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).     

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.     

Ethanol production by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and fermentation

Rice straw was successfully converted to ethanol by separate enzymatic hydrolysis and fermentation by Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. The hydrolysis temperature and pH of commercial cellulase and β-glucosidase enzymes were first investigated and their best performance obtained at 45 °C and pH 5.0. The pretreatment of the straw with dilute-acid hydrolysis resulted in 0.72 g g^?1 sugar yield during 48 h enzymatic hydrolysis, which was higher than steam-pretreated (0.60 g g^?1) and untreated straw (0.46 g g^?1). Furthermore, increasing the concentration of the dilute-acid pretreated straw from 20 to 50 and 100 g L^?1 resulted in 13% and 16% lower sugar yield, respectively. Anaerobic cultivation of the hydrolyzates with M. indicus resulted in 0.36–0.43 g g^?1 ethanol, 0.11–0.17 g g^?1 biomass, and 0.04–0.06 g g^?1 glycerol, which is comparable with the corresponding yields by S. cerevisiae (0.37–0.45 g g^?1 ethanol, 0.04–0.10 g g^?1 biomass and 0.05–0.07 glycerol). These two fungi produced no other major metabolite from the straw and completed the cultivation in less than 25 h. However, R. oryzae produced lactic acid as the major by-product with yield of 0.05–0.09 g g^?1. This fungus had ethanol, biomass and glycerol yields of 0.33–0.41, 0.06–0.12, and 0.03–0.04 g g^?1, respectively.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Integrated process to produce biohydrogen from wheat straw by enzymatic saccharification and dark fermentation

In this study, different pretreatment methods, including lyophilization, hydrothermal pretreatment, and ultrasound combined with dilute alkali post-cooking, were investigated to enhance the efficiency of enzymatic saccharification and biohydrogen production of the wheat straw. All pretreatment methods could effectively remove lignin and hemicellulose while retaining cellulose, further enhancing the biomass accessibility for subsequently enzymatic saccharification and biohydrogen production. A reducing sugar concentration of 13.18 g/L was acquired when wheat straw was treated with ultrasound and dilute alkali cooking (R_U). The sequential fermentative hydrogen yield of the substrate R_U was 133.6 mL/g total solids (TS), which was 5.6-fold larger than that of the raw material (23.9 mL/g TS). The study confirmed that ultrasound combined with dilute alkali cooking was an effective method, which not only provided significant guideline for improving biohydrogen production but also presented helpful direction for the efficient pretreatment of other lignocellulosic biomass.     

Evaluation the efficacy of extrusion pretreatment via enzymatic digestibility and simultaneous saccharification & fermentation with rapeseed straw

The efficacy of extrusion pretreatment was evaluated by enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with straw of rapeseed, Brassica napus, an agricultural residue. An acceptable pretreatment result was obtained at a barrel temperature of 165 °C, acid concentration of 20 g L⁻¹, liquid feeding rate of 13.4 cm³ min⁻¹, solid feeding rate of 1.0 g min⁻¹, screw rotation speed of 6.3 rad s⁻¹, and residence time of 10.2 min, with a yield of xmg, representing the sum of the corresponding hydrolyzed sugars; xylose, mannose and galactose, of 794.3 g kg⁻¹ and a glucose release of 21.0 g kg⁻¹. These were calculated to be 963.0 g kg⁻¹ and 910.3 g kg⁻¹ based on cellulose and hemicellulose recoveries,respectively. The highest enzymatic digestibility of 781.0 g kg⁻¹was higher than that obtained from the batch pretreatment with dilute acid by 1.4-fold. The SSF process afforded an ethanol concentration of 16.0 g L⁻¹, corresponding to an ethanol yield of 790 g kg⁻¹ based on the total available cellulose in the pretreated rapeseed straw.     

