Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Hydrogen production from vegetable waste by bioaugmentation of indigenous fermentative communities

This work adopted an innovative approach for fermentative H₂ production from common domestic organic waste, at 28 °C, in the absence of pretreatment: the self-fermentation of non-sterile vegetable waste and the bioaugmentation of microbial indigenous fermenting communities. For this purpose, three new H₂-producing strains, Buttiauxella sp. 4, Rahnella sp. 10 and Raoultella sp. 47, isolated and enriched from vegetable waste, were individually tested on two types of vegetable waste and compared with a bacterial artificial consortium composed of the three strains put together. The three single strains were also characterized for their ability to produce H₂ on different sugars, such as xylose, arabinose and cellobiose, as these are key products of hydrolysis of cellulose and hemicelluloses. H₂ production occurred from self-fermentation with yields ranging from 18.08 to 21.95 ml H₂/g VS. All bacterial inocula promoted a significant increase of the H₂ yield and the H₂ production rate, compared to the self-fermentation. The inocula of the artificial consortium yielded the maximum H₂ production of 85.65 ml H₂/g VS with the highest H₂ production rate of 2.56 ml H₂/h.     

Batch fermentative hydrogen production utilising sago (Metroxylon sp.) starch processing effluent by enriched sago sludge consortia

Sago starch processing effluent (SSPE) is an ideal bio-resource that can be utilised as a substrate for fermentative reactions due to its relatively high organic content. Annually in Malaysia, about 2.5 million tonnes of effluent are generated from the processing of sago starch. In this study, the potential use of SSPE as a substrate for fermentative hydrogen production was confirmed under all the experimental conditions studied. The maximum hydrogen production and volumetric hydrogen production rate were 575 mL H₂/L SSPE and 57.54 mL H₂/hr.L SSPE, respectively, from cultures with an initial pH of 7 and substrate concentration of 11 g soluble carbohydrate/L SSPE. The final soluble metabolites were comprised mainly of acetate (24–43%), butyrate (4–20%), propionate (1–7%) and ethanol (44–66%), suggesting an acetic acid-ethanol type fermentation pathway.     

Hydrogen production from high slat medium by co-culture of Rhodovulum sulfidophilum and dark fermentative microflora

Potential of pH decrease is one of the major obstacle in stable operation in coculture of dark- and photo-fermentative bacteria for hydrogen production. In this study, a dark fermentative bacterial consort and acid-tolerant marine photo-fermentative bacterium, Rhodovulum sulfidophilum TH-102, were individual or co-cultured in high salt medium for hydrogen production. All co-cultures produced more hydrogen than the individual culture of photo or dark fermentation. The dark/photo bacterial ratios were 1:5, 1:10, 1:15 and 1:20, respectively. Among the coculture ratios, bacterial ratio 1:10 produced the highest hydrogen yield (1694 ± 21 mL/L). The addition of the photo-fermentative bacterium to the dark fermentation consort stabilized the pH value and decreased the oxidation-reduction potential of the co-culture system and extended the hydrogen production period. Carbon fixation by the photo-fermentative bacterium may play some role in improving the hydrogen yield of the co-culture system.     

Hydrogen production potentials and fermentative characteristics of various substrates with different heat-pretreated natural microflora

Batch tests were carried out to investigate the effects of heat-pretreated inocula on the fermentative hydrogen production characteristics of various types of substrates. A total of 8 different inocula and 4 different substrates (starch, glycerol, oil and peptone) were used. Heat pretreatment of the inocula was conducted in order to harvest spore-forming clostridial bacteria. Significant hydrogen production potentials were observed from starch (20.5–174.4 ml H₂/g-COD_starch) and glycerol (11.5–38.1 ml H₂/g-COD_glycerol); however, almost no hydrogen was produced from oil and peptone. When starch was used as a substrate, two different fermentation patterns were observed, according to the inocula: butyric acid-type and ethanol-type fermentation. Polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE) analysis was conducted to compare the bacterial structures cultivated on the starch medium. Different species of clostridial bacteria were observed between the butyric acid-type and ethanol-type fermentation cultures. When glycerol was used as a substrate, 1,3-propanediol was the main by-product with each inoculum. The results of the present study suggest that simultaneous production of ethanol or 1,3-propanediol in addition to hydrogen is a more promising strategy than conventional hydrogen production in acidogenesis.     

