Enhanced Saccharification and Fermentation of Rice Straw by Reducing the Concentration of Phenolic Compounds Using an Immobilized Enzyme Cocktail.

The present study reports the partial purification and characterisation of a novel xylanase from Bacillus subtilis subsp. subtilis JJBS250 followed by its applicability in the saccharification of rice straw. Bacterial xylanase was partially purified by gel filtration chromatography using Sephadax G-50 matrix that displayed optimal activity at 60 °C and pH 7.0 with half-life values of 1420 and 718 min at 60 °C and 80 °C, respectively. Bacterial xylanase showed high tolerance to ethanol with Km and Vmax values of 1.62 mg/ml and 23.92 U/ml, respectively, for beechwood xylan. Maximum amount of reducing sugars was obtained from sodium carbonate pretreated rice straw (151.65 mg/g substrate) as compared to untreated biomass (90.49 mg/g substrate) using 20 U/g enzyme dose at 60 °C and pH 7.0 after 48 h. Furthermore, the saccharification of pretreated rice straw was improved by synergistic action of bacterial and fungal xylanases. Analysis of rice straw using scanning electron microscopy (SEM), x-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy clearly showed the morphological changes and delignification in rice straw after pretreatment. Bacterial xylanase exhibiting novel properties including ethanol tolerance and high thermostability is highly suitable for the production of second generation biofuels in simultaneous saccharification and fermentation process using lignocellulosic substrates.

Optimization of cellulase production by Myceliophthora thermophila BJTLRMDU3 was studied in solid state fermentation. Sequential use of Plackett-Burman and response surface methodology (RSM) resulted in the production of 98.81, 243.19, and 316.48 U/g dry moldy residue (DMR) of FPase, CMCase, and β-glucosidase, respectively. Statistical optimization has resulted in more than 4.0-fold increase in the production of cellulases. Optimization of saccharification of untreated and pretreated rice straw was carried out by cellulolytic enzymes of thermophilic mold M. thermophila. The liberation of reducing sugars was higher using fungal cellulases in ammonia-pretreated rice straw as compared with untreated biomass. Maximum liberation of reducing sugars was attained at 60 °C (224.24 mg/g substrate) and pH 5.0 (246.33 mg/g substrate) after an incubation time of 24 h (345.61 mg/g substrate) using enzyme dose of 20 U/g in ammonia-pretreated rice straw. Supplementation of xylanase further enhanced the saccharification (488.78 mg/g substrate) of pretreated rice straw. Analysis using high-performance liquid chromatography (HPLC) indicated the presence of various monomeric and oligomeric sugars in the enzymatic hydrolysate of pretreated rice straw. Both, Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) revealed the structural changes in rice straw after ammonia pretreatment.

The utilization of rice straw for biofuel production is limited by its composition. The pretreatment process is required to improve the enzymatic accessibility of polysaccharides in the biomass prior to enzymatic saccharification. In this study, simultaneous biological pretreatment and saccharification (SPS) of rice straw starting from laccase production by Panus neostrigosus I9 was operated in a 2-L fermenter. It was found that fungal physiology was strongly influenced by the agitation, and that the highest laccase production was obtained at an agitation speed of 750 rpm (209.96 ± 0.34 U/L). The dilution rate of 0.05 h−1 was set in continuous fermentation which resulted in laccase activity of 678.49 ± 20.39 U/L, approximately three times higher than that in batch culture. Response surface methodology (RSM) was applied to achieve the condition for maximum percentage of delignification. The maximum percentage of delignification of 45.55% was accomplished after pretreatment of rice straw with laccase enzyme 39.40 U/g rice straw at 43.70 °C for 11.19 h. Reducing sugar of 3.85 ± 0.15 g/L was obtained from the digested rice straw in a SPS reactor, while non-pretreated rice straw gave only 1.13 ± 0.10 g/L within 12 h of incubation. The results indicated that simultaneous biological pretreatment and saccharification (SPS) of rice straw by laccase helped to improve the accessibility of cellulose by cellulolytic enzymes.

