Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

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.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

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%.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

High yield simultaneous hydrogen and ethanol production under extreme-thermophilic (70 °C) mixed culture environment

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO₄ (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H₂/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.     

Biohydrogen production using corn stalk employing Bacillus licheniformis MSU AGM 2 strain

Stalk of corn (Zea mays L.), a commonly available crop residue in tropical and subtropical countries and found in plenty. In this study, we demonstrated that the pretreated corn stalk waste could be used to produce hydrogen. The isolated Bacillus licheniformis MSU AGM 2 strain (HM214759) from the pretreated paper mill effluent produced hydrogen under optimized conditions: carbon source (1 g/l), nitrogen source (12.5 g/l), temperature (35 °C) and pH (6.0). Alkaline pretreatment with 2% NaOH removed lignin by 48% from the corn stalk waste. Pretreated corn stalk ranges from 1 to 5 g/l were tested for the effective bacterial growth and hydrogen production. Kinetic parameters analyzed in 1 l bioreactor showed the maximum hydrogen production and hydrogen yield with 185 ml/l and 82.5 ml/g substrate, respectively. Growth profile and modified Gompertz model at the above mentioned condition fitted well (R², 0.93). Hence the anaerobic fermentation by the isolated strain had increased the hydrogen evolution rate with formic acid, acetate and butyrate concentrations at the end of the fermentation.     

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$0.53?\delta ?0.83$).     

Biohydrogen production from wastewater by Clostridium beijerinckii: Effect of pH and substrate concentration

An investigation of biological hydrogen production from glucose by Clostridium beijerinckii was conducted in a synthetic wastewater solution. A study examining the effect of initial pH (range 5.7–6.5) and substrate loading (range 1–3 g COD/L) on the specific conversion and hydrogen production rate has shown interaction behaviour between the two independent variables. Highest conversion of 10.3 mL H₂/(g COD/L) was achieved at pH of 6.1 and glucose concentration of 3 g COD/L, whereas the highest production rate of 71 mL H₂/(h L) was measured at pH 6.3 and substrate loading of 2.5 g COD/L. In general, there appears to be a strong trend of increasing hydrogen production rate with an increase in both substrate concentration and pH. Butyrate (14–63%), formate (10–45%) and ethanol (16–40%) were the main soluble products with other volatile fatty acids and alcohols present in smaller quantities.     

Control strategies for hydrogen production through co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53

This study evaluated hydrogen production by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 with different control strategies. To enhance cooperation of dark and photo-fermentation bacteria during hydrogen production process, the glucose concentration, phosphate buffer concentration and initial pH were controlled at 6 g/l, 50 mmol/l and 7.5, respectively. The maximum yield and rate of hydrogen production were 3.10 mol H₂/mol glucose and 17.2 mmol H₂/l/h, respectively. Ethanol from E. harbinense B49 in acetate medium can enhance hydrogen production by R. faecalis RLD-53 except the ratio of ethanol to acetate (R_E/A) among 0.8 to 1.0. Control of the proper phosphate buffer concentration (50 mmol/l) not only increased acetic acid production by E. harbinense B49, but also maintained stable pH of co-culture system. Therefore, the results showed that co-culture of E. harbinense B49 and immobilized R. faecalis RLD-53 was a promising way of converting glucose into hydrogen.     

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.     

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

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

Enhancement of biohydrogen producing using ultrasonication

Ultrasonication was evaluated as a pretreatment for biological hydrogen production from glucose in batch studies, in comparison with heat-shock pretreatment, acid pretreatment, and base pretreatment. The optimized sonication energy for hydrogen production using anaerobic digester sludge was 79 kJ/gTS. Sonication with temperature control (less than 30 °C) increased volumetric hydrogen production by 120% over the untreated sludge, and by 40% over the heat-shock and acid pretreated sludge, with a marginal (∼10%) increase in hydrogen production rate. Upon comparing the molar hydrogen yield in sonicated sludge with and without temperature control, the deleterious effect of heat on some hydrogen producers as reflected by a 30% decrease in yield to 1.03 mol H₂/mol glucose is evident. Sonication with temperature control affected a 45% increase in molar hydrogen yield to 1.55 mol H₂/mol glucose over heat-shock pretreatment at 70 °C for 30 min and acidification to pH 3.0 for 24 h at 4 °C. Sonication with temperature control produced a biomass yield of 0.13 g VSS/g COD, as compared to 0.24 g VSS/g COD for the untreated sludge. The hydrogen yield increased linearly with the molar acetate to butyrate ratio and decreased linearly with the biomass yield.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

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.     

Biological hydrogen production in an anaerobic sequencing batch reactor: pH and cyclic duration effects

An anaerobic sequencing batch reactor (ASBR) was used to evaluate biological hydrogen production from carbohydrate-rich organic wastes. The goal of the proposed project was to investigate the effects of pH (4.9, 5.5, 6.1, and 6.7), and cyclic duration (4, 6, and 8 h) on hydrogen production. With the ASBR operated at 16-h HRT, 25 g COD/L, and 4-h cyclic duration, the results showed that the maximum hydrogen yield of 2.53 mol H₂/mol sucrose_consumed appeared at pH 4.9. The carbohydrate removal efficiency declined to 56% at pH 4.9, which indirectly resulted in the reduction of total volatile fatty acid production. Acetate fermentation was the dominant metabolic pathway at pH 4.9. The concentration of mixed liquor volatile suspended solid (MLVSS) also showed a decrease from nearly 15,000 mg/L between pHs 6.1 and 6.7 to 6000 mg/L at pH 4.9. Investigation of the effect of cyclic duration found that hydrogen yield reached the maximum of 1.86 mol H₂/mol sucrose_consumed at 4-h cyclic duration while ASBR was operating at 16-h HRT, 15 g COD/L, and pH 4.9. The experimental results showed that MLVSS concentration increased from 6200 mg/L at 4-h cyclic duration to 8500 mg/L at 8-h cyclic duration. However, there was no significant change in effluent volatile suspended solid concentration. The results of butyrate to acetate ratio showed that using this ratio to correlate the performance of hydrogen production is not appropriate due to the growth of homoacetogens. In ASBR, the operation is subject to four different phases of each cycle, and only the complete mix condition can be achieved at react phase. The pH and cyclic duration under the unique operations profoundly impact fermentative hydrogen production.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.