Continuous dark fermentative hydrogen production by mesophilic microflora: Principles and progress

Continuous, dark fermentative hydrogen production technology using mixed microflora at mesophilic temperatures may be suitable for commercial development. Clostridial-based cultures from natural sources have been widely used, but more information on the need for heat treatment of inocula and conditions leading to germination and sporulation are required. The amount of nutrients given in the literature vary widely. Hydrogen production is reported to proceed without methane production in the reactor in the pH range 4.5–6.7, with hydraulic retention times optimally between a few hours and 3 days depending on substrate. Higher substrate concentrations should be more energy-efficient but there are product inhibition limitations, for example from unionised butyric acid. Inhibition by H₂ can be reduced by stirring, sparging or extraction through membranes. Of the reactor types investigated, while granules have the best performance with soluble substrate, for particulate feedstock biofilm reactors or continuous stirred tank reactors may be most successful. A second stage is required to utilise the fermentation end products which, when cost-effective reactors are developed, may be photofermentation or microbial fuel cell technologies. Anaerobic digestion is a currently-available technology and the two-stage process is reported to give greater conversion efficiency than anaerobic digestion alone.     

Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures

Batch tests were conducted to evaluate the enhancement of hydrogen/ethanol (EtOH) productivity using cow dung microflora to ferment α-cellulose and saccharification products (glucose and xylose). Hydrogen/ethanol production was evaluated based on hydrogen/ethanol yields (HY/EY) under 55 °C at various initial pH conditions (5.5–9.0). Our test results indicate that cow dung sludge is a good mixed natural-microflora seed source for producing biohydrogen/ethanol from cellulose and xylose. The heat-pretreatment, commonly used to produce hydrogen more efficiently from hexose, applied to mixed anaerobic cultures did not help cow dung culture convert cellulose and xylose into hydrogen/ethanol. Instead of heat-pretreatment, the mixed culture received enrichments cultivated at 55 °C for 4 days. Positive results were observed: hydrogen/ethanol production from fermenting cellulose and xylose was effectively enhanced at increases of 4.8 (ethanol) to 8 (hydrogen) and 2.4 (ethanol) to 15.6 (hydrogen) folds, respectively. In which, the ethanol concentration produced from xylose reached 4–4.4 g/L, an output comparable to that of using heat-treated sewage sludge and better than that (1.25–3 g/L) using pure cultures. Our test results show that for the enriched cultures the initial cultivation pH can affect hydrogen/ethanol production including HY, EY and liquid fermentation product concentration and distribution. These results were also concurred using a denaturing gradient gel electrophoresis analysis saying that both cultivation pH and substrate can affect the enriched cow dung culture microbial communities. The enriched cow dung culture had an optimal initial cultivation pH range of 7.6–8.0 with peak HY/EY values of 2.8 mmol-H₂/g-cellulose, 5.8 mmol-EtOH/g-cellulose, 0.3 mol-H₂/mol-xylose and 1 mol-EtOH/mol-xylose. However, a pH change of 0.5 units from the optimal values reduced hydrogen/ethanol production efficiency by 20%. Strategies based on the experimental results for optimal hydrogen/ethanol production from cellulose and xylose using cow dung microflora are proposed.     

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.     

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.     

Modeling fermentative hydrogen production from sucrose supplemented with dairy manure

Our previous studies had shown that fermentative hydrogen production from sucrose could be improved with dairy manure as a supplement. In addition to contributing to nearly 10% more hydrogen yield at ambient temperature, dairy manure was shown to be capable of providing the required nutritional needs, buffering capacity, and hydrogen-producing organisms, improving the practical viability of fermentative hydrogen production. In this report, we present a kinetic model for fermentative hydrogen production from sucrose supplemented with dairy manure. This model includes hydrogen production from sucrose as well as from the soluble products hydrolyzed from particulate manure. The integrated model was calibrated using experimental data from one batch reactor and validated with dissolved COD, hydrogen, and volatile fatty acid data from four other reactors. Predictions by this model agreed well with the temporal trends in the experimental data, with r² averaging 0.85 for dissolved COD; 0.94 for total COD; 0.84 for hydrogen; 0.84 for acetic acid; and 0.89 for butyric acid; quality of fit in the case of propionic acid was lower with r² averaging 0.57.     

