Comparative study on biohydrogen production by newly isolated Clostridium butyricum SP4 and Clostridium beijerinckii SP6

The hydrogen-producing bacteria SP4 and SP6 were isolated from the compost and identified by 16S rRNA gene sequencing as Clostridium butyricum and Clostridium beijerinckii, respectively. A comparative study on the biohydrogen-producing activity of the isolated strains was carried out using mono-, di- and tri-saccharides belonging to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose). To assess the biotechnological significance, real wastewater rich in sugars (cheese whey, confectionery wastewater, sugar beet processing wastewater) was also used as a substrate. C. butyricum SP4 fermented sugars with a yield of 0.93–1.52 mol H₂/mol hexose (pentose); the maximum yield was obtained from fructose, the minimum – from raffinose and cellobiose. The most preferred substrate for C. beijerinckii SP6 was sucrose with a yield of 1.76 mol H₂/mol hexose, while cellobiose yielded only 0.64 mol H₂/mol hexose. Overall, the efficiency of converting wastewater to H₂ by C. butyricum SP4 was also slightly lower (66–93 ml H₂/g chemical oxygen demand (COD)) than that of C. beijerinckii SP6 (76–103 ml H₂/g COD). Even though the main soluble metabolite products (SMPs) for both isolates were acetate and butyrate, C. butyricum SP4 also produced a significant amount of ethanol (up to 21.5% of SMPs) and formate (up to 32.5% of SMPs), and C. beijerinckii SP6 – lactate (up to 25% of SMPs). A distinctive feature of C. beijerinckii SP6 was a significantly lower (almost 2 times) yield of SMPs, while C. butyricum SP4 had a higher rate of H₂ production according to the results obtained from the kinetic study using the modified Gompertz equation and the first order equation. Analysis of Spearman's rank correlation coefficients revealed a statistically significant relationship between the kinetic parameters of H₂ production and the concentration of butyrate and the final pH of the medium for C. butyricum SP4, and with the concentration of ethanol for C. beijerinckii SP6. These findings provide valuable information on the metabolic capabilities of the most studied hydrogen-producing representatives of the Clostridium genus for their use in optimizing the technology for biohydrogen production by dark fermentation of various organic wastes.     

Dark fermentative hydrogen production from food waste: Effect of biomass ash supplementation

This work aimed to investigate the effects of supplementing two distinct types of ash, namely fly ash (FA) and bottom ash (BA) on the dark fermentation (DF) process of food waste (FW) for H₂ production. Both types of biomass combustion ash (BCA) were collected in an industrial bubbling fluidized bed combustor, using residual forest biomass as fuel. Results indicated that adding BCA at different doses of 1, 2 and 4 g/L could effectively enhance H₂ generation when compared to the control test without BCA addition. This stimulatory effect was attributed to the crucial role of metal elements released from BCA such as sodium, potassium, calcium, magnesium, and iron in the provision of buffering capacity and inorganic nutrients for the functioning of hydrogen-forming bacteria. The highest H₂ yield of 169 mL per g of volatile solids (VS) were obtained by adding only a small amount of BA (1 g/L) to the reactive system, representing a significant increment of 1070% compared to the control reactor. Furthermore, a significant decrease in the bacterial lag phase time from 26 h to 2.7 h, as well as about a 12-fold increase in the energy recovery as H₂ gas was observed at BA dosage of 1 g/L in comparison with the control reactor. Overall, this study suggested that a proper addition of BCA could promote the DF process of FW and enhance biohydrogen production.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

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.     

Fermentative hydrogen production from wastewaters: A review and prognosis

Biohydrogen is a promising candidate which can replace a part of our fossil fuels need in day-to-day life due its perceived environmental benefits and availability through dark fermentation of organic substrates. Moreover, advances in biohydrogen production technologies based on organic wastewater conversion could solve the issues related to food security, climate change, energy security and clean development in the future. An evaluation of studies reported on biohydrogen production from different wastewaters will be of immense importance in economizing production technologies. Here we have reviewed biohydrogen production yields and rates from different wastewaters using sludges and microbial consortiums and evaluated the feasibility of biohydrogen production from unexplored wastewaters and development of integrated bioenergy process. Biohydrogen production has been observed in the range of substrate concentration 0.25–160 g COD/L, pH 4–8, temperature 23–60 °C, HRT 0.5–72 h with various types of reactor configuration. The most efficient hydrogen production has been obtained at an organic loading rate (OLR) 320 g COD/L/d, substrate concentration 40 g COD/L, HRT 3 h, pH 5.5–6.0, temperature 35 °C in a continuously-stirred tank reactor system using mixed cultures and fed with condensed molasses fermentation soluble wastewater. The net energy efficiency analysis showed vinasse wastewater has the highest positive net energy gain followed by glycerin wastewater and domestic sewage as 140.39, 68.65, 51.84 kJ/g COD feedstock with the hydrogen yield (HY) of 10 mmol/g COD respectively.     

