Sequencing batch reactor enhances bacterial hydrolysis of starch promoting continuous bio-hydrogen production from starch feedstock

Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H₂ fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H₂ from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 °C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H₂ production with the starch hydrolysate, the highest H₂ production rate and yield was 0.52 L/h/L and 13.2 mmol H₂/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH₄HCO₃) concentration was 2.62 g/L, attaining a good H₂ production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H₂ fermentation while using dark fermentation effluents as the substrate.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation

Biohydrogen is usually produced via dark fermentation, which generates CO₂ emissions and produces soluble metabolites (e.g., volatile fatty acids) with high chemical oxygen demand (COD) as the by-products, which require further treatments. In this study, mixotrophic culture of an isolated microalga (Chlorella vulgaris ESP6) was utilized to simultaneously consume CO₂ and COD by-products from dark fermentation, converting them to valuable microalgae biomass. Light intensity and food to microorganism (F/M) ratio were adjusted to 150 μmol m⁻² s⁻¹ and F/M ratio, 4.5, respectively, to improve the efficiency of assimilating the soluble metabolites. The mixotrophic microalgae culture could reduce the CO₂ content of dark fermentation effluent from 34% to 5% with nearly 100% consumption of soluble metabolites (mainly butyrate and acetate) in 9 days. The obtained microalgal biomass was hydrolyzed with 1.5% HCl and subsequently used as the substrate for bioH₂ production with Clostridium butyricum CGS5, giving a cumulative H₂ production of 1276 ml/L, a H₂ production rate of 240 ml/L/h, and a H₂ yield of 0.94 mol/mol sugar.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.     

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.     

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

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

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

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

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%.     

Performance and population analysis of hydrogen production from sugarcane juice by non-sterile continuous stirred tank reactor augmented with Clostridium butyricum

Non-sterile operation of continuous stirred tank reactor (CSTR) augmented with Clostridium butyricum and fed with sugarcane juice was studied at various hydraulic retention time (HRT). The maximum hydrogen production rate and yield of 3.38 mmol H₂/L/h and 1.0 mol H₂/mol hexose consumed, respectively, were achieved at HRT 4 h. The relationship of the augmented microorganism and normal flora in the fermentation system under non-sterile condition were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Initially, at HRT 36 h, other species related to Lactobacillus harbinensis and Klebseilla pneumoniae were present as a major group in the reactor. When HRT was decreased to 12, 6 and 4 h, C. butyricum was present with a competition between L. harbinensis and K. pneumoniae. Results indicated that augmented C. butyricum could compete with contaminated microorganisms during non-sterile operation at low HRT (12-4 h) with the support of normal flora (K. pneumoniae).     

Enhanced enzymatic conversion with freeze pretreatment of rice straw

Production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years, because of its low cost and great potential availability. The pretreatment process is important for increasing the enzymatic digestibility of lignocellulosic materials. Enzymatic conversion with freeze pretreatment of rice straw was evaluated in this study. The freeze pretreatment was found to significantly increase the enzyme digestibility of rice straw from 48% to 84%. According to the results, enzymatic hydrolysis of unpretreated rice straw with 150 U cellulase and 100 U xylanase for 48 h yielded 226.77 g kg⁻¹ and 93.84 g kg⁻¹ substrate-reducing sugars respectively. However, the reducing sugar yields from freeze pretreatment under the same conditions were 417.27 g kg⁻¹ and 138.77 g kg⁻¹ substrate, respectively. In addition, hydrolyzates analysis showed that the highest glucose yield obtained during the enzymatic hydrolysis step in the present study was 371.91 g kg⁻¹ of dry rice straw, following pretreatment. Therefore, the enhanced enzymatic conversion with freeze pretreatment of rice straw was observed in this study. This indicated that freeze pretreatment was highly effective for enzymatic hydrolysis and low environmental impact.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Fermentative hydrogen production from hydrolyzed cellulosic feedstock prepared with a thermophilic anaerobic bacterial isolate

Hydrogen gas was produced via dark fermentation from natural cellulosic materials and α-cellulose via a two-step process, in which the cellulosic substrates were first hydrolyzed by an isolated cellulolytic bacterium Clostridium strain TCW1, and the resulting hydrolysates were then used as substrate for fermentative H₂ production. The TCW1 strain was able to hydrolyze all the cellulosic materials examined to produce reducing sugars (RS), attaining the best reducing sugar production yield of 0.65 g reducing sugar/g substrate from hydrolysis of α-cellulose. The hydrolysates of those cellulosic materials were successfully converted to H₂ via dark fermentation using seven H₂-producing bacterial isolates. The bioH₂ production performance was highly dependent on the type of cellulosic feedstock used, the initial reducing sugar concentration (C_RS,o) (ranging from 0.7 to 4.5 mg/l), as well as the composition of sugar and soluble metabolites present in the cellulosic hydrolysates. It was found that Clostridium butyricum CGS5 displayed the highest H₂-producing efficiency with a cumulative H₂ production of 270 ml/l from α-cellulose hydrolysate (C_RS,o = 4.52 mg/l) and a H₂ yield of 7.40 mmol/g RS (or 6.66 mmol/g substrate) from napier grass hydrolysate (C_RS,o = 1.22 g/l).     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

