Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures

Batch tests were conducted to evaluate the enhancement of hydrogen/ethanol (EtOH) productivity using cow dung microflora to ferment α-cellulose and saccharification products (glucose and xylose). Hydrogen/ethanol production was evaluated based on hydrogen/ethanol yields (HY/EY) under 55 °C at various initial pH conditions (5.5–9.0). Our test results indicate that cow dung sludge is a good mixed natural-microflora seed source for producing biohydrogen/ethanol from cellulose and xylose. The heat-pretreatment, commonly used to produce hydrogen more efficiently from hexose, applied to mixed anaerobic cultures did not help cow dung culture convert cellulose and xylose into hydrogen/ethanol. Instead of heat-pretreatment, the mixed culture received enrichments cultivated at 55 °C for 4 days. Positive results were observed: hydrogen/ethanol production from fermenting cellulose and xylose was effectively enhanced at increases of 4.8 (ethanol) to 8 (hydrogen) and 2.4 (ethanol) to 15.6 (hydrogen) folds, respectively. In which, the ethanol concentration produced from xylose reached 4–4.4 g/L, an output comparable to that of using heat-treated sewage sludge and better than that (1.25–3 g/L) using pure cultures. Our test results show that for the enriched cultures the initial cultivation pH can affect hydrogen/ethanol production including HY, EY and liquid fermentation product concentration and distribution. These results were also concurred using a denaturing gradient gel electrophoresis analysis saying that both cultivation pH and substrate can affect the enriched cow dung culture microbial communities. The enriched cow dung culture had an optimal initial cultivation pH range of 7.6–8.0 with peak HY/EY values of 2.8 mmol-H₂/g-cellulose, 5.8 mmol-EtOH/g-cellulose, 0.3 mol-H₂/mol-xylose and 1 mol-EtOH/mol-xylose. However, a pH change of 0.5 units from the optimal values reduced hydrogen/ethanol production efficiency by 20%. Strategies based on the experimental results for optimal hydrogen/ethanol production from cellulose and xylose using cow dung microflora are proposed.     

Effect of microwave irradiation pretreatment of cow dung compost on bio-hydrogen process from corn stalk by dark fermentation

The bio-hydrogen production potential from corn stalk was significantly affected by microwave irradiation pretreatment of cow dung compost in batch tests. The maximum hydrogen yield of 144.3 ml/g-corn stalk and hydrogen production rate of 3.6 ml/g-corn stalk h⁻¹ were observed using the pretreated compost by microwave radiation of 1.5 min at fixed Na₂CO₃ dosage of 800 mg/l, Fe dosage of 400 mg/l, substrate concentration of 20 g/l, which increased about 99.6% and 85.2% compared with that of the control. The effects of microwave irradiation on microbial characteristics were further discussed by Atomic Force Microscope (AFM), determination of protein content and PCR-DGGE. The four dominant hydrogen-producing strains had been isolated and confirmed to be Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis and Enterococcus faecium, respectively. The diversity and symbiosis relations of the mixed bacteria were also observed in fermentation hydrogen production process.     

Bioconversion of corncob to hydrogen using anaerobic mixed microflora

Biohydrogen production from corncob using natural anaerobic microflora was reported for the first time. The optimum pretreatment condition for the corncob was determined to be 100 °C, 30 min, and 1% HCl (w/w). The maximum hydrogen yield of 107.9 ml/g-TVS and hydrogen production rate of 4.20 ml/g-TVS h⁻¹ was obtained under the condition of 10 g/l substrate concentration and initial pH 8.0. Butyrate and acetate were the dominant metabolic by-products of hydrogen fermentation. Chemical composition analysis, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to study the mechanism of degrading corncob for hydrogen production. The amorphous domains of cellulose and hemicellulose were hydrolyzed into fermentable saccharides through acid pretreatment and the microorganisms had a devastating effect on the crystallinity of the cellulose. The hydrogen yield from pretreated corncob was much higher than from raw corncob. Therefore, the acid pretreatment played a crucial role on hydrogen production from corncob.     

