Hydrogen-producing Bioreactor Research Articles

Evaluation of inhibitory compounds produced by bacteria isolated from a hydrogen-producing bioreactor during the self-fermentation of wheat straw.

The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.

The duo Clostridium and Lactobacillus linked to hydrogen production from a lignocellulosic substrate.

The study aimed to identify interspecies interactions within a native microbial community present in a hydrogen-producing bioreactor fed with two wheat straw cultivars. The relationships between the microbial community members were studied building a canonical correspondence analysis and corroborated through in vitro assays. The results showed that the bioreactor reached a stable hydrogen production of ca. 86 mL/kg·d in which the cultivar change did not affect the average performance. Lactobacillus and Clostridium dominated throughout the whole operation period where butyric acid was the main metabolite. A canonical correspondence analysis correlated positively Lactobacillus with hydrogen productivity and hydrogen-producing bacteria like Clostridium and Ruminococaceae. Agar diffusion testing of isolated strains confirmed that Lactobacillus inhibited the growth of Enterococcus, but not of Clostridium. We suggest that the positive interaction between Lactobacillus and Clostridium is generated by a division of labor for degrading the lignocellulosic substrate in which Lactobacillus produces lactic acid from the sugar fermentation while Clostridium quickly uses this lactic acid to produce hydrogen and butyric acid. The significance of this work lies in the fact that different methodological approaches confirm a positive association in the duo Lactobacillus-Clostridium in a bioreactor with stable hydrogen production from a complex substrate.

Anaerobic hydrogen and methane production from low-strength beverage wastewater

In this study, the low-strength effluent from an equalization tank of the wastewater treatment plant in a beverage factory was used for the production of hydrogen and methane. The COD concentration of this low-strength wastewater was 2.9 ± 2.0 g COD/L. In a two-phase anaerobic fermentation system, the hydrogen-producing bioreactor was operated at HRT 8 h, while the methane-producing reactor operated at HRT 24 h. The maximum MPR, methane yield (MY), methane content and COD removal were 72 ± 31 mL/L-d, 58 ± 12 mL/g COD, 92 ± 2% and 78%, respectively. Energy efficiency in this study was calculated as follows, the maximum heating value was 2.2 × 108 kcal/y. The annual carbon-emission reduction was 8.8 × 104 kg CO2/y, 5.2 × 104 kg CO2/y, 7.2 × 104 kg CO2/y when energy-equivalent coal, natural gas or fuel was used, respectively.

Temperature-sensitive PSII: a novel approach for sustained photosynthetic hydrogen production.

The need for energy and the associated burden are ever growing. It is crucial to develop new technologies for generating clean and efficient energy for society to avoid upcoming energetic and environmental crises. Sunlight is the most abundant source of energy on the planet. Consequently, it has captured our interest. Certain microalgae possess the ability to capture solar energy and transfer it to the energy carrier, H2. H2 is a valuable fuel, because its combustion produces only one by-product: water. However, the establishment of an efficient biophotolytic H2 production system is hindered by three main obstacles: (1) the hydrogen-evolving enzyme, [FeFe]-hydrogenase, is highly sensitive to oxygen; (2) energy conversion efficiencies are not economically viable; and (3) hydrogen-producing organisms are sensitive to stressful conditions in large-scale production systems. This study aimed to circumvent the oxygen sensitivity of this process with a cyclic hydrogen production system. This approach required a mutant that responded to high temperatures by reducing oxygen evolution. To that end, we randomly mutagenized the green microalgae, Chlamydomonas reinhardtii, to generate mutants that exhibited temperature-sensitive photoautotrophic growth. The selected mutants were further characterized by their ability to evolve oxygen and hydrogen at 25 and 37 °C. We identified four candidate mutants for this project. We characterized these mutants with PSII fluorescence, P700 absorbance, and immunoblotting analyses. Finally, we demonstrated that these mutants could function in a prototype hydrogen-producing bioreactor. These mutant microalgae represent a novel approach for sustained hydrogen production.