Enhancing thermophilic dark fermentative hydrogen production at high glucose concentrations via bioaugmentation with Thermotoga neapolitana

The aim of the present study was to investigate the effect of gradually increasing glucose concentrations (from 5.6 to 111 mmol L⁻¹) on the fermentative H₂ production with and without bioaugmentation. A stirred tank reactor (STR) was operated at 70 °C and inoculated with a hyperthermophilic mixed culture or a hyperthermophilic mixed culture bioaugmented with Thermotoga neapolitana. With both the unaugmented (control) and augmented cultures, the H₂ production rate was improved when the initial glucose concentration was increased. In contrast, the highest H₂ yield (1.68 mol H₂ mol⁻¹ glucose consumed) was obtained with the augmented culture at the lowest glucose concentration of 5.6 mmol L⁻¹ and was 37.5% higher than that obtained with the unaugmented culture at the same feed glucose concentration. Overall, H₂ production rates and yields were higher in the bioaugmented cultures than in the unaugmented cultures whatever the glucose concentration. Quantitative polymerase chain reaction targeting T. neapolitana hydA gene and MiSeq sequencing proved that Thermotoga was not only present in the augmented cultures but also the most abundant at the highest glucose concentrations.     

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Predictive and explicative models of fermentative hydrogen production from solid organic waste: Role of butyrate and lactate pathways

Solid organic waste represents an abundant, cheap, and available source of biodegradable substrates not yet exploited to produce biohydrogen by dark fermentation. The impact of the composition of solid organic waste on microbial metabolic pathways and subsequently on biohydrogen production, has not been clearly elucidated. The aim of this study is to determine the compositional features of different substrates that influence bioH2 production. For this, we measured Biological hydrogen potentials (BHP) on 26 different substrates and performed a multivariate statistical analysis of the experimental data using a partial least square regression. The results showed that the BHP values correlated well with the initial carbohydrate content measured after mild hydrolysis. A predictive model explaining more than 89% of the experimental variability was then built to predict the maximal biohydrogen yield with a high accuracy and for a large spectrum of organic waste. An explicative model showed that only carbohydrates, butyrate and lactate concentrations were significant variables explaining more than 98% of biohydrogen yield variability. Interestingly, an interaction term between carbohydrates and lactate concentrations was required to explain microbial pathways producing hydrogen.     

Dark fermentative biohydrogen production from confectionery wastewater in continuous-flow reactors

The production of biohydrogen from industrial wastewater through the dark fermentation (DF) process has attracted increased interest in recent years. To implement a DF process on a large scale, a thorough knowledge of laboratory scale process control is required. The operating parameters and design features of the reactors have a great influence on the efficiency of the process. In this work, the possibility of continuous production of biohydrogen from confectionery wastewater was evaluated. The DF process was carried out at 37 ± 1 °C in two different reactors: an upflow anaerobic filter (AF) and a fluidized bed reactor (AFB). Polyurethane foam (PU) was used to immobilize the biomass. The DF process was studied at four hydraulic retention times (HRT) (1.5, 2.5, 7.5 and 15 days) and the corresponding organic loading rates (OLR) (9.21, 6.12, 2.04 and 1.02 g COD_init/(L day)). The highest hydrogen yield (HY) (44.73 ml/g COD_init) and hydrogen production rate (HPR) (92.5 ml/(L day)) was observed in AFB at HRT of 7.5 days and 2.5 days, respectively. The highest concentration of hydrogen in biogas was 34% in AF and 36% in AFB at HRT of 7.5 days. In contrast to AF, the COD removal efficiency in AFB increased with increasing HRT. The pH of the effluent was low (3.95–4.38). However, due to the use of PU for biomass immobilization, it is possible that there were local zones in the reactor that were optimal for the functioning of not only acidogens, but also methanogens. This was evidenced by a rather high content of methane in biogas (2.5% in AF and 9.6% in AFB at HRT of 15 days). These results provide valuable data for optimizing the continuous DF of wastewater from confectionery and other food industries to produce biohydrogen or biohythane.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Lactic acid as a substrate for fermentative hydrogen production

