Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture

Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H₂ from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H₂ yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H₂ mol⁻¹ xylose_consumed and 3.2 ± 0.16 mol-H₂ mol⁻¹ glucose_consumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H₂ yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H₂ mol⁻¹ sucrose_consumed. The effects of stirring speed and different pH levels on growth and H₂ production were studied in the CSABR for highly efficient H₂ production. Growth and H₂ production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H₂ production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H₂ L⁻¹ h⁻¹ at 32, 52, 67, and 86 h, respectively.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

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).     

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.     

Pretreatment enhanced structural disruption,enzymatic hydrolysis,fermentative hydrogen production from rice straw

Rice straw (RS) is one of the major lignocellulosic wastes in the world and an abundant feedstock for producing biofuels and chemicals. However, RS is difficult to decompose. In this study, NaOH/urea and electrohydrolysis pretreated RS were used to enhance the structural disruption, enzymatic hydrolysis, and fermentative hydrogen production. Scanning electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy analyses demonstrated that both NaOH/urea and electrohydrolysis pretreatments could effectively disrupt the lignin structure and increase the cellulose crystallinity of RS. Following pretreatment, RS was hydrolyzed by cellulase. After 96 h of enzymatic hydrolysis, NaOH/urea- and electrohydrolysis-pretreated RS produced 3.2- and 1.7-fold higher total reducing sugars than the unpretreated RS (232.95 ± 3.60 mg/g), respectively. Finally, the obtained RS hydrolysates were used for fermentative hydrogen production. NaOH/urea- and electrohydrolysis-pretreatment hydrolysates produced 125.0 and 163.0 mL H₂/g RS, respectively, which is much higher than the hydrogen yield of unpretreated hydrolysates.     

Biohydrogen production from rice straw hydrolyzate in a continuously external circulating bioreactor

The biohydrogen production from rice straw hydrolyzate in a continuously external circulating bioreactor (CECBR) was carried out in this study. The rice straw hydrolyzate was obtained by a concentrated sulphuric acid pretreatment. The original hydrolyzate concentration was 40–50 g total sugar/L. The feeding concentration of hydrolyzate was adjusted to 20 g total sugar/L by tap water. The working volume of CECBR was 300 mL with a height of 22.5 and a width of 7.5 cm respectively. The positions of external circulating ports were 13 and 5 cm high of CECBR with volumetric circulating rate of 9.6 L/min.     

Hydrogen production from carrot pulp by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

Hydrogen was produced from carrot pulp hydrolysate, untreated carrot pulp and (mixtures of) glucose and fructose by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana in pH-controlled bioreactors. Carrot pulp hydrolysate was obtained after enzymatic hydrolysis of the polysaccharide fraction in carrot pulp. The main sugars in the hydrolysate were glucose, fructose, and sucrose.     

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.     

Acidified glycerol pretreatment for enhanced ethanol production from rice straw

Rice straw was pretreated using an industrial grade glycerol for ethanol production. The pretreatment was conducted at 130–210 °C for 1–24 h with 5% solid loading. The glucan content in the regenerated rice straw increased with increasing pretreatment temperature and time. The production of fermentable sugars initially increased as the pretreatment temperature and reaction time increased, but then decreased somewhat at the higher temperatures and with longer reaction duration. The highest amount of reducing sugar produced by the enzymatic hydrolysis was achieved at 190 °C for 10 h with 5% solid loading, optimal condition for the glycerol pretreatment of rice straw. Furthermore, it was observed that glycerol pretreatment with the addition of HCl improved the digestibility of fermentable sugars by 4–5 times that of untreated samples. Fermentation of hydrolysates resulted in an ethanol yield of 0.44 g/g sugar, corresponding to a theoretical yield of 84.3%. It was concluded that acidified glycerol is one of the good candidates of the organic solvent for the pretreatment of lignocellulosic biomass.     

Low concentration of NaOH/Urea pretreated rice straw at low temperature for enhanced hydrogen production

In lignocellulose-to-hydrogen bioconversion, reducing the concentration of chemical agents in pretreatment is of great interest. In this study, rice straw (RS) pretreated at reduced NaOH and urea (NU) concentrations was evaluated. Results showed that the composition of RS exhibited excellent pretreatment performance at a reduced concentration of NU. When the concentration of NaOH was decreased to 3 wt% in combination with 6 wt% urea, the lignin was reduced by 59.52% with a cellulose and hemicellulose loss of less than 17%. Moreover, extending the pretreatment time at a low concentration of NU could effectively promote the biodegradability of RS. Upon fermentation by Thermoanaerobacterium thermosaccharolyticum M18 for H₂ production, the H₂ production increased up to 213.06 mL/g with a substrate treated by 3 wt% NaOH/6 wt% urea at low solid loading for 15 d, which was 16.31% higher than the counterpart subjected to a 7 wt% NaOH/12 wt% urea pretreatment. The present results suggest the NU pretreatment can be carried out at low concentrations to improve the conversion of RS into bio-H₂ production.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Clostridium strain co-cultures for biohydrogen production enhancement from condensed molasses fermentation solubles

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.     