Metabolic shift and electron discharge pattern of anaerobic consortia as a function of pretreatment method applied during fermentative hydrogen production

We have made an attempt to evaluate the variation in the electron discharge (ED) pattern of anaerobic consortia as a function of pretreatment viz., chemical, heat-shock, acid and oxygen-shock in comparison with untreated mixed consortia during fermentative hydrogen (H₂) production. Experiments were performed with dairy wastewater as substrate using anaerobic mixed consortia as biocatalyst (pretreated individually and in combination). Cyclic voltammetry (CV) elucidated significant variation in the ED pattern of mixed consortia along with H₂ production and substrate degradation (SD) as a function of pretreatment method applied. Higher ED was observed with all pretreated consortia which can be attributed to the stable proton (H⁺) shuttling due to the suppression of methanogenic activity. Oxygen-shock method and untreated consortia showed lower H₂ production and higher SD among the variations studied, while, combined pretreated consortia resulted higher H₂ production and lower SD. Lower ED observed with untreated consortia suggests the H⁺ reduction during methanogenesis rather than the inter-conversion of metabolites, which is presumed to be necessary for H₂ production. ED observed with combined pretreated consortia corroborated well with the observed H₂ production. Redox pairs were visualized on the voltammograms with almost all the experimental variations studied except untreated consortia. The potentials (E₀) of redox pairs observed were corresponding to intracellular electron carriers viz., NAD⁺/NADH (E₀ −0.32 V) and FAD⁺/FADH₂ (E₀ −0.24 V).     

Stoichiometric analysis of biological hydrogen production by fermentative bacteria

In this study, biohydrogen production from glucose by two fermentative bacteria (Clostridium butyricum, a typical strictly anaerobic bacterium, and Klebsiella pneumoniae, a well-studied facultative anaerobic and nitrogen-fixing bacterium) are stiochiometrically analyzed according to energy (ATP), reducing equivalent and mass balances. The theoretical analysis reveals that the maximum yield of hydrogen on glucose by Clostridium butyricum is 3.26 mol/mol when all acetyl-CoA entering into the acetate pathway (α=1$\alpha =1$), which is higher than that by Klebsiella pneumoniae under strictly anaerobic conditions. In the latter case, the maximum yield by Klebsiella pneumoniae is 2.86 mol hydrogen per mol glucose when five sevenths of acetyl-CoA is transformed to acetate. However, under microaerobic condition the maximum yield of hydrogen on glucose by Klebsiella pneumoniae could reach 6.68 mol/mol if all acetyl-CoA entered into tricarboxylic acid (TCA) cycle (γ=1$\gamma =1$) and a quantity of 53% of the reducing equivalents generated in the metabolism were completely oxidized by molecular oxygen. On the other hand, the relationship between hydrogen production and biomass formation is distinct by Clostridium butyricum from that by Klebsiella pneumoniae.   The former yield of hydrogen on glucose increases as biomass. In contrast, the latter one decreases as biomass in a certain range of molar fraction of acetate in total acetyl-CoA metabolism (5/7?β?0$5/7?\beta ?0$). Microaerobic condition is favorable for high hydrogen production with low biomass formation by Klebsiella pneumoniae   in a certain range of the molar fraction of all reducing equivalents oxidized completely by molecular oxygen (0.53?δ?0.83$0.53?\delta ?0.83$).     

Effect of temperature on fermentative hydrogen production by mixed cultures

Effect of temperatures ranging from 20 °C to 55 °C on fermentative hydrogen production by mixed cultures was investigated in batch tests. The experimental results showed that, at initial pH 7.0, during the fermentative hydrogen production using glucose as substrate, the substrate degradation efficiency and hydrogen production potential increased with increasing temperatures from 20 °C to 40 °C. The maximal substrate degradation efficiency was 98.1%, the maximal hydrogen production potential was 269.9 mL, the maximal hydrogen yield was 275.1 mL/g glucose and the shortest lag time was 7.0 h. The temperature for fermentative hydrogen production by mixed cultures was optimized to be 40 °C. The expanded Ratkowsky models could be used to describe the effect of temperatures on the hydrogen production potential, maximum hydrogen production rate and the lag time during fermentative hydrogen production.     

