Biohydrogen Production from Palm Oil Mill Effluent by Locally Isolated Clostridium Butyricum Eb6

Hydrogen is a renewable, clean source of energy which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. Potential biomass that could be the substrates for biohydrogen generation include food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol. The objectives of this study were to isolate biohydrogen producing bacteria, to maximize the biohydrogen production in a synthetic medium and palm oil mill effluent (POME) and to improve the strain by overexpressing the hydrogenase gene in the host cell. A biohydrogen producer was successfully isolated from anaerobic POME sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced biohydrogen during active cell growth. Controlled study was done on synthetic medium with 10 g/L glucose resulted in biohydrogen production at 948ml H2/L-medium and volumetric biohydrogen production rate of 172 mL H2/L-medium/h at initial pH 5.5. The supplementation of yeast extract at 4 g/L was found to have a significant effect with the highest biohydrogen production of 992 mL H2/L-medium. The effect of pH on biohydrogen production from POME was investigated, with the optimum biohydrogen production ability at pH 5.5. The maximum biohydrogen production and maximum volumetric biohydrogen production rate were at 3195 mL H2/L-medium and 1034 mL H2/L-medium/h, respectively. The biohydrogen content in the biogas produced was in the range of 60 - 70%. Optimization of biohydrogen production using synthetic medium was done on pH, glucose and iron concentration according to response surface methods (RSM) analysis. By central composite design (CCD) results, pH, glucose concentration and iron concentration were shown to significantly influence the biohydrogen gas production individually, interactively and quadratively (P<0.05) with some exception. The CCD results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO4 was the optimum condition for biohydrogen production which gave a yield of biohydrogen at 2.2 mol H2/mol glucose. For the confirmation experiment model, t-test result showed that experimental data curve had a high confidence at 95% with t = 2.225. Based on the results of this study, optimization of the culture condition for C. butyricum EB6 significantly increased the biohydrogen production.Clostridium butyricum EB6 successfully produced hydrogen gas from POME. Central composite design and response surface methodology were applied to determine the optimum conditions for biohydrogen production (Pc) and maximum biohydrogen production rate (Rmax) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the biohydrogen production and production rate individually and interactively. The optimum conditions for biohydrogen production (Pc) was pH 5.69, temperature 36ºC and 92 g COD/L, with an estimated value of 306 mL H2/g carbohydrate. The optimum conditions for maximum biohydrogen production rate (Rmax) was pH 6.52, temperature 41ºC and 60 g COD/L, with an estimated value of 914 ml H2/ h. An overlay study was carried out to get an overall model optimization. The optimized conditions for the overall model was pH 6.05, temperature 36ºC and 94 g COD/L. [Fe]-hydrogenase (hydA) gene of C. butyricum EB6 was successfully amplified from the genomic DNA. Sequencing results of the hydA gene was identified with open reading frames of 1725 bp which encodes hydA of 574 amino acids with approximate size of 64 kDaltons. The hydA of C. butyricum was found 80.5% similar to hydA of C. acetobutylicum P262 and closely similar to Clostridia hydrogenase. A modified method of electroporation on C. butyricum EB6 was established for transformation of hydA. A hydA-expressing recombinant EB6 was successfully obtained with higher biohydrogen production from 4.2 L-H2/ L-medium to 4.8 L-H2/ L-medium compared to the wild type

Green Hydrogen Production from Residual Lignocellulosic Biomass via Dark Fermentation: Maximizing Hydrogen Yield via Optimal Pretreatment Method and Substrate-to-Inoculum Ratio

