Different feedback effects of aqueous end products on hydrogen production of Clostridium tyrobutyricum

Feedback inhibition is one of the main challenges of fermentative hydrogen production. In this study, the effects of butyrate and acetate on hydrogen production of Clostridium tyrobutyricum were investigated. Substrate consumption and hydrogen production were accelerated when acetate ≤15 g/L was fed. Exogenous acetate induced acetate assimilation and increased the metabolic flux of butyrate synthesis. Exogenous butyrate significantly decreased biomass formation, and slowed substrate consumption and hydrogen production. Metabolic and gene expression analyses showed that butyrate impaired glycolysis and acetate production pathway. The increased butyrate/acetate molar ratio was deemed as a strategy for cells to alleviate pH decrease and reduce the inhibition of undissociated butyric acid. Inhibition model analyses indicated butyrate was the main inhibitor in butyrate-type hydrogen production. This study demonstrates the different feedback effects of acetate and butyrate on hydrogen production of C. tyrobutyricum and provides strategies to relieve the feedback inhibition for efficient hydrogen production.     

Inhibitory effect of ethanol,acetic acid,propionic acid and butyric acid on fermentative hydrogen production

The inhibitory effect of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate. The experimental results showed that, at 35 °C and initial pH 7.0, during the fermentative hydrogen production, the substrate degradation efficiency, hydrogen production potential, hydrogen yield and hydrogen production rate all trended to decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effect of added ethanol on fermentative hydrogen production was smaller than those of added acetic acid, propionic acid and butyric acid. The modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study successfully. The modified Logistic model could describe the progress of cumulative hydrogen production.     

Effect of operation parameters on anaerobic fermentation using cow dung as a source of microorganisms

Anaerobic fermentation by microorganisms is a promising method of hydrogen production for it can be conducted at mild conditions. In this paper, a series of tests were carried out to investigate the effect of pH, hydraulic retention time (HRT), temperature (T) and substrate concentration on anaerobic dark fermentation. Glucose was utilized as model substrate. The Taguchi orthogonal array was applied in the experimental design and a verification experiment was tested. The results showed the optimal parameters for hydrogen production were pH 5.0, HRT 8.34 h, T 33.5 °C and substrate concentration of 14 g/L, with hydrogen yield of 2.15 mol H₂/mol glucose. Butyric-type fermentation occurred in most tests. According to the analysis of effluent contents, at pH 5.5, 5.0, 4.0, the effluent contained mostly butyric acid (43.1–56.6%), followed by acetic acid (24.6–29.8%); at HRT 4.17, 6.26, 8.34 h, the effluent contained mostly butyric acid (43.0–53.6%). Increasing temperature from 29 to 39.5 °C resulted in the decrease of butyric acid percentage but increase of ethanol percentage. Substrate concentration had little effect on product constitution.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort

A newly enriched marine phototrophic bacterial consort was studied for its capability of hydrogen production in batch cultivations using butyrate as the sole carbon source. Analyses of denaturing gradient gel electrophoresis (DGGE) profiles showed that the mixed bacterial consort consisted mainly of Ectothiorhodospira, Sporolactobacillus, and Rhodovulum. Important parameters investigated include temperature, light intensity, initial pH, and butyrate concentration. The pH of the culture medium significantly increased as fermentation proceeded. Optimal cell growth was observed at temperature of 25–35 °C, light intensity of 80–120 μmol photons/m² s, initial pH of 8, butyrate concentration of 20–40 mmol/l. Optimal conditions for hydrogen production were 30 °C, light intensity of 80 μmol photons/m² s, initial pH 8. The increase of butyrate concentration (10–50 mmol/l) resulted in higher hydrogen production, but the yield of hydrogen production (mol H₂/mol butyrate) gradually decreased with increasing butyrate concentration. The maximal hydrogen yield and hydrogen production rate were estimated to be 2.52 ± 0.12 mol H₂/mol butyrate and 19.40 ± 2.32 ml/l h, respectively. These results indicate that optimization of the culture conditions resulted in a simultaneous increase in biohydrogen production and cell growth.     

Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.     

