Effect of lignocellulose-derived inhibitors on growth and hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16

In the process of producing H₂ from lignocellulosic materials, inhibitory compounds could be potentially formed during pre-treatment. This work experimentally investigated the effect of lignocellulose-derived inhibitors on growth and hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16. Representative compounds presented in corn stover acid hydrolysate were added in various concentrations, individually or in various combinations and subsequently inhibitions on growth and H₂ production were quantified. Acetate sodium was not inhibitory to T. thermosaccharolyticum W16, rather than it was stimulatory to the growth and H₂ production. Alternatively, furfural, hydroxymethylfurfural (HMF), vanillin and syringaldehyde were potent inhibitors of growth and hydrogen production even though these compounds showed inhibitory effect depending on their concentrations. Synergistic inhibitory effects were exhibited in the introduction of combinations of inhibitors to the medium and in hydrolysate with concentrated inhibitors. Fermentation results from hydrolysates revealed that to increase the efficiency of this bioprocess from corn stover hydrolysate, the inhibitory compounds concentration must be reduced to the levels present in the raw hydrolysate.     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

Bioaugmentation with Thermoanaerobacterium thermosaccharolyticum W16 to enhance thermophilic hydrogen production using corn stover hydrolysate

In the present work, with corn stover hydrolysate as the substrate, an efficient hydrogen-producing thermophile, Thermoanaerobacterium thermosaccharolyticum W16, was added to three kinds of seed sludge (rotten corn stover (RCS), cow dung compost (CDC), and sludge from anaerobic digestion (SAD)) to investigate the effect of bioaugmentation on thermophilic hydrogen production. Batch test results indicate that the bioaugmentation with a small amount of the strain T. thermosaccharolyticum W16 (5% of total microbes) increased the hydrogen yield to varying degrees (RCS: from 8.78 to 9.90 mmol H₂/g utilized sugar; CDC: from 8.18 to 8.42 mmol H₂/g utilized sugar; SAD: from 8.55 to 9.17 mmol H₂/g utilized sugar). The bioaugmentation process also influenced the soluble metabolites composition towards more acetate and less butyrate production for RCS, and more acetate and less ethanol accumulation for SAD. Microbial community analysis indicates that Thermoanaerobacterium spp. and Clostridium spp. dominated microbial community in all situations and might be mainly responsible for thermophilic hydrogen generation. For RCS and SAD, the bioaugmentation obviously increased the relative abundance of the strain T. thermosaccharolyticum W16 in microbial community, which might be the main reason for the improvement of hydrogen production in these cases.     

Biohydrogen production from xylose by Thermoanaerobacterium thermosaccharolyticum KKU19 isolated from hot spring sediment

A thermophilic hydrogen producer was isolated from hot spring sediment and identified as Thermoanaerobacterium thermosaccharolyticum KKU19 by biochemical tests and 16S rRNA gene sequence analysis. The strain KKU19 showed the ability to utilize various kinds of carbon sources. Xylose was the preferred carbon source while peptone was the preferred organic nitrogen source. The optimum conditions for hydrogen production and cell growth on xylose were an initial pH of 6.50, temperature of 60 °C, a carbon to nitrogen ratio of 20:1, and a xylose concentration of 10.00 g/L. This resulted in a maximum cumulative hydrogen production, hydrogen production rate and hydrogen yield of 3020 ± 210 mL H₂/L, 3.95 ± 0.20 mmol H₂/L h and 2.09 ± 0.02 mol H₂/mol xylose consumed, respectively. Acetic and butyric acids were the main soluble metabolite products suggesting acetate and butyrate type fermentation.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Improvement of hydrogen production by newly isolated Thermoanaerobacterium thermosaccharolyticum IIT BT-ST1