Master's thesis in Environmental engineeringThis thesis presents an investigation into the biohydrogen production potential of Lignocellulosic Aquatic Residue (LAR), a byproduct of an industrial process. A detailed examination of the substrate and inoculum characterization, pretreatment methods, biohydrogen production via dark fermentation at different Substrate-to-Inoculum Ratios (SIRs), and kinetics modelling was conducted. The study aims to illustrate that LAR can serve as an effective substrate for renewable biohydrogen production via dark fermentation. After mild acid hydrolysis and lipid extraction pretreatment, LAR showed a high carbohydrate and lipid content. However, the pretreatment process needs to be optimized to avoid the introduction or release of inhibitory compounds since no gas production was observed from those pretreated LAR. Further examination revealed an optimal SIR of 2.7, where Hydrogen Yield (HY) of LAR reached around 280 mL H2 g −1 VS. A Continuous Flow Stirred-Tank Reactor (CFSTR) was built to upscale the biohydrogen production, which produced promising preliminary results. Energy output estimation indicated that biohydrogen production from LAR could contribute between 2.6 to 3.5 TWh per year, equating to 1.2 to 1.6 % of Norway’s total energy demand. This approach turns an otherwise waste product into a source of renewable energy. These findings suggest that the utilization of LAR for biohydrogen production via dark fermentation holds significant potential for future green energy solutions. Continued research is necessary to optimize pretreatment methods, operational conditions, and to fully understand this unique biomass resource

Food waste and food processing waste for biohydrogen production: a review

Food waste and food processing wastes which are abundant in nature and rich in carbon content can be attractive renewable substrates for sustainable biohydrogen production due to wide economic prospects in industries. Many studies utilizing common food wastes such as dining hall or restaurant waste and wastes generated from food processing industries have shown good percentages of hydrogen in gas composition, production yield and rate. The carbon composition in food waste also plays a crucial role in determining high biohydrogen yield. Physicochemical factors such as pre-treatment to seed culture, pH, temperature (mesophilic/thermophilic) and etc. are also important to ensure the dominance of hydrogen-producing bacteria in dark fermentation. This review demonstrates the potential of food waste and food processing waste for biohydrogen production and provides a brief overview of several physicochemical factors that affect biohydrogen production in dark fermentation. The economic viability of biohydrogen production from food waste is also discussed

Electro-extractive fermentation for efficient biohydrogen production

Electrodialysis, an electrochemical membrane technique, was found to prolong and enhance the production of biohydrogen and purified organic acids via the anaerobic fermentation of glucose by Escherichia coli. Through the design of a model electrodialysis medium using cationic buffer, pH was precisely controlled electrokinetically, i.e. by the regulated extraction of acidic products with coulombic efficiencies of organic acid recovery in the range 50–70% maintained over continuous 30-day experiments. Contrary to\ud previous reports, E. coli produced H2 after aerobic growth in minimal medium without inducers and with a mixture of organic acids dominated by butyrate. The selective separation of organic acids from fermentation provides a potential nitrogen-free carbon source for further biohydrogen production in a parallel photofermentation. A parallel study incorporated this fermentation system into an integrated biohydrogen refinery (IBR) for the conversion of organic waste to hydrogen and energy

Characterization of Different Biomass for Biohydrogen Production

The aim of this project is to study the potential of biomass resource available in the country for use in biohydrogen production. The objectives of the research project are to characterize different biomass sources based on biomass properties. The biomass properties being studied are moisture content, calorific value, elemental composition, ash content, volatile matter content and fixed carbon content. In general, two main analyses are conducted: Ultimate and Proximate analysis. The main equipments to be used are CHNS Analyzer, Moisture Analyzer, Bomb Calorimeter and Thermal Gravimetric Analyzer. The study consists of a series of experiments and analysis relating to the properties of the biomass samples to look into the prospect for biohydrogen production. Biomass means any plant-derived organic matter available on a renewable basis. Biomass is basically organic material cultivated energy crops derived from agriculture or woodbased operations to produce solid, liquid or gaseous fuels. Biohydrogen is the process of producing hydrogen from biological processes or biomass. Biohydrogen production has become an important study since it is known that fossil fuels resources in the world is depleting at ahigh rate. The analysis would help characterize different biomass samples for the purpose of biohydrogen production and help realize the potential of biohydrogen production in the country