Screening for sugar and ethanol processing characteristics from anatomical fractions of wheat stover

Due to concerns with stover collection systems, soil sustainability, and processing costs to produce ethanol, there are opportunities to investigate the optimal plant fractions to collect. Wheat stover fractions were separated by hand and analyzed for glucan, xylan, acid-soluble lignin, acid-insoluble lignin, and ash composition. Internodes had the highest glucan content (38.2% zero percent moisture basis) and the other fractions varied between 29.9% and 33.4%. The stover fractions were pretreated with either 0%, 0.4%, or 0.8% NaOH for 2 h at room temperature, washed, autoclaved, and saccharified. In addition, acid pretreated samples underwent simultaneous saccharification and fermentation (SSF) to ethanol. In general, the acid and alkaline pretreatments produced similar trends with leaves requiring very little pretreatment to achieve high conversion rates (greater than 80%). Chaff responded very well to pretreatment and high conversion efficiencies resulted when pretreated under alkaline or acidic conditions. Nodes and internodes were more recalcitrant than the other anatomical fractions. Pretreatment with 0.8% sulfuric acid (0.24 g sulfuric acid/g biomass) did not result in a significantly higher conversion of glucan to ethanol as the native material. Pretreatment with 0.8% NaOH (0.06 g NaOH/g biomass) at room temperature for 2 h resulted in high conversion efficiencies for all plant fractions, greater than 73% of the available glucan. These differences in pretreatment susceptibilities suggest that a biomass collection system that removes specific portions of wheat stover could result in significant differences in ethanol production costs.     

Tandem-ring mill pulverization benefits for enzymatic saccharification of biomass

A vibration mill, called a “tandem-ring mill”, which has cog-ring media in place of the ball medium of a conventional vibration mill, was developed to achieve high-impact pulverization of lignocellulosic biomass for bio-ethanol production. This study investigated pulverization characteristics of various biomass samples of Japanese cedar, eucalyptus, rice straw, rice husk, and reeds using a batch-type tandem-ring mill for 60 min and 100 min pulverizations. The pulverized biomasses were characterized by the mean particle diameter, crystallinity index, and enzymatic saccharification. The mean particle diameter of biomass was decreased rapidly in the first 20 min. Then the biomass mean particle diameter reached around 40 μm by flocculation. Furthermore, the crystallinity indexes of biomasses were decreased by pulverization. Especially, woody biomasses of cedar and eucalyptus were decreased rapidly around 10%, nearly a non-crystalline state. Woody biomasses, Japanese cedar, and eucalyptus showed high saccharification efficiency. Similarly, grass biomasses, rice straw, and rice husks showed high saccharification, aside from reeds. Therefore pretreatment of various biomasses using a tandem-ring mill was suitable for enzymatic saccharification.     

Acidified glycerol pretreatment for enhanced ethanol production from rice straw

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

Evaluation of high throughput screening methods in picking up differences between cultivars of lignocellulosic biomass for ethanol production

We present a unique evaluation of three advanced high throughput pretreatment and enzymatic hydrolysis systems (HTPH-systems) for screening of lignocellulosic biomass for enzymatic saccharification. Straw from 20 cultivars of winter wheat from two sites in Denmark was hydrothermally pretreated and enzymatically processed in each of the separately engineered HTPH-systems at 1) University of California, Riverside, 2) National Renewable Energy Laboratory (NREL), Colorado, and 3) University of Copenhagen (CPH). All three systems were able to detect significant differences between the cultivars in the release of fermentable sugars, with average cellulose conversions of 57%, 64%, and 71% from Riverside, NREL and CPH, respectively. The best correlation of glucose yields was found between the Riverside and NREL systems (R² = 0.2139), and the best correlation for xylose yields was found between Riverside and CPH (R² = 0.4269). All three systems identified Flair as the highest yielding cultivar and Dinosor, Glasgow, and Robigus as low yielding cultivars. Despite different conditions in the three HTPH-systems, the approach of microscale screening for phenotypically less recalcitrant feedstock seems sufficiently robust to be used as a generic analytical platform.     

Optimization of dilute acid and hot water pretreatment of different lignocellulosic biomass: A comparative study

Pretreatment of biomass to alter their recalcitrant structures is an essential step to obtain high yield of products via bioconversion processes. In this study, main emphasis was to compare the results evaluated in terms of total reducing sugars (TRS) yield after acid and hot water pre-treatment process performed with laboratory scale equipment using different lignocellulosic biomass. The biomass chosen for this purpose i.e. sugarcane bagasse and bamboo were collected from Guwahati, Assam and their physico-chemical characteristics were examined using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and thermo gravimetric analysis (TGA) including proximate and ultimate analysis. Crystalinity of the biomass used was observed to be 33.15% and 31.29% for sugarcane bagasse and bamboo respectively. Hot water and dilute acid pretreatment allows selective solubility of hemicellulose which improves the accessibility of enzymes for cellulose hydrolysis. The highest yield of TRS was observed at run order 8 for both acid and hot water pretreatment (23.49 and 26.50 gL⁻¹) with respect to sugarcane bagasse. But, the pretreatment results obtained for bamboo was slightly different to that of sugarcane bagasse. The highest yield of TRS was obtained at run order 8 for acid (15.6 gL⁻¹) and run order 10 for hot water (17.98 gL⁻¹) pretreatment respectively. Irrespective of biomass type, hot water pretreatment process produced more TRS than acid pretreatment process.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

