High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Fermentative hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture

In this study, production of hydrogen (H₂) from glucose, xylose, galactose, mannose, arabinose and rhamnose by a strain isolated from activated sludge was investigated. The strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter amalonaticus strain SA01 (99%). Optimal cultivation parameters for H₂ production and growth such as pH and temperature were investigated. H₂ yields from glucose at optimal conditions (pH 6.0 and 34 °C) were 1.82 ± 0.02 mol-H₂/mol-glucose. Strain CMC-1 fermented galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation period lowered residual formate level in the media and improved H₂ production for galactose, mannose and xylose (1.68 ± 0.24, 1.93 ± 0.14 and 1.63 ± 0.07 mol-H₂/mol-substrate respectively).     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H₂ production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H₂ yield and specific H₂ production rates with xylose and glucose are 1.63 mol-H₂/mol xylose and 11.14-H₂ mmol/h g-cdw, and 2.02 mol-H₂/mol-glucose and 9.37 mmol-H₂/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H₂ yield and specific H₂ production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H₂/mol-mixed sugar and 12.56 mmol-H₂/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.     

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H₂) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L⁻¹ furfural and 0.25 g L⁻¹ HMF, the maximum H₂ yields observed were 1.89 ± 0.27 mol mol⁻¹ glucose and 1.75 ± 0.22 mol mol⁻¹ glucose, respectively. In cultures with maximum H₂ yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.     

Bioconversion of crude glycerol from biodiesel production to hydrogen

This study evaluates the potential of bioconversion of crude glycerol, discharged from biodiesel production plant, to hydrogen (H₂) by an enriched microbial community. Microbial community was enriched from activated sludge in a medium amended with 2.5 g/L of crude glycerol. Optimal cultivation parameters for H₂ production such as initial pH, cultivation temperature and substrate concentration were investigated. H₂ yields from raw glycerol at optimal conditions (pH 6.5; 40 °C and 1 g/L raw glycerol) were 1.1 ± 0.1 mol-H₂/mol-glycerol_consumed. H₂ production was associated with acetate-butyrate type fermentation, along with ethanol as one of the end products. Kinetic experiments on H₂ production from pure and crude glycerol indicated the absence of any inhibitory effects from the impurities present in crude glycerol. The community analysis revealed that the enriched microbial consortium was dominated mainly by Clostridium species.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

Statistical optimization of factors affecting biohydrogen production from xylose fermentation using inhibited mixed anaerobic cultures

The role of different chemical and physical factors in enhancing biohydrogen production from xylose using a mixed anaerobic culture was examined under mesophilic conditions. A fractional factorial design (FFD) 3^(k–p) was used to optimize pH, the oleic acid (OA) concentration and the biomass concentration. The FFD analysis indicated that the hydrogen (H₂) yield was affected by 3 single factors as well as by 2 factor interactions. Under optimum conditions (1600 mg L⁻¹ of oleic acid (OA) and 1900 mg L⁻¹ VSS and pH 6.7), the H₂ yield reached 2.64 ± 0.12 mol mol⁻¹ of xylose (80% of the theoretical yield). Based on the ANOVA and Pareto chart analysis, the linear and quadratic OA and pH terms were significant and the linear and quadratic VSS terms were insignificant. Normally distribution of the residuals was confirmed from the Anderson-Darling (AD) plot. The studentized residuals versus the predicted values plot clearly demonstrated that the data points were randomly scattered.     

Continuous H2 production during fermentation of the carbon components of lignocellulose hydrolysates: Insight into the influence of pH conditions on the conversion efficiency of individual sugars

Sugars released from lignocellulose biomass are a promising substrate for biohydrogen production. This study evaluates the effect of pH controlled between 4.0 and 7.5 on continuous dark-fermentative H₂ production from the mixture of cellobiose, xylose and arabinose. High hydrogen production rate was obtained for pH values between 6.0 and 7.0 with a maximum of 7.41 ± 0.16 L/L-d at pH 7.0. On the other hand, the highest H₂ yields of around 1.74 ± 0.02 mol/mol_consumed were obtained at pH 4.5, 5.0 and 6.0. Cellobiose was completely utilized in nearly the entire pH range, while the highest consumption of xylose and arabinose was obtained at pH 6.0 and 7.0, respectively. It shows the challenges in selecting optimum pH for fermentation of mixed sugars. Significant impact of pH conditions on the microbial structure was observed. Between pH 4.0 and 7.0 Clostridium genus dominated the consortium, while above pH 7.0 relative abundance of Bacillus genus increased significantly.     

Thermophilic hydrogen production from cellulose with rumen fluid enrichment cultures: Effects of different heat treatments

Elevated temperatures (52, 60 and 65 °C) were used to enrich hydrogen producers on cellulose from cow rumen fluid. Methanogens were inhibited with two different heat treatments. Hydrogen production was considerable at 60 °C with the highest H₂ yield of 0.44 mol-H₂ mol-hexose⁻¹ (1.93 mol-H₂ mol-hexose-degraded⁻¹) as obtained without heat treatment and with acetate and ethanol as the main fermentation products. H₂ production rates and yields were controlled by cellulose degradation that was at the highest 21%. The optimum temperature and pH for H₂ production of the rumen fluid enrichment culture were 62 °C and 7.3, respectively. The enrichments at 52 and 60 °C contained mainly bacteria from Clostridia family. At 52 °C, the bacterial diversity was larger and was not affected by heat treatments. Bacterial diversity at 60 °C remained similar between heat treatments, but decreased during enrichment. At 60 °C, the dominant microorganism was Clostridium stercorarium subsp. leptospartum.     