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

Stoichiometric analysis of biological hydrogen production by fermentative bacteria

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

Challenges for biohydrogen production via direct lignocellulose fermentation

Direct cellulose fermentation by cellulolytic anaerobic bacteria offers potential to generate renewable hydrogen (H₂) from inexpensive “waste” cellulosic feedstocks. The rates and yields of H₂ production via direct cellulose fermentation are low and must be increased significantly if this technology is to become a viable method for generating usable H₂. A much more comprehensive understanding of the relationships between gene and gene product expression, end-product synthesis patterns, and the factors that regulate carbon and electron balance, within the context of the bioreactor conditions must be achieved if we are to improve molar yields of H₂ during cellulose fermentation. Strategies to increase yields of H₂ production from cellulose include manipulation of carbon and electron flow via end-product inhibition (metabolic shift), metabolic engineering at the genetic level, synergistic co-cultures, and bioprocess engineering and bioreactor designs that maintain a neutral pH during fermentation and ensure rapid removal of H₂ and CO₂ from the aqueous phase.     

Biohydrogen production from ricebran using Clostridium saccharoperbutylacetonicum N1-4

Treated ricebran hydrolysate was fermented anaerobically using Clostridium saccharoperbutylacetonicum N1-4 at an initial pH of 6 ± 0.2 and an operating temperature of 30 °C for production of hydrogen. The effects of different pretreatment methods on the liberation of sugar from 100 g of ricebran per litre of medium (distilled water) were investigated. In addition, the effects of the pretreatment method on ricebran hydrolysates of different initial ricebran concentrations on liberated sugar as well as the effects of the initial inoculum concentration, ricebran (substrate) concentration, and FeSO₄·7H₂O concentration on the yield as well as the productivity of hydrogen were investigated. The combination of enzymatic hydrolysis and a boiling pretreatment method produced the most fermentable sugar, 29.03 ± 0.0 g/L from 100 g of ricebran per litre of medium (distilled water), while the amount of sugar liberated by ricebran hydrolysates of different initial ricebran concentrations upon pretreatment monotonically increased with the initial ricebran concentration. The increment in substrate, inoculum, and FeSO₄·7H₂O concentrations had a significantly positive effect (p < 0.05) on both the yield and productivity of hydrogen. The maximum hydrogen gas yield (Y_P/S) and productivity of 3.37 mol-H₂ per mol-sugar consumed and 7.58 mmol/(L h), respectively, were obtained from ricebran hydrolysate with a 100 g/L ricebran concentration (equivalent to 28.59 ± 1.27 g sugar/L). In other experiments, 0.03 g/L FeSO₄·7H₂O and 1.5 g/L inoculum resulted in the best hydrogen gas yield and productivity from ricebran hydrolysates.     

Ethanol production from enzymatic hydrolysis of sugarcane bagasse pretreated with lime and alkaline hydrogen peroxide

In this work we evaluated ethanol production from enzymatic hydrolysis of sugarcane bagasse. Two pretreatments agents, lime and alkaline hydrogen peroxide, were compared in their performance to improve the susceptibility of bagasse to enzymatic action. Mild conditions of temperature, pressure and absence of acids were chosen to diminish costs and to avoid sugars degradation and consequent inhibitors formation. The bagasse was used as it comes from the sugar/ethanol industries, without grinding or sieving, and hydrolysis was performed with low enzymes loading (3.50 FPU g⁻¹ dry pretreated biomass of cellulase and 1.00 CBU g⁻¹ dry pretreated biomass of ??-glucosidase). The pretreatment with alkaline hydrogen peroxide led to the higher glucose yield: 691 mg g⁻¹ of glucose for pretreated bagasse after hydrolysis of bagasse pretreated for 1 h at 25 °C with 7.35% (v/v) of peroxide. Fermentation of the hydrolyzates from the two pretreatments were carried out and compared with fermentation of a glucose solution. Ethanol yields from the hydrolyzates were similar to that obtained by fermentation of the glucose solution. Although the preliminary results obtained in this work are promising for both pretreatments considered, reflecting their potential for application, further studies, considering higher biomass concentrations and economic aspects should be performed before extending the conclusions to an industrial process.     

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

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