Improving enzymatic hydrolysis of wheat straw using ionic liquid 1-ethyl-3-methyl imidazolium diethyl phosphate pretreatment

This study aims to establish a cellulose pretreatment process using ionic liquids (ILs) for efficient enzymatic hydrolysis. The IL 1-ethyl-3-methyl imidazolium diethyl phosphate ([EMIM]DEP) was selected in view of its low viscous and the potential of accelerating enzymatic hydrolysis, and it could be recyclable. The yield of reducing sugars from wheat straw pretreated with this IL at 130 °C for 30 min reached 54.8% after being enzymatically hydrolyzed for 12 h. Wheat straw regenerated were hydrolyzed more easily than that treated with water. The fermentability of the hydrolyzates, obtained after enzymatic saccharification of the regenerated wheat straw, was evaluated using Saccharomyces cerevisiae. This microbe could ferment glucose efficiently, and the ethanol production was 0.43 g/g glucose within 26 h. In conclusion, the IL [EMIM]DEP shows promise as pretreatment solvent for wheat straw, although its cost should be reduced and in-depth exploration of this subject is needed.     

Decomposition of Lignocellulose by Cyathus stercoreus (Schw.) de Toni NRRL 6473, a “White Rot” Fungus from Cattle Dung

Cyathus stercoreus (Schw.) de Toni NRRL 6473, isolated from aged and fragmented cattle dung collected from a Michigan pasture, effected substantial losses in lignin (45%) from wheat straw during a 62-day fermentation (25°C). The basidiomycete also improved wheat straw digestibility by freeing α-cellulose for enzymatic hydrolysis to glucose (230 mg of glucose per 1,000 mg of fermented residue). The rationale for selecting C. stercoreus in attempting to biologically modify the lignin and cellulose components in wheat straw or other gramineous agricultural residues was based on the expectation that this organism is ecologically specialized to enzymatically attack the substructures of native lignins in grasses.     

Efficiency of pretreatments for optimal enzymatic saccharification of soybean fiber

The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H₂SO₄), sodium hydroxide (NaOH), and ammonium hydroxide (NH₃OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144 μm_{pp (peak-to-peak)} for 30 and 60 s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H₂SO₄ [1% (w/w), 90 °C, 1.5 h], NaOH [15% (w/w), 65 °C, 17 h], and NH₃OH [15% (w/w), 65 °C, 17 h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH₃OH pretreatment resulted in the highest saccharification yield (63%) after 48 h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified.     

Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes

Rice straw was pretreated using aqueous-ammonia solution at moderate temperatures to enable production of the maximum amount of fermentable sugars from enzymatic hydrolysis. The effects of various operating variables including pretreatment temperature, pretreatment time, the concentration of ammonia and the solid-to-liquid ratio on the degree of lignin removal and the enzymatic digestibility were optimized using response surface methodology. The optimal reaction conditions, which resulted in an enzymatic digestibility of 71.1%, were found to be 69 °C, 10 h and an ammonia concentration of 21% (w/w). The effects of different commercial cellulases and the additional effect of a non-cellulolytic enzyme, xylanase, were also evaluated. Additionally, simultaneous saccharification and fermentation was conducted with rice straw to assess the ethanol production yield and productivity.     

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.     

Optimization of microwave-assisted FeCl3 pretreatment conditions of rice straw and utilization of Trichoderma viride and Bacillus pumilus for production of reducing sugars

In this study, Box-Behnken design (BBD) and response surface methodology (RSM) were used to optimize microwave-assisted FeCl₃ pretreatment conditions of rice straw with respect to FeCl₃ concentration, microwave intensity, irradiation time and substrate concentration. When rice straw was pretreated at the optimal conditions of FeCl₃ concentration, 0.14 mol/L; microwave intensity, 160 °C; irradiation time, 19 min; substrate concentration, 109 g/L; and inoculated with Trichoderma viride and Bacillus pumilus, the production of reducing sugars was 6.62 g/L. This yield was 2.9 times higher than that obtained with untreated rice straw. The microorganisms degraded 37.8% of pretreated rice straw after 72 h. The structural characteristic analyses suggest that microwave-assisted FeCl₃ pretreatment damaged the silicified waxy surface of rice straw, disrupted almost all the ether linkages between lignin and carbohydrates, and removed lignin.     

DiSC (direct saccharification of culms) process for bioethanol production from rice straw

A simple process (the direct-saccharification-of-culms (DiSC) process) to produce ethanol from rice straw culms, accumulating significant amounts of soft carbohydrates (SCs: glucose, fructose, sucrose, starch and β-1,3-1,4-glucan) was developed. This study focused on fully mature culms of cv. Leafstar, containing 69.2% (w/w of dried culms) hexoses from SCs and cellulose. Commercially-available wind-separation equipment successfully prepared a culm-rich fraction with a SC recovery of 83.1% (w/w) from rice straw flakes (54.1% of total weight of rice straw). The fraction was suspended in water (20%, w/w) for starch liquefaction, and the suspension was subjected to a simultaneous saccharification and fermentation with yeast, yielding 5.6% (w/v) ethanol (86% of the theoretical yield from whole hexoses in the fraction) after 24 h fermentation. Thus, the DiSC process produced highly-concentrated ethanol from rice straw in a one vat process without any harsh thermo-chemical pretreatments.     

