Trace metals supplementation enhanced microbiota and biohythane production by two-stage thermophilic fermentation

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L⁻¹, respectively) with maximum H₂ and CH₄ yields of 139 mL H₂ gVS⁻¹ and 454 mL CH₄ gVS⁻¹, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H₂ stage and enhancing number methanogens in the CH₄ stage.     

Effect of pH control on biohythane production and microbial structure in an innovative multistage anaerobic hythane reactor (MAHR)

An innovative multistage anaerobic hythane reactor (MAHR) which combines an internal biofilm (M_H) and an external up-flow sludge blanket (M_M) was proposed to produce biohythane from wastewater. The effect of pH on its biohythane production and microbial diversity was performed. Results showed that the maximum hydrogen production rate (4.900 L/L/d) was achieved at a pH of 6.0, in comparison to a maximum methane production rate of 10.271 L/L/d at a pH of 6.5. In addition, a suitable hythane (H₂/(H₂+CH₄) of 16.06%) production can be achieved in M_H after the initial pH was adjusted from 7.0 to 6.5, and a relatively high methane yield (271.34 mL CH₄/gCOD) was obtained in M_M. Illumina Miseq sequencing results revealed that decreasing pH led to an increase of the acidogenesis families (Eubacteriaceae, Ruminococcaceae) in M_H and an increase of hydrogenotrophic methanogens (Methanobacteriaceae) in M_M. The Methanosaetaceae gradually occupied a major portion after a long period of recovery. This work demonstrated the unique advantages of MAHR for the biohythane production under optimal pH conditions.     

Hydrogen and methane production from untreated rice straw and raw sewage sludge under thermophilic anaerobic conditions

Bioenergy produced from co-digestion of sewage sludge (SS) and rice straw (RS) as raw materials, without pretreatment and additional nutrients, was compared for the one-stage system for producing methane (CH₄) and the two-stage system for combined production of hydrogen (H₂) and CH₄ in batch experiments under thermophilic conditions. In the first stage H₂ fermentation process using untreated RS with raw SS, we obtained a high H₂ yield (21 ml/g-VS) and stable H₂ content (60.9%). Direct utilization of post-H₂ fermentation residues readily produced biogas, and significantly enhanced the CH₄ yield (266 ml/g-VS) with stable CH₄ content (75–80%) during the second stage CH₄ fermentation process. Overall, volatile solids removal (60.4%) and total bioenergy yield (8804 J/g-VS) for the two-stage system were 37.9% and 59.6% higher, respectively, than the one-stage system. The efficient production of bioenergy is believed to be due to a synergistically improved second stage process exploiting the well-digested post-H₂ generation residues over the one-stage system.     

Hydrogen and methane yields of untreated, water-extracted and acid (HCl) treated maize in one- and two-stage batch assays

In the present study, two-stage H₂ and CH₄ production was compared with one-stage CH₄ production from maize subjected to water extraction and acid (HCl) treatment. In addition, the effect of duration (2 and 14 days) of the first-stage H₂ process on the H₂ yields and subsequent CH₄ yields from the second-stage was also investigated. Results showed that the average H₂ yields from untreated maize were 5.6 and 9.9 ml/g volatile solids added (VS_added) after 2 and 14 days, respectively. On the other hand, H₂ yields from water-extracted and HCl-treated maize were 18.0 and 20.5 ml/gVS_added (14 d), respectively. On comparison to one-stage CH₄ assays, the average increase in CH₄ yields from two-stage assays with 2 d H₂ stage were 7, 9 and 27% for untreated, water-extracted and HCl-treated maize, respectively.     

One-stage H2 and CH4 and two-stage H2 + CH4 production from grass silage and from solid and liquid fractions of NaOH pre-treated grass silage

In the present study, mesophilic CH₄ production from grass silage in a one-stage process was compared with the combined thermophilic H₂ and mesophilic CH₄ production in a two-stage process. In addition, solid and liquid fractions separated from NaOH pre-treated grass silage were also used as substrates. Results showed that higher CH₄ yield was obtained from grass silage in a two-stage process (467 ml g⁻¹ volatile solids (VS)_original) compared with a one-stage process (431 ml g⁻¹ VS_original). Similarly, CH₄ yield from solid fraction increased from 252 to 413 ml g⁻¹ VS_original whereas CH₄ yield from liquid fraction decreased from 82 to 60 ml g⁻¹ VS_original in a two-stage compared to a one-stage process. NaOH pre-treatment increased combined H₂ yield by 15% (from 5.54 to 6.46 ml g⁻¹ VS_original). In contrast, NaOH pre-treatment decreased the combined CH₄ yield by 23%. Compared to the energy value of CH₄ yield obtained, the energy value of H₂ yield remained low. According to this study, highest CH₄ yield (495 ml g⁻¹ VS_original) could be obtained, if grass silage was first pre-treated with NaOH, and the separated solid fraction was digested in a two-stage (thermophilic H₂ and mesophilic CH₄) process while the liquid fraction could be treated directly in a one-stage CH₄ process.     

