Hydrogen production by co-cultures of Clostridium butyricum and Rhodospeudomonas palustris: Optimization of yield using response surface methodology

Both dark and photo-fermentation can be used for biological hydrogen production; either performed separately, in two-stage systems, or in co-culture. A single stage process is less laborious and costly; however, the two types of microorganisms have different nutritional requirements requiring optimization of culture conditions. Here a response surface methodology (RSM) with a Box-Behnken design was used to optimize microorganism ratio and substrate and buffer concentrations, and to evaluate their interactive effects for maximization of hydrogen yield. Clostridium butyricum and Rhodopseudomonas palustris were grown on a potato starch/glucose base medium at 30 °C under continuous illumination (40 W m^?2 light intensity). The highest hydrogen yield, 6.4 ± 1.3 mol H₂/mol glucose, was obtained with a substrate concentration of 15 g/L, buffer concentration of 50 mM, and microorganism ratio of 3. The observed strong interaction between buffer and substrate concentration is most likely due to the need to optimize the pH for co-cultures.     

Enhanced hydrogen production performance of Rubrivivax gelatinosus M002 using mixed carbon sources

A new isolated photosynthetic bacterium, Rubrivivax gelatinosus M002, can produce hydrogen with glucose or lactate as sole carbon source, and grow on butyrate and acetate without hydrogen evolution. Experiments on studying its hydrogen production performance from glucose mixed with acetate, butyrate or lactate were carried out. The results showed that the hydrogen yield increased significantly and the pH value of the photo-fermentations could retain around 7 in these mixed carbon sources cultures. A hydrogen yield of 9.9 mol H₂/mol-glucose was observed when 20 mM acetate and 15 mM glucose was co-fed as substrate. The maximum hydrogen production rate was 44 mL/(L·h), which was 37.5% higher than the highest rate obtained with glucose as sole carbon source. The results suggest an alternative way for high-yield hydrogen production with mixed carbon source in one-step process instead of two-step fermentation process.     

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.     

Near stoichiometric reforming of biodiesel derived crude glycerol to hydrogen by photofermentation

Biodiesel manufacture produces crude glycerol as a major byproduct. At the scale estimated for future biodiesel production, extensive quantities of crude glycerol fraction will be generated, creating a large waste stream with potentially significant environmental impacts. The magnitude of projected future crude glycerol supplies suggests that its conversion to a biofuel is the only viable route to producing a product that does not cause market saturation. Previously it was shown that crude glycerol could be converted to hydrogen, a possible future clean energy carrier, by photofermentation using Rhodopseudomonas palustris through photofermentation. Here, the effects of nitrogen source and different concentrations of crude glycerol on this process were assessed. At 20 mM glycerol, 4 mM glutamate, 6.1 mol hydrogen/mole of crude glycerol were obtained under optimal conditions, a yield of 87% of the theoretical, and significantly higher than what was achieved previously.     

Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

Improvement of hydrogen production with Rhodopseudomonas palustris CQK-01 by Ar gas sparging

For improving photo-biohydrogen production, a novel gas bubble column photobioreactor with Ar gas sparging was developed for biohydrogen production by purple non-sulfur phototrophic bacteria, Rhodoseudomonas palustris CQK-01. The dissolved hydrogen concentration was in-situ measured by a hydrogen microsensor. Experimental results demonstrated that Ar gas sparging dramatically decreased the dissolved hydrogen concentration, resulting in an improvement in the photo-biohydrogen production performance. Furthermore, effects of the gas flow rate and the time interval of gas sparging were investigated. The results showed that with an increase in the gas flow rate, the hydrogen production performance increased initially due to the reduced dissolved hydrogen concentration and enhanced mass transport, and then it decreased as a result of an increased shear stress. Meanwhile, the short sparging time interval resulted in a low accumulation of dissolved hydrogen in the bioreactor, hence high hydrogen production performance. The optimal hydrogen production rate (5.86 mmol/l/h) and hydrogen yield (3.38 mol H₂/mol glucose) were obtained at the gas flow rate of 10 ml/min, respectively.     

