Hydrogen production from agricultural waste by dark fermentation: A review

The degradation of the natural environment and the energy crisis are two vital issues for sustainable development worldwide. Hydrogen is considered as one of the most promising candidates as a substitute for fossil fuels. In this context, biological processes are considered as the most environmentally friendly alternatives for satisfying future hydrogen demands. In particular, biohydrogen production from agricultural waste is very advantageous since agri-wastes are abundant, cheap, renewable and highly biodegradable. Considering that such wastes are complex substrates and can be degraded biologically by complex microbial ecosystems, the present paper focuses on dark fermentation as a key technology for producing hydrogen from crop residues, livestock waste and food waste. In this review, recent findings on biohydrogen production from agricultural wastes by dark fermentation are reported. Key operational parameters such as pH, partial pressure, temperature and microbial actors are discussed to facilitate further research in this domain.     

Enhancement of fermentative biohydrogen production from textile desizing wastewater via coagulation-pretreatment

A real textile desizing wastewater (TDW) was coagulation-pretreated to enhance its potential of biohydrogen production. Batch fermentation showed that the hydrogen production was efficiently enhanced (550 and 120% increments for hydrogen production rate and hydrogen yield, respectively) and the production performance was substrate-concentration dependent. A peak hydrogen production rate of 3.9 L/L-d and hydrogen yield of 1.52 mol/mol hexose were obtained while using coagulant GGEFloc-653 at a dosage of 1 g/L to pretreat TDW with the concentration of 15 g total sugar/L. The coagulation-pretreatment could have butyrate-type fermentation with high biohydrogen production and the removed some toxic materials that might drive the metabolic pathways to those not favoring biohydrogen production. Based on the data obtained, strategies to operate the coagulation and biohydogen fermentation are suggested. Moreover, fermentation effluent utilization such as for two-stage biogas production and further biohythane (a mixture of H₂ and CH₄) generation are also elucidated.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

Ultrasonication for biohydrogen production from food waste

The applicability of ultrasonication for enhancement of hydrogen production from food wastes was evaluated in three different systems. System A is a conventional continuously-stirred tank reactor (CSTR) fed raw food waste; system B is a conventional continuously-stirred tank reactor fed sonicated food waste, and system C (US patent-pending) is the sonicated biological hydrogen reactor (SBHR) which comprised a CSTR connected with an ultrasonic probe at the bottom of the reactor. In this study, the increase in hydrogen production rate relative to the control (system A) due to sonication of the feed before the digestion was 27%, compared to 90% in the SBHR. Similarly, the CSTR with sonicated feed exhibited a 23% increase in hydrogen yield as mol H₂/mol hexose_consumed compared to a 62% increase in the SBHR relative to the control (system A). The VSS destruction in the SBHR was higher than those in the CSTR and CSTR with sonicated feed by 50% and 60%, respectively.     

Effect of ammonia on biohydrogen production from food waste via anaerobic fermentation

The effect of different additive ammonia (0–10 g/l as nitrogen) on hydrogen production from the anaerobic batch mesophilic fermentation of food waste was studied at two feed-to-microorganism ratios (F/M), 3.9 and 8.0. Anaerobic sludge taken from an anaerobic digester was used as inoculum. The hydrogen yield at F/M 3.9 and 8.0 without additive ammonia was 77.2 and 51.0 ml-H₂/gVS, respectively. At F/M 3.9, the hydrogen production was enhanced by adding additive ammonia in the system when the total ammonia nitrogen (TAN) concentration was no higher than 6.0 g/l. A maximum hydrogen yield of 121.4 ml-H₂/gVS was obtained at a TAN concentration of 3.5 g/l. At F/M 8.0, the enhancement of hydrogen production was found in a narrower range of additive TAN concentrations, with a highest yield of 60.9 ml-H₂/gVS at the TAN of 1.5 g/l. Hydrogen production was inhibited at higher additive TAN concentrations for both F/M ratios. This study provides a novel strategy for controlling ammonia for production of hydrogen from food waste via anaerobic fermentation.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Characteristics of the process of biohydrogen production from simple and complex substrates with different biopolymer composition

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 °C. Peptone and sunflower oil were characterized by the lowest production of H₂, namely 5.0 and 2.3 ml H₂/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H₂/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H₂/g COD or 143.4 ml H₂/g carbohydrates; from pig feed – 32.1 ml H₂/g COD or 91.6 ml H₂/g carbohydrates; and from sewage sludge – 9.3 ml H₂/g COD or 98.0 ml H₂/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H₂, its content in biogas, as well as the ratio of H₂/soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate.     