Pilot-scale of biohythane production from palm oil mill effluent by two-stage thermophilic anaerobic fermentation

The pilot-scale of two-stage thermophilic (55 °C) for biohythane production from palm oil mill effluent (POME) was operated at hydraulic retention time (HRT) of 2 days and organic loading rate (OLR) of 27.5 gCOD/L⋅d) for first stage and HRT of 10 days and OLR of 5.5 gCOD/L⋅d for second stage. Biohythane production rate was 1.93 L-gas/L⋅d with biogas containing 11% H₂, 37% CO₂, and 52% CH₄. Recirculation of methane effluent mixed with POME at a ratio of 1:1 can control pH in the first stage at an optimal range of 5.0–6.5. Microbial community in hydrogen stage dominated by Thermoanaerobacterium sp., while methane stage dominated by Methanosarcina sp. The H₂/CH₄ ratio of biohythane was 0.13–0.18 which suitable for vehicle fuel. Biohythane production from POME could be promising cleaner biofuel with flexible and controllable H₂/CH₄ ratio.     

An approach of auxiliary carbohydrate source on stabilized biohythane production and energy recovery by two-stage anaerobic process from swine manure

In this study, a two-stage biohythane production system was used to treat swine manure to solve the high Chemical Oxygen Demand (COD) concentration and verify the total energy recovery between the two-stage and a traditional single-stage system. Experiments were carried out in single-stage methane production, two-stage biohythane production in long Hydraulic Retention Time (HRT), and short HRT. The COD removal efficiency and energy recovery were finally compared between single-stage (CH₄ fermenter) and two-stage (H₂+ CH₄ fermenter) systems. The results showed that the methane production rate of 53.2 ± 2.7 mL/d.L, the COD removal efficiency of 29.6 ± 5.8%, and total energy recovery of 2.9 ± 0.1 kJ/L.d was obtained in the single-stage of methane production system with HRT 11.08 d, pH 7, and temperature 55 °C, respectively. In the two-stage of hydrogen and methane productions system, the hydrogen production rate of 1.8 ± 0.7 mL/d.L, the methane production rate of 65.7 ± 2.5 mL/d.L, the COD removal rate was 97.8 ± 1.7%, and the total energy recovery of 3.6 ± 0.1 kJ/L.d was obtained and stabilized when the sugary wastewater content gradually reduced to 0%. This study shows that the methane production rate increases 20%, COD removal efficiency increases to 97.8 ± 1.7%, and total energy recovery increases 30%. At the same time, the single-stage (CH₄ fermenter) switched to a two-stage (H₂+ CH₄ fermenter) system. The two-stage anaerobic biohythane production system successfully treated the high organic swine manure and obtained a higher energy recovery against the traditional single-stage of the biomethane production system.     

The effects of fuel variability on the electrical performance and durability of a solid oxide fuel cell operating on biohythane

Biohythane is typically composed of 60/30/10 vol% CH₄/CO₂/H₂ and can be produced via two-stage anaerobic digestion of renewable and low carbon biomass with much greater efficiency compared with CH₄/CO₂ biogas. This work investigates the effects of fuel variability on the electrical performance and fuel processing of a commercially available anode supported solid oxide fuel cell (SOFC) operating on biohythane mixtures at 750 °C. Cell electrical performance was characterised using current-voltage curves and electrochemical impedance spectroscopy. Fuel processing was characterised using quadrupole mass spectroscopy. It is shown that when H₂/CO₂ is blended with CH₄ to make biohythane, the SOFC efficiency is significantly increased, high SOFC durability is achieved, and there are considerable savings in CH₄ consumption. Enhanced electrical performance was due to the additional presence of H₂ and promotion of CH₄ dry reforming, the reverse Boudouard and reverse water-gas shift reactions. These processes alleviated carbon deposition and promoted electrochemical oxidation of H₂ as the primary power production pathway. Substituting 50 vol% CH₄ with 25/75 vol% H₂/CO₂ was shown to increase cell power output by 81.6% at 0.8 V compared with pure CH₄. This corresponded to a 3.4-fold increase in the overall energy conversion efficiency and a 72% decrease in CH₄ consumption. A 260 h durability test demonstrated very high cell durability when operating on a typical 60/30/10 vol% CH₄/CO₂/H₂ biohythane mixture under high fuel utilisation due to inhibition of carbon deposition. Overall, this work suggests that decarbonising gas grids by substituting natural gas with renewably produced H₂/CO₂ mixtures (rather than pure H₂ derived from fossil fuels), and utilising in SOFC technology, gives considerable gains in energy conversion efficiency and carbon emissions savings.     

