Co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum for enhancing hydrogen production via thermophilic fermentation of cornstalk waste

A strategic method utilizing the co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum has been developed to improve hydrogen production via the thermophilic fermentation of cornstalk waste. The hydrogen yield in the co-culture fermentation process reached 68.2 mL/g-cornstalk which was 94.1% higher than that in the mono-culture. The hydrogen fermentation process was successfully scaled-up from 125 mL anaerobic bottles to an 8 L continuous stirred tank reactor, and the hydrogen production from cornstalk waste was significantly improved in the bioreactor system due to efficient mixing and mass transfer. The hydrogen yield in the bioreactor reached 74.9 mL/g-cornstalk which was 9.8% higher than that in the 125 mL anaerobic bottle. The present work indicates that the direct microbial conversion of lignocellulosic waste by co-culturing C. thermocellum and C. thermosaccharolyticum is a promising avenue for enhancing hydrogen production.     

Biohydrogen production from immobilized cells and suspended sludge systems with condensed molasses fermentation solubles

The anaerobic fermentation using the condensed molasses fermentation solubles (CMS) as substrate in a continuously stirred anaerobic bioreactor (CSABR) was carried out for optimal hydrogen production performance of biohydrogen production rate and yield, where as two kinds of bioreactors used. One is a suspended sludge bioreactor (SSB) which used suspended seed sludge. The other bioreactor is an immobilized cell bioreactor (ICB) which used immobilized cells and mix the same seed sludge in the SSB as the source of the bacteria. It was found that the hydrogen production rate increased with a decrease in the hydraulic retention time (HRT), when substrate concentration was 40 g COD/L in an SSB as well as maximum hydrogen production rate of 14.04 ± 2.08 L/d/L obtained at HRT 0.5 h (ca. 5.78 times value of HRT 4 h) in the SSB system. The hydrogen production rate at low dilution rate (HRT > 4 h), in the ICB is better than SSB, meanwhile at a high dilution rate (HRT < 4 h), due to the presence of enriched granules in the SSB (12.30 g VSS/L), absent in the ICB (9.89 g VSS/L), the hydrogen production rate was 7.60 ± 1.05 L/d/L (ca. 1.23 times higher than HRT 4 h), which was lower than the rate in the SSB. Eventually, the hydrogen production rate increased by increasing the substrate concentrations from 40 to 60 g COD/L within the HRT range of 2–4 h in both the SSB as well as in ICB systems.     

Production of hydrogen from sewage biosolids in a continuously fed bioreactor: Effect of hydraulic retention time and sparging

Hydrogen was produced from primary sewage biosolids via mesophilic anaerobic fermentation in a continuously fed bioreactor. Prior to fermentation the sewage biosolids were heated to 70 °C for 1 h to inactivate methanogens and during fermentation a cellulose degrading enzyme was added to improve substrate availability. Hydraulic retention times (HRT) of 18, 24, 36 and 48 h were evaluated for the duration of hydrogen production. Without sparging a hydraulic retention time of 24 h resulted in the longest period of hydrogen production (3 days), during which a hydrogen yield of 21.9 L H₂ kg⁻¹ VS added to the bioreactor was achieved. Methods of preventing the decline of hydrogen production during continuous fermentation were evaluated. Of the techniques evaluated using nitrogen gas to sparge the bioreactor contents proved to be more effective than flushing just the headspace of the bioreactor. Sparging at 0.06 L L min⁻¹ successfully prevented a decline in hydrogen production and resulted in a yield of 27.0  L H₂ kg⁻¹ VS added, over a period of greater than 12 days or 12 HRT. The use of sparging also delayed the build up of acetic acid in the bioreactor, suggesting that it serves to inhibit homoacetogenesis and thus maintain hydrogen production.     

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

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.     

An integrated biological hydrogen production process based on ethanol-type fermentation and bipolar membrane electrodialysis

An integrated bio-hydrogen production system involving fermentative hydrogen production and product separation is proposed. In this process, microorganisms conduct ethanol-type fermentation and generate H₂ gas in anaerobic bioreactor, and acetate is removed from fermentation broth by using a two chamber bipolar membrane electrodialysis as separation unit. A comparative study of fermentative hydrogen production of Ethanoligenens harbinese B49 in the integrated system with traditional fermentation process was carried out. Compared to traditional process, accumulated H₂ elevated 23%, glucose utilization ratio increased by 135% and cell growth increased by 27% in the integrated system. The specific hydrogen production rate reached 2.2 mol H₂/mol glucose, indicating that separation of acetate from fermentation system has a great role in promoting hydrogen producing capacity. Bipolar membrane electrodialysis showed high acetate separation efficiency and low glucose loss rate. In the integrated system, pH could be used to direct electrodialysis operation, since it has an exponential correlation with acetate concentration in fermentation broth. These results provide a new method for achieving efficient and stable H₂ production with simultaneous glucose recovery and acetate inhibition release.     

Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater

Production of biohydrogen using dark fermentation has received much attention owing to the fact that hydrogen can be generated from renewable organics including waste materials. The key to successful application of anaerobic fermentation is to uncouple the liquid retention time and the biomass retention time in the reactor system. Various reactor designs based on biomass retention within the reactor system have been developed. This paper presents our research work on bioreactor designs and operation for biohydrogen production. Comparisons between immobilized-cell systems and suspended-cell systems based on biomass growth in the forms of granule, biofilm and flocs were made. Reactor configurations including column- and tank-based reactors were also assessed. Experimental results indicated that formation of granules or biofilms substantially enhanced biomass retention which was found to be proportional to the hydrogen production rate. Rapid hydrogen-producing culture growth and high organic loading rate might limit the application of biofilm biohydrogen production, since excessive growth of fermentative biomass would result in washout of support carrier. It follows that column-based granular sludge process is a preferred choice of process for continuous biohydrogen production from organic wastewater, indicating maximum hydrogen yield of 1.7 mol-H₂/mol-glucose and hydrogen production rate of 6.8 L-H₂/L-reactor h.     

维生素B4对秸秆类生物质光发酵产氢的影响

以玉米秸秆类生物质为产氢原料,研究维生素B4对HAU-M1光合细菌生长和产氢过程的影响规律。结果表明,当维生素B4浓度为75 mg/L时,光合细菌生长情况最好,细菌干重最大值为0.934 g/L;维生素B4浓度为100 mg/L时,氢气累积产量达178 mL,比对照组显著提高了43.8%,对光合细菌产氢的促进效果最好;添加维生素B4对HAU-M1光合细菌发酵产氢过程的pH值影响显著,可减弱发酵液酸化,有利于光合细菌发酵产氢。显见,维生素B4对HAU-M1光合细菌生长及秸秆类生物质光合产氢具有明显的促进作用,可为进一步研究开发秸秆类生物质光合细菌发酵产氢工艺技术提供科学参考。     

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.     

Kinetic analysis of biohydrogen production from anaerobically treated POME in bioreactor under optimized condition

In this study, the biohydrogen production from POME was performed under mesophillic conditions by mixed culture in a 2 L bioreactor using the optimized conditions obtained previously. The effect of controlling pH initially or throughout the fermentation was also examined. The fermentation performance was monitored by comparing P, R_m, λ, and P_s in both systems. In this present study, the reactor system showed higher hydrogen production potential values with the utilization of pH control. Hydrogen production potential was increased two folds when the reactor system was equipped with pH control rather than just fixed the initial pH at 5.8. The biohydrogen production under controlled pH occurred after 7 h fermentation resulting in maximum Ps and Rm of 1.32 L/L POME and 0.144 L/L.h, respectively.     

Energy balance of dark anaerobic fermentation as a tool for sustainability analysis

A process aimed at producing energy needs to produce more energy than the energy necessary to run the process itself in order to be energetically sustainable. In this paper, an energy balance of a batch anaerobic bioreactor has been defined and calculated, both for different operative conditions and for different reactor scales, in order to analyze the sustainability of hydrogen production through dark anaerobic fermentation. Energy production in the form of hydrogen and methane, energy to warm up the fermentation broth, energy loss during fermentation and energy for mixing and pumping have been considered in the energy balance. Experimental data and literature data for mesophilic microorganism consortia have been used to calculate the energy balance. The energy production of a mesophilic microorganism consortium in a batch reactor has been studied in the 16–50 °C temperature range. The hydrogen batch dark fermentation resulted to only have a positive net production of energy over a minimal reactor dimension in summer conditions with an energy recovery strategy. The best working temperature resulted to be 20 °C with 20% of available energy. Hydrogen batch dark fermentation may be coupled with other processes to obtain a positive net energy by recovering energy from the end products of hydrogen dark fermentation. As an example, methane fermentation has been considered to energetically valorize the end products of hydrogen fermentation. The combined process resulted in a positive net energy over the whole range of tested reactor dimension with 45–90% of available energy.     