Effect of sulfate on hydrogen production from the organic fraction of municipal solid waste using co-culture of E. coli and Enterobacter aerogenes

In the present study, the effect of sulfate on the hydrogen production from the organic fraction of municipal solid (OFMSW) waste using co-culture of Enterobacter aerogenes and E. coli has been studied under varying pH conditions. The presence of sulfate in the feedstock declines hydrogen production efficiency. To evaluate the effect of sulfate on hydrogen production from OFMSW, COD/sulfate ratio of 17.5, 15.0, 12.5, 10.0, 7.5, 5.0 and 2.5 were applied at different pH conditions (i.e. pH 5.5, 6.0 and 6.5). The hydrogen production continuously declined with the decreasing COD/sulfate ratio and increase in pH. The cumulative hydrogen production decreased from 220.8 ± 10.5 mL in control to a minimum of 98.3 ± 10.5 mL, 74.4 ± 10.4 mL, and 44.6 ± 2.6 mL at pH 5.5, 6.0 and 6.5 respectively. The major content of gaseous composition included hydrogen and CO₂ at higher COD/sulfate ratio and low pH, while H₂S formation started with the decrease in COD/sulfate ratio and increase in the pH. Similarly, sulfate removal efficiency was found to be influenced by COD/sulfate ratio and pH condition. Soluble metabolite analysis revealed that total volatile fatty acid concentration was not affected by sulfate addition. Thus, Sulfate removal is essential prior to fermentation in order to improve hydrogen yield.     

Evaluation of hydrogen producing cultures using pretreated food waste

In order to enhance bio-hydrogen production from food waste, pretreatment methods are widely used. The influence of the initial pH and autoclaving were investigated in batch experiments. Fermentative studies showed that pure cultures like Clostridium beijerinckii could directly utilize raw food waste to produce hydrogen, while other cultures (Clostridium butyricum and Clostridium pasteurianum) could produce hydrogen only after pH adjustment. In this case, the optimal starting pH of the culture was found to be 7. Autoclaving could further enhance hydrogen yields due to increased hydrolysis of food waste. The maximum hydrogen yield was achieved by C. butyricum (38.9 mL-H₂/g-VS_added) after autoclaving food waste with pH adjustment at 7. In addition, the ratio acetic to butyric acid was decreased by autoclaving pretreatment, because butyrate metabolic pathway was favored in the fermentation process. However, suitable pH for bacteria growth and the low ammonia production could be achieved from autoclaving food waste.     

Enzymatic routes to hydrogen and organic acids production from banana waste fermentation by autochthonous bacteria: Optimization of pH and temperature

The Central Composite Rotational Design (CCRD) was employed to find the optimum pH (5.09–7.91) and temperature (27.1–46,9 °C) for hydrogen production in banana waste (BW) fermentation by autochthonous microbial biomass. The P and Rm ranged between 6.06 and 62.43 mL H₂ and 1.13–12.56 mL H₂.h^?1, respectively. The temperature 37 °C and pH 7.0 were the optimum conditions for P (70.19 mL H₂) and Rm (12.43 mL H₂.h^?1) as predicted by the mathematical model. Fructose and glucose are the primary alternative carbon sources in banana waste-fed batch reactors. The high concentration of lactic acid and H₂ production was associated to Lactobacillus (52–81%) and Clostridium (14–35%). However, the most important finding was about butyric acid (HBu). This acid is the better indicator of hydrogen production than acetic acid (HAc). The pH effected carbohydrates fermentation and organic acids production. The genes encoding the enzymes related to galactose, sucrose, fructose, arabinose and xylose metabolism were predominant.     

Effect of extrinsic lactic acid on fermentative hydrogen production

In this paper we report the effect of extrinsic lactic acid on hydrogen production from a starch-containing medium by a mixed culture. Study of the effect of addition of four metabolites, namely ethanol, lactic acid, butyric acid and acetic acid illustrated that lactic acid had a positive effect on both the maximum hydrogen production and hydrogen production rate. The addition of 10 mM lactic acid to a batch containing starch increased the hydrogen production rate and hydrogen production yield from 4.31 to 8.23 mL/h and 5.70 to 9.08 mmol H₂/g starch, respectively. This enhancement in hydrogen production rate and yield was associated with a shift from acetic acid and ethanol formation to formation of butyric acid as the predominant metabolite. The increase in hydrogen production yield was attributed to the increase in the available residual NADH for hydrogen production. When lactic acid was used as the sole carbon source, no significant hydrogen production was observed.     

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.     

Effect of pH and sulfate concentration on hydrogen production using anaerobic mixed microflora

The effects of varying sulfate concentrations with pH on continuous fermentative hydrogen production were studied using anaerobic mixed cultures growing on a glucose substrate in a chemostat reactor. The maximum hydrogen production rate was 2.8 L/day at pH 5.5 and sulfate concentration of 3000 mg/L. Hydrogen production and residual sulfate level decreased with increasing the pH from 5.5 to 6.2. The volatile fatty acids (VFAs) and ethanol fractions in the effluent were in the order of butyric acid (HBu) > acetic acid (HAc) > ethanol > propionic acid (HPr). Fluorescence In Situ Hybridization (FISH) analysis revealed the presence of hydrogen producing bacteria (HPB) under all pH ranges while sulfate reducing bacteria (SRB) were present at pH 5.8 and 6.2. The inhibition in hydrogen production by SRB at pH 6.2 diminished entirely by lowering to pH 5.5, at which activity of SRB is substantially suppressed.     