Vacuum promotes metabolic shifts and increases biogenic hydrogen production in dark fermentation systems

The successful operation of any type of hydrogen-producing bioreactor depends on the performance of the microorganisms present in the system. Both substrate and partial gas pressures are crucial factors affecting dark fermentation metabolic pathways. The main objective of this study was to evaluate the impact of both factors on hydrogen production using anaerobic granular sludge as inoculum and, secondly, to study the metabolic shifts of an anaerobic community subjected to low partial gas pressures. With this goal in mind, seven different wastewater (four synthetic media, two industrial wastewater, and one domestic effluent) and the effect of applying vacuum on the systems were analyzed. The application of vacuum promoted an increase in the diversity of hydrogenproducing bacteria, such as Clostridium, and promoted the dominance of acetoclastic- over hydrogenotrophic methanogens. The application of different media promoted a wide variety of metabolic pathways. Nevertheless, reduction of the hydrogen partial pressure by application of vacuum lead to further oxidation of reaction intermediates irrespective of the medium used, which resulted in higher hydrogen and methane production, and improved the COD removal. Interestingly, vacuum greatly promoted biogenic hydrogen production from a real wastewater, which opens possibilities for future application of dark fermentation systems to enhance biohydrogen yields.

Evaluation of recycling the effluent of hydrogen fermentation for biobutanol production: Kinetic study with butyrate and sucrose concentrations

Butyrate in the effluent of hydrogen-producing bioreactor is a potential feed for biobutanol production. For recycling butyrate, this study investigated the kinetics of biobutanol production by Clostridium beijerinckii NRRL B592 from different paired concentrations of butyrate and sucrose in a series of batch reactors. Results show that the lag time of butanol production increased with higher concentration of either sucrose or butyrate. In regression analyses, the maximum specific butanol production potential of 6.49gg−1 of dry cell was projected for 31.9gL−1 sucrose and 1.3gL−1 butyrate, and the maximum specific butanol production rate of 0.87gd−1g−1 of dry cell was predicted for 25.0gL−1 sucrose and 2.6gL−1 butyrate. The specific butanol production potential will decrease if more butyrate is added to the reactor. However, both sucrose and butyrate concentrations are weighted equally on the specific butanol production rate. This observation also is true on butanol yield. The maximum butanol yield of 0.49molmol−1 was projected for 25.0gL−1 sucrose and 2.3gL−1 butyrate. In addition, a confirmation study found butanol yield increased from 0.2 to 0.3molmol−1 when butyrate addition increased from 0 to 1gL−1 under low sugar concentration (3.8gL−1 sucrose). The existence of butyrate increases the activity of biobutanol production and reduces the fermentable sugar concentration needed for acetone–butanol–ethanol fermentation.

Temperature-sensitive PSII and promiscuous PSI as a possible solution for sustainable photosynthetic hydrogen production

Sustainable hydrogen production in cyanobacteria becomes feasible as a result of our recent studies of the structure of photosystem I encoding operon in a marine phage. We demonstrated that the fused PsaJF subunit from the phage, substituted for the two separate subunits in Synechocystis, enabled the mutated PSI to accept electrons from additional electron donors such as respiratory cytochromes. In this way, a type of photorespiration was created in which the cell consumes organic material through respiratory processes and PSI serves as a terminal electron acceptor, substituting for cytochrome oxidase. We designed a hydrogen-producing bioreactor in which this type of photorespiration could utilize the organic material of the cell as an electron source for H2 production. We propose, in parallel, to engineer cyanobacterial and/or algal strains with a temperature-sensitive PSII and enhanced respiration rates to achieve efficient and sustainable hydrogen production. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

ADM1-based modeling of methane production from acidified sweet sorghum extract in a two stage process

The present study focused on the application of the Anaerobic Digestion Model 1 on the methane production from acidified sorghum extract generated from a hydrogen producing bioreactor in a two-stage anaerobic process. The kinetic parameters for hydrogen and volatile fatty acids consumption were estimated through fitting of the model equations to the data obtained from batch experiments. The simulation of the continuous reactor performance at all HRTs tested (20, 15, and 10 d) was very satisfactory. Specifically, the largest deviation of the theoretical predictions against the experimental data was 12% for the methane production rate at the HRT of 20 d while the deviation values for the 15 and 10 d HRT were 1.9% and 1.1%, respectively. The model predictions regarding pH, methane percentage in the gas phase and COD removal were in very good agreement with the experimental data with a deviation less than 5% for all steady states. Therefore, the ADM1 is a valuable tool for process design in the case of a two-stage anaerobic process as well.