Hydrogen production by non-photosynthetic bacteria

This paper is devoted to the identification of hydrogen-producing non-photosynthetic bacteria and a discussion of the following three areas of possible research: (1) hydrogen from sewage treatment plants; (2) hydrogen from rumen bacteria; and (3) large-scale production of hydrogen through the genetic manipulation of hydrogen-producing non-photosynthetic bacteria.     

Sodium inhibition of fermentative hydrogen production

A continuous-stirred-tank reactor (CSTR) was fed with low-sodium influent containing 0.27 g of Na⁺/L for 70 days (Phase I), and then subjected to higher concentrations of Na⁺/L, i.e. 2.41 (Phase II), 5.36 (Phase III), and 10.14 g (Phase IV-1). At the quasi-steady state of each phase, biomass was sampled for an acute sodium toxicity assay. Unlike the control biomass, which exhibited a monotonic decrease of specific H₂ production activity (SHPA) with increasing sodium concentration from 0.27 to 21.00 g Na⁺/L, the acclimated biomass maintained their activity up to 6.00 g Na⁺/L. Soluble microbial product analysis revealed that a sudden increase of the exterior sodium concentration changed the metabolic pathway such that it became favorable to lactate production while depressing butyrate production. Meanwhile, when the biomass was allowed for sufficient time to adapt to the chronic toxicity condition, the volumetric H₂ production rate (VHPR) was maintained above 4.05 L H₂/L/d at up to Phase III. However, an irrecoverable H₂ production drop was observed at Phase IV-1 with a significant increase of lactate and propionate production. Although the sodium concentration decreased to 8.12 (Phase IV-2), 6.61 (Phase IV-3), and 5.36 g Na⁺/L (Phase V) at further operation, the performance was never recovered. A PCR-DGGE analysis revealed that lactic acid bacteria (LAB) and propionic acid bacteria (PAB) were only detected at Phases IV and V, which are not capable of producing H₂.     

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

Harnessing of biohydrogen from wastewater treatment using mixed fermentative consortia: Process evaluation towards optimization

Utilizing wastewater as a potential source for renewable energy generation through biological routes has instigated considerable interest recently due to its sustainable nature. An attempt was made in this communication to review and summarize the work carried out in our laboratory on dark fermentation process of biohydrogen (H₂) production utilizing wastewater as primary substrate under acidogenic mixed microenvironment towards optimization of dynamic process. Process was evaluated based on the nature and composition of wastewater, substrate loading rates, reactor configuration, operation mode, pH microenvironment and pretreatment procedures adopted for mixed anaerobic culture to selectively enrich acidogenic H₂ producing consortia. The fermentative conversion of the substrate to H₂ is possible by a series of complex biochemical reactions manifested by selective bacterial groups. In spite of striking advantages, the main challenge of fermentative H₂ production is that, relatively low energy from the organic source was obtained in the form of H₂. Further utilization of unutilized carbon sources present in wastewater for additional H₂ production will sustain the practical applicability of the process. In this direction, enhancing H₂ production by adapting various strategies, viz., self-immobilization of mixed consortia (onto mesoporous material and activated carbon), integration with terminal methanogenic and photo-biological processes and bioaugmentation with selectively enriched acidogenic consortia were discussed. Application of acidogenic microenvironment for in situ production of bioelectricity through wastewater treatment employing microbial fuel cell (MFC) was also presented.     

Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures

Dark fermentation using mixed cultures is an attractive biological process for producing hydrogen (H₂) from lignocellulosic biomass at a low cost. Physicochemical pretreatment is generally used to convert lignocellulosic materials into monosaccharides. However, the processes also involved release degradation byproducts which can, in turn, inhibit microbial growth and metabolism and, hence, impact substrate conversion. In this study, the impact on H₂ production of lignocellulose-derived compounds (i.e. furan derivatives, phenolic compounds and lignins) was assessed along with their effect on bacterial communities and metabolisms. Batch tests were carried out using xylose as model substrate (1.67 mol_H2 mol_xylose⁻¹ in the control test). All the putative inhibitory compounds showed a significant negative impact on H₂ production performance (ranging from 0.34 to 1.39 mol_H2 mol_xylose⁻¹). The H₂ yields were impacted more strongly by furan derivatives (0.40–0.51 mol_H2 mol_xylose⁻¹) than by phenolic compounds (1.28–1.39 mol_H2 mol_xylose⁻¹). Except for the batch tests supplemented with lignins, the lag phase was shorter for inhibitors having the highest molecular weight (8 days versus 22 days for the lowest MW). Variability of the lag phase was clearly related to a shift in bacterial community structure, as shown by multivariate ordination statistics. The decrease in H₂ yield was associated with a decrease in the relative abundance of several H₂-producing clostridial species. Interestingly, Clostridium beijerinkii was found to be more resistant to the inhibitors, making this bacterium an ideal candidate for H₂ production from hydrolyzates of lignocellulosic biomass.     

