Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility

Four commonly used chemical pretreatment processes based on dilute acid, lime, aqueous ammonia steeping followed by dilute acid hydrolysis, and sodium hydroxide, were evaluated to provide comparative performance data. An obverse correlation between lignin removal and enzymatic digestibility of pretreated corn stover was observed. Compared with other three pretreatments, pretreatment of corn stover with 2% NaOH substantially increased the lignin removal and enhanced the accessibility and digestibility of cellulose. The hydrolysis yield of NaOH-pretreated corn stover reached 81.2% by 48 h at 8.0% substrate concentration and cellulase dosage of 20 FPU g⁻¹ substrate. Chemical analysis showed that the enzymatic hydrolysate from NaOH-pretreated corn stover contained higher content of fermentable sugars and less inhibitors, which is suitable for subsequent fermentation process to produce ethanol. The research results are meaningful in bioconversion and utilization of renewable lignocellulosic biomass.     

Development of AFEX-based consolidated bioprocessing on wheat straw for biohydrogen production using anaerobic microflora

Consolidated bioprocessing (CBP) of wheat straw (WS) pretreated via ammonia fiber expansion (AFEX) for hydrogen production was discussed in this study. Results indicate that rotted wood crumb is a good mixed natural-microflora seed source for biohydrogen production from WS. The hydrogen-producing capability of the inoculum could be improved by an enrichment treatment. In order to enhance the digestibility of WS, AFEX pretreatment conditions were investigated. The optimal conditions were determined to be 100 °C, 1:1 (w/w) ammonia to biomass loading, and 30min residence time. The yield of H₂ was 67.8% higher using AFEX-pretreated WS than using untreated material. Moreover, degradation products derived from AFEX pretreatment exerted no impact on hydrogen fermentation if controlled below 1.5% substrate-loading-equivalent. In addition, the maximum AFEX-WS conversion and H₂ production were achieved at substrate loading of 0.8%. This work indicates that CBP offers a promising platform for direct and cost-effective cellulosic hydrogen production compared to separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF).     

Reutilization of carbon sources through sugar recovery from waste rice straw

Rice straw was utilized for the cultivation of Phanerochaete chrysoporium to produce cellobiose dehydrogenase. The composition of the rice straw after fermentation was found to be 28.77% glucan, 19.05% xylan and 54.81% other lignin containing sugars. The glucan and xylan content decreased due to the consumption of glucan and xylan by P. chrysoporium. After fermentation, the rice straw waste was subjected to chemical pretreatment to remove lignin. The effect of dilute acid pretreatment was not notable because of the glucose loss. However, when the rice straw after fermentation was treated with aqueous ammonia, the composition changed to 44.73% glucan, 25.43% xylan and 29.52% other lignin containing sugars. The aqueous ammonia pretreatment was optimized and an ammonia concentration, reaction time and temperature of 20%, 6 h and 60 °C, respectively, were determined to be the optimal pretreatment conditions After removal of lignin, the initial reaction rate was increased to 0.009583 g/L s, which was about 3 fold higher than the rice straw after fermentation. X-ray diffractometry was performed to investigate the crystallinity index, and the XRD results showed that biological treatment and the combination of both biological treatment and chemical pretreatment decreased the crystallinity index.     

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H₂ production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H₂ yield and specific H₂ production rates with xylose and glucose are 1.63 mol-H₂/mol xylose and 11.14-H₂ mmol/h g-cdw, and 2.02 mol-H₂/mol-glucose and 9.37 mmol-H₂/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H₂ yield and specific H₂ production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H₂/mol-mixed sugar and 12.56 mmol-H₂/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.     