Methane free biohydrogen production from carbon monoxide using a continuously operated moving bed biofilm reactor

Biological water-gas shift (WGS) reaction is a green and sustainable alternative to thermochemical-catalytic WGS process for hydrogen production from carbon monoxide (CO). However, CO tolerant carboxydotrophic microbes for hydrogen production and scaling up the technology using a bioreactor system present challenges in successful application of this technology. This study demonstrated the capability of anaerobic microbial consortium for biohydrogen production from CO using a moving bed biofilm reactor (MBBR). The CO conversion pathway followed by the anaerobic biomass was first elucidated by inhibiting the methanogens present using 2-bromoethanesulfonate (BES) at an optimum concentration of 10 mmol/L. An increase in inlet CO concentration to the MBBR enhanced the H₂ production, but the CO conversion efficiency was low. More than 80% CO conversion efficiency was obtained only for a low inlet CO concentration. A maximum H₂ concentration of 19.5 mmol/L along with 2 mmol/L of acetate were obtained for 36 mmol/L of inlet CO concentration in the bioreactor. The carbon flux analysis showed that the CO was mainly utilized for methane free H₂ production, and only <10% of carbon flux was diverted towards acetate formation. Overall, this study demonstrated that MBBR system can be used for steady state biohydrogen production over a prolonged operation period.     

Biohydrogen production from wheat straw hydrolysate using Caldicellulosiruptor saccharolyticus followed by biogas production in a two-step uncoupled process

A two-step, un-coupled process producing hydrogen (H₂) from wheat straw using Caldicellulosiruptor saccharolyticus in a ‘Continuously stirred tank reactor’ (CSTR) followed by anaerobic digestion of its effluent to produce methane (CH₄) was investigated. C. saccharolyticus was able to convert wheat straw hydrolysate to hydrogen at maximum production rate of approximately 5.2 L H₂/L/Day. The organic compounds in the effluent collected from the CSTR were successfully converted to CH₄ through anaerobic digestion performed in an ‘Up-flow anaerobic sludge bioreactor’ (UASB) reactor at a maximum production rate of 2.6 L CH₄/L/day. The maximum energy output of the process (10.9 kJ/g of straw) was about 57% of the total energy, and 67% of the energy contributed by the sugar fraction, contained in the wheat straw. Sparging the hydrogenogenic CSTR with the flue gas of the UASB reactor ((60% v/v) CH₄ and (40% v/v) CO₂) decreased the H₂ production rate by 44%, which was due to the significant presence of CO₂. The presence of CH₄ alone, like N₂, was indifferent to growth and H₂ production by C. saccharolyticus. Hence, sparging with upgraded CH₄ would guarantee successful hydrogen production from lignocellulosic biomass prior to anaerobic digestion and thus, reasonably high conversion efficiency can be achieved.     

Biohydrogen production from starch wastewater and application in fuel cell

Biohydrogen was produced from starch in wastewater by anaerobic fermentation. The effects of parameters, such as pH, starch concentration were investigated and optimum operating conditions were determined. The optimal pH and starch concentration for hydrogen production at 37 °C were 6.5 and 5 g/L, respectively with a maximum hydrogen yield of 186 ml/g-starch. The produced biogas contains 99% of hydrogen after passing through KOH solution to remove CO₂. The anaerobic fermentation installation was integrated with a proton-exchange-membrane fuel cell (PEMFC) system for on-line electricity generation. This combination system of biohydrogen and fuel cell achieved a power output of 0.428 W at 0.65 V per cell.     

Accelerated startup of biological hydrogen production process by addition of Ethanoligenens harbinense B49 in a biofilm-based column reactor

Two biofilm-based column reactors with walnut shell (WS) as carrier media were applied for fermentative hydrogen production using glucose as substrate by mixed microbial cultures at the temperature of 35 °C. Pure hydrogen producing bacteria Ethanoligenens harbinense B49 was supplemented into the reacting system periodically or continuously to enhance hydrogen production ability at the startup period. The results showed that the bioreactors supplemented with E. harbinense B49 performed better than the reactor without bacteria addition. Continuous addition mode was recommended, since the hydrogen production performance was better and the operation was easily to be accomplished. The optimal addition amount of pure bacteria was also investigated. The optimal bacterial addition amount was found to be 2.5% which led a better hydrogen production rate. In addition, the bioreactor supplemented with pure bacteria continuously presented a high hydrogen production ability as the specific hydrogen production rate (SHPR) maximized at 1.36 L/g-VSS·d; whereas, the bioreactor without bacteria addition obtained a maximum specific hydrogen production rate of 1.10 L/g-VSS·d. The addition of E. harbinense B49 favored the transformation to ethanol type fermentation in the bioreactor. Thereby, the startup period had been accelerated remarkably.     