Optimization of saccharification of rice straw plays a crucial role in harnessing maximum fermentable sugars through enzymatic hydrolysis of this complex polymer. The present study was aimed to evaluate the applicability of A. tubingensis NKBP-55 cellulolytic cocktail for generation of fermentable sugars and to carry out parametric optimization of the saccharification process followed by batch ethanolic fermentation of the rice straw hydrolysate. Also, evaluation of the alterations (composition, crystallinity and structural morphology) occurring in the wall polymer structure after sequential treatments was studied. The cellulolytic cocktail, under the statistically optimized conditions (rice straw 4% w/v and pH 5.0) liberated 578 mg fermentable sugars (glucose 463 mg, xylose 114.35 and cellobiose 0.71 mg) per gram of pretreated rice straw showing 2.17-fold enhancement. Candida shehatae NCIM 3501 utilized both glucose and xylose and produced 16.15 g/L bioethanol along with yield (YP/S) 0.50 after 24 h of fermentation. Its bioethanol production was observed to be better than Saccharomyces cerevisiae (13.39 g/L) and their co-culture (15.57 g/L). Sequential changes brought about by the enzymatic treatment of the solid substrate revealed by X-ray diffraction and FTIR analyses showed decrease in the absolute crystallinity (CrI) (54.80 to 48.31%) and total crystallinity index (TCI) (1.002 to 0.991) and confirmed the degradation of the crystalline domain of cellulose by enzyme. The study demonstrated the potential of cellulolytic cocktail produced by A. tubingensis NKBP-55 in rice straw saccharification and its efficient fermentation by conventional and xylose-fermenting yeast in batch ethanolic fermentation.

Simultaneous production of cellulase and ferulic acid esterase by Penicillium decumbens with rice straw as the sole carbon source

Saccharification and detoxification of Na2CO3 pretreated rice straw with on-site manufactured enzymes secreted by Aspergillus fumigatus to enhance bioethanol yield

Multiple effects of swelling by sodium bicarbonate after delignification on enzymatic saccharification of rice straw

A two-step process for efficient enzymatic saccharification of rice straw

Open rice straw burning has created hazardous effects on the environment and human health. Biological pretreatment of rice straw has been proven as an environmental-benign and economical feasible process for the production of biofuels and value-added products under milder and greener conditions by avoiding harmful chemicals and toxic reagents. A consolidated bioprocess for the production of cellulolytic enzymes and biological pretreatment was developed to achieve a cost-effective saccharification of rice straw. Biodegradation of biomass was slightly enhanced with increase in cultivation time of microbial pretreatment. Furthermore, the modification in biomass due to biological pretreatment weakened the interaction between hemicellulose and lignin that resulted in significant reduction in lignin using Bacillus subtilis subsp. subtilis JJBS300, Myceliophthora thermophila BJTLRMDU3, and Aspergillus oryzae SBS50 for biological pretreatment of rice straw. Bacterial culture produced maximum FPase, CMCase, and β-glucosidase of 28, 17, and 20.36 U/g DMR after 2 days during pretreatment process, whereas M. thermophila produced maximum FPase (85.10 U/g DMR), CMCase (96.89 U/g DMR), and β-glucosidase (91.92 U/g DMR) after 9 days. The mold A.oryzae SBS50 also produced maximum FPase (57.41 U/g DMR), CMCase (55.36 U/g DMR), and β-glucosidase (48.04 U/g DMR) after 9 days of pretreatment. Maximum amount of reducing sugars of 52.41, 86.74, and 49.59 mg/g substrate were liberated from 6-, 5-, and 3-day-old biological pretreated rice straw by B. subtilis, M. thermophila, and A. oryzae, respectively, after enzymatic hydrolysis for 6 h at 60 oC and pH 5.0. Laccase-pretreated rice straw followed by enzymastic saccharification resulted in liberation of 162.82 mg/g reducing sugars at pH 5.0 and 60 °C after 6h using 20 U/g cellulase. Simultaneous laccase pretreatment and saccharification (SLPS) process further enhanced the liberated reducing sugars, i.e., 179.47 mg/g substrate. FTIR, XRD, and SEM analyses showed marked morphological changes as a result of delignification after biological pretreatment of rice straw. Biological pretreatment being an environmental-benign process causing no harm to the environment in comparison to physical and chemical pretreatments of lignocellulosic biomass could be a better pretreatment strategy for bioremediation of lignocellulosic substrates.

In this study, sono-assisted dilute sulfuric acid process was evaluated for its viability of simultaneous pretreatment and saccharification of rice straw. Three critical factors for simultaneous pretreatment and saccharification process, such as sonication time (30–50 min), temperature (70–90 °C), and acid concentration (5–10 %), were optimized to maximize reducing sugar yield using Box-Behnken design and response surface methodology. The response surface methodology model was found to be adequately fitted to the obtained data. Simultaneous pretreatment and saccharification factors were optimized at sonication of 50 min, 80 °C and an acid concentration of 10 % yielding the maximum sugar content (31.78 g/100 g of biomass). Scanning electron microscopy revealed that the smooth surface of raw biomass was altered into a rough and porous surface as a result of sugar release, which showed the prospective feasibility of simultaneous pretreatment and saccharification process. This process integration may lead to develop economical bioethanol production facility. However, further research is required to make this process industrially viable.