Continuous hydrogen and methane production in a two-stage cheese whey fermentation system

The feasibility of integrating biological hydrogen and methane production in a two-stage process using mixed cultures and cheese whey powder (CWP) as substrate was studied. The effect of operational parameters such as hydraulic retention time (HRT) and organic loading rate (OLR) on the volumetric hydrogen (VHPR) and methane (VMPR) production rates was assessed. The highest VHPR was 28 L H2/L/d, obtained during stable operation in a CSTR at HRT and OLR of 6 h and 142 g lactose/ L/d, respectively. Moreover, hydrogen (13 L/L/d) was produced even at HRT as low as 3.5 h and OLR of 163 g lactose/L/d, nonetheless, the reactor operation was not stable. Regarding methane production in an UASB reactor, the acidified effluent from the hydrogen-producing bioreactor was efficiently treated obtaining COD removals above 90% at OLR and HRT of 20 g COD/L/d and 6 h, respectively. The two-stage process for continuous production of hydrogen and methane recovered over 70% of the energy present in the substrate. This study demonstrated that hydrogen production can be efficiently coupled to methane production in a two-stage system and that CWP is an adequate substrate for energy production.

Butanol production from the effluent of hydrogen fermentation

The purpose of the study was to recover butanol from the effluent of the hydrogen-producing bioreactor containing acetate, butyrate, and carbohydrate. The butanol production by Clostridium beijerinckii NRRL B592 was evaluated under both unsterilized and sterilized conditions for examining the potential of butanol production for the practical application. Sucrose of 10 g/L and butyrate of 2 g/L coupled with acetate buffer were used to mimic the effluent. Sucrose was completely consumed in the both unsterilized and sterilized conditions during acetone-butanol-ethanol (ABE) fermentation. However, the results illustrate that the carbohydrate consumption rate in the unsterilized condition was higher than that in the sterilized condition. The maximum butanol concentrations of 3,500 and 3,750 mg/L were achieved in the sterilized and unsterilized conditions, respectively. Meanwhile, it was found that the acetate and the butyrate concentrations of 600 and 1,500 mg/L, and 300 and 1,000 mg/L were ingested to yield butanol in the sterilized condition and in the unsterilized condition, respectively. The results concluded that high levels of acetate and butyrate could eliminate the interference of other microbial populations, resulting in the enrichment of C. beijerinckii NRRL B592 in the fermentor. The butanol production by C. beijerinckii NRRL B592 could be, therefore, produced from the effluent of the hydrogen-producing bioreactor. It promised that the microbial butanol production is one of attractive bioprocesses to recover energy from wastes.

Fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor

Microbial products formed in biological wastewater treatment systems are closely related to system performance and status, and many of them have fluorescence spectral characteristics. In this work, the fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor were studied using three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy. Since the components of the microbial products are complex, the parallel factor analysis (PARAFAC) method was used to extract the real spectra from the overlapped spectra. Two principal components were identified from the EEM spectra. The peaks at excitation-emission maxima of 280/350 and 350/440 nm were, respectively, attributed to the fluorescence of proteins and NADH. Their real concentrations were quantified using the PARAFAC coupled with the second-order calibration method. Results show that the formation rate of proteins was correlated to the production rate of hydrogen and volatile fatty acids, as well as the substrate degradation rate. A close correlation between the hydrogen partial pressure and the two fluorophores was found out. This study provides a reliable and convenient approach, which could be potentially used for monitoring the wastewater treatment reactor performance through measuring the fluorescence spectra of the supernatant.