The properties of pellets from mixing bamboo and rice straw

Rice straw pellets are the main type of biomass solid fuel and have great potential as a bioenergy resource of the future in China. But it also showed important problems because of its high content of ashes and its low gross calorific value, reducing the possibility to be used in domestic heating. It was certified that mixing different types of biomass materials was helpful to improve the properties of pellets. To improve properties of rice straw pellets and investigate the effect of mixing bamboo and rice straw on the pellet properties, some properties of pellets, manufactured using different mixing ratio of bamboo and rice straw particles, were determined in this research. It can be concluded from this research that physical properties of all pellets meet the requirements of Pellet Fuels Institute Standard Specification for Residential/Commercial Densified except for bulk density of pellets, manufactured using mixing ratio (≤3:2) of bamboo and rice straw. The inorganic ash and gross calorific value of rice straw pellets cannot meet the requirement of Pellet Fuels Institute Standard Specification for Residential/Commercial Densified (8.0%) and the minimum requirement for making commercial pellets of DIN 51731 (>17,500 J/g). Both properties are improved through mixing bamboo particles and rice straw particles. It is significant that inorganic ash content and gross calorific value of pellets, manufactured using mixing ratio (≥3:2) of bamboo and rice straw, were lower than 8.0% and higher than 17,500 J/g, respectively. This also shows that mixing different biomass materials is an effective way to optimize properties of biomass solid fuel. All pellets after improvement are proposed as biomass solid fuel and have the potential to be developed as commercial pellets on an industrial scale in China.     

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.     

Converting Eucalyptus biomass into ethanol: Financial and sensitivity analysis in a co-current dilute acid process. Part II

The technical and financial performance of high yield Eucalyptus biomass in a co-current dilute acid pretreatment followed by enzymatic hydrolysis process was simulated using WinGEMS^® and Excel^®. Average ethanol yield per dry Mg of Eucalyptus biomass was approximately 347.6 L of ethanol (with average carbohydrate content in the biomass around 66.1%) at a cost of $0.49 L⁻¹ of ethanol, cash cost of ∼ $0.46 L⁻¹ and CAPEX of $1.03 L⁻¹ of ethanol. The main cost drivers are: biomass, enzyme, tax, fuel (gasoline), depreciation and labor. Profitability of the process is very sensitive to biomass cost, carbohydrate content (%) in biomass and enzyme cost. Biomass delivered cost was simulated and financially evaluated in Part I; here in Part II the conversion of this raw material into cellulosic ethanol using the dilute acid process is evaluated.     

Evaluation of fuel ethanol production from aqueous ammonia-treated rice straw via simultaneous saccharification and fermentation

Rice straw (RS) has been considered a promising feedstock for ethanol production in Asia. However, the recalcitrance of biomass, particularly the presence of lignin, hinders the enzymatic saccharification of polysaccharides in RS and consequently decreases the ethanol yield. Here, we used aqueous ammonia pretreatment to remove lignin from RS (aRS). The reaction conditions were a solid:liquid ratio of 1:12, an ammonia concentration of 27% (w w⁻¹), room temperature, and a 2-week incubation. We evaluated enzymatic digestibility and the ethanol production yield. A 42% reduction in lignin content increased the glucan conversion of aRS to glucose from 20 to 71% using a combination of Cellic Ctec2 cellulases and Cellic Htec2 xylanases at enzyme loads of 15 FPU +100 XU g⁻¹ solid. Scanning electron microscopy highlighted the extensive removal of external fibres and increased porosity of aRS, which aided the accessibility of cellulose for enzymes. Using the same enzyme dosage and a solid load of 100 g L⁻¹, simultaneous saccharification and fermentation using a monoculture of Saccharomyces cerevisiae and co-culture with Candida tropicalis yielded ethanol concentrations of 22 and 25 g L⁻¹, corresponding to fermentation efficiencies of 96 and 86% fermentation, respectively. The volumetric ethanol productivities for these systems were 0.45 and 0.52 g L⁻¹ h⁻¹. However, the ethanol yield based on the theoretical glucose and xylose concentrations was lower for the co-culture (0.44 g g⁻¹) than the monoculture (0.49 g g⁻¹) due to the low xylose consumption. Further research should optimise fermentation variables or select/improve microbial strains capable of fermenting xylose to increase the overall ethanol production yield.     

