Thermophilic hydrogen fermentation from Korean rice straw by Thermotoga neapolitana

Rice straw, a low-cost lignocellulosic biomass was used as feedstock for thermophilic hydrogen fermentation by Thermotoga neapolitana. Hydrogen production, the growth and cellulose digestibility of the hyperthermophile in batch mode from untreated as well as chemically pretreated (ammonia and dilute sulfuric acid) Korean rice straws were investigated. Pretreatment method using combination of 10% ammonia and 1.0% dilute sulfuric acid was developed to increase the digestibility of rice straw for the hyperthermophilic H₂ fermentation and to decrease the time consumption. In a typical fermentation using raw rice straw, 29% of the substrate was digested and 2.3 mmol H₂/g straw of hydrogen yield was consistently obtained. Compared with the pretreatments using only ammonia or dilute sulfuric acid, the combined pretreatment method using both chemical agents significantly increases the digestibility of rice straw with 85.4% of substrate consumption. H₂ production on rice straw from this combined pretreatment showed the highest yield (2.7 mmol H₂/g straw) and the highest sugar conversions (72.9% of glucose and 95.7% of xylose).     

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.     

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.     

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.     

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.     

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%.     

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.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.     

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.     

Application of Plackett–Burman experimental design to optimize biohydrogen fermentation by E. coli (XL1-BLUE)

Escherichia coli is attractive for biotechnological hydrogen production. Compared to other biohydrogen producing bacteria e.g. Clostridium species, E. coli is able to tolerate oxygen, fast growing and well-characterized in physiological and biochemical terms. According to the well known metabolic pathways of E. coli, the hydrogen production from different substrates is dependent on the membrane-boundary formate-hydrogen lyase (FHL) enzyme complex. The efficiency and economic success of hydrogen fermentation are influenced by the applied operational conditions. In this work the optimal conditions (composition of broth, inoculum size, stirring speed) for biohydogen fermentation using E. coli (XL1-BLUE) were investigated by experimental design. We found that among the several variables only formate compound plays a key role in hydrogen formation and the optimal conditions for biohydrogen production were identified as follows: 30 mM formate, 5 g/l yeast extract, 10 g/l tryptone, 3.33 g/l NaCl, 0.05 g dry cell weight/l initial cell density and 220 rpm stirring rate, where productivity and yield were 426 ml H₂ l⁻¹ d⁻¹ and 0.41 mol H₂/mol formate, respectively.     

Pretreatment of Miscanthus for hydrogen production by Thermotoga elfii

Pretreatment methods for the production of fermentable substrates from Miscanthus, a lignocellulosic biomass, were investigated. Results demonstrated an inverse relationship between lignin content and the efficiency of enzymatic hydrolysis of polysaccharides. High delignification values were obtained by the combination of mechanical, i.e. extrusion or milling, and chemical pretreatment (sodium hydroxide). An optimized process consisted of a one-step extrusion-NaOH pretreatment at moderate temperature (70°C). A mass balance of this process in combination with enzymatic hydrolysis showed the following: pretreatment resulted in 77% delignification, a cellulose yield of more than 95% and 44% hydrolysis of hemicellulose. After enzymatic hydrolysis 69% and 38% of the initial cellulose and hemicellulose fraction, respectively, was converted into glucose, xylose and arabinose. Of the initial biomass, 33% was converted into monosaccharides. Normal growth of Thermotoga elfii on hydrolysate was observed and high amounts of hydrogen were produced.     

Strategy for enhancing photo-hydrogen production yield by repeated fed-batch cultures

In order to obtain the high H₂ yield, photo-hydrogen production by Rhodopseudomonas faecalis strain RLD-53 in fed-batch culture using acetate as the sole carbon was studied. In repeated fed-batch culture, biomass increased rapidly from 0.05 to 0.65 g/l within 120 h, with the specific maximal growth rate estimated 0.68 × 10⁻³ g/h. After 120 h, biomass increased slowly and after each time feeding biomass increased slightly. The specific cumulative H₂ volumes in each phase were 2791.3, 1161.7, 1445.5 and 840.6 ml H₂/l-culture, respectively. The average H₂ yield was 3.17 mol H₂/mol acetate based on the whole process while the average substrate conversion efficiency reached 79.3%. Specific maximum H₂ production rate and H₂ content was 37.2 ml H₂/l/h and 95.5%, respectively. The results demonstrated the repeated fed-batch mode obtained higher efficiency for hydrogen production, feeding acetate concentration and control of pH were important to fed-batch culture hydrogen production.     

