Anaerobic extremely thermophilic carboxydotrophic bacteria in hydrotherms of Kuril Islands

A new group of extremely thermophilic, obligately anaerobic, carboxydotrophic eubacteria is described. The organisms are characterized by a novel type of chemotrophic metabolism in thermophilic environments. They grow at temperatures up to 80–85°C chemolithotrophically with 100% CO in the gas phase as the sole energy source. The CO oxidation is coupled to H₂ and CO₂ formation according to the equation CO+H₂O → H₂+CO₂. No other products of metabolism are produced. The group of CO-utilizing, H₂-producing anaerobes includes diverse bacteria. They are non-sporeforming rods differing in morphology, CO uptake rates, habitats, and maximum growth temperatures. The new carboxydotrophic thermophilic anaerobes are widely distributed in freshwater and coastal marine hydrotherms of the Kuril Islands. Offprint requests to: V. A. Svetlichny.     

Thermophilic biohydrogen production: how far are we?

Apart from being applied as an energy carrier, hydrogen is in increasing demand as a commodity. Currently, the majority of hydrogen (H₂) is produced from fossil fuels, but from an environmental perspective, sustainable H₂ production should be considered. One of the possible ways of hydrogen production is through fermentation, in particular, at elevated temperature, i.e. thermophilic biohydrogen production. This short review recapitulates the current status in thermophilic biohydrogen production through fermentation of commercially viable substrates produced from readily available renewable resources, such as agricultural residues. The route to commercially viable biohydrogen production is a multidisciplinary enterprise. Microbiological studies have pointed out certain desirable physiological characteristics in H₂-producing microorganisms. More process-oriented research has identified best applicable reactor types and cultivation conditions. Techno-economic and life cycle analyses have identified key process bottlenecks with respect to economic feasibility and its environmental impact. The review has further identified current limitations and gaps in the knowledge, and also deliberates directions for future research and development of thermophilic biohydrogen production.     

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Hydrogen and methane production from desugared molasses by a two‐stage thermophilic anaerobic process was investigated in a series of two up‐flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen‐producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H₂/day/L, corresponding to a yield of 132 mL H₂/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH₄/day/L, corresponding to a yield of 239 mL CH₄/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H_2, 38.7% CO₂, and 44.8% CH₄, containing approximately 15% energy by hydrogen is viable to be bio‐hythane.     

Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H₂-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H₂ under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H₂ production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H₂/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H₂ producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H₂ production is quite similar.     

Current status of the metabolic engineering of microorganisms for biohydrogen production

The improvement of H₂ production capabilities of hydrogen (H₂)-producing microorganisms is a challenging issue. Microorganisms have evolved for fast growth and substrate utilization rather than H₂ production. To develop good H₂-producing biocatalysts, many studies have focused on the redirection and/or reconstruction of cellular metabolisms. These studies included the elimination of enzymes and carbon pathways interfering or competing with H₂ production, the incorporation of non-native metabolic pathways leading to H₂ production, the utilization of various carbon substrates, the rectification of H₂-producting enzymes (nitrogenase and hydrogenase) and photophosphorylation systems, and in silico pathway flux analysis, among others. Owing to these studies, significant improvements in the yield and rate of H₂ production, and in the stability of H₂ production activity, were reached. This review presents and discusses the recent developments in biohydrogen production, with a focus on metabolic pathway engineering.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

An Anaerobic Chemostat That Permits the Collection and Measurement of Fermentation Gases

A chemostat was designed to allow anaerobic growth in the culture vessel in the absence of a continuous stream of O₂-free gas. Produced gases were collected within the culture and collection vessels, and pressure build-up was prevented by allowing gases to expand into a collapsed football bladder. The culture overflow was collected in a flask, held at 0 C, that was emptied by applying a positive CO₂ pressure to the system. Ruminococcus albus, a H₂ and CO₂-producing anaerobe, was used to test the operation of the apparatus. H₂ production was measured by sampling the various gas spaces of known volume and measuring H₂ concentration by gas chromatography. Measurement of accumulated fermentation gases and the effects of the accumulation on fermentations can be studied with the apparatus.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Development of a system for the on‐line measurement of carbon dioxide production in microbioreactors: Application to aerobic batch cultivations of Candida utilis