Efficient H₂ producing bacterial strain Thermoanaerobacterium thermosaccharolyticum IIT BT-ST1 was isolated from the anaerobic digester. Taguchi design of experiment was applied to evaluate the influence of the temperature, pH, glucose, FeSO₄ and yeast extract on H₂ production with three levels of orthogonal array in the experimental design. Temperature showed most significant influence on the H₂ production process. Investigation of mutual interaction between the process parameters was studied employing Box–Behnken design. Experimentally optimized process parameters (60 °C, pH 6.5, 20 mM FeSO₄, 4 g L⁻¹ yeast extract and 12 g L⁻¹ glucose) gave the maximum H₂ production of 3930 mL L⁻¹ in 24 h, which have close resemblance with the theoretical values. Continuous H₂ production using packed bed reactor was studied. Maximum H₂ production rate of 1691 mL L⁻¹ h⁻¹ at a dilution rate of 0.6 h⁻¹ was observed which is about 10 times higher than the batch process.     

Thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19: Optimization of media composition

The composition of media for thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19 were optimized in order to maximize the hydrogen production potential (P_s). Results from Plackett-Burman design indicated that FeSO₄, CaCl₂, NaHCO₃, and MgCl₂ had a significantly effect (P ≤ 0.05) on P_s. The optimum media composition obtained from the response surface methodology (RSM) with central composite design (CCD), using the hydrolysate at a total sugar concentration of 8.98 g/L, were (all in mg/L): FeSO₄, 1454.65; MgCl_2, 511.36; CaCl_2, 278.62; and NaHCO_3, 2186.41 in which the P_s of 2397 mL H₂/L were obtained. Verification experiment using the optimum media composition in a continuous stirred tank reactor indicated a highly reproducible result in which the P_s of 2608 mL H₂/L was achieved at a hydraulic retention time of 32 h. The results demonstrated that the media composition obtained from the batch experiment using RSM with CCD can be practically applied to continuously produce hydrogen from the hydrolysate with the least error.     

An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate

Anaerobic sequencing batch reactor (ASBR) process offers great potential for H₂ production from wastewaters. In this study, an ASBR was used at first time for enhanced continuous H₂ production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times (HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at 65 mmol sugars L⁻¹. Results showed that increasing the HRT from 6 to 12 h led to the H₂ production rate increased from 6.7 to the maximum of 9.6 mmol H₂ L⁻¹ h⁻¹ and the substrate conversion reached 90.3%, although the H₂ yield remained at the same level of 1.7 mol H₂ mol⁻¹ substrate. Taking into account both H₂ production and substrate utilization efficiencies, the optimum HRT for continuous H₂ production via an ASBR was determined at 12 h. Compared with other continuous H₂ production processes, ASBR yield higher H₂ production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H₂ production from lignocellulosic biomass.     

Co-digestion of oil palm trunk hydrolysate with slaughterhouse wastewater for thermophilic bio-hydrogen production by Thermoanaerobacterium thermosaccharolyticm KKU19

The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H₂SO₄ method using 1.56% (w/v) H₂SO₄ and 7.50 min reaction time at 450 W. The Plackett–Burman method was used to screen the key factors that influenced the hydrogen production potential (P_s). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO₄ concentration influenced the P_s. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum P_s of 2604 ± 86 mL H₂/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO₄ concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The P_s obtained when the hydrolysate was supplemented with SHW (2604mL ± 86 mL H₂/L substrate) was comparable to the P_s obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 ± 87 mL H₂/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source.     

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.     

Improved hydrogen production via thermophilic fermentation of corn stover by microwave-assisted acid pretreatment

A microwave-assisted acid pretreatment (MAP) strategy has been developed to enhance hydrogen production via thermophilic fermentation of corn stover. Pretreatment of corn stover by combining microwave irradiation and acidification resulted in the increased release of soluble substances and made the corn stover more accessible to microorganisms when compared to thermal acid pretreatment (TAP). MAP showed obvious advantages in short duration and high efficiency of lignocellulosic hydrolysis. Analysis of the particle size and specific surface area of corn stover as well as observation of its cellular microstructure were used to elucidate the enhancement mechanism of the hydrolysis process by microwave assistance. The cumulative hydrogen volume reached 182.2 ml when corn stover was pretreated by MAP with 0.3 N H₂SO₄ for 45 min, and the corresponding hydrogen yield reached 1.53 mol H₂/mol-glucose equivalents converted to organic end products. The present work demonstrates that MAP has potential to enhance the bioconversion efficiency of lignocellulosic waste to renewable biofuel.     