Soil was used as seed without pretreatment for the fermentation of lactate in order to produce H₂. When the pH was kept at a constant level, H₂ production occurred in three steps. The first was the production of small amounts of H₂ during the initial growth of the microflora. Then, after a reasonably long period of inactivity during the cultivation stage, a large amount of H₂ was produced in additional two steps. It was found that the reduction of the pH after the initial growth phase had a positive effect on the H₂ yield. After cell growth at pH 6 and cultivation at pH 5, a total H₂ yield of 154 mmol l⁻¹ was achieved. It was found that acetate was essential for the rapid H₂ formation. On the other hand, even small initial concentrations of butyrate impeded the H₂ formation.     

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.     

Silage as source of bacteria and electrons for dark fermentative hydrogen production

In this study, grass silage was used both as a source of bacteria and as a substrate for dark fermentative hydrogen production. Silage is produced by lactic acid fermentation controlled by end point pH (<4.0). In this study, the fermentation of silage was successfully continued and directed to hydrogen production by neutralizing the pH. Highest hydrogen yield of 37.8 ± 5.8 mL H₂/g silage was obtained at 25 g/L of silage. The main soluble metabolites were acetate and butyrate with the final concentrations of 1.5 ± 0.2 and 0.5 ± 0.0 g/L, respectively. Bacteria present (at 25 g silage/L) included Ruminobacillus xylanolyticum, Acetanaerobacterium elongatum and Clostridium populeti and were involved in silage fermentation to hydrogen. In summary, this work demonstrates that grass silage becomes amenable to hydrogen fermentation by indigenous silage bacteria through pH neutralization.     

Dark fermentative hydrogen gas production from molasses using hot spring microflora

This study reports hydrogen gas (H₂) production from molasses by hot spring microflora in three stages. During the first two stages most convenient temperature, inoculation percentage (INP) ensuring the highest H₂ yield and rate were determined using suspended culture. Then, H₂ was produced by the same culture immobilized on porous ceramic rings at three different hydraulic retention times. For the suspended culture experiments, the most effective H₂ production resulting 202.32 mL H₂/g COD was obtained at 37 °C with 10 INP. The highest H₂ formation of 534.35 mLH₂/d was realized for the biofilm culture at 0.53-day hydraulic retention time and H₂ production using hot spring microflora in biofilm form was found to be promising. The pH of the experiments remained stable around 5.5–6.5 without a requirement for pH adjustment during the fermentation.     

Effect of enzyme addition on fermentative hydrogen production from wheat straw

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H₂), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H₂ usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H₂ production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H₂ production from untreated wheat straw ranged from 5.18 to 10.52 mL-H₂ g-VS⁻¹. Whatever the experimental enzyme addition procedure, a two-fold increase in H₂ production yields ranging from 11.06 to 19.63 mL-H₂ g-VS⁻¹ was observed after enzymatic treatment of the wheat straw. The high variability in H₂ yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H₂ production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw⁻¹.     

Optimization and modelling of dark fermentative hydrogen production from cheese whey by Enterobacter aerogenes 2822

In India, annually about 3.3–5 million tons of cheese whey is produced which may causes serious problems for the environment if left untreated. In this study, pretreated cheese whey was utilized to produce hydrogen via dark fermentation by Enterobacter aerogenes 2822 cells in 2 L double walled cylindrical bioreactor having working volume of 1.5 L. Effect of change in total carbohydrate concentration in cheese whey (CW_TC, 20–45 g L^?1), temperature (T, 25–37 °C) and pH (5.5–7.5) was investigated on volumetric hydrogen production rate (VHPR) using Box Behnken design (BBD). Experimental VHPR of 24.7 mL L^?1 h^?1 was attained at an optimum concentration of 32.5 g L^?1 CW_TC, 31 °C T and 6.5 pH, which was in good correlation with predicted rate of 23.2 mL L^?1 h^?1. Mathematical models based on Monod and logistic equations were developed to describe the kinetics of substrate consumption and growth profile of E. aerogenes 2822 under optimum conditions. While for the modelling of fermentative hydrogen production in batch mode, Modified Gompertz equation and Leudeking-Piret models were used which gave proper simulated fitting. These results will add significant values to cheese whey by converting it into a clean form of bioenergy.     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.