An efficient dark fermentative hydrogen production by GMV control of pH

This study proposes that the on-line pH control via a model-based adaptive controller markedly improves the dark fermentative hydrogen production. According to the dynamic behavior of the dark fermentation process, pH, which rapidly declines with the beginning of the biogas production, should be precisely controlled around its optimal value in a narrow range. The success of on-line pH control was guaranteed by performing the preliminary simulation studies by experimental data obtained from dynamic analysis to determine ARMAX model order with Recursive Least Squares parameter estimation method and then to control the pH with Generalized Minimum Variance (GMV) controller. On-line control of pH at the optimal value of 6.0 during the 25 h dark fermentation process resulted in 5.4 times higher biogas production, 6.2 times higher biogas production potential, nearly doubled the duration of fermentation, and 18.4% biogas production rate increment in comparison with the uncontrolled pH case.     

发酵产氢面临的问题及对策

氢是一种高效、清洁、可再生的燃料，通过发酵的方式产生氢气，已经成为国内外研究的热点。目前，这一新兴技术的研究取得了可喜的成果。文章在综合国内外发酵产氢技术的基础上，通过比较沼气发酵和产氢发酵的技术成果，揭示了挥发性有机酸反馈抑制是制约生物法发酵产氢的关键因素，提出了如何提高氢的转化率和发酵稳定性的措施和对策。     

Thermophilic fermentative hydrogen production from various carbon sources by anaerobic mixed cultures

In the present work, various carbon sources, xylose, glucose, galactose, sucrose, cellobiose, and starch were tested for thermophilic (60 °C) fermentative hydrogen production (FHP) by using the anaerobic mixed culture. An inoculum was obtained from a continuously-stirred tank reactor (CSTR) operated at pH 5.5 and HRT 12 h, and fed with tofu processing waste. The dominant species in the CSTR were found to be Thermoanaerobacterium thermosaccharolyticum and Clostridium thermosaccharolyticum, which are well known thermophilic H₂-producers in anaerobic-state, and have the ability to utilize a wide range of carbohydrates. When initial pH was adjusted to 6.8 ± 0.1 but not controlled during fermentation, vigorous pH drop began within 5 h, and finally reached 4.0–4.5 in all carbon sources. Although over 90% of substrate removal was achieved for all carbon sources except cellobiose (71.7%), the fermentation performances were profoundly different with each other. Glucose, galactose, and sucrose exhibited relatively higher H₂ yields whereas lower H₂ yields were observed for xylose, cellobiose, and starch. On the other hand, when pH was controlled (pH ≥ 5.5), the fermentation performance was enhanced in all carbon sources but to a different extent. A substantial increase in H₂ production was observed for cellobiose, a 1.9-fold increase of H₂ yield along with a substrate removal increase to 93.8%, but a negligible increase for xylose. H₂ production capabilities of all carbon sources tested were as follows: sucrose > galactose > glucose > cellobiose > starch > xylose. The maximum H₂ yield of 3.17 mol H₂/mol hexose_added achieved from sucrose is equivalent to a 26.5% conversion of energy content in sucrose to H₂. Acetic and butyric acids were the main liquid-state metabolites of all carbon sources while lactic acid was detected only in cellobiose, starch and xylose exhibiting relatively lower H₂ yields.     

Dark fermentative biohydrogen production by Acinetobacter junii-AH4 utilizing various industry wastewaters

Hydrogen (H₂) is one of the most promising renewable energy sources, anaerobic bacterial H₂ fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H₂ is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H₂ production was achieved by using the bacteria Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H₂ production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H₂ and Acinetobacter junii-AH4 has shown the highest cumulative H₂ of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H₂/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H₂ production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H₂.