Effect of pH shock on single-stage biohythane production using gel-entrapped anaerobic microorganisms

A novel single-reactor system having entrapped anaerobic microorganisms has been developed to co-produce H₂ and CH₄. pH is one of the key operating and environmental parameters affecting the performance of a bio-system. This work aimed to investigate the pH shock effects on the novel biohythane system. The experiments were suddenly changing the original cultivation pH value of 6 into 4, 5, 7 or 8 for 4 h. The results indicate that a short pH shock could be used to regulate H₂/CH₄ composition without notably affecting biogas yield and chemical oxygen demand (COD) removal. Peak biohythane production was obtained after the pH shock to 8, having H₂/CH₄ yields of 11.5 ± 1.6/44.8 ± 3.1 mL/g COD. During pseudo steady-state conditions of effective cultivation periods, the values of H₂ content in biohythane and COD removal efficiency were in ranges of 20–39% and 71–79%, respectively. The significances and applications of the experimental results have been discussed. The novelty of this work is elucidating a less-discussed field-operation problem of pH perturbances for a newly-developed biohythane system.     

Hydrogen and methane production from lipid-extracted microalgal biomass residues

A two-stage process to produce hydrogen and methane from lipid-extracted microalgal biomass residues (LMBRs) was developed. The biogas production and energy efficiency were compared between one- and two-stage processes. The two-stage process generated 46 ± 2.4 mL H₂/g-volatile solid (VS), and 393.6 ± 19.5 mL CH₄/g-VS. The methane yield was 22% higher than the one in the one-stage process. Energy efficiency increased from 51% in the one-stage process to 65% in the two-stage process. Additionally, it was found that repeated batch cultivation was a useful method to cultivate the cultures to improve the methane production rate and reduce the fermentation time. In the repeated batch cultivation, the methane yield slightly decreased if the ammonia levels rose, suggesting that the accumulation of ammonia could affect methane production.     

Design of a novel biohythane process with high H2 and CH4 production rates

A biohythane process based on wheat straw including: i) pretreatment, ii) H₂ production using Caldicellulosiruptor saccharolyticus, iii) CH₄ production using an undefined consortium, and iv) gas upgrading using an amine solution, was assessed through process modelling including cost and energy analysis. According to simulations, a biohythane gas with the composition 46–57% H₂, 43–54% CH₄ and 0.4% CO₂, could be produced at high production rates (2.8–6.1 L/L/d), with 93% chemical oxygen demand (COD) reduction, and a net energy yield of 7.4–7.7 kJ/g dry straw. The model was calibrated and verified using experimental data from dark fermentation (DF) of wheat straw hydrolysate, and anaerobic digestion of DF effluent. In addition, the effect of gas recirculation was investigated by both wet experiments and simulation. Sparging improved H₂ productivities and yields, but negatively affected the net energy gain and cost of the overall process.     

Characterization of microbial community in the two-stage process for hydrogen and methane production from food waste

The structure of a microbial community in the two-stage process for H₂ and CH₄ production from food waste was investigated by a molecular biological approach. The process was a continuous combined thermophilic acidogenic hydrogenesis and mesophilic (RUN1) or thermophilic (RUN2) methanogenesis with recirculation of the digested sludge. A two-phase process suggested in this study effectively separate H₂-producing bacteria from methanogenic archaea by optimization of design parameters such as pH, hydraulic retention time (HRT) and temperature. Galore microbial diversity was found in the thermophilic acidogenic hydrogenesis, Clostridium sp. strain Z6 and Thermoanaerobacterium thermosaccharolyticum were considered to be the dominant thermophilic H₂-producing bacteria. The hydrogenotrophic methanogens were inhibited in thermophilic methanogenesis, whereas archaeal rDNAs were higher in the thermophilic methanogenesis than those in mesophilic methanogenesis. The yields of H₂ and CH₄ were in equal range depending on the characteristics of food waste, whereas effluent water quality indicators were different obviously in RUN1 and RUN2. The results indicated that hydrolysis and removal of food waste were higher in RUN2 than RUN1.     