For effective saccharification of rice straw we focused on enzyme preparations from wood-rotting fungi that have the ability to degrade cell wall polysaccharides and lignin. We tested extracellular enzyme preparations from 14 species of fungi for saccharification activity and examined the factor for saccharification by statistical analysis. An enzyme preparation from Schizophyllum commune had the highest saccharification activity of rice straw. This preparation contained highly active endo-β-xylanase, endo-β-glucanase (CMCase), β-d-glucosidase and acetylxylan esterase. Correlation analysis of the 14 enzyme preparations demonstrated that acetylxylan esterase was closely related to saccharification activity in rice straw. Multiple regression analysis also showed that acetylxylan esterase had an important role in saccharification. Ligninolytic enzymes, which are characteristic of white-rot fungi, did not contribute to saccharification activity of rice straw. Deacetylation is an essential factor for saccharification of rice straw and enzyme preparations for saccharification need to contain highly active acetylxylan esterase as well as highly active cellulolytic and xylanolytic enzymes, but not ligninolytic ones.

Catalytic hydrothermal saccharification of rice straw was carried out in the presence of sulfonated mesoporous silica (SBA-15) -based solid acid catalysts. Effects of reaction time, reaction temperature and weight ratio of solid acid catalyst to rice straw on the saccharification of rice straw were investigated. With increasing reaction time and reaction temperature, the yield of monosaccharide initially increased and then decreased, but the yield of byproducts increased. With increasing weight ratio of solid acid catalyst to rice straw up to 25 %, the yield of monosaccharide initially increased and then remained almost constant. The yield of monosaccharide was 38 % for the saccharifi cation of rice straw carried out at 180 ℃ for 1 h with the weight ratio of rice straw/distilled water/ solid acid catalyst equal to 3/30/0.5 (g/g/g). The composition of rice straw and crystallinity index of cellulose in rice straw before and after the saccharifi cation were determined, respectively. Based on the composition analysis, most xylan and glucan derived from hemicellulose were decomposed, but glucan derived from cellulose was little decomposed via catalytic hydrolysis at 180 ℃ and below. On the other hand, xylan and glucan derived from hemicellulose were almost completely decomposed, and part of glucan derived from cellulose was also decomposed at 200 ℃ and above. At the same time, some ash and lignin in rice straw were removed in all the reactions.

Rice straw is an agricultural by-product that generally used as a roughage. The fermentation process could increase the nutrient availability of rice straw. Urea addition in the fermentation process is expected to increase the digestibility. The current study aimed to evaluate the nutrient content and in vitro digestibility of fermented rice straw by different levels of urea addition. Microstar LA2 was used for the fermentation process in rice straw by 21 days. Completely randomized design with four treatments and five replications was applied in the current study. The treatments were K (fermented rice straw without urea addition), U1 (fermented rice straw with 0.15% urea addition), U2 (fermented rice straw with 0.3% urea addition) and U3 (fermented rice straw with 0.45% urea addition). The observed parameters were the nutrient content, in vitro gas production, in vitro true digestibility (IVTD) and rumen fermentation product. The results showed that there was no significant difference in ether extract (EE), neutral detergent fiber (NDF) and acid detergent fiber (ADF) for all treatments. However, adding urea on the rice straw fermentation process could increase total gas production after 24h time of incubation (P<0.05). Adding 0.15% urea could increase gas production caused by fermentation of the insoluble fraction (GPNSF) by 31.62% (P<0.05). There was no significant difference between U1, U2, and U3 on GPNSF variables. Adding a minimum 0.15% urea on the rice straw fermentation process also could increase metabolizable energy (ME), organic matter digestibility (OMD), microbial protein (MP) and single-chain fatty acid (SCFA) production. It could be concluded that adding 0.15% urea on the fermentation process is enough to increase the nutrient availability and in vitro digestibility of rice straw.

Prospects of utilizing seawater as a reaction medium for pretreatment and saccharification of rice straw

Simultaneous delignification and saccharification of rice straw as a lignocellulosic biomass by immobilized Thrichoderma viride sp. to enhance enzymatic sugar production