We developed and applied a conductometric method for the quantitative online measurement of the carbon dioxide (CO₂) production during batch cultivations of Candida utilis on a 100‐μL scale. The applied method for the CO₂ measurement consisted of absorption of the produced CO₂ from the exhaust gas of the microbioreactor in an alkali solution, of which the conductivity was measured on‐line. The measured conductivity change of the alkali solution showed a linear relation with the total amount of CO₂ absorbed. After calibration of the CO₂ measurement system, it was connected to a well of a 96‐well microtiter plate. The mixing in the well was achieved by a magnetic stirrer. Using online measurement of the CO₂ production during the cultivation, we show reproducible exponential batch growth of C. utilis on a 100‐μL scale. The CO₂ production measurements obtained from the microcultivation were compared with the CO₂ production measurement in a 4‐L bioreactor equipped with a conventional off‐gas analyzer. The measurements showed that on‐line measurement of the CO₂ production rate in microbioreactors can provide essential data for quantitative physiological studies and provide better understanding of microscale cultivations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

Hydrogen production by microalgae

The production of H₂ gas from water and sunlightusing microalgae, `biophotolysis', has been a subjectof applied research since the early 1970s. A numberof approaches have been investigated, but most provedto have fundamental limitations or requireunpredictable research breakthroughs. Examples areprocesses based on nitrogen-fixing microalgae andthose producing H<sub>2</sub> and O<sub>2</sub> simultaneously fromwater (`direct biophotolysis'). The most plausibleprocesses for future applied R & D are those whichcouple separate stages of microalgal photosynthesisand fermentations (`indirect biophotolysis'). Theseinvolve fixation of CO₂ into storagecarbohydrates followed by their conversion to H₂by the reversible hydrogenase, both in dark andpossibly light-driven anaerobic metabolic processes. Based on a preliminary engineering and economicanalysis, biophotolysis processes must achieve closeto an overall 10% solar energy conversion efficiencyto be competitive with alternatives sources ofrenewable H₂, such as photovoltaic-electrolysisprocesses. Such high solar conversion efficiencies inphotosynthetic CO₂ fixation could be reached bygenetically reducing the number of light harvesting(antenna) chlorophylls and other pigments inmicroalgae. Similarly, greatly increased yields ofH₂ from dark fermentation by microalgae could beobtained through application of the techniques ofmetabolic engineering. Another challenge is toscale-up biohydrogen processes with economicallyviable bioreactors.Solar energy driven microalgae processes forbiohydrogen production are potentially large-scale,but also involve long-term and economically high-riskR&D. In the nearer-term, it may be possible tocombine microalgal H₂ production with wastewatertreatment.     

Biological production of H2, CH4 and CO2 in the deep subsurface of the Iberian Pyrite Belt

Most of the terrestrial deep subsurfaces are oligotrophic environments in which some gases, mainly H₂, CH₄ and CO₂, play an important role as energy and/or carbon sources. In this work, we assessed their biotic and abiotic origin in samples from subsurface hard-rock cores of the Iberian Pyrite Belt (IPB) at three different depths (414, 497 and 520 m). One set of samples was sterilized (abiotic control) and all samples were incubated under anaerobic conditions. Our results showed that H₂, CH₄ and CO₂ remained low and constant in the sterilized controls while their levels were 4, 4.1 and 2.5 times higher respectively, in the unsterilized samples compared to the abiotic controls. The δ¹³C_CH4-values measured in the samples (range −31.2 to −43.0 ‰) reveals carbon isotopic signatures that are within the range for biological methane production. Possible microorganisms responsible for the biotic production of the gases were assessed by CARD-FISH. The analysis of sequenced genomes of detected microorganisms within the subsurface of the IPB allowed to identify possible metabolic activities involved in H₂ (Rhodoplanes, Shewanella and Desulfosporosinus), CH₄ (Methanobacteriales) and CO₂ production. The obtained results suggest that part of the H₂, CH₄ and CO₂ detected in the deep subsurface has a biological origin.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

Interspecific hydrogen transfer during methanol degradation by Sporomusa acidovorans and hydrogenophilic anaerobes

In the presence of active hydrogenophilic sulfate-reducing bacteria, the homoacetogenic bacterium Sporomusa acidovorans did not produce acetate during methanol degradation. H₂S and presumably CO₂ were the only end products. Since the sulfate-reducer did not degrade methnol or acetate, the sulfidogenesis from methanol was related to a complete interspecific hydrogen transfer between both species.In coculture with hydrogenophilic methanogenic bacteria (Methanobacterium formicicum, Methanospirillum hungatei), the interspecific hydrogen transfer with S. acidovorans was incomplete. Beside CH₄ and presumably CO₂, acetate was produced. The results suggested that H₂-production and H₂-consumption were involved during anaerobic methanol degradation by S. acidovorans and the hydrogenophilic anaerobes play an important role during methanol degradation by homoacetogenic bacteria in anoxic environments.     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium

Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified β-oxidation to H₂/CO₂ and acetate. These intermediates are converted to CH₄/CO₂ by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO₂/H₂ and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H₂-producing syntroph–methanogen partnership that may serve to improve community stability.