Evaluation of biohydrogen production from glucose and protein at neutral initial pH

Organic wastes are considered as potential substances for economical biohydrogen production, because the carbohydrate and protein are main components. Previous investigations indicate that an optimum hydrogen production appear in acidic conditions to carbohydrates, or in alkali condition to protein. However, in practice, the treatment of these organic wastes by anaerobic fermentation usually carries out at neutral pH condition, in which biohydrogen production is only a middle process. So, the purpose of this paper is to evaluate the biohydrogen production at neutral pH condition from carbohydrates or protein. Batch tests were conducted to investigate the differences in biohydrogen production by anaerobic fermentation at neutral initial pH using carbohydrate and protein (glucose and peptone) as the sole carbon source. The experimental results showed that the maximal hydrogen yields of two substrates were about 0.14 ml H₂/mg glucose and 0.077 ml H₂/mg protein, respectively, at neutral initial pH. Although the hydrogen yields of glucose is far greater than that of protein at neutral pH, they were lower than previous results of hydrogen production in acidic condition to carbohydrate or in alkali condition to protein. This result shows that the neutral pH is not an optimal condition for biohydrogen production. In this experiment of biohydrogen production, a phenomenon has been observed that the hydrogen production and hydrogen consumption occurred simultaneously in the fermentation of protein, whereas the hydrogen production occurred only in the fermentation of glucose. Furthermore, the different evaluation of the main components of the organic liquid by-products produced by fermentation of each substrate implied that the biohydrogen production pathways of these two substrates were different. Molecular analysis indicated that the dominant microorganisms during the anaerobic fermentation of these two substrates are greatly different.     

Fermentative biohydrogen production by mixed anaerobic consortia: Impact of glucose to xylose ratio

Glucose and xylose are the dominant monomeric carbohydrates present in agricultural materials which can be used as potential building blocks for various biotechnological products including biofuels production. Hence, the imperative role of glucose to xylose ratio on fermentative biohydrogen production by mixed anaerobic consortia was investigated. Microbial catabolic H₂ and VFA production studies revealed that xylose is a preferred carbon source compared to glucose when used individually. A maximum of 1550 and 1650 ml of cumulative H₂ production was observed with supplementation of glucose and xylose at a concentration of 5.5 and 5.0 g L⁻¹, respectively. A triphasic pattern of H₂ production was observed only with studied xylose concentration range. pH impact data revealed effective H₂ production at pH 6.0 and 6.5 with xylose and glucose as carbon sources, respectively. Co-substrate related biohydrogen fermentation studies indicated that glucose to xylose ratio influence H₂ and as well as VFA production. An optimum cumulative H₂ production of 1900 ml for 5 g L⁻¹ substrate was noticed with fermentation medium supplemented with glucose to xylose ratio of 2:3 at pH 6. Overall, biohydrogen producing microbial consortia developed from buffalo dung could be more effective for H₂ production from lignocellulosic hydrolysates however; maintenance of glucose to xylose ratio, inoculum concentration and medium pH would be essential requirements.     

How can hydrothermal treatment impact the performance of continuous two-stage fermentation for hydrogen and methane co-generation?

Hydrothermal treatment can facilitate hydrolysis of biomass wastes such as algae and livestock manures, by converting high-molecular weight carbohydrates and proteins to monosaccharides and amino acids. However, further decomposition and reciprocal reaction of monosaccharides and amino acids are usually accompanied with hydrothermal treatment, which have negative impacts on microbial fermentation performance. In this study, glucose and glycine were used as model substrates during hydrothermal treatment coupled with semi-continuous hydrogen and methane fermentation. The results showed that thermal decomposition of glucose was stronger than glycine, due to the binary interactions between carbonyl group and amino group. Acidic condition could suppress conversion of intermediate compounds to polymers, thereby improving 5-HMF concentration to 7.59 g/L. Hydrothermal by-products had adverse impacts on hydrogen fermentation stability, resulting in a wide fluctuation of hydrogen production rate of around 0.55 L/L/d. Adding sulfuric acid for treatment would increase the competition of sulphate reducing bacteria, and cause a stuck methane fermentation. Additionally, by-products degradation promoted the growth of hydrogenotrophic and mixotrophic methanogens.     

