These shifts occurred both at pH 4.5 and 5.2. Sparging the reactor with nitrogen to reduce hydrogen in the off-gas from 50% to 7% gave stable operation with a hydrogen yield of 1.9 moles hydrogen /mole hexose consumed over an 18-day period.     

Thermophilic biohydrogen production by an anaerobic heat treated-hot spring culture

Batch experiments were conducted to investigate the thermophilic biohydrogen production using an enrichment culture from a Turkish hot spring. Following the enrichment, the culture was heat treated at 100 °C for 10 min to select for spore-forming bacteria. H₂ production was accompanied by production of acetate, butyrate, lactate and ethanol. H₂ production was associated by acetate–butyrate type fermentation while accumulation of lactate and ethanol negatively affected the H₂ yield. H₂ production was highest in the temperature range from 49.6 to 54.8 °C and optimum values for initial pH and concentrations of iron, yeast extract and glucose were 6.5, 40 mg/l, 4–13.5 g/l, respectively. PCR–DGGE profiling showed that the heat treated culture consisted of species closely affiliated to genus Thermoanaerobacterium.     

Thermoanaerobium acetigenum spec. nov., a new anaerobic,extremely thermophilic,xylanolytic non-spore-forming bacterium isolated from an Icelandic hot spring

An anaerobic, extremely thermophilic, xylanolytic nonspore-forming bacterium, strain X6B, was isolated from a 70°C Icelandic hot spring sediment. The bacterium was rod-shaped, 3.6–5.9 m long and 0.7 to 1.0 m wide, and cells grew singly, in pairs, and occasionally formed chains. The bacterium was nonmotile with no flagella. Cells from mid-to late exponential gowth-phase cultures stained gram-negative but had a gram-positive like cell wall structure in transmission electron photomicrographs. The bacterium grew between 50°C and 78°C with an optimum temperature at about 65°C to 68°C. Growth occurred between pH 5.2 and 8.5 with an optimum pH close to 7. During growth on beech wood xylan, glucose and d-xylose, the isolate produced CO₂, acetate and H₂ as major fermentation products, and a small amounts of ethanol; lactate was not produced. X6B did not reduce acetone to isopropanol or sulphate or thiosulfate to sulfide. The base composition of X6B's cellular DNA was 35.7 mol% guanine + cytosine. The properties of this strain do not fit any previously described species. The name proposed for the isolated bacterium was Thermoanaerobium acetigenum, spec. nov.     

A dual-substrate steady-state model for biological hydrogen production in an anaerobic hydrogen fermentation process

Biological hydrogen production from anaerobic waste fermentation possesses potential benefits in simultaneously reducing organic wastes and generating sustainable energy sources. Three kinetic-based steady-state models for anaerobic fermentation of multiple substrates, including glucose and peptone, were evaluated. Experimental results obtained from a continuous stirred tank reactor (CSTR) were primarily used for model evaluation. The dual-substrate steady-state model developed and the associated kinetic parameters estimated in this study successfully described the anaerobic growth of hydrogen-producing bacteria. The model was able to capture the general trends of consumption of substrates and accumulation of products, including formate, acetate, butyrate, and hydrogen, at dilution rates (D) between 0.06 and 0.69/h. According to the model, the adverse effects of endogeneous and peptone metabolism on net hydrogen production can be minimized by increasing D. For the operational conditions of D > 0.69/h, however, substantial washout of hydrogen-producing bacteria from the CSTR was observed, and it resulted in a rapid drop in hydrogen production rate as well.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

微生物产氢研究的进展

氢能由于其清洁、高效、可再生的特点而成为一种最有吸引力的化石燃料的替代能源。与传统的热化学和电化学制氢技术相比,生物制氢具有低能耗、少污染等特点。本文主要对各种微生物的生物产氢方法作一综合概括,着重介绍光合紫色非硫细菌(PNS)产氢研究的最新进展。     

Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell

Hydrogen gas production from cellulose was investigated using an integrated hydrogen production process consisting of a dark fermentation reactor and microbial fuel cells (MFCs) as power sources for a microbial electrolysis cell (MEC). Two MFCs (each 25 mL) connected in series to an MEC (72 mL) produced a maximum of 0.43 V using fermentation effluent as a feed, achieving a hydrogen production rate from the MEC of 0.48 m³ H₂/m³/d (based on the MEC volume), and a yield of 33.2 mmol H₂/g COD removed in the MEC. The overall hydrogen production for the integrated system (fermentation, MFC and MEC) was increased by 41% compared with fermentation alone to 14.3 mmol H₂/g cellulose, with a total hydrogen production rate of 0.24 m³ H₂/m³/d and an overall energy recovery efficiency of 23% (based on cellulose removed) without the need for any external electrical energy input.     

Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland

The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO₂, and H₂. The temperature optimum for growth was between 70 and 75° C, and growth occurred in the range of 50–75° C. The pH range for growth was 4.7–8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H₂ or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed. Received: 7 October 1996 / Accepted: 14 March 1997     

Fe(III) as an electron acceptor for H2 oxidation in thermophilic anaerobic enrichment cultures from geothermal areas

Six sustainable enrichment cultures of thermophilic H₂-oxidizing microorganisms utilizing Fe(III) as an electron acceptor were obtained from geothermally heated environments located on two continents (America, Eurasia) and on islands in the Northern (Iceland) and Southern (Fiji) hemispheres, demonstrating the wide distribution of these microorganisms. The main products of amorphic Fe(III) oxide reduction were magnetite and siderite. The observed temperature range for Fe(III) reduction in growing cultures was from 55°C to 87°C, extending the known limits for growth of Fe(III)-reducing microorganisms producing extracellular magnetite to nearly 90°C. Received: August 13, 1996 / Accepted: January 17, 1997     

Characterization of a thermophilic sulfur oxidizing enrichment culture dominated by a Sulfolobus sp. obtained from an underground hot spring for use in extreme bioleaching conditions

A thermoacidophilic elemental sulfur and chalcopyrite oxidizing enrichment culture VS2 was obtained from hot spring run-off sediments of an underground mine. It contained only archaeal species, namely a Sulfolobus metallicus-related organism (96% similarity in partial 16S rRNA gene) and Thermoplasma acidophilum (98% similarity in partial 16S rRNA gene). The VS2 culture grew in a temperature range of 35–76°C. Sulfur oxidation by VS2 was optimal at 70°C, with the highest oxidation rate being 99 mg S⁰ l⁻¹ day⁻¹. At 50°C, the highest sulfur oxidation rate was 89 mg l⁻¹ day⁻¹ (in the presence of 5 g Cl⁻ l⁻¹). Sulfur oxidation was not significantly affected by 0.02–0.1 g l⁻¹ yeast extract or saline water (total salinity of 0.6 M) that simulated mine water at field application sites with availability of only saline water. Chloride ions at a concentration above 10 g l⁻¹ inhibited sulfur oxidation. Both granular and powdered forms of sulfur were bioavailable, but the oxidation rate of granular sulfur was less than 50% of the powdered form. Chalcopyrite concentrate oxidation (1% w/v) by the VS2 resulted in a 90% Cu yield in 30 days.     

Characteristics of hydrogen and methane production from cornstalks by an augmented two- or three-stage anaerobic fermentation process

This paper presents the co-production of hydrogen and methane from cornstalks by a two- or three-stage anaerobic fermentation process augmented with effective artificial microbial community. Two-stage fermentation by using the anaerobic sludge and DGGE analysis showed that effective and stable strains should be introduced into the system. We introduced Enterobacter aerogens or Clostridium paraputrificum into the hydrogen stage, and C. paraputrificum was proven to be more effective. In the three-stage process consisting of the improved hydrolysis, hydrogen and methane production stages, the highest soluble sugars (0.482 kg/kg cornstalks) were obtained after the introduction of Clostridium thermocellum in the hydrolysis stage, under the thermophilic (55 °C) and acidic (pH 5.0) conditions. Hydrolysates from 1 kg of cornstalks could produce 2.61 mol (63.7 l) hydrogen by augmentation with C. paraputrificum and 4.69 mol (114.6 l) methane by anaerobic granular sludge, corresponding to 54.1% energy recovery.     

Photoproduction of hydrogen by <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> from thermophilic fermentation effluent

Rhodobacter capsulatus was used for the phototrophic hydrogen production on effluent solution derived from the thermophilic fermentation of Miscanthus hydrolysate by Thermotoga neapolitana. Pretreatments such as centrifugation, dilution, buffer addition, pH adjustment and sterilization were suggested for the effluent before being fed to the photofermentation. Batch-wise experiments showed that R. capsulatus grows and produces hydrogen on the pretreated effluent solution. Moreover, it was found that the hydrogen yield increased from 0.3 to 1.0 L/L_culture by addition of iron to the effluent solution.