Effects of grinding processes on enzymatic degradation of wheat straw

The effectiveness of wheat straw fine to ultra-fine grindings at pilot scale was studied. The produced powders were characterised by their particle-size distribution (laser diffraction), crystallinity (WAXS) and enzymatic degradability (Trichoderma reesei enzymatic cocktail). A large range of wheat-straw powders was produced: from coarse (median particle size ∼800 μm) to fine particles (∼50 μm) using sieve-based grindings, then ultra-fine particles ∼20 μm by jet milling and ∼10 μm by ball milling. The wheat straw degradability was enhanced by the decrease of particle size until a limit: ∼100 μm, up to 36% total carbohydrate and 40% glucose hydrolysis yields. Ball milling samples overcame this limit up to 46% total carbohydrate and 72% glucose yields as a consequence of cellulose crystallinity reduction (from 22% to 13%). Ball milling appeared to be an effective pretreatment with similar glucose yield and superior carbohydrate yield compared to steam explosion pretreatment.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil

Agro-industrial wastes of cattle dung, vinegar-production residue and rice straw were solid-state fermented by inoculation with Trichoderma harzianum SQR-T037 (SQR-T037) for production of bioorganic fertilizers containing SQR-T037 and 6-pentyl-α-pyrone (6PAP) to control Fusarium wilt of cucumber in a continuously cropped soil. Fermentation days, temperature, inoculum and vinegar-production residue demonstrated significant effects on the SQR-T037 biomass and the yield of 6PAP, based on fractional factorial design. Three optimum conditions for producing the maximum SQR-T037 biomass and 6PAP yield were predicted by central composite design and validated. Bioorganic fertilizer containing 8.46 log₁₀ ITS copies g⁻¹ dry weight of SQR-T037 and 1291.73 mg kg⁻¹ dry weight of 6PAP, and having the highest (p < 0.05) biocontrol efficacy, was achieved at 36.7 fermentation days, 25.9 °C temperature, 7.6% inoculum content, 41.0% vinegar-production residue, 20.0% rice straw and 39.0% cattle dung. This is a way to offer a high value-added use for agro-industrial wastes.     

Evaluation of Fusarium oxysporum cellulolytic system for an efficient hydrolysis of hydrothermally treated wheat straw

The crude multienzyme extract produced by Fusarium oxysporum cultivated under submerged conditions in 20 L bioreactor using brewers spent grain and corn cobs in a ratio 2:1 as the carbon source was evaluated with regard to an efficient saccharification of hydrothermally treated wheat straw. Several factors concerning the obtained hydrolysis yield and reaction rate were investigated. The takeout of product sugars (in situ) was effective at reducing end-product inhibition and lead to a bioconversion about 80% of the theoretical. A kinetic model incorporating dynamic adsorption, enzymatic hydrolysis, and product inhibition was developed. The model predicted very satisfactorily the experimental data.     

The optimized CO2-added ammonia explosion pretreatment for bioethanol production from rice straw

A CO₂-added ammonia explosion pretreatment was performed for bioethanol production from rice straw. The pretreatment conditions, such as ammonia concentration, CO₂ loading level, residence time, and temperature were optimized using response surface methodology. The response for optimization was defined as the glucose conversion rate. The optimized pretreatment conditions resulting in maximal glucose yield (93.6 %) were determined as 14.3 % of ammonia concentration, 2.2 MPa of CO₂ loading level, 165.1 °C of temperature, and 69.8 min of residence time. Scanning electron microscopy analysis showed that pretreatment of rice straw strongly increased the surface area and pore size, thus increasing enzymatic accessibility for enzymatic saccharification. Finally, an ethanol yield of 97 % was achieved via simultaneous saccharification and fermentation. Thus, the present study suggests that CO₂-added ammonia pretreatment is an appropriate process for bioethanol production from rice straw.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed (Ulva lactuca) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.     

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g_p/g_s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO₂ released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

Structure and saccharification of rice straw pretreated with sulfur trioxide micro-thermal explosion collaborative dilutes alkali

In this paper, a sulfur trioxide collaborative dilutes alkali method has been developed to pre-treat rice straw and it has been studied that the pre-treated rice straw structure affected the saccharification of the rice straw hydrolyzed by cellulose enzymatic hydrolysis. The results show that the reaction of the sulfur trioxide with rice straw resulted in the internal micro-thermal explosion, and the saccharification rate was 91% based on the pretreated rice straw with sulfur trioxide for 4h following 1% w/v NaOH treatment for 7h at 50°C.     

Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis

Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses).Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L⁻¹. However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L⁻¹ by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L⁻¹, with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L⁻¹ h⁻¹, 0.4 g g⁻¹ and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498.     

Fungal pretreatment: An alternative in second-generation ethanol from wheat straw

The potential of a fungal pretreatment combined with a mild alkali treatment to replace or complement current physico-chemical methods for ethanol production from wheat straw has been investigated. Changes in substrate composition, secretion of ligninolytic enzymes, enzymatic hydrolysis efficiency and ethanol yield after 7, 14 and 21 days of solid-state fermentation were evaluated. Most fungi degraded lignin with variable selectivity degrees, although only eight of them improved sugar recovery compared to untreated samples. Glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lacteus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of 62% in both cases. Conversions from glucose to ethanol reached around 90%, showing that no inhibitors were generated during this pretreatment. No close correlations were found between ligninolytic enzymes production and sugar yields.