The photosynthetic hydrogen production performance of a newly isolated Rhodobacter capsulatus JL1 with various carbon sources

Rhodobacter capsulatus is a photosynthetic bacterium with the ability to produce H₂ under photosynthetic condition. In this study, a new strain JL1 isolated from lake water was identified as Rhodobacter capsulatus by phylogenetic analysis of 16S ribosomal DNA (rDNA) sequence. Initial medium pH and l-glutamate (nitrogen source) concentration were optimized. At optimum pH 7.0 and 7 mmol/L l-glutamate, R. capsulatus JL1 could grow and produce hydrogen on the carbon sources of acetate, butyrate, glucose, xylose and fructose with the maximum substrate to H₂ conversion efficiencies of 67.5%, 26.6%, 46.1%, 46.2% and 46.6%, respectively. The maximum H₂ production rate, 124 ± 0.6 mL/(L·h), was obtained using 20 mmol-glucose/L as the carbon source. The addition of appropriate acetic acid to the tests with low concentration of glucose was able to improve the H₂ yield. Under the optimum operation parameters, the maximum H₂ yield and H₂ production rate of R. capsulatus JL1 from 16.4 g-corn straw/L-culture were 2966.5 ± 43.2 mL/L and 71.1 ± 4.5 mL/(L·h), while the chemical oxygen demand (COD) removal rate was up to 49.6%. This study indicates that R. capsulatus JL1 can serve as good candidate strain for H₂ production with organic waste water as well as effluent of dark-fermentation.     

Photofermentation of malate for biohydrogen production— A modeling approach

A kinetic model for photofermentative biohydrogen production is developed in this study to predict the dynamics of the process. The proposed model contains 17 parameters to describe cell growth, substrate consumption, and hydrogen evolution as well as inhibition of the process by biomass, light intensity, and substrate. Batch experimental results from the literature were used to calibrate and validate the model with malic acid as a model substrate, using Rhodobacter sphaeroides as a model biomass. Temporal hydrogen evolution and cell growth predicted by the proposed model agreed well with the experimentally measured data obtained from four literature reports, with statistically significant correlation coefficients exceeding 0.9. Based on sensitivity analysis performed with the validated model, only six of the 17 parameters were found to be significant. Model simulations indicated that the range of optimal light intensity for maximum hydrogen yield from malate by R. sphaeroides was 150–250 W/m².     

Photobiological hydrogen production: photochemical efficiency and bioreactor design

Biological production of hydrogen can be carried out by photoautotrophic or photoheterotrophic organisms. Here, the photosystems of both processes are described.The main drawback of the photoautotrophic hydrogen production process is oxygen inhibition. The few efficiencies reported on the conversion of light energy into hydrogen energy are low, less than 1.5% on a solar spectrum basis. However, these can be increased to 3–10%, by the immediate removal of produced oxygen.The photochemical efficiency of hydrogen production can be calculated theoretically, and is estimated to be 10% (on solar spectrum basis) for the photoheterotrophic process. With use of the theoretical photochemical efficiency, and the climatic data on sunlight irradiance at a certain location at a certain moment of the year, the theoretical maximum hydrogen production can be estimated.Data on H₂ yields and photochemical efficiency from experiments reported in the literature are summarized. Photochemical efficiencies, essentially based on artificial light, can reach 10% or even more, but only at low light intensities, with associated low-H₂ production rates.Some reflections on possible photobioreactors lead to two types of (modified) photobioreactors that might be successful for a large-scale biological hydrogen production.     

Different role of focA and focB encoding formate channels for hydrogen production by Escherichia coli during glucose or glycerol fermentation

The dependence of H₂ production on the formate channels, FocA and FocB, by Escherichia coli at pH 5.5, 6.5 and 7.5 was shown using focA and focB mutants and comparing with the wild type. Moreover, effect of exogenous addition of formate (10 mM) on H₂ production was allotted. The results acquired propose that during glucose fermentation formate import can occur through FocB at different pHs; external formate drives FocA to import direction. However, during glycerol fermentation formate might be imported through FocB, whereas formate is exported preferentially through FocA at pH 7.5.     

Hydrogen production by co-cultures of Lactobacillus and a photosynthetic bacterium, Rhodobacter sphaeroides RV

Hydrogen production with glucose by using co-immobilized cultures of a lactic acid bacterium, Lactobacillus delbrueckii NBRC13953, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels was studied. Glucose was converted to hydrogen gas in a yield of 7.1 mol of hydrogen per mole of glucose at a maximum under illuminated conditions.