Ferric oxide/date seed activated carbon nanocomposites mediated dark fermentation of date fruit wastes for enriched biohydrogen production

Biohydrogen production from biomass waste, not only addresses the energy demand in a renewable manner but also resolves the safe disposal issues associated with these biowastes. Also, scalable and low-cost techniques to enhance biohydrogen production have gained more attraction and are highly explored. In this research work, date-palm fruit wastes have been studied for their biohydrogen production potential using Enterobacter aerogenes by dark fermentation. Hydrogen yield and productivity were improved through the addition of iron oxide nanoparticles (Fe₃O₄ NPs) and its date seed activated carbon nanocomposites (Fe₃O₄/DSAC) to the fermentation media. Studies on discrete inclusions of these NPs showed that the appropriate dosage of NPs promoted, while higher dosages repressed the hydrogen production performance. Optimal dosage and fermentation time was observed as 150 mg/L and 24 h for both the additives. Fe₃O₄/DSAC nanocomposites showed better hydrogen production enhancement than Fe₃O₄ NPs. Maximum hydrogen yield of 238.7 mL/g was obtained for the 150 mg/L nanocomposites, which was 65.7% higher than that of the standalone Fe₃O₄ NPs and three folds higher than the yield of the control run without any NPs inclusion (78.4 mL/g). Metabolites analysis showed that the hydrogen evolution followed the ethanol-acetate pathway. Formation levels of longer chain propionate and butyrate co-metabolites were significantly low in the presence of Fe₃O₄/DSAC than Fe₃O₄. The carbon support in the nanocomposites acted as an adsorbent-buffer, which favored the medium pH in-addition to the stimulatory effects of Fe₃O₄ NPs. Cell growth and specific hydrogenase activity analysis were also performed to supplement the hydrogen production results. Gompertz and modified Logistic kinetic models were employed for kinetic modeling of experimental hydrogen production values. The Fe₃O₄/DSAC nanocomposites exhibited significant application potential for the production of biohydrogen from date fruit wastes.     

High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant

A mesophilic high hydrogen producing strain DMHC-10 was isolated from a lab scale anaerobic reactor being operated on distillery wastewater for hydrogen production. DMHC-10 was identified as Clostridium sp. on the basis of 16S rRNA gene sequencing. Various medium components (carbon and nitrogen sources) and environmental factors (initial pH, temperature of incubation) were optimized for hydrogen production by Clostridium sp. DMHC-10. The strain, in late exponential growth phase, showed maximum hydrogen production (3.35 mol-H₂ mol⁻¹ glucose utilized) at 37 °C, pH 5.0 in a medium supplemented with organic nitrogen source. Butyric acid to acetic acid ratio was ca. 2.3. Hydrogen production declined when organic nitrogen was replaced with inorganic nitrogen.     

The effect of HRT on biohydrogen production from acid hydrolyzed waste wheat in a continuously operated packed bed reactor

Biohydrogen production in a continuously operated up flow packed bed reactor was investigated at different hydraulic retention time (HRT) varying between 2 h and 13 h scouring sponge pad. The substrate was sugar solution obtained from hydrolysis of waste wheat at pH = 2 and 90 °C in an autoclave for 15 min. Experimental results indicated that hydrogen production volume and yield increases with decreasing HRT. The highest volumetric hydrogen production rate and yield were obtained as VHPR = 1.75 L H₂/L d and Y_H2 = 1.6 moL H₂/mol TS, respectively, at HRT = 2 h. Yields and rates at HRT = 2 h were almost two times of that obtained at HRT = 13 h. It can be concluded that metal mesh covered plastic scouring sponge pad is a suitable microorganism support particle to obtain high hydrogen yield and rate at short HRTs by dark fermentation.     