Comparative net energy ratio analysis of pellet produced from steam pretreated biomass from agricultural residues and energy crops

A process model was developed to determine the net energy ratio (NER) for the production of pellets from steam pretreated agricultural residue (wheat straw) and energy crops (i.e., switchgrass in this case). The NER is a ratio of the net energy output to the total net energy input from non-renewable energy sources into a system. Scenarios were developed to measure the effects of temperature and level of steam pretreatment on the NER of steam pretreated wheat straw and switchgrass pellets. The NERs for the base case at 6 kg h⁻¹ are 1.76 and 1.37 for steam-pretreated wheat straw and switchgrass-based pellets, respectively. The reason behind the difference is that more energy is required to dry switchgrass pellets than wheat straw pellets. The sensitivity analysis for the model shows that the optimum temperature for steam pretreatment is 160 °C with 50% pretreatment (i.e. 50 % steam treated material is blended with the raw biomass and then pelletised). The uncertainty results for NER for steam pretreated wheat straw and switch grass pellets are 1.62 ± 0.10 and 1.42 ± 0.11, respectively.     

Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw

The cellobiohydrolase II (CBH II) gene cbh2 from Trichoderma reesei was cloned and its codons were optimized in accordance with the codon usage frequencies of the host Pichia pastoris. The AOX1 strong promoter inducible by methanol was employed to efficiently express the foreign gene cbh2 in P. pastoris. It was found that 5.84 ± 0.42 U cm⁻³ CBH II was obtained at 96 h using the synthetic cbh2 gene whose codons were optimized, 2.02-fold higher than using the native cbh2 gene (2.89 ± 0.32 U cm⁻³), indicating that the codon optimization strategy was an effective approach to enhance the heterologous expression of CBH II in P. pastoris. The product of recombinant P. pastoris CBH II had an approximate molecular weight 58 kDa. Its optimal pH and temperature were 5.0 and 50 °C, respectively. The recombinant CBH II was used to enhance the yields of the enzymatic hydrolysis of the corn stover and rice straw pretreated with sodium hydroxide by improving the exo-exo-synergism between CBH II and CBH I in T. reesei cellulase. The yields 94.7% and 83.3% were achieved in the enzymatic hydrolysis of corn stover and rice straw pretreated by sodium hydroxide, respectively.     

Valorization of Eucalyptus wood by glycerol-organosolv pretreatment within the biorefinery concept: An integrated and intensified approach

The efficient utilization of lignocellulosic biomass and the reduction of production cost are mandatory to attain a cost-effective lignocellulose-to-ethanol process. The selection of suitable pretreatment that allows an effective fractionation of biomass and the use of pretreated material at high-solid loadings on saccharification and fermentation (SSF) processes are considered promising strategies for that purpose. Eucalyptus globulus wood was fractionated by organosolv process at 200 °C for 69 min using 56% of glycerol-water. A 99% of cellulose remained in pretreated biomass and 65% of lignin was solubilized. Precipitated lignin was characterized for chemical composition and thermal behavior, showing similar features to commercial lignin. In order to produce lignocellulosic ethanol at high-gravity, a full factory design was carried to assess the liquid to solid ratio (3–9 g/g) and enzyme to solid ratio (8–16 FPU/g) on SSF of delignified Eucalyptus. High ethanol concentration (94 g/L) corresponding to 77% of conversion at 16FPU/g and LSR = 3 g/g using an industrial and thermotolerant Saccharomyces cerevisiae strain was successfully produced from pretreated biomass. Process integration of a suitable pretreatment, which allows for whole biomass valorization, with intensified saccharification-fermentation stages was shown to be feasible strategy for the co-production of high ethanol titers, oligosaccharides and lignin paving the way for cost-effective Eucalyptus biorefinery.