Isolation of hydrogen generating microflora from cow dung for seeding anaerobic digester

The effectiveness of using cow dung as a source for isolating hydrogen generating microflora was investigated under varying isolating conditions based on viz.: pH adjustment and pH adjustment coupled with heat treatment. The viability of the isolated microflora was tested in an anaerobic jar with respect to biogas generation, hydrogen content and pH. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH of 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. In the case of microflora isolated from cow dung with 10% solids, by subjecting to pH adjustment coupled with heat treatment resulted in biogas free from methane content during the fermentation at pH 4, 5 and 6, respectively. At the end of 120 h of fermentation for a reactor pH at 4, 5 and 6 the cumulative biogas generation was 1685, 2610 and 2353 ml, whereas the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

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.     

Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures

Biohydrogen production from the cornstalk wastes with acidification pretreatment was reported in this paper. Batch tests were carried out to analyze influences of several environmental factors on biohydrogen production from cornstalk wastes. Two predominant bacterial morphologies, namely spore-forming rod shape bacteria and micrococcus were screened, purified, and identified after enriched from a hydrogen-producing fermentor with cow dung composts. The maximum cumulative H₂ yield of 149.69 ml H₂ g⁻¹ TVS was obtained at initial pH 7.0 and substrate concentration 15 g l⁻¹, the value is about 46-fold as compared with that of raw cornstalk wastes. The maximum hydrogen production rate was 7.6 ml H2 h⁻¹. The hydrogen concentration in biogas was 45–56% (v/v) and there was no significant methane observed in the biogas throughout this study. In addition, biodegradation characteristics of the substrate by microorganisms were also discussed. During the conversion of cornstalk wastes into hydrogen, the acetate, propionate, butyrate, and the ethanol were main by-products in the metabolism of hydrogen fermentation. The test results showed that the acidification pretreatment of the substrate plays a crucial role in conversion of the cornstalk wastes into biohydrogen gas by the cow dung composts generating hydrogen.     

Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria

Beer lees are the main by-product of the brewing industry. Biohydrogen production from beer lees using anaerobic mixed bacteria was investigated in this study, and the effects of acidic pretreatment, initial pH value and ferrous iron concentration on hydrogen production were studied at 35 °C in batch experiments. The hydrogen yield was significantly enhanced by optimizing environmental factors such as hydrochloric acid (HCl) pretreatment of substrate, initial pH value and ferrous iron concentration. The optimal environmental factors of substrate pretreated with 2% HCl, pH = 7.0 and 113.67 mg/l Fe²⁺ were observed. A maximum cumulative hydrogen yield of 53.03 ml/g-dry beer lees was achieved, which was approximately 17-fold greater than that in raw beer lees. In addition, the degradation efficiency of the total reducing sugar, and the contents of hemicellulose, cellulose, lignin and metabolites are presented, which showed a strong dependence on the environmental factors.     

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.     

Microwave-assisted alkali pretreatment of rice straw to promote enzymatic hydrolysis and hydrogen production in dark- and photo-fermentation

Rice straw was pretreated by microwave-assisted alkali to improve saccharification in enzymatic hydrolysis and hydrogen yield in combined dark- and photo-fermentation in this paper. A maximum reducing sugar yield of 69.3 g/100 g TVS was obtained when 50 g/l rice straw was pretreated by microwave heating for 15 min at 140 °C in 0.5% NaOH solution and then enzymatically hydrolyzed for 96 h. When hydrolyzed rice straw was used for hydrogen production by anaerobic bacteria in dark-fermentation, a maximum hydrogen yield of 155 ml/g TVS was obtained. The residual solution (mainly acetate and butyrate) from dark-fermentation was reutilized for hydrogen production by immobilized photosynthetic bacteria in photo-fermentation. By combination of dark- and photo-fermentation, the maximum hydrogen yield was greatly enhanced to 463 ml/g TVS, which is 43.2% of the theoretical hydrogen yield.     

Biohydrogen production from apple pomace by anaerobic fermentation with river sludge

The biological hydrogen (bio-H₂) production from apple pomace (AP) by fermentation using natural mixed microorganisms in batch process was studied under various experimental conditions. The river sludge was used as a seed after being boiled for 15 min. The results show that the optimal pretreatment for AP was to soak it in the ammonia liquor of 6% for 24 h at room temperature. An optimal fermentation condition for bio-H₂ production was proposed that the pretreated AP at 37 °C, the initial pH of 7.0 and the fermentation concentration of 15 g/l could produce a maximum cumulative H₂ yield (CHYm) of 101.08 ml/g total solid (TS) with an average H₂ production rate (AHPR) of 8.08 ml/g TS/h. During the conversion of AP into H₂, acetic acid, ethanol, propionic acid and butyric acid were main liquid end-products.     

