Hydrogen-Producing Microflora and Fe–Fe Hydrogenase Diversities in Seaweed Bed Associated with Marine Hot Springs of Kalianda, Indonesia

Microbial fermentation is a promising technology for hydrogen (H₂) production. H₂ producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H₂ through Fe–Fe hydrogenases (H₂ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H₂ production. A H₂-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe–Fe H₂ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H₂-producing microflora showing 90.0–99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H₂ producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe–Fe H₂ases, seven phylotypes were obtained, showing 63–97 % identities to known Fe–Fe H₂ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H₂-producing Clostridia, Halothermothrix orenii-like Fe–Fe H₂ases (80 % identity), and cellulolytic H₂-producing Clostridia, C. papyrosolvens-like Fe–Fe H₂ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe–Fe H₂ases surveys suggested that the thermophilic and halotolerant H₂-producing microflora in seaweed bed of hot spring area represented previously unknown H₂ producers, and have potential application for H₂ production.     

Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background

Pure bacterial strains give better yields when producing H₂ than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂ production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.

Results

Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20?L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂ production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂ yields were obtained for starch fermentations, and the highest yield of 2.91?mol?H₂/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5?L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂ at higher rates than the pure Clostridium cultures, without negatively affecting the H₂ yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂ during 13?days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.

Conclusions

The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H₂ using glucose and starch as carbon sources. The artificial co-cultures produced H₂ at higher rates than the pure strains, while the H₂ yields were only slightly affected.     

Comparative study of biological hydrogen production by pure strains and consortia of facultative and strict anaerobic bacteria

In this paper, a simple and rapid method was developed in order to assess in comparative tests the production of binary biogas mixtures containing CO₂ and another gaseous compound such as hydrogen or methane. This method was validated and experimented for the characterisation of the biochemical hydrogen potential of different pure strains and mixed cultures of hydrogen-producing bacteria (HPB) growing on glucose.The experimental results compared the hydrogen production yield of 19 different pure strains and sludges: facultative and strict anaerobic HPB strains along with anaerobic digester sludges thermally pre-treated or not. Significant yields variations were recorded even between different strains of the same species by i.e. about 20% for three Clostridium butyricum strains. The pure Clostridium butyricum and pasteurianum strains achieved the highest yields i.e. up to 1.36 mol H₂/mol glucose compared to the yields achieved by the sludges and the tested Escherichia and Citrobacter strains.     

Spatial succession and metabolic properties of functional microbial communities in an anaerobic baffled reactor

The spatial successions of bacterial and archaeal communities in anaerobic digestion were investigated in a glucose-degrading five-compartment anaerobic baffled reactor (ABR). The distributions of H₂-producing acetogens, H₂-utilizing acetogens and methanogens in different anaerobic-digestion stages were quantitatively analyzed using functional probes. The results show that the acidogenesis stage and acetogenesis stage were located in the first two compartments, while the methanogenesis were located in the last two compartments. In acidogenesis/acetogenesis stage of anaerobic digestion, H₂-producing acetogens (19.7%) and H₂-utilizing acetogens (8.3%) were the dominant bacterial community. While in methanogenesis stage, methanogens became the dominant (40.2%) with H₂-producing acetogens and H₂-utilizing acetogens only accounting for 6.6% and 4.8%, respectively. With the bacterial population decreasing from 7.2 ± 0.5 × 10¹² cells mL⁻¹ to 0.6 ± 0.3 × 10¹² cells mL⁻¹ along water flowing direction, their diversity increased from 2.79 to 299. The acidogenic bacteria, such as Lactococcus sp., Uncultured Firmicutes bacterium, and Uncultured Clostridium sp., etc., dominated in the acidogenesis/acetogenesis stage, while Uncultured Desulfobacterales bacterium became dominant in the methanogenesis stage. A two-stage anaerobic process may be suitable for easily degradable organic matters removal.     

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO2/H2 blend

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.     

Clostridium vulturis sp. nov., isolated from the intestine of the cinereous vulture (Aegypius monachus)

A Gram-stain positive, strict anaerobe, spore-forming, motile rod-shaped bacterial strain with peritrichous flagella, designated YMB-57^T, was isolated from the intestine of a cinereous vulture (Aegypius monachus) in Korea. StrainYMB-57^T was found to show optimal growth at 37 °C, pH 7.5 and 1.0 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain YMB-57^T belongs to the genus Clostridium and is most closely related to the type strains of Clostridium subterminale (96.9 % sequence similarity), Clostridium thiosulfatireducens (96.7 %) and Clostridium sulfidigenes (96.6 %). The main fermentation end-products identified following growth in PYG medium were acetate, butyrate, ethanol, propanol, carbon dioxide and hydrogen. Peptone was converted to ethanol, and butanol, whereas glucose was fermented to ethanol. The major cellular fatty acids were identified as C_16:0, C_18:1 ω9c, and C_18:1 ω9c DMA and the DNA G+C content was determined to be 34.0 mol%. Phenotypic and phylogenetic differences indicate that strain YMB-57^T is distinct from other Clostridium species. It is proposed that strain YMB-57^T be classified as the type strain of a novel species of the genus Clostridium, with the name Clostridium vulturis sp. nov. The type strain is YMB-57^T (=KCTC 15114^T = JCM 17998^T).     