Biohydrogen production from food waste: Influence of the inoculum-to-substrate ratio

In this study, the influence of the inoculum-to-substrate ratio (ISR) on dark fermentative hydrogen production from food waste (FW) was evaluated. ISR values ranging from 0.05 to 0.25 g VSinoculum/g VSsubstrate were investigated by performing batch tests at T = 39 °C and pH = 6.5, the latter being the optimal value identified based on a previous study. The ISR was found to affect the fermentation process, clearly showing that an adequate ISR is essential in order to optimise the process kinetics and the H2 yield. An ISR of 0.14 proved to optimum, leading to a maximum H2 yield of 88.8 L H2/kg VSFW and a maximum production rate of 10.8 L H2/kg VSFW∙h. The analysis of the fermentation products indicated that the observed highest H2 production mostly derived from the typical acetate/butyrate-type fermentation

Bio-hythane production from food waste by dark fermentation coupled with anaerobic digestion process: A long-term pilot scale experience

In this paper are presented the results of the investigation on optimal process operational conditions of thermophilic dark fermentation and anaerobic digestion of food waste, testing a long term run, applying an organic loading rate of 16.3 kgTVS/m3d in the first phase and 4.8 kgTVS/m3d in the second phase. The hydraulic retention times were maintained at 3.3 days and 12.6 days, respectively, for the first and second phase. Recirculation of anaerobic digested sludge, after a mild solid separation, was applied to the dark fermentation reactor in order to control the pH in the optimal hydrogen production range of 5-6. It was confirmed the possibility to obtain a stable hydrogen production, without using external chemicals for pH control, in a long term test, with a specific hydrogen production of 66.7 l per kg of total volatile solid (TVS) fed and a specific biogas production in the second phase of 0.72 m3 per kgTVS fed; the produced biogas presented a typical composition with a stable presence of hydrogen and methane in the biogas mixture around 6 and 58%, respectively, carbon dioxide being the rest

Effects of several inocula on the biochemical hydrogen potential of sludge-vinasse co-digestion

The influence of the inoculum on the Biochemical Hydrogen Potential test (BHP) was investigated. Thermophilic BHP from sludge-vinasses co-digestion (50:50) was studied employing three types of inocula: Acidogenic Inoculum, Sludge Inoculum and Thermal Sludge Inoculum. The maximum hydrogen yield was obtained with a sludge inoculum (177 mL H2/g VSadded). This yield was 21 and 36% higher than for acidogenic inoculum and thermal sludge inoculum, respectively. The results revealed that the choice of inoculum had significant impact on the hydrogen yield and the sludge inoculum is the most beneficial for BHP tests. The percentages between Eubacteria:Archaea increased from 59.2:40.8 to 92.0:9.0 during BHP tests using the sludge inoculum while it remained stablish in the others cases around 50:50. Furthermore, hydrogen production was accompanied by the generation of volatile fatty acids, mainly acetic, butyric and propionic acids. There were no differences in the rate of hydrogen production in any of the BHP

Review of Continuous Fermentative Hydrogen-Producing Bioreactors from Complex Wastewater

In recent years, the production of hydrogen through dark fermentation has become increasingly popular because it is a sustainable approach to produce clean energy. Thus, an evaluation of studies reported on hydrogen production from different complex wastewaters will be of immense importance in economizing production technologies. This work presents a review of the advances in the bioreactor and bioprocess design for biohydrogen production from different complex wastewaters. The biohydrogen production is discussed emphasizing the production metabolic pathways, bioreactor configuration and operation, organic loading rate (OLR), pretreatment of wastewater, as well as microbial diversity. Also, in this review, various bioreactor configurations and performance parameters including H2 yield (HY) and hydrogen production rate (HPR) are evaluated and presented. The work concludes with challenges and prospects of biohydrogen production and claims for more systematic and comprehensive studies on the subject