Populational and metabolic shifts induced by acetate,butyrate and lactate in dark fermentation

Dark fermentation is subject to inhibition by end products. In this study, the effects of acetate, butyrate and lactate on fermentation routes of glucose were investigated for concentrations ranging from 25 to 400 mM. Whatever the acid considered, an inhibition threshold of hydrogen production was observed at acid concentration as low as 50 mM. 300 mM of acetate, 200 mM of butyrate and 400 mM of lactate were critical concentrations resulting exclusively in lactate production. At these high concentrations, bacterial communities shifted from Clostridiaceae to Lactobacillaceae family after acetate or lactate addition, and to Bacillaceae after butyrate addition. At lower acid concentrations, the nature and the concentration of the added acid shaped metabolic and populational changes. Specifically, Clostridium butyricum was able to grow up to 250 mM, 150 mM and 300 mM of acetate, butyrate and lactate respectively, but was suspected to shift its metabolism towards lactate production.     

Revivability of fermentative hydrogen producing bioreactors

In this study we investigated the revivability of a continuous biological hydrogen producing reactor after a period of feed interruption. Before the feed interruption, the hydrogen production yield was 1.36 mol H₂/mol glucose with butyric acid and acetic acid as the main metabolic products. However, after feed interruption, butyric acid formation completely stopped and the hydrogen yield decreased to 0.29 mol H₂/mol glucose. Lactic acid, ethanol and acetic acid became the main metabolites after re-start up. Reduction of organic loading rate together with increasing the pH after the feed interruption resulted in an increase in the hydrogen yield to 0.7 mol H₂/mol glucose. The microbial community dynamics showed complete elimination of Clostridium affiliated strains and predominance of Lactobacillus affiliated strains after the re-start up of the reactor.     

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.     

Evaluating interactive toxic impact of heavy metals and variations of microbial community during fermentative hydrogen production

This study investigated the stimulating or inhibitory effect of Zn, Cu, Cd, Pb, and their mixtures on fermentative hydrogen production. Heavy metals inhibited the production process at all concentrations except 0.50 ppm of Zn. Consistent with the half of the maximum inhibitory concentration (IC₅₀), Cu + Pb caused the greatest inhibition as it is more toxic than other heavy metal mixtures. As the concentration of heavy metal increased, the HBu/HAc ratio (butyrate/acetate) tended to decrease and the amount of hydrogen production decreased. Clostridium sp., Klebsiella sp., Dysgonomas sp., Enterobacter sp., and other hydrogen-producing bacteria were observed by polymerase chain reaction (PCR) – analysis by denaturing gradient gel electrophoresis (DGGE) during the fermentative hydrogen process. A significant relationship was observed between the richness index and the hydrogen production (p-value = 0.01 < 0.05) from the Pielou index. The Pielou index (E′) and the Shannon-Weaver index (H’) had no significant relationship (p-value = 0.12 > 0.05).     

Bio-hydrogen production by a new isolated strain Rhodopseudomonas sp. WR-17 using main metabolites of three typical dark fermentation type

In this work, a new strain WR-17 was isolated for photo-fermentative hydrogen production and its hydrogen production capacity was investigated by utilizing main liquid byproducts of three dark fermentation types in batch culture. Experimental results indicated that strain WR-17 was identified as genus Rhodopseudomonas and maximum hydrogen yield of 2.42 mol H₂/mol acetate was obtained when the acetate was used as sole carbon source. Strain WR-17 had an excellent ability of using mixed short chain acids of three typical fermentations such as acetate and ethanol, acetate and butyrate, acetate and propionate. Result demonstrated that the metabolites of butyric acid-type fermentation as substrate is fitting to produce hydrogen and maximum cumulative hydrogen volume of 2156 ml/L-medium was obtained when acetate of 30 mmol/L and butyrate of 15 mmol/L were used. Therefore, butyric acid-type fermentation has great potential for further obtaining high hydrogen yield by the combining photo-fermentation.     