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.     

Evaluation of the potential hydrogen production by diazotrophic Burkholderia species

Hydrogen (H₂) is considered one of the most promising fuels for sustainable energy. Because nitrogenase produces H₂ as a normal by-product, we tested the N₂-fixing bacterial strains Burkholderia unamae and Burkholderia tropica to determine their H₂ production capacities. To maximize H₂ production, several culture conditions were tested and optimized, including atmospheric conditions, carbon sources and chemical compounds such as enzyme cofactors and sugar cane molasses. The results showed that both strains were capable of H₂ production. The culture medium with the highest H₂ yield was composed of 1% v/v molasses enriched with Na₂MoO₄ (0.2 g/L), FeSO₄ (0.2 g/L) and cysteine (0.02 g/L) under a partial vacuum (air 20% v/v) without Ar final atmosphere. Under these conditions, the highest H₂ production rate obtained was 24.64 mmol H₂/L for B. unamae. The present study contributes an optimization process for H₂ production in N₂-fixing Burkholderia species. We propose further research and development to improve H₂ production rates in order to make biohydrogen a tangible reality.     

Nutrient and alkalinity removal by corn grain, stover and cob harvest in Upper Midwest USA

In the USA, most corn stover currently remains in fields as crop residue that provides soil erosion control and maintains soil organic carbon levels. This stover is a potential biofuel feedstock for direct combustion, pyrolysis, and ethanol fermentation. At a research site in south central Wisconsin, the northern edge of the US Corn Belt, corn grain harvest averaged 9.8 Mg ha⁻¹ DM over a 6-year period, 1997 to 2002. Removal of all stover could recover an additional 7.2 Mg ha⁻¹ y⁻¹ DM and, in the process, remove an additional 47, 6, 81 and 197 kg ha⁻¹ y⁻¹ of N, P, K and calcium carbonate equivalent, respectively. The fertilizer replacement cost for stover removal is 32 $ Mg⁻¹ DM, which is 95% of the fertilizer value of the grain. However, most of the N, P, K and alkalinity of the stover is found in the leaves, stalk, and husks, not in the cob. At our study site, complete stover removal would export 235 $ ha⁻¹ y⁻¹ of fertilizer and limestone, mainly as K, while cob export would be worth 20 $ ha⁻¹ y⁻¹ in nutrient equivalents. Based on this research, removal of cobs only is equivalent to 16.6% of total stover removal but with a greatly reduced fertilizer replacement cost of 17 $ Mg⁻¹ DM and the same energy density.     

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.     

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.     

Evaluation of hydrogen production from corn cob with the mesophilic bacterium Clostridium hydrogeniproducens HR-1

Corn cob is a promising hydrogen fermentation substrate, not only because of its abundant and low cost, but also because of its high cellulose and hemicellulose content. However, little information is available on the use of corn cob as a feedstock for hydrogen production. In this study, corn cob was hydrolyzed by cellulase after acid steam-explosion, alkali soaking, or steam-explosion pretreatment. The liquid products of pretreatment and the enzymatic hydrolysates were then used as carbon sources for hydrogen production by Clostridium hydrogeniproducens HR-1. Pretreatment followed by enzymatic hydrolysis yielded 720, 670, and 530 mg reducing sugars/g corn cob, and the hydrogen yield from corn cob reached 119, 100, and 83 ml H₂/g corn cob, which is 55.9%, 46.7%, and 38.8% of the theoretical hydrogen yield from corn cob using C. hydrogeniproducens HR-1, respectively.     

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.