A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OL

Caldicellulosiruptor owensensis strain OL^T (DSM 13100) is an obligately anaerobic, extreme thermophilic bacterium that is capable of utilizing a broad range of carbohydrates and producing H₂ as a metabolic by-product. The performance of C. owensensis on glucose and xylose was analyzed in lab-scale bioreactors to assess its potential use in biohydrogen production. Acetate, H₂, and CO₂ were the main end products during exponential growth of the organism on either sugar. Lactate production was triggered during the transition into the stationary phase and was associated with an increase in the levels of specific l-lactate dehydrogenase activity. In addition, minor amounts of ethanol and propionate could be detected. H₂ and acetate yields were lower on xylose than on glucose, marking an opposite trend to biomass and lactate yields. The influence of elevated H₂ partial pressure on product distribution was more dramatic in xylose-fermenting cultures. Replacement of yeast extract in the medium with a standard vitamins solution improved H₂ yield on both sugars, where it reached 100% of the theoretical maximum, i.e. 4 mol per mol hexose, on glucose. By using the defined medium, both the maximum specific growth rate and the maximum volumetric H₂ production rate of C. owensensis increased significantly on glucose and almost doubled on xylose. Screening other sugars besides glucose and xylose revealed a clear sugar-dependent product-distribution pattern and a direct correlation between biomass and lactate yields, which might be explained considering energy metabolism of the cells. The organism is proposed as a new candidate for biohydrogen production at high yields.     

The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process

In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H₂/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.     

Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6

A local bacterial isolate from palm oil mill effluent (POME) sludge, identified as Clostridium butyricum EB6, was used for biohydrogen production. Optimization of biohydrogen production was performed via statistical analysis, namely response surface methodology (RSM), with respect to pH, glucose and iron concentration. The results show that pH, glucose concentration and iron concentration significantly influenced the biohydrogen gas production individually, interactively and quadratically (P < 0.05). The center composite design (CCD) results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO₄ were the optimal conditions for biohydrogen production, yielding 2.2 mol H₂/mol glucose. In confirmation of the experimental model, t-test results showed that curve fitted to the experimental data had a high confidence level, at 95% with t = 2.225. Based on the results of this study, optimization of the culture conditions for C. butyricum EB6 significantly increased the production of biohydrogen.     

Hydrogen production by Clostridium acetobutylicum ATCC 824 and megaplasmid-deficient mutant M5 evaluated using a large headspace volume technique

Biohydrogen production is measured using a variety of techniques, ranging from low cost intermittent gas release methods where yields are usually reduced due to high partial pressures of hydrogen, to expensive respirometers that can eliminate pressure buildup. A new large headspace volume technique was developed that reduces the potential for hydrogen gas inhibition without the need for a respirometer. We tested this method with two strains of clostridia, Clostridium acetobutylicum ATCC 824 and its mutant M5 that lacks a megaplasmid responsible for butanol and acetone production, and a mixed culture (heat-treated sludge). The hydrogen yield using M5 (2.64 mol-H₂/mol-glucose) was 47% higher than that of the parent strain (1.79 mol-H₂/mol-glucose), and 118% larger than that obtained in tests with the sludge inoculum (1.21 mol-H₂/mol-glucose). The increased yield for M5 was primarily due to a decrease in biomass synthesis (38%) compared to the parent strain. Hydrogen yields measured using this new method were on average 14% higher than those obtained using a conventional respirometric method. These findings indicate enhanced biohydrogen production from the megaplasmid-deficient mutant of C. acetobutylicum ATCC 824, and that an intermittent gas-sampling technique can effectively measure high hydrogen gas by using a large headspace volume.     

Hydrogen production by anaerobic co-digestion of rice straw and sewage sludge

In this study, the rich carbon content of rice straw and the high nitrogen content of sewage sludge make the straw a good potential substrate and the sludge a viable inoculum for biohydrogen production. Two treatment conditions for the sewage sludge (raw and heat-treated) were used in the present experiments. Batch test using a mixture of rice straw and sewage sludge were carried out to investigate the optimum carbon to nitrogen (C/N) ratio for effective biohydrogen production. The experimental results indicate that untreated sludge could be used as the inoculum for efficient hydrogen production when mixed with the appropriate proportion of rice straw. According to our results, biogas and hydrogen production in all raw sludge cases ramped up more quickly and also exhibited longer and higher hydrogen production in comparison with heat-treated cases. At the C/N ratio of 25 in untreated sludge, hydrogen production was 33% higher than heat-treated one. Additionally, under the same conditions, high and stable hydrogen content (58%) and the maximal hydrogen yield (0.74 mmol H₂/g-VS added straw) were obtained.     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