Enhancement of batch biohydrogen production from prehydrolysate of acid treated oil palm empty fruit bunch

Carbohydrates from hydrolyzed biomass has been a potential feedstock for fermentative hydrogen production. In this study, oil palm empty fruit bunch (OPEFB) was treated by sulfuric acid in different concentrations at 120 °C for 15 min in the autoclave. The optimal condition for pretreatment was obtained when OPEFB was hydrolyzing at 6% (w/v) sulfuric acid concentration, which gave the highest total sugar of 26.89 g/L and 78.51% of sugar production yield. However, the best conversion efficiency of OPEFB pretreatment was 39.47 at sulfuric acid concentration of 4%. A series of batch fermentation were performed to determine the effect of pH in fermentation media and the potential of this prehydrolysate was used as a substrate for fermentative hydrogen production under optimum pretreatment conditions. The prehydrolysate of OPEFB was efficiently converted to hydrogen via fermentation by acclimatized mixed consortia. The maximum hydrogen production was 690 mL H₂ L⁻¹ medium, which corresponded to the yield of 1.98 molH₂/mol_xylose achieved at pH 5.5 with initial total sugar concentration of 5 g/L. Therefore, the results implied that OPEFB prehydrolysate is prospective substrate for efficient fermentative hydrogen conducted at low controlled pH. No methane gas was detected throughout the fermentation.     

Engineering strategies for the enhanced photo-H2 production using effluents of dark fermentation processes as substrate

The major obstacle of combining dark and photo fermentation for high-yield biohydrogen production is substrate inhibition while using dark fermentation effluent as the sole substrate. To solve this problem, the dark fermentation broth was diluted with different dilution ratio to improve photo-H₂ production performance of an indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5. The best photo-H₂ production performance occurred at a dilution ratio of 1:2, giving a highest overall H₂ production rate of 10.72 ml/l/h and a higher overall H₂ yield of 6.14 mol H₂/mol sucrose. The maximum H₂ content was about 88.1% during the dilution ratio of 1:2. The photo-H₂ production performance was further improved by supplying yeast extract and glutamic acid as the nutrient. The results indicate that the overall H₂ production rate and H₂ yield increased to 17.02 ml/l/h and 10.25 mol H₂/mol sucrose, respectively. Using a novel solar-energy-excited optical fiber photobioreactor (SEEOFP) with supplementing tungsten filament lamp (TL) irradiation, the overall H₂ production rate was improved to 17.86 ml/l/h. Meanwhile, the power consumption by combining SEEOFP and TL was about 37.1% lower than using TL alone. This study demonstrates that using optimal light sources and proper dilution of dark fermentation effluent, the performance of photo-H₂ production can be markedly enhanced along with a reduction of power consumption.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

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

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

Insights into biological delignification of rice straw by Trametes hirsuta and Myrothecium roridum and comparison of saccharification yields with dilute acid pretreatment

Rice straw is the most abundant agricultural residue on a global scale and is widely available as feedstock for cellulosic fuel production. However, it is highly recalcitrant to biochemical deconstruction and also generates inhibitors that affect enzymatic saccharification. Rice straw from eastern Arkansas was subjected to dilute acid pretreatment (160 °C, 48 min and 1.0% sulfuric acid) and solid-state fermentation with two lignocellulolytic fungi, Trametes hirsuta and Myrothecium roridum, and their saccharification efficacies were compared. T. hirsuta and M. roridum were tested separately; pretreatment of rice straw with either strain for seven days resulted in 19 and 70% enrichment of its holocellulose content, respectively. However, liquid chromatography analysis of the alkali extracts showed significant differences in cell wall degradation by T. hirsuta and M. roridum. T. hirsuta removed 15% more phenolic compounds and 38% more glucan than M. roridum, while M. roridum removed 77% more xylan than T. hirsuta. Fungal and dilute acid pretreated biomass was then hydrolyzed using Accellerase^® 1500, a saccharification cocktail. Saccharification efficiency of M. roridum was 37% higher than that of dilute acid pretreatment of rice straw, requiring 8% lower enzyme loading and 50% shorter enzymatic hydrolysis duration. Alkali extraction of fungal pretreated biomass also yielded 10–15 g kg⁻¹ of acid precipitable polymeric lignin (APPL), which is a valuable co-product for biorefineries. In comparison to dilute acid pretreatment, fungal pretreatment could be a cost-effective alternative for the degradation of recalcitrant biomass, such as rice straw.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

Effect of sodium sulfite on acid pretreatment of wheat straw with respect to its final conversion to ethanol