Optimization of biogas production from wheat straw stillage in UASB reactor

In the present study, thermophilic anaerobic digestion of wheat straw stillage was investigated. Methane potential of stillage was determined in batch experiments at two different substrate concentrations. Results showed that higher methane yields of 324 ml/g-(volatile solids) VS_added were obtained at stillage concentrations of 12.8 g-VS/L than at 25.6 g-VS/l. Continuous anaerobic digestion of stillage was performed in an up-flow anaerobic sludge blanket (UASB) reactor at 55 °C with 2 days hydraulic retention time. Results showed that both substrate concentration and organic loading rate (OLR) influenced process performance and methane yields. Maximum methane yield of 155 ml CH₄/g-COD was obtained at stillage mixtures with water of 25% (v/v) in the feed and at an OLR of 17.1 g-COD/(l.d). Soluble chemical oxygen demand (SCOD) removal at this OLR was 76% (w/w). Increase in OLR to 41.2 g-COD/(l.d) and/or stillage concentration in the feed to 33–50% (v/v) resulted in low methane yields or complete process failure. The results showed that thermophilic anaerobic digestion of wheat straw stillage alone for methane production is feasible in UASB reactor at an OLR of 17.1 g-COD/(l.d) and at substrate concentration of 25% in the feed. The produced methane could improve the process energy and economics of a bioethanol plant and also enable to utilize the stillage in a sustainable manner.     

Fermentative hydrogen production by the newly isolated Enterobacter asburiae SNU-1

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.     

Enhanced bio-hydrogen production by anaerobic fermentation of apple pomace with enzyme hydrolysis

A series of batch experiments were conducted to investigate the effects of the HCl-pretreated concentrations, enzyme hydrolysis time and temperature, the cellulase dosage, the ultrasonic time and the fermentation substrate concentration on hydrogen (H₂) production from the anaerobic fermentation of apple pomace (AP). The natural mixed microorganisms from river sludge was used as the seed after being boiled for 15 min. A maximum cumulative H₂ yield (CHYm) of 134.04 ml/g total solid (TS) and an average H₂ production rate (AHPR) of 12.00 ml/g TS/h were obtained from the fermentation of the enzyme-hydrolyzed AAP (a AP soaked with 6% ammonia liquor for 24 h) at the substrate concentration of 15 g/L. The optimal enzyme hydrolysis conditions were proposed as follows: AAP with a cellulase dosage of 12.5 mg/g TS at the hydrolysis substrate concentration of 20 g/L after the ultrasonic irradiation for 20 min was hydrolyzed by the enzyme catalysis at 45 °C and initial pH 5.0 for 48 h.     

The effect of Ni,Fe and Mg concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53

In the present study, the effect of Ni²⁺ (0–10 μmol/l), Fe²⁺ (0–200 μmol/l) and Mg²⁺ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni²⁺ was able to enhance the hydrogen production rate and the hydrogen yield; Fe²⁺ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni²⁺ and Fe²⁺ at a higher concentration inhibited cell growth. When Ni²⁺ and Fe²⁺ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H₂/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg²⁺ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H₂/mol acetate, but it was favorable to cell growth.     

Biological hydrogen production from steam-exploded straw by simultaneous saccharification and fermentation

Hydrogen was produced by simultaneous saccharification and fermentation from steam-exploded corn straw (SECS) using Clostridium butyricum AS1.209. Effect of various process parameters, such as solid to liquid ratio, enzyme loading and initial pH, etc., were examined with respect to maximum hydrogen productivity which was obtained by fitting the cumulative hydrogen production data to a modified Gompertz equation. Maximum specific hydrogen production rate and maximal hydrogen yield were 126 ml/g VSS d and 68 ml/g SECS, respectively. The yield of soluble metabolites was 197.7 mg/g SECS. Acetic acid accounted for 46% of the total was the most abundant product and this shows that hydrogen production from SECS was essentially acetate-type fermentation. Hydrogen production by simultaneous saccharification and fermentation of SECS has the predominance of short lag-stage and high maximum specific hydrogen production rate and it was a promising method for hydrogen production and straw biomass conversion.     

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.