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.     

木糖厌氧发酵产氢特性研究

以木糖作为厌氧发酵产氢底物,热预处理(100℃,处理20 min)的厌氧颗粒污泥作为接种物,研究了中温条件(37℃)下厌氧发酵产氢特性.结果表明,当反应进行至50 h时,累积产氢量最大,为81.11 mL;乙酸、丁酸和乙醇是液相末端产物中的主要物质,其中乙酸和丁酸的浓度分别为1290 mg/L和1225 mg/L,发酵类型是典型的丁酸型发酵;反应体系的pH值开始降低,最后稳定在4.40左右,形成一个稳定的缓冲体系.     

Modeling of fermentative hydrogen production from sweet sorghum extract based on modified ADM1

The Anaerobic digestion model 1 (ADM1) framework can be used to predict fermentative hydrogen production, since the latter is directly related to the acidogenic stage of the anaerobic digestion process. In this study, the ADM1 model framework was used to simulate and predict the process of fermentative hydrogen production from the extractable sugars of sweet sorghum biomass. Kinetic parameters for sugars’ consumption and yield coefficients of acetic, propionic and butyric acid production were estimated using the experimental data obtained from the steady states of a CSTR. Batch experiments were used for kinetic parameter validation. Since the ADM1 does not account for metabolic products such as lactic acid and ethanol that are crucial during the fermentative hydrogen production process, the structure of the model was modified to include lactate and ethanol among the metabolites and to improve the predictions. The modified ADM1 simulated satisfactorily batch experiments although further modifications could be made in order to further improve the predictions for the hydrogenogenic process.     

Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: Effects of temperature and pH

Fermentative hydrogen production from cassava stillage was conducted to investigate the influences of temperature (37 °C, 60 °C, 70 °C) and initial pH (4–10) in batch experiments. Although the seed sludge was mesophilic anaerobic sludge, maximum hydrogen yield (53.8 ml H₂/gVS) was obtained under thermophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme-thermophilic (70 °C) conditions respectively. The difference was mainly due to the different VFA and ethanol distributions. Higher hydrogen production corresponded with higher ratios of butyrate/acetate and butyrate/propionate. Similar hydrogen yields of 66.3 and 67.8 ml H₂/gVS were obtained at initial pH 5 and 6 respectively under thermophilic condition. The total amount of VFA and ethanol increased from 3536 to 7899 mg/l with the increase of initial pH from 4 to 10. Initial pH 6 was considered as the optimal pH due to its 19% higher total VFA and ethanol concentration than that of pH 5. Homoacetogenesis and methonogenesis were very dependent on the initial pH and temperature even when the inoculum was heat-pretreated. Moreover, a difference between measured and theoretical hydrogen was observed in this study, which could be attributed to homoacetogenesis, methanogenesis and the degradation of protein.     

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.     

Value analysis for commercialization of fermentative hydrogen production from biomass

In addition to producing hydrogen gas, biohydrogen production is also used to process wastewater. Therefore, this study specifically conducted value analyses of two different scenarios of fermentative hydrogen production from a biomass system: to increase the value of a wastewater treatment system and to specifically carry out hydrogen production. The analytical results showed that fermentative hydrogen production from a biomass system would increase the value of a wastewater treatment system and make its commercialization more feasible. In contrast, fermentative hydrogen production from a biomass system designed specifically for producing hydrogen gas would have a lower system value, which indicated that it is not yet ready for commercialization. The main obstacle to be overcome in promoting biohydrogen production technology and system application is the lack of sales channels for the system's products such as hydrogen gas and electricity. Thus, in order to realize its commercialization, this paper suggests that governments provide investment subsidies for the use of biohydrogen production technology and establish a buy-back tariff system for fuel cells.     