Mesophilic biohydrogen production from calcium hydroxide treated wheat straw

The use of Ca(OH)₂ pre-treatment to improve fermentative biohydrogen yields, from wheat straw was investigated. Wheat straw was pre-treated with 7.4% (w/w) Ca(OH)₂ at ambient temperature (20 °C) for 2, 5, 8, and 12 days, prior to 35 °C fermentation with sewage sludge inoculum. Biohydrogen yields were evaluated during dark fermentation and simultaneous saccharification fermentation (SSF) of total pre-treated straw material and compared to those from separated solid and hydrolysate fractions. Ca(OH)₂ pre-treatment followed by SSF, exhibited a synergetic relationship. On average, 58.78 mL-H₂ g-VS⁻¹ was produced from SSF of pre-treated and filtered solids. This was accompanied by approximately a 10-fold increase in volatile fatty acid production, compared to the untreated control. By omitting pre-treatment hydrolysate liquors from SSF, H₂ production increased on average by 35.8%, per VS of harvested straw. Additional inhibition studies indicated that CaCO₃, formed as a result of pre-treatment pH control, could promote homoacetogenesis and reduce biohydrogen yields.     

Ultrasonic pretreatment for an enhancement of biohydrogen production from complex food waste

The efficacy of ultrasonication pretreatment method for complex food waste prior to anaerobic digestion is evaluated for enhancement of H₂ yield (HY) and rate (R). The RSM results showed that the ultimate H₂ production increased with increasing TS content and ultrasonication time (UT). Desirability function integrated with RSM predicted an optimum condition of TS and UT as: 8% TS and 12 min, for maximization of HY and R. The highest HY, 149 mL/g VS_added, and R, 5.23 mL/h, were achieved during the verification test at optimized conditions. Furthermore, a significant decreased lag phase followed by highest molar HBu/HAc ratio (2.2) was also achieved at optimized conditions with lowest specific energy input (13,500 kJ/kg TS). The significant relative enhancement of HY, 75%, and R, 104%, implies that ultrasonically pretreated complex food waste with higher TS loading is about 1.7–2.1 times more effective for enhanced bioH₂ production compared to unsonicated food waste.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Co-digestion of corn (nejayote) and brewery wastewater at different ratios and pH conditions for biohydrogen production

The capacity of a heat-treated sludge (HTS) to produce hydrogen from the mono- and co-digestion of corn (NW, pH 13.1) and brewery (BW, pH 3.8) wastewater was evaluated. The co-digestion of NW and BW was conducted with ratios (NW/BW) from 40/60 to 80/20 (vol/vol) at pH 6 and under different initial pH values (from 5.8 to 12.3) according to the substrates mixtures. With the initial pH adjusted to 6, the highest production (302 mL) occurred for the mono-digestion of NW, but hydrogen was produced in all incubations. For incubations under variable pH values, the highest hydrogen production was obtained with the ratio 60/40 (270 mL), followed by the ratio 65/35 (260 mL) with pH values of 10.8 and 10.4, respectively. The initial pH influenced the kinetic parameters, especially on maximum production and lag phase. For the ratio 60/40, with an alkaline pH value (10.4), the lag phase was delayed up to 122 h, but the highest volume of hydrogen was obtained with this condition. The Clostridium genus, present in all samples, could be associated as the main responsible for hydrogen production. Besides, the presence of Burkholderia genus, previously related to hydrogen production, was identified as the main involved in the culture at pH 10.8.     