Inhibitory effect of chloroform on fermentative hydrogen and methane production from lipid-extracted microalgae

To improve the sustainability of microalgae as a bioenergy feedstock, lipid-extracted microalgae (LEM) are often further treated by anaerobic digestion (AD). However, the residual chloroform used for extracting lipids as a solvent could inhibit this process, an aspect that has not been studied to date. In this study, the inhibitory effect of chloroform on H₂ and CH₄ production was investigated by performing batch tests. To prepare the feedstock, Chlorella vulgaris was ultrasonicated and the supernatant was discarded after centrifugation. In case of H₂ production, it was found that the H₂ yield fell to almost half that of the control (15.6 mL H₂/g COD_added) at 100 mg CHCl₃/L. The reason for the decrease of the H₂ yield with the increase of chloroform level was due to the change of metabolites from acetate and butyrate to lactate via a non-hydrogenic reaction. In comparison with H₂ production, a much more severe inhibitory effect of chloroform on CH₄ production was observed. The inhibitor concentration (IC_{30, 60, and 90}) on H₂ production was 138, 319, and 622 mg CHCl₃/L, respectively, while concentrations of 15, 37, and 86 mg CHCl₃/L were obtained on CH₄ production. When the chloroform concentration was ≥25 mg/L on CH₄ production, more than 2 g COD/L of organic acids remained, resulting in a decrease of CH₄ yield. These findings indicate that the residual chloroform in LEM should be seriously considered to prevent possible microbial inhibition when designing a process for additional energy recovery from microalgae via AD.     

Effect of food to microorganisms (F/M) ratio on biohythane production via single-stage dark fermentation

Hythane is a mixture of hydrogen and methane gases which are generally produced in separate ways. This work studied mesophilic biohythane gas (H₂+CH₄+CO₂) production in a bioreactor via single-stage dark fermentation. The fermentation was conducted in batch mode using mixed anaerobic microflora and food waste and condensed molasses fermentation soluble to elucidate the effects of food to microorganisms (F/M) ratio (ranging from 0.2 to 38.2) on gas production, metabolite variation, kinetics and biohythane-composition indicator performances. The experimental results indicate that the F/M ratio and fermentation time affect biohythane production efficiency with values of peak maximum hydrogen production rate 9.60 L/L-d, maximum methane production rate 0.72 L/L-d, and hydrogen yield (HY) of 6.17 mol H₂/kg COD_added. Depending on the F/M ratios, the H₂, CH₄ and CO₂ biogas components were 10–60%, 5–20% and 35–70%, respectively. Prospects for the further real application for single-stage biohythane fermentation based on the experimental data are proposed. This work characterizes an important reactor operation factor F/M ratio for innovative single-stage dark fermentation.     

Green hythane production from food waste: Integration of dark-fermentation and methanogenic process towards biogas up-gradation

This study presents an integration of acidogenesis (dark-fermentation) and methanogenesis for green hythane/biohythane production from food waste in two stages (S–I and S-II) and phases (P–I and P-II) of operational variations. The regulatory influence of biocatalyst and redox environment on anaerobic fermentation was evaluated  through a rapid protocol in the context of biogas up-gradation with reference to bio-hydrogen (bio-H₂), biomethane (CH₄), bio-hythane (H₂+CH₄) and their composition (H₂/(H₂+CH₄)) as major markers. Bioreactors with two different parent cultures (heat-shock pretreated and untreated) were operated at pH 6 and 7 in two phases to overcome the impediment of single-phase operation aiming for maximum energy recovery from the untreated substrate of P–I. Integration of S–I with S-II was beneficial to achieve 1.22 times higher cumulative bio-hythane production (4.25 L) compared to S–I (3.47 L) condition alone. The bio-hythane composition mimics the H₂ enriched CNG (H-CNG) and showed the potential to be implemented for biogas up-gradation as a tool.     

Enhancement of biohythane production from solid waste by co-digestion with palm oil mill effluent in two-stage thermophilic fermentation

Improvement of biohythane production from oil palm industry solid waste residues by co-digestion with palm oil mill effluent (POME) in two-stage thermophilic fermentation was investigated. A two-stage co-digestion of solid waste with POME has biohythane production of 26.5–34 m³/ton waste. The co-digestion of solid waste with POME increased biohythane production of 67–114% compared to digestion POME alone. Co-digestion of solid waste with POME enhanced hydrolysis constant (k_h) from 0.07 to 0.113 to 0.120–0.223 d⁻¹. The hydrolysis constant (k_h) of co-digestion was 10 times higher than the single digestion of solid waste. Clostridium sp. was predominated in the hydrogen stage, while Methanosphaera sp. was predominant in methane stage. The co-digestion of solid waste with readily biodegradable organic matter (POME) could significantly increase biohythane production with achieving the significant cost reduction for pretreatment of solid wastes.     