This study investigates the mesophilic biohydrogen production from glucose using a strictly anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced batch reactor (TBSBR) packed with a Lantec HD Q-PAC^® packing material (132 ft²/ft³ specific surface). The reactor was operated for 62 days. The main parameters measured here were hydrogen composition, hydrogen production rate and soluble metabolic products. pH, temperature, recirculation flow rate and inlet glucose concentration at 10 g/L were the controlled parameters. The maximum specific hydrogen production rate and the hydrogen yield found from this study were 146 mmol H₂/L.d and 1.67 mol H₂/mol glucose. The maximum hydrogen composition was 83%. Following a thermal treatment, the culture was active without adding fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen production rate were improved. For all sequences, the soluble metabolites were dominated by the presence of butyric and acetic acids compared to other volatile fatty acids. The results from the standard biohydrogen production (BHP) test which was conducted using samples from TBSBR as inoculum confirmed that the culture generated more biogas and hydrogen compared to the pure strain of C. butyricum CWBI1009. The effect of biofilm activity was studied by completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For three subsequent sequences, similar results were recorded as in the previous sequences with 40% removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was evidenced in this packing material, which is characterized by a relatively high specific surface area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard BHP test was conducted, resulting in no hydrogen production.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 ± 0.07 mol H₂/mol glucose (mean ± S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 ± 0.03 mol H₂/mol glucose, 0.17 ± 0.01 mol H₂/mol glucose, 0.11 ± 0.01 mol H₂/mol glucose and 0.20 ± 0.04 mol H₂/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp.. However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Fermentative hydrogen production by a new alkaliphilic Clostridium sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay,New Caledonia

The hydrogen-producing strain PROH2 pertaining to the genus Clostridium was successfully isolated from a shallow submarine hydrothermal chimney (Prony Bay, New Caledonia) driven by serpentinization processes. Cell biomass and hydrogen production performances during fermentation by strain PROH2 were studied in a series of batch experiments under various conditions of pH, temperature, NaCl and glucose concentrations. The highest hydrogen yield, 2.71 mol H₂/mol glucose, was observed at initial pH 9.5, 37 °C, and glucose concentration 2 g/L, and was comparable to that reported for neutrophilic clostridial species. Hydrogen production by strain PROH2 reached the maximum production rate (0.55 mM-H₂/h) at the late exponential phase. Yeast extract was required for growth of strain PROH2 and improved significantly its hydrogen production performances. The isolate could utilize various energy sources including cellobiose, galactose, glucose, maltose, sucrose and trehalose to produce hydrogen. The pattern of end-products of metabolism was also affected by the type of energy sources and culture conditions used. These results indicate that Clostridium sp. strain PROH2 is a good candidate for producing hydrogen under alkaline and mesothermic conditions.     

Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria

Glycerol is an inevitable by-product from biodiesel synthesis process and could be a promising feedstock for fermentative hydrogen production. In this study, the feasibility of using crude glycerol from biodiesel industry for biohydrogen production was evaluated using seven isolated hydrogen-producing bacterial strains (Clostridium butyricum, Clostridium pasteurianum, and Klebsiella sp.). Among the strains examined, C. pasteurianum CH4 exhibited the best biohydrogen-producing performance under the optimal conditions of: temperature, 35 °C; initial pH, 7.0; agitation rate, 200 rpm; glycerol concentration, 10 g/l. When using pure glycerol as carbon source for continuous hydrogen fermentation, the average H₂ production rate and H₂ yield were 103.1 ± 8.1 ml/h/l and 0.50 ± 0.02 mol H₂/mol glycerol, respectively. In contrast, when using crude glycerol as the carbon source, the H₂ production rate and H₂ yield was improved to 166.0 ± 8.7 ml/h/l and 0.77 ± 0.05 mol H₂/mol glycerol, respectively. This work demonstrated the high potential of using biodiesel by-product, glycerol, for cost-effective biohydrogen production.     

Effect of microwave pretreatment of mixed culture on biohydrogen production from waste of sweet produced from Benincasa hispida

To enhance the production of biohydrogen from biomass, various pretreatment methods play important role. In this study, effect of microwave irradiation on the culture was studied on biohydrogen production from Benincasa hispida (Petha) solid waste at different powers for a fixed interval of time. The highest power studied was 800 W with a frequency of 2450 MHz. The amount of soluble sugars found in the waste was 13.9 mg/L having the chemical oxygen demand (COD) of 3000 mg/L. Studies have been performed in batch reactors using mixed consortia and results were also compared with the reactor operated at the normal conditions i.e. without any inoculum pretreatment. Maximum hydrogen produced was 14 mmol H₂ per mol of soluble sugar consumed in the reactor in which the inoculum was exposed to 320 W of microwave for 5 min. SEM analysis of this microwave pretreated culture was done.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum

Clostridium acetobutylicum is an attractive industrial microorganism for biochemical production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, metabolically engineered C. acetobutylicum carrying glucose-6-phosphate dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, than the wild type. Furthermore, when pH and glucose concentration were optimized for the CA-hydA strain, enhanced hydrogen productivity of 25.8% was achieved in 7 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production.     

Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18

Pantoea agglomerans BH18, isolated from mangrove sludge, could produce hydrogen under marine culture condition. To improve the hydrogen-producing capacity of this strain, we constructed a stable transposon-mutagenized library of P. agglomerans BH18. A Tn7-based transposon was randomly inserted into genomic DNA of P. agglomerans BH18. Mutants were identified by kanamycin resistance and amplification of the inserted transposon sequences. A transposon mutant, named as strain TB212, was screened for the highest hydrogen production ability. The total volume of hydrogen gas evolved by this mutant strain TB212 was 60% higher than that of the wild type. The mutant strain TB212 was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum initial pH of 7.0, and hydrogen production was 2.52 ± 0.02 mol H₂/mol glucose (mean ± S.E.) under marine culture condition. The mutant strain TB212 could produce hydrogen at the salt concentration from 3 to 6%. It was concluded that the transposon-mutagenized library may be a useful tool for investigation of high efficiency hydrogen-producing bacteria.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

Enhanced biohydrogen production from date seeds by Clostridium thermocellum ATCC 27405

Biohydrogen production from waste lignocellulosic biomass serves the dual purpose of converting waste into valuable products and alleviates waste disposal issues. In this study, waste date seeds were valorized for biohydrogen production via consolidated bioprocessing by Clostridium thermocellum ATCC 27405. Effect of various surfactants (PEG1000, surfactin, Triton X-100) and sodium carbonate (buffering agent) on biohydrogen production from the acid pre-treated substrate was examined. Among the various surfactants, addition of Triton X-100 resulted in the maximum biohydrogen yield of 103.97 mmol/L at an optimal dosage of 0.75% w/v. Triton X-100 supplementation favoured the production of ethanol and acetate as co-metabolites than butyrate. Addition of Na₂CO₃ to date seed fermentation medium at a concentration of 15 mM enhanced the biohydrogen production by 33.16%. Also, Na₂CO₃ buffering supported the glycolytic pathway and subsequent ethanol production than acetate/butyrate formation. Combined effect of the optimal dosages of Triton X-100 and Na₂CO₃ resulted in high hydrogen productivity up to 72 h (0.443 mmol/g h of H₂) with a total increase in hydrogen yield of 40.6% at the end of 168 h, as compared to fermentation supplemented with Triton X-100 alone. Further analysis revealed that the combined effects of the additives resulted in better substrate degradation, favourable pH window and cell growth promotion which ensured enhanced hydrogen productivity and yield. Thus, the study highlights a novel stimulatory approach for enhanced biohydrogen production from a new substrate.     

Stoichiometric analysis of biological hydrogen production by fermentative bacteria

In this study, biohydrogen production from glucose by two fermentative bacteria (Clostridium butyricum, a typical strictly anaerobic bacterium, and Klebsiella pneumoniae, a well-studied facultative anaerobic and nitrogen-fixing bacterium) are stiochiometrically analyzed according to energy (ATP), reducing equivalent and mass balances. The theoretical analysis reveals that the maximum yield of hydrogen on glucose by Clostridium butyricum is 3.26 mol/mol when all acetyl-CoA entering into the acetate pathway (α=1$\alpha =1$), which is higher than that by Klebsiella pneumoniae under strictly anaerobic conditions. In the latter case, the maximum yield by Klebsiella pneumoniae is 2.86 mol hydrogen per mol glucose when five sevenths of acetyl-CoA is transformed to acetate. However, under microaerobic condition the maximum yield of hydrogen on glucose by Klebsiella pneumoniae could reach 6.68 mol/mol if all acetyl-CoA entered into tricarboxylic acid (TCA) cycle (γ=1$\gamma =1$) and a quantity of 53% of the reducing equivalents generated in the metabolism were completely oxidized by molecular oxygen. On the other hand, the relationship between hydrogen production and biomass formation is distinct by Clostridium butyricum from that by Klebsiella pneumoniae.   The former yield of hydrogen on glucose increases as biomass. In contrast, the latter one decreases as biomass in a certain range of molar fraction of acetate in total acetyl-CoA metabolism (5/7?β?0$5/7?\beta ?0$). Microaerobic condition is favorable for high hydrogen production with low biomass formation by Klebsiella pneumoniae   in a certain range of the molar fraction of all reducing equivalents oxidized completely by molecular oxygen (0.53?δ?0.83$0.53?\delta ?0.83$).