A pilot-scale high-rate biohydrogen production system with mixed microflora

A pilot-scale high-rate dark fermentative hydrogen production plant has been established in the campus of Feng Chia University to develop biohydrogen production pilot-plant technology. This pilot-plant system is composed of two feedstock storage tanks (0.75 m³ each), a nutrient storage tank (0.75 m³), a mixing tank (0.6 m³), an agitated granular sludge bed fermentor (working volume 0.4 m³), a gas-liquid-solid separator (0.4 m³) and a control panel. The seed mixed microflora was obtained from a lab-scale agitated granular sludge bed bioreactor. This pilot-scale fermentor was operated for 67 days at 35 °C, an organic loading rate (OLR) of 40-240 kg COD/m³/d, and the influent sucrose concentration of 20 and 40 kg COD/m³. Both biogas and hydrogen production rates increased with increasing OLR. However, the biomass concentration (volatile suspended solids, VSS) only increased with an increasing OLR at an OLR range of 40-120 kg COD/m³/d, whereas it decreased when OLR was too high (i.e., 240 kg COD/m³/d). The biogas consisted mainly of H₂ and CO₂ with a H₂ content range of 23.2-37.8%. At an OLR of 240 kg COD/m³/d, the hydrogen content in biogas reached its maximum value of 37% with a hydrogen production rate (HPR) of 15.59 m³/m³/d and a hydrogen yield of 1.04 mol H₂/mol sucrose. This HPR value is much higher than 5.26 m³/m³/d (fermented molasses substrate) and 1.56 m³/m³/d (glucose substrate) reported by other pilot-scale systems. Moreover, HPR was also greatly affected by pH. At an optimal pH of 5.5, the bacterial community became simple, while the efficient hydrogen producer Clostridium pasteurianum was dominant. The factors of energy output compared with the energy input (E_f) ranged from 13.65 to 28.68 on biohydrogen, which is higher than the E_f value on corn ethanol, biodiesel and sugarcane ethanol but in the similar range of cellulosic ethanol.     

Effect of system optimizing conditions on biohydrogen production from herbal wastewater by slaughterhouse sludge

The study evaluates the biohydrogen production from herbal wastewater as the substrate by the enriched mixed slaughterhouse sludge as the seed source. In the following experiments, batch-fermentations are carried out with the optimum substrate concentrations, fermentation pH and fermentation temperature to observe the effects of H₂ production, hydrogen yield and other fermentation end products at different conditions. The hydrogen production is increased as substrate concentration increased up to 8 g COD/L WW, but drastically decreased at 10 g COD/L WW. When the pH of fermentation is controlled to 6.5, a maximum amount of hydrogen yield could be obtained. The hydrogen production is maximum at 50 °C (930 ± 30 mL/L WW) compared to 30 °C (436 ± 16 mL/L WW). Acid-forming pathway with butyric acid as a major metabolite dominated the metabolic flow during the hydrogen production. The experimental results indicated that effective hydrogen production from the herbal wastewater could be obtained by thermophilic acidogenesis at proper operational conditions.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Mathematical modelling for biohydrogen production by Clostridium beijerinckii

Hydrogen is an energy source that can be produced by Clostridium sporogenes microorganism. In the present work, modeling of dark fermentation using Clostridium beijerinckii and dextrose as substrate was performed to evaluate how the gases and liquid by-products affect the biological process. A mathematical model was developed according to ADM1. The developed model takes into account biochemical reactions, physicochemical equilibrium as well as mass transfer processes during dark fermentation. Findings revealed that Clostridium beijerinckii reached a yield as high as 3.58 mol of H₂/mol of dextrose and generates by-products in the aqueous phase that may either be used as raw materials in a chemical process. Clostridium beijerinckii is very sensitive to acid media (pH < 5.0) and shows a low rate of biohydrogen production (even the absence of metabolic activity) at pH lower than 4.5. The developed model is able to predict (R² > 0.95) dextrose consumption profile, cumulative biohydrogen production and the maximum concentrations of liquid by-products.     

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h^?1), a higher butyric acid production of 6.8 g·L^?1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h^?1, with a constant hydrogen production of 507 mL·h^?1, corresponding to a volumetric rate of 6.10 L·L^?1 d^?1, and a yield of 2.39 mol of H₂ per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.