Glycerol fermentation to hydrogen by Thermotoga maritima: Proposed pathway and bioenergetic considerations

The production of biohydrogen from glycerol, by the hyperthermophilic bacterium Thermotoga maritima DSM 3109, was investigated in batch and chemostat systems. T. maritima converted glycerol to mainly acetate, CO₂ and H₂. Maximal hydrogen yields of 2.84 and 2.41 hydrogen per glycerol were observed for batch and chemostat cultivations, respectively. For batch cultivations: i) hydrogen production rates decreased with increasing initial glycerol concentration, ii) growth and hydrogen production was optimal in the pH range of 7–7.5, and iii) a yeast extract concentration of 2 g/l led to optimal hydrogen production. Stable growth could be maintained in a chemostat, however, when dilution rates exceeded 0.025 h⁻¹ glycerol conversion was incomplete. A detailed overview of the catabolic pathway involved in glycerol fermentation to hydrogen by T. maritima is given. Based on comparative genomics the ability to grow on glycerol can be considered as a general trait of Thermotoga species. The exceptional bioenergetics of hydrogen formation from glycerol is discussed.     

Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

Two-stage co-hydrolysis of rice straw by Trichoderma reesei ZM4-F3 and Pseudomonas aeruginosa BSZ-07

Rhamnolipid biosurfactant was added to rice straw hydrolysis system to enhance the production of reducing sugars. Differing from the traditional method, on-site production of rhamnolipid made the rice straw decomposing fungus Trichoderma reesei ZM4-F3 and rhamnolipid producing bacteria Pseudomonas aeruginosa BSZ-07 work together. As the growth periods of these two strains are 96 h and 48 h, respectively, a new two-stage co-hydrolysis bioprocess was achieved. T. reesei ZM4-F3 was cultivated for rice straw hydrolysis in the first 48 h at suitable conditions. For the next 48 h, the results showed that the temperature of 34 °C and pH value of 5.5 were the optimum conditions, and the optimum adding inoculation concentration ratio of P. aeruginosa BSZ-07 to T. reesei ZM4-F3 was 4%. Under these conditions, the co-hydrolysis sample could improve the production of reducing sugars to 2.57 g l⁻¹, 15.20% higher than that of the control. The increased enzyme stability was indicated to be one of the mechanisms of the stimulatory effect of rhamnolipid on rice straw hydrolysis system. Compared with Tween-80 and sodium dodecylsulphate, rhamnolipid biosurfactant exhibited a better stimulatory effect on rice straw hydrolysis. Since the two-stage co-hydrolysis system could leave out the rhamnolipid purification process, thus reducing the biosurfactant production cost effectively, it seems to be a new prospective bioprocess for rice straw hydrolysis.     

Single-stage photo-fermentative hydrogen production from hydrolyzed straw biomass using Rhodobacter sphaeroides

The hydrogen production utilizing photosynthetic and anaerobic bacteria in two-stage approach has many drawbacks, such as shortage of raw materials and complexity of operations. Accordingly, we aimed to develop a simple one-stage H₂ production protocol using the depolymerization of maize straw cellulose as a cheap carbon source. R. sphaeroides HY01 and its mutant (Hup^?) were studied regarding their H₂ production under different culture conditions. Further study using two model sugars, their combination, and straw hydrolysate as carbon sources was conducted to determine the effects of substrate on H₂ production. When using the straw hydrolysate as carbon source, the pH remained in a range of 7.1–7.6, whereas it dropped to 5.4–7.4 when using the model sugars, and the former biomass value was greater. The H₂ production performance of the mutant was significantly better than that of HY01. One-step photo-fermentative H₂ production was superior when using straw hydrolysate as opposed to the simple model sugars, and its yield was up to 4.62 mol H₂·mol^?1 reducing sugar.     

Assessment of the structural factors controlling the enzymatic saccharification of rice straw cellulose

In this study, the structural factors controlling the enzymatic saccharification of rice straw cellulose were examined by preparing structure-modified rice straw samples such as dewaxed, alkali-treated, oxidized, and swollen rice straw. It was found that the initial enzymatic saccharification rate of the various structure-modified samples was largely controlled by the initial cellulose surface area of the cellulose unit. Although the presence of lignin limited the cellulose surface area, there was no strong relationship between the lignin content and the initial reaction. On the other hand, the long-term enzymatic saccharification of rice straw cellulose highly depended on the lignin removal rate (lignin content). It was also found that silica is not a crucial factor in controlling the enzymatic saccharification.     

Review on fermentative biohydrogen production from water hyacinth,wheat straw and rice straw with focus on recent perspectives

Hydrogen (H₂) is often considered as the best option to store energy coming from renewable sources. Hydrogen production from lignocellulosic biomass via fermentation offers low cost and environmental friendly method in terms of energy balance and provides a sustainable pathway for utilization of huge amount of unused biomass. In this regard, special attention on potential of different lignocellulosic biomass is required. In this paper, the fermentative hydrogen production from three carbohydrates-rich biomass: water hyacinth, wheat straw and rice straw is comprehensively reviewed. In other point of view, usage of H₂ has a 10% growth annually that will reach to 8–10% of total energy in 2025. Furthermore, research on recent trends of fermentative hydrogen production is crucial and vital. However, the majority of the published researches in the last decade confirmed that some challenges exists which are the process optimization, effecting parameters and commercialization aspects.     

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.     

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.