Developing a thermophilic hydrogen-producing microbial consortia from geothermal spring for efficient utilization of xylose and glucose mixed substrates and oil palm trunk hydrolysate

Xylose and glucose are the major sugar components of lignocellulosic hydrolysate. This study aims to develop thermophilic hydrogen-producing consortia from eight sediments-rich samples of geothermal springs in Southern Thailand by repeated batch cultivation at 60 °C with glucose, xylose and xylose-glucose mixed substrates. Significant hydrogen production potentials were obtained from thermophilic enriched cultures encoded as PGR and YLT with the maximum hydrogen yields of 241.4 and 231.6 mL H₂/g sugar_consumed, respectively. After repeated batch cultivation the hydrogen yield from xylose-glucose mixed substrate of PGR increased to 375 mL H₂/g sugar_consumed which was 30% higher than that of YLT (287 mL H₂/g sugar_consumed). Soluble metabolites from xylose-glucose mixed substrates were composed mostly of butyric acid (20.6-21.8 mM), acetic acid (7.2-13.5 mM), lactic acid (8.2-11.7 mM) and butanol (4.4-13.0 mM). Denaturing gradient gel electrophoresis (DGGE) profiles illustrated small difference in microbial patterns of PGR enriched with glucose, xylose-glucose mixed substrate and xylose. The dominant populations were affiliated with low G + C content Gram-positive bacteria, Thermoanaerobacterium sp., Thermoanaerobacter sp., Caloramater sp. and Anoxybacillus sp. based on the 16S rRNA gene. Cultivation of the enriched culture PGR in oil palm trunk hydrolysate, the maximum hydrogen yield of 301 mL H₂/g sugar_consumed was achieved at hydrolysate concentration of 40% (v/v). At higher concentration to 80% (v/v), the hydrogen fermentation process was inhibited. Therefore, the efficient thermophilic hydrogen-producing consortia PGR has successfully developed and has great potential for production of biohydrogen from mixed sugars hydrolysate.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Determining optimum conditions for hydrogen production from glucose by an anaerobic culture using response surface methodology (RSM)

Hydrogen fermentation is a very complex process and is greatly influenced by many factors. Previous studies have demonstrated that temperature, pH and substrate are important factors controlling biological H₂ production. Response surface methodology with central composite design was used in this study to optimize H₂ production from glucose by an anaerobic culture. The individual and interactive effects of pH, temperature and glucose concentration on H₂ production were also evaluated. The optimum conditions for maximum H₂ yield of 1.75 mol-H₂ mol-glucose⁻¹ were found as temperature 38.8 °C, pH 5.7 and glucose concentration 9.7 g L⁻¹. The linear effects of temperature and pH as well as their quadratic effects on H₂ yield were significant, while the interactive effects of three parameters were minor.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

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.     

Enzymatic routes to hydrogen and organic acids production from banana waste fermentation by autochthonous bacteria: Optimization of pH and temperature

The Central Composite Rotational Design (CCRD) was employed to find the optimum pH (5.09–7.91) and temperature (27.1–46,9 °C) for hydrogen production in banana waste (BW) fermentation by autochthonous microbial biomass. The P and Rm ranged between 6.06 and 62.43 mL H₂ and 1.13–12.56 mL H₂.h^?1, respectively. The temperature 37 °C and pH 7.0 were the optimum conditions for P (70.19 mL H₂) and Rm (12.43 mL H₂.h^?1) as predicted by the mathematical model. Fructose and glucose are the primary alternative carbon sources in banana waste-fed batch reactors. The high concentration of lactic acid and H₂ production was associated to Lactobacillus (52–81%) and Clostridium (14–35%). However, the most important finding was about butyric acid (HBu). This acid is the better indicator of hydrogen production than acetic acid (HAc). The pH effected carbohydrates fermentation and organic acids production. The genes encoding the enzymes related to galactose, sucrose, fructose, arabinose and xylose metabolism were predominant.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Towards the integration of dark- and photo-fermentative waste treatment. 4. Repeated batch sequential dark- and photofermentation using starch as substrate

In this study we demonstrated the technical feasibility of a prolonged, sequential two-stage integrated process under a repeated batch mode of starch fermentation. In this durable scheme, the photobioreactor with purple bacteria in the second stage was fed directly with dark culture from the first stage without centrifugation, filtration, or sterilization (not demonstrated previously). After preliminary optimization, both the dark- and the photo-stages were performed under repeated batch modes with different process parameters. Continuous H₂ production in this system was observed at a H₂ yield of up to 1.4 and 3.9 mole mole⁻¹ hexose during the dark- and photo-stage, respectively (for a total of 5.3 mole mole⁻¹ hexose), and rates of 0.9 and 0.5 L L⁻¹ d⁻¹, respectively. Prolonged repeated batch H₂ production was maintained for up to 90 days in each stage and was rather stable under non-aseptic conditions. Potential for improvements in these results are discussed.