Isolation of Key Methanogens for Global Methane Emission from Rice Paddy Fields: a Novel Isolate Affiliated with the Clone Cluster Rice Cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H₂) continuously provided by heterotrophic H₂-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H₂-producing syntroph, Syntrophobacter fumaroxidans, as the H₂ supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Novel Thermophilic Sulfate-Reducing Bacteria from a Geothermally Active Underground Mine in Japan

Thermophilic sulfate-reducing bacteria were enriched from samples obtained from a geothermal underground mine in Japan. The enrichment cultures contained bacteria affiliated with the genera Desulfotomaculum, Thermanaeromonas, Thermincola, Thermovenabulum, Moorella, “Natronoanaerobium,” and Clostridium. Two novel thermophilic sulfate-reducing strains, RL50JIII and RL80JIV, affiliated with the genera Desulfotomaculum and Thermanaeromonas, respectively, were isolated.     

Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H₂, it can produce acids (lactic, acetic and butyric acids) and CO₂, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H₂ yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H₂ yield compared with the wild type strain. However, under low partial pressure of H₂, the hbd-deficient strain showed increased H₂ production with the simultaneous decrease of ethanol production, indicating that H₂ production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H₂ production by C. butyricum.     

Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis

It is well known that some intestinal bacteria, such as Escherichia coli, can produce a remarkable amount of molecular hydrogen (H₂). Although the antioxidant effects of H₂ are well documented, the present study examined whether H₂ released from intestinally colonized bacteria could affect Concanavalin A (ConA)-induced mouse hepatitis. Systemic antibiotics significantly decreased the level of H₂ in both liver and intestines along with suppression of intestinal bacteria. As determined by the levels of AST, ALT, TNF-α and IFN-γ in serum, suppression of intestinal bacterial flora by antibiotics increased the severity of ConA-induced hepatitis, while reconstitution of intestinal flora with H₂-producing E. coli, but not H₂-deficient mutant E. coli, down-regulated the ConA-induced liver inflammation. Furthermore, in vitro production of both TNF-α and IFN-γ by ConA-stimulated spleen lymphocytes was significantly inhibited by the introduction of H₂. These results indicate that H₂ released from intestinal bacteria can suppress inflammation induced in liver by ConA.     

Effects of heat treatment on hydrogen production potential and microbial community of thermophilic compost enrichment cultures

Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70 °C. Highest cellulose degradation and highest H₂ yield were 57% and 1.4 mol-H₂ mol-hexose⁻¹ (2.4 mol-H₂ mol-hexose-degraded⁻¹), respectively, obtained at 52 °C with the heat-treated (80 °C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation.     

Composition of extracellular polymeric substances influences the autoaggregation capability of hydrogen-producing bacterium Ethanoligenens harbinense

Non-spore-forming Ethanoligenens, a novel genus of hydrogen-producing bacteria, is endowed with great application potential in biohydrogen production due to acidophilic and autoaggregating growth. In order to elucidate the mechanism of autoaggregation of Ethanoligenens harbinense, extracellular polymeric substances (EPS) from YUAN-3 had been extracted and analyzed. The EPS was mainly produced during the exponential phase and with protein, carbohydrate and DNA as its main components, with yields of 21.0 ± 0.8 mg/g-cell dry weight (CDW), 16.9 ± 0.8 mg/g-CDW and 3.5 ± 0.5 mg/g-CDW, respectively. Compared with the EPS composition of semi-autoaggregating hydrogen-producing bacteria W1 and non-autoaggregating hydrogen-producing bacteria B49, carbohydrate and protein played an important part in the autoaggregation of YUAN-3.     

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 °C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H₂ mol⁻¹ glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter.     

Isolation and Characterization of a New Clostridium sp. That Performs Effective Cellulosic Waste Digestion in a Thermophilic Methanogenic Bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55°C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.     

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO₂. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H⁺-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na⁺-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.     

Prolonged cultivation of an anaerobic bacterial community producing hydrogen

This paper studies various methods of long-term maintenance of the process of hydrogen evolution during the growth of an anaerobic bacterial community on a starch-containing medium. Continuos fermentation with periodic feeding and effluent removal for 72 days, allow to obtain from 0.10 to 0.23 H₂/l of medium/day. The regime of regular transfer lasted more than 100 days, forming an average of 0.81 l H₂/l of medium/day. The advantages and disadvantages of different methods of microbial hydrogen production during a dark starch fermentation process are presented. From the obtained H₂-producing microbial community, we isolated an anaerobic spore-forming bacterium (strain BF). Phylogenetic analysis of the 16S PNA gene sequence of the new strain showed that according to its genotype it belongs to the Clostridium butyricum species.     

In situ hydrogen utilization for high fraction acetate production in mixed culture hollow-fiber membrane biofilm reactor

Syngas fermentation is a promising route for resource recovery. Acetate is an important industrial chemical product and also an attractive precursor for liquid biofuels production. This study demonstrated high fraction acetate production from syngas (H₂ and CO₂) in a hollow-fiber membrane biofilm reactor, in which the hydrogen utilizing efficiency reached 100 % during the operational period. The maximum concentration of acetate in batch mode was 12.5 g/L, while the acetate concentration in continuous mode with a hydraulic retention time of 9 days was 3.6?±?0.1 g/L. Since butyrate concentration was rather low and below 0.1 g/L, the acetate fraction was higher than 99 % in both batch and continuous modes. Microbial community analysis showed that the biofilm was dominated by Clostridium spp., such as Clostridium ljungdahlii and Clostridium drakei, the percentage of which was 70.5 %. This study demonstrates a potential technology for the in situ utilization of syngas and valuable chemical production.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Ruminococcus hydrogenotrophicus sp. nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces

A new H₂/CO₂-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject. The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli. The isolates grew autotrophically by metabolizing H₂/CO₂ to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds. The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate. The G+C content of DNA of strain S5a33 was 45.2 mol%. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed. The type strain of R. hydrogenotrophicus is S5a33 (DSM 10507). Furthermore, H₂/CO₂ acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus). Received: 11 April 1996 / Accepted: 11 June 1996