Microbial lipid production by the oleaginous yeast Cryptococcus curvatus O3 grown in fed-batch culture

This study investigates the capability of the oleaginous yeast Cryptococcus curvatus O3 to synthesize microbial lipids using glucose as its sole carbon source. Both glucose concentration and varying nitrogen sources have a significant effect on cell growth and microbial lipid accumulation in batch and fed-batch cultures. When cultivated in a shaking flask at 30 °C with glucose as sole carbon source, the cellular biomass and lipid content reached 51.8 kg m⁻³ and 651 g kg⁻¹, respectively. The fed-batch culture in a 30 × 10⁻³ m³ stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m⁻³, 827 g kg⁻¹, and 0.47 kg m⁻³ h⁻¹, respectively. These data indicate that C. curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production. Gas chromatography analysis of the synthesized microbial lipids revealed that the major constituents are long-chain fatty acids, such as palmitic acid, stearic acid, oleic acid, and linoleic acid. The results suggest that the microbial lipids produced by C. curvatus O3 can be used to produce biodiesel.     

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.     

Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production

Volatile fatty acids (VFA) from dark fermentation hydrogen production were tested as carbon sources for the culture of oleaginous yeast Cryptococcus curvatus, which is a promising feedstock for biofuel production. The optimal acetate concentration and pH were investigated when potassium acetate was used as the sole carbon source. Comparisons were then made when hydrogen production effluent (HPE) from synthetic wastewater was tested as feedstock. A pH-stat culture fed with acetic acid ultimately produced 168 g/L biomass, with a lipid content of 75.0%. No inhibitor to yeast growth was produced in the hydrogen production process. However, inhibition occurred in culture with HPE from food waste (FW), indicating that inhibitors may be present in the original raw food waste. This inhibition could be avoided by a process that uses glucose as the initial carbon source and then is continuously fed with FW-HPE. The biomass productivity in this continuous culture process reached 0.34 g/L/h, but the lipid content was only 13.5%. These results suggest that FW-HPE alone is not an optimal feedstock, but HPE derived from nitrogen-deficient waste streams could be good feedstocks. This study provides preliminary evidence for the feasibility of using organic waste for the co-production of hydrogen and lipid.     

Photo-bioproduction of hydrogen by Chlamydomonas reinhardtii using a semi-continuous process regime

Photo-bioproduction of hydrogen by using green algae, Chlamydomonas reinhardtii, was investigated in batch and semi-continuous process regimes, in a continuous stirred type photobioreactor. Batch cultivation was carried out for 35 days which was one of the longest cited in literature. Total hydrogen production with batch culture reached 316 ml. The observations from the batch culture provided useful data about the production process. Three important observations were made from the batch cultivation. One was the requirement of a 2 day-lag time for the start of the hydrogen production. Second one was the fact that the maximum hydrogen production is reached at around day 4. Third one was the decline of hydrogen production after a week. Semi-continuous regime was preferred rather than a continuous one based on these data. Semi-continuous cultivation was continued for 127 days yielding a total hydrogen production of 1108 ml. In the semi-continuous process, the effects of parameters such as dilution ratio, dilution frequency and fresh medium addition were studied. The range of these parameters was also decided, based on the batch cultivation data. Each experiment testing for different parameters lasted for 7 days and thus five consecutive sets were completed in 35 days.The results showed a direct correlation between the amount and frequency of dilution and hydrogen production. Semi-continuous regime gave the opportunity of dividing the continuous production in consecutive batches and the process was in good relation with batch regime.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.     

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H₂) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H₂ yield (HY) of 0.65 mol H₂/mol acetate- and 2.50 mol H₂/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H₂/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H₂ production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H₂ production. When pH was controlled to 7.0, strain KD131 wt evolved more H₂ by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H₂ production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH₄)₂SO₄. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H₂ production in expending reducing power.     

Fermentative hydrogen production with xylose by Clostridium and Klebsiella species in anaerobic batch reactors

Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H₂ and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H₂/mol xylose), 8% (0.5 mol H₂/mol xylose), 10% (0.6 mol H₂/mol xylose) and 14% (0.8 mol H₂/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.