Effects of hydraulic retention time on anaerobic hydrogenation performance and microbial ecology of bioreactors fed with glucose–peptone and starch–peptone

This study evaluated anaerobic hydrogenation performance and microbial ecology in bioreactors operated at different hydraulic retention time (HRT) conditions and fed with glucose–peptone (GP) and starch–peptone (SP). The maximum hydrogen production rates for GP- and SP-fed bioreactors were found to be 1247 and 412 mmol-H₂/L/d at HRT of 2 and 3 h, respectively. At HRT > 8 h, hydrogen consumption due to peptone fermentation could occur and thus reduced hydrogen yield from carbohydrate fermentation. Results of cloning/sequencing and denaturant gradient gel electrophoresis (DGGE) indicated that Clostridium sporogenes and Clostridium celerecrescens were dominant hydrogen-producing bacteria in the GP-fed bioreactor, presumably due to their capability on protein hydrolysis. In the SP-fed bioreactor, Lactobacillus plantarum, Propionispira arboris, and Clostridium butyricum were found to be dominant populations, but the presence of P. arboris at HRT > 3 h might be responsible for a lower hydrogen yield from starch fermentation. As a result, optimizing HRT operation for bioreactors was considered an important asset in order to minimize hydrogen-consuming activities and thus maximize net hydrogen production. The limitation of simple parameters such as butyrate to acetate ratio (B/A ratio) in predicting hydrogen production was recognized in this study for bioreactors fed with multiple substrates. It is suggested that microbial ecology analysis, in addition to chemical analysis, should be performed when complex substrates and mixed cultures are used in hydrogen-producing bioreactors.     

A pilot study of biohythane production from cornstalk via two-stage anaerobic fermentation

This study aimed to study the feasibility and stability of biohythane production from cornstalk via two-stage anaerobic fermentation without hydrolysis step in a semi-continuous pilot scale system. The present study applied a 1 m³ continuous stirred tank reactor for biohydrogen production and a 0.5 m³ up-flow anaerobic sludge bed for biomethane production. During the entire operation, a hydrogen production yield of 25.02 L/kg TS and hydrogen production rate of 0.46 L/L/d was achieved in first-stage. In addition, a methane yield of 95.38 L/kg TS and methane production rate of 4.06 L/L/d was achieved in second-stage by using the liquid effluent after first-stage. The percentage of hydrogen in the biohythane gas was 18.47% which suitable for vehicle fuel. Moreover, it was feasible to use the solid residue as a growth medium in seedlings to improve energy and carbon recovery. The results suggest that biohythane production from cornstalk could be a promising biofuel avenue.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

基于UASB反应器的厨余厌氧发酵产氢研究

针对目前厨余连续流发酵产氢处理负荷不高、产氢率较低的难题,采用UASB反应器进行厨余发酵产氢研究。在温度为30℃,进水COD浓度为2 000~10 000 mg/L,水力停留时间为2~6 h条件下,产氢速率最大达到17.04 L/(L.d)。反应器内有颗粒污泥的形成,平均生物量达到6.17 g/L,为氢气的产生提供了有利保障。当出水pH为4.2~4.4,碱度为260~340 mg/L的条件下,乙醇和乙酸占挥发酸总量的89.2%,形成稳定的乙醇型发酵类型,反应器最高处理负荷COD达到60 kg/(m3.d)。试验结果表明,UASB反应器具有更高的产氢效能和更加稳定的产氢效果,能够为厨余发酵产氢提供有利的保障。     

Anaerobic fermentative system based scheme for green energy sustainable houses

The green energy sustainable house based on bio-hydrogen and bio-methane energy technologies proposed in this study employs dark fermentation technology to complete a scheme for green energy sustainable house that includes energy production, storage, distribution control, load applications, recycling, waste treatment, and reuse. In order to resolve the problem of wastewater discharge from hydrogen production in green energy sustainable houses, this study proposes wastewater chemical oxygen demand (COD) treatment research, and suggests the use of two-stage anaerobic treatment to produce two types of bio-energy i.e. hydrogen and methane, while simultaneously reducing COD levels.Methane production employed a condensed molasses fermentation solubles (CMS) and hydrogen fermentation tank effluent as a substrate to test the COD reducing efficiency and overall efficiency of methane production. It was found that if CMS is used during the hydrolysis and acidogenesis stages, the maximum carbohydrate degradation rate will be approximately 70% (F/M ratio of 1.9-2.3), and the COD removal rate will increase from 15 to 20% (F/M ratio of 1.9-2.3) to 68% (F/M ratio of 0.5). This study showed that the total gas (H₂ and CH₄) production yield from effluent of hydrogen fermentation tank (56.2 KJ/mol substrate) is greater than the value for CMS.In this study, a 3.2 m³ anaerobic hydrogen reactor is evaluated to provide a family with 3-4 kW of power. When acclimatization is performed under conditions of 20 g COD/L substrate and hydraulic retention time (HRT) of 8 h, the COD removal rate can reach approximately 50%. If a methane-generating reactor with a 95% COD removal rate is used to degrade effluent from the hydrogen reaction tank, it will be possible to reduce the COD of organic effluent to under 500 mg/L. Since this water quality is not far from that of ordinary untreated household wastewater (approximately 300-500 mg COD/L), the effluent can be discharged into a community sewer system and treated in a community sewage treatment facility.