Biological hydrogen production in an anaerobic sequencing batch reactor: pH and cyclic duration effects

An anaerobic sequencing batch reactor (ASBR) was used to evaluate biological hydrogen production from carbohydrate-rich organic wastes. The goal of the proposed project was to investigate the effects of pH (4.9, 5.5, 6.1, and 6.7), and cyclic duration (4, 6, and 8 h) on hydrogen production. With the ASBR operated at 16-h HRT, 25 g COD/L, and 4-h cyclic duration, the results showed that the maximum hydrogen yield of 2.53 mol H₂/mol sucrose_consumed appeared at pH 4.9. The carbohydrate removal efficiency declined to 56% at pH 4.9, which indirectly resulted in the reduction of total volatile fatty acid production. Acetate fermentation was the dominant metabolic pathway at pH 4.9. The concentration of mixed liquor volatile suspended solid (MLVSS) also showed a decrease from nearly 15,000 mg/L between pHs 6.1 and 6.7 to 6000 mg/L at pH 4.9. Investigation of the effect of cyclic duration found that hydrogen yield reached the maximum of 1.86 mol H₂/mol sucrose_consumed at 4-h cyclic duration while ASBR was operating at 16-h HRT, 15 g COD/L, and pH 4.9. The experimental results showed that MLVSS concentration increased from 6200 mg/L at 4-h cyclic duration to 8500 mg/L at 8-h cyclic duration. However, there was no significant change in effluent volatile suspended solid concentration. The results of butyrate to acetate ratio showed that using this ratio to correlate the performance of hydrogen production is not appropriate due to the growth of homoacetogens. In ASBR, the operation is subject to four different phases of each cycle, and only the complete mix condition can be achieved at react phase. The pH and cyclic duration under the unique operations profoundly impact fermentative hydrogen production.     

Hydrogen production from rice winery wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures

Continuous production of hydrogen from the anaerobic acidogenesis of a high-strength rice winery wastewater by a mixed bacterial flora was demonstrated. The experiment was conducted in a 3.0-l upflow reactor to investigate individual effects of hydraulic retention time (HRT) (2–24 h), chemical oxygen demand (COD) concentration in wastewater (14–36 g COD/l), pH (4.5–6.0) and temperature (20–55°C) on bio-hydrogen production from the wastewater. The biogas produced under all test conditions was composed of mostly hydrogen (53–61%) and carbon dioxide (37–45%), but contained no detectable methane. Specific hydrogen production rate increased with wastewater concentration and temperature, but with a decrease in HRT. An optimum hydrogen production rate of 9.33 lH₂/gVSSd was achieved at an HRT of 2 h, COD of 34 g/l, pH 5.5 and 55°C. The hydrogen yield was in the range of 1.37–2.14 mol/mol-hexose. In addition to acetate, propionate and butyrate, ethanol was also present in the effluent as an aqueous product. The distribution of these compounds in the effluent was more sensitive to wastewater concentration, pH and temperature, but was less sensitive to HRT. This upflow reactor was shown to be a promising biosystem for hydrogen production from high-strength wastewaters by mixed anaerobic cultures.     

Enhanced biohydrogen production from date seeds by Clostridium thermocellum ATCC 27405

Biohydrogen production from waste lignocellulosic biomass serves the dual purpose of converting waste into valuable products and alleviates waste disposal issues. In this study, waste date seeds were valorized for biohydrogen production via consolidated bioprocessing by Clostridium thermocellum ATCC 27405. Effect of various surfactants (PEG1000, surfactin, Triton X-100) and sodium carbonate (buffering agent) on biohydrogen production from the acid pre-treated substrate was examined. Among the various surfactants, addition of Triton X-100 resulted in the maximum biohydrogen yield of 103.97 mmol/L at an optimal dosage of 0.75% w/v. Triton X-100 supplementation favoured the production of ethanol and acetate as co-metabolites than butyrate. Addition of Na₂CO₃ to date seed fermentation medium at a concentration of 15 mM enhanced the biohydrogen production by 33.16%. Also, Na₂CO₃ buffering supported the glycolytic pathway and subsequent ethanol production than acetate/butyrate formation. Combined effect of the optimal dosages of Triton X-100 and Na₂CO₃ resulted in high hydrogen productivity up to 72 h (0.443 mmol/g h of H₂) with a total increase in hydrogen yield of 40.6% at the end of 168 h, as compared to fermentation supplemented with Triton X-100 alone. Further analysis revealed that the combined effects of the additives resulted in better substrate degradation, favourable pH window and cell growth promotion which ensured enhanced hydrogen productivity and yield. Thus, the study highlights a novel stimulatory approach for enhanced biohydrogen production from a new substrate.