Effect of gas sparging on continuous fermentative hydrogen production

The effect of gas sparging on continuous fermentative H₂ production was investigated in completely stirred-tank reactors (CSTR) using internal biogas, N₂ and CO₂ with various flow rates (100, 200, 300 and 400 ml/min). The sparging with external gases of N₂ and CO₂ showed higher H₂ yield than the control of no sparging and internal biogas sparging. It indicated that the decrease of H₂ partial pressure by external gas sparging had a beneficial effect on H₂ fermentation. Especially, CO₂ sparging was more effective in the reactor performance than N₂ sparging, accompanied by higher production of H₂ and butyrate. The best performance was obtained by CO₂ sparging at 300 ml/min, resulting in the highest H₂ yield of hexose_consumed and the maximum specific H₂ production rate of 6.89 L H₂/g VSS/day. Compared to N₂ sparging, there might be another positive effect in CO₂ sparging apart from lowering H₂ partial pressure. High CO₂ partial pressure had little effect on H₂-producing bacteria but inhibitory effect on other microorganisms such as acetogens and lactic acid bacteria which were competitive with H₂-producing bacteria. Only H₂-producng bacteria, such as Clostridium tyrobutyricum, C. proteolyticum and C. acidisoli were isolated under CO₂ sparging conditions based on 16S rDNA analysis by PCR-DGGE.     

Biohydrogen production from specified risk materials co-digested with cattle manure

Biohydrogen production from the anaerobic digestion of specified risk materials (SRM) co-digested with cattle manure was assessed in a 3 × 5 factorial design. Total organic loading rates (OLR) of 10, 20, and 40 g L⁻¹ volatile solids (VS) were tested using manure:SRM (wt/wt) mixtures of 100:0 (control), 90:10, 80:20, 60:40, and 50:50 using five 2 L continuously stirred biodigesters operating at 55 °C. Gas samples were taken daily to determine hydrogen production, and slurry samples were analyzed daily for volatile fatty acid (VFA) concentration, total ammonia nitrogen (TAN), and VS degradation. Hydrogen production (mL g⁻¹ VS fed) varied quadratically according to OLR (P < 0.01), with maximum production at OLR20, while production decreased linearly (P < 0.0001) as SRM concentration increased. Reduced hydrogen production associated with SRM inclusion at >10% VS may be attributed to a rapid increase in TAN (r = −0.55) or other inhibitors such as long chain fatty acids. Reduced hydrogen production (P < 0.01) at OLR40 versus OLR20 may be related to increased rate of VFA accumulation and final VFA concentration (P < 0.001), as well as inhibition due to hydrogen accumulation (P < 0.001). Biohydrogen production from SRM co-digested with cattle manure may not be feasible on an industrial scale due to reduced hydrogen production with increasing levels of SRM.     

Recent advance in inhibition of dark fermentative hydrogen production

Dark fermentative hydrogen production is an effective and feasible technology for biological hydrogen production. However, this technology has not been commercially applied yet. One of the major reasons is that several inhibitory factors limit hydrogen production and the commercial potential. In this review paper, the various inhibitory factors which influence the dark fermentation hydrogen production were systematically analyzed and summarized, including inorganic inhibitors (heavy metal ions, light metal ions, ammonia, sulfate and hydrogen gas), organic inhibitors (volatile fatty acids, furan derivative and phenolic components), and bio-inhibitors (bacteriocins and thiosulfinate). The inhibitory concentration and mechanism were discussed in-depth and comprehensively. The strategies for mitigating these inhibitory factors were also introduced and discussed. Suggestion for future study in this aspect was proposed to promote the scale-up and commercial application of dark fermentative hydrogen production.     

Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract

The aim of the present study was to assess the influence of substrate concentration on the fermentative hydrogen production from sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time (HRT) of 12 h and carbohydrate concentrations ranging from 9.89 to 20.99 g/L, in glucose equivalents. The maximum hydrogen production rate and yield were obtained at the concentration of 17.50 g carbohydrates/L and were 2.93 ± 0.09 L H₂/L reactor/d and 0.74 ± 0.02 mol H₂/mol glucose consumed, corresponding to 8.81 ± 0.02 L H₂/kg sweet sorghum, respectively. The main metabolic product at all steady states was butyric acid, while ethanol production was high at high substrate concentrations. The experiments showed that hydrogen productivity depends significantly on the initial carbohydrate concentration, which also influences the distribution of the metabolic products.