A pretreatment process that combines dilute acid and sodium sulfite has been applied to wheat straw to study the effect of temperature (120–180 °C) and sodium sulfite concentration (0–3%) on the yield of glucose in subsequent enzymatic hydrolysis and ethanol production by fermentation. The results were compared with both dilute acid pretreatment (without Na₂SO₃ addition) and hot water pretreatment. Formation of furfural and hydroxymethylfurural, which can inhibit ethanol-producing microorganisms, were measured and the ethanol yield in a subsequent fermentation was evaluated. The results indicate that a combination of 180 °C, 30 min, 1% H₂SO₄ and 2.4% Na₂SO₃ during pretreatment produced the highest ethanol yield; 17.3 g/100 g dry weight of initial biomass, which corresponds to 75% of the theoretical yield from glucose. 28 mg of furan inhibitors (sum of furfural and hydroxymethylfurfural) per gram dry weight of initial wheat straw were generated under this condition. Increasing sulfite loading up to 2.4% decreased inhibitor formation, leading to increased delignification and preservation of cellulose from dissolution. On the other hand, an elevated temperature in combination with low pH reduced the amount of solid phase after pretreatment, increased the level of inhibitors and reduced the concentration of ethanol produced by fermentation.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

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.     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Enhanced hydrogen and volatile fatty acid production from sweet sorghum stalks by two-steps dark fermentation with dilute acid treatment in between

This study investigated the potential of hydrogen and volatile fatty acid coproduction from two steps dark fermentation with dilute acid treatments of the residual slurry after 1st step fermentation. Sweet sorghum stalks (SS) was used as substrate along with Clostridium thermosaccharolyticum as production microbe. Residual lignocelluloses after 1st step fermentation were treated for 1 h by sulfuric acid concentration of 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5% (w/v) with different reaction temperature of 120, 90 and 60 °C were studied. The optimum severity conditions for the highest yield of products found from the treatment acid concentration of 1.5% (w/v) at 120 °C for 10 g/L of substrate concentration. Experimental data showed that two-step fermentation increased 76% hydrogen, 84% acetic acid and 113% of butyric acid production from single step. Maximum yields of hydrogen, acetic acid and butyric acid were 5.77 mmol/g-substrate, 2.17 g/L and 2.07 g/L respectively. This two-step fermentation for hydrogen and VFA production using the whole slurry would be a promising approach to SS biorefinery.     

Dilute-acid pretreatment of barley straw for biological hydrogen production using Caldicellulosiruptor saccharolyticus

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H₃PO₄ and H₂SO₄, and to a lesser extent, HNO₃. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.     

Effect of enzyme addition on fermentative hydrogen production from wheat straw

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H₂), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H₂ usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H₂ production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H₂ production from untreated wheat straw ranged from 5.18 to 10.52 mL-H₂ g-VS⁻¹. Whatever the experimental enzyme addition procedure, a two-fold increase in H₂ production yields ranging from 11.06 to 19.63 mL-H₂ g-VS⁻¹ was observed after enzymatic treatment of the wheat straw. The high variability in H₂ yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H₂ production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw⁻¹.     

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.     

Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).     

Hydrogen and methane production from untreated rice straw and raw sewage sludge under thermophilic anaerobic conditions

Bioenergy produced from co-digestion of sewage sludge (SS) and rice straw (RS) as raw materials, without pretreatment and additional nutrients, was compared for the one-stage system for producing methane (CH₄) and the two-stage system for combined production of hydrogen (H₂) and CH₄ in batch experiments under thermophilic conditions. In the first stage H₂ fermentation process using untreated RS with raw SS, we obtained a high H₂ yield (21 ml/g-VS) and stable H₂ content (60.9%). Direct utilization of post-H₂ fermentation residues readily produced biogas, and significantly enhanced the CH₄ yield (266 ml/g-VS) with stable CH₄ content (75–80%) during the second stage CH₄ fermentation process. Overall, volatile solids removal (60.4%) and total bioenergy yield (8804 J/g-VS) for the two-stage system were 37.9% and 59.6% higher, respectively, than the one-stage system. The efficient production of bioenergy is believed to be due to a synergistically improved second stage process exploiting the well-digested post-H₂ generation residues over the one-stage system.