Interactions between Clostridium sp. and other facultative anaerobes in a self-formed granular sludge hydrogen-producing bioreactor

The interactions of Clostridium sp. and other non-hydrogen producing bacteria directly influence anaerobic hydrogen production. In this study, bacteria in a sucrose-feeding hydrogen-producing bioreactor were investigated via 16S rDNA-based analysis. Results showed that Clostridium pasteurianum, Klebsiella sp., and Streptococcus sp. were the predominant microorganisms. The Streptococcus sp. cells were found to localize inside hydrogen-producing granular sludge and were surrounded by clostridia. Significant oxygen consumption was found in the Klebsiella sp. pure culture experiment, in which oxidation reduction potential (ORP) dropped from 100 to −500 mV during the log phase within 2 h. Oxygen consumption by Streptococcus sp. was not significant, and it accumulated EPS under anaerobic conditions. Results suggest that Klebsiella sp. first utilized the oxygen to form anaerobic conditions in this system. Streptococcus sp., on the other hand, produced EPS complexes to strengthen the sludge granule followed by the mass growth of Clostridium sp.

Fermentative effluents from hydrogen producing bioreactor as substrate for poly(β-OH) butyrate production with simultaneous treatment: An integrated approach

The feasibility of bioplastics production as poly(beta-OH)butyrate (PHB) was studied with individual volatile fatty acids (VFA) and acid-rich effluents from a biohydrogen producing reactor (HBR) as primary substrates employing aerobic consortia as biocatalyst under anoxic microenvironment. Butyrate as substrate showed higher PHB productivity (33%) followed by acetate (32%), acids mixture (16%) and propionate (11%) among synthetic VFA studied. Acid-rich effluents from HBR yielded higher PHB productivity (25%) especially at lower substrate loading conditions. Decrement observed in PHB production (from 25% to 6%) with increase in substrate load might be due to the presence of high concentration of residual carbon along with acid metabolites. Neutral redox operation showed effective PHB production compared to acidic and basic conditions due to associated higher metabolic activity of the biocatalyst. The integrated approach helped to treat additional COD from acid-rich HBR effluents apart from by-product recovery.

Succession of the Bacterial Community and Dynamics of Hydrogen Producers in a Hydrogen-Producing Bioreactor

Variation in the hydrogen production rate was consistent with the succession of dominant bacteria during the batch fermentation process. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes and quantitative analysis of the hydA genes at both the DNA and mRNA levels confirmed that Clostridium perfringens was the most dominant hydrogen producer in the bioreactor.

Ecologically engineered system (EES) designed to integrate floating, emergent and submerged macrophytes for the treatment of domestic sewage and acid rich fermented-distillery wastewater: Evaluation of long term performance

An ecologically engineered system (EES) was designed to mimic the natural cleansing functions of wetlands to bring about wastewater treatment. EES consisted of three tanks containing diverse biota viz., aquatic macrophytes, submerged plants, emergent plants and filter feeders connected in series. The designed system was evaluated for 216 days by operating in continuous mode (20 l/day) to treat both sewage (DS) and fermented-distillery wastewater (FDW, from hydrogen producing bioreactor). Floating macrophyte system (Tank 1) was more effective in removing COD and nitrates. Submerged and emergent integrated macrophyte system (Tank 2) showed an effective removal of volatile fatty acids (VFAs) along with COD. Filter-feeding system (Tank 3) visualized the removal of COD, VFA, turbidity and color. On the whole the system can treat effectively DS (COD, 68.06%; nitrate, 22.41%; turbidity, 59.81%) and FDW (COD, 72.92%; nitrate, 23.15%; color, 46.0%). The designed EES can be considered as an economical approach for the treatment of both sewage and fermented wastewaters.

Statistical optimization of process parameters on biohydrogen production from glucose by Clostridium sp. Fanp2

Statistically based experimental designs were applied to optimizing process parameters for hydrogen production from glucose by Clostridium sp. Fanp2 which was isolated from effluent sludge of anaerobic hydrogen-producing bioreactor. The important factors influencing hydrogen production, which identified by initial screening method of Plackett–Burman, were glucose, phosphate buffer and vitamin solution. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Box–Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and identify optimal values that bring maximum hydrogen production. Experimental results showed that glucose, vitamin solution and phosphate buffer concentration all had an individual significant influence on the specific hydrogen production potential ( P s). Simultaneously, glucose and vitamin solution, glucose and phosphate buffer were interdependent. The optimal conditions for the maximal P s were: glucose 23.75 g/l, phosphate buffer 0.159 M and vitamin solution 13.3 ml/l. Using this statistical optimization method, the hydrogen production from glucose was increased from 2248.5 to 4165.9 ml H 2/l.