Biohythane production from swine manure and pineapple waste in a single-stage two-chamber digester using gel-entrapped anaerobic microorganisms

A mixture of swine manure and pineapple waste was used to check the feasibility of producing biohythane in a newly-developed single-stage anaerobic fermentation system that having immobilized H₂ and CH₄-producing microbes in a two-chamber digester. Tested hydraulic retention times (HRT) were from 96 h to 6 h. HRT 6 h resulted in peak gas production performance with hydrogen production rate 1240 and methane production rate 812 mL/L-d. Besides, the synergistic function of generation and consumption of volatile fatty acids in this hybrid biosystem had a significant impact on biohythane composition with acetate and butyrate being the dominant liquid metabolites. Chemical oxygen demand and ammonium removal efficiencies were 52.4 and 78.8%, respectively during steady-state conditions. Based on the experimental findings, prospects for field applications of single-stage biohythane fermentation were suggested.     

Pretreatment of second and third generation feedstock for enhanced biohythane production: Challenges,recent trends and perspectives

In the context of biofuel production and achieving sustainable bioeconomy, the use of lignocellulosic and algae biomass in anaerobic fermentation processes yields biohythane that has a typical composition of 10–15% H₂, 50–55% CH₄ and 30–40% CO₂. Using organic biomass-based substrates has been shown to minimize environmental impacts due to the versatile production of high-value products under normal operating conditions that are practically achievable. However, the biohythane yield depends on different factors such as the biomass type, the organic loading rate, soluble metabolic products formed, the type of fermentation (single/dual stage) and the pretreatment strategy adopted for the biomass. Different pretreatment strategies based on physical, chemical and biological processes have been proposed in the literature. In this review, improvements in biohythane yield as a result of these pretreatment strategies, the need/effect of inoculum enrichment, the effects of pH, temperature, trace element addition and organic loading rate has been reviewed. Finally, the major developments of improving biohythane yield due to the addition of co-substrates and the current trends are discussed.     

Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor

The objective of this study was to evaluate the production of hydrogen in a two-stage CSTR system – both reactors having the same volume – and compare its performance with a conventional one-stage process. The lab-scale two-stage and one-stage systems were operated at five pHs and five hydraulic retention time (HRTs). The maximum volumetric hydrogen productivity and yield obtained with the two-stage system were 5.8 mmol L⁻¹ h⁻¹ and 2.7 mol H₂ mol glucose⁻¹, respectively, at an HRT of 12 h and pH 5.5. Overall, the two-stage system showed, at steady state, a better performance that the one-stage system for all the evaluated pHs. However, a comparison between the one-stage system, operating at 6 h of HRT, and the first reactor of the two-stage system at the same HRT did not show any significant difference, highlighting the positive impact of having a two-stage process. The determination of the ratio between the experimental measured H₂ in the gas phase and the theoretical H₂ generated in the liquid phase (discrepancy factor) indicated that an important part of the hydrogen produced in the first reactor was transferred into the second reactor instead of being desorbed in the headspace. Therefore, the improving of hydrogen production in the two-stage system is rather attributed to the increased transfer of hydrogen from liquid to gas than an actual total hydrogen production increase.     

Hydrogen and methane potential based on the nature of food waste materials in a two-stage thermophilic fermentation process

The purpose of this study is to investigate the biological H₂ and CH₄ potential based on the nature of organic waste materials in a two-stage thermophilic fermentation process. Three varieties of actual waste specifically potato, kitchen garbage and bean curd manufacturing waste (okara) were selected. The production rates for H₂ and CH₄ were as follows: potato, 2.1 and 1.2 l/l/d; garbage, 1.7 and 1.5 l/l/d; okara, 0.4 and 1.4 l/l/d in the continuous processes. The H₂ and CH₄ yields were 20–85 ml H₂/g VS_added and 329–364 ml CH₄/g VS_added, respectively. The H₂ yield increased and the CH₄ yield decreased in the order of potato, kitchen garbage and okara. The H₂ yield was shown to be not only dependent on the proportion of carbohydrate but also on the hydrolysis pH of the organic waste, which was influenced by the nature of the organic waste materials. Higher yields of H₂ or CH₄ were obtained when the hydrolysis pH of the organic waste was close to the optimum pH range of H₂-producing bacteria or methanogenic archaea in the two-stage fermentation processes.