Delignification Of Rice Straw Research Articles

Enhancing Bioethanol Production from Rice Straw through Environmentally Friendly Delignification Using Versatile Peroxidase.

Rice straw (RS), an agricultural residue rich in carbohydrates, has substantial potential for bioethanol production. However, the presence of lignin impedes access to these carbohydrates, hindering efficient carbohydrate-to-bioethanol conversion. Here, we expressed versatile peroxidase (VP), a lignin-degrading enzyme, in Pichia pastoris and used it to delignify RS at 30 °C using a membrane bioreactor that continuously discarded the degraded lignin. Klason lignin analysis revealed that VP-treatment led to 35% delignification of RS. We then investigated the delignified RS by SEC, FTIR, and SEM. The results revealed the changes of RS caused by VP-mediated delignification. Additionally, we compared the saccharification and fermentation yields between RSs treated with and without VP, VP-RS, and Ctrl-RS, respectively. This examination unveiled an improvement in glucose and bioethanol production, VP-RS exhibiting up to 1.5-fold and 1.4-fold production, respectively. These findings underscore the potential of VP for delignifying RS and enhancing bioethanol production through an eco-friendly approach.

Characterization of thermostable recombinant laccase F from Trametes hirsuta and its application in delignification of rice straw

Characterization of thermostable recombinant laccase F from Trametes hirsuta and its application in delignification of rice straw

Designing of recombinant hydrolytic enzymes cocktail for effective saccharification of delignified rice straw

Enzymatic saccharification is the most crucial and challenging step in lignocellulosic biorefineries for bioethanol production. The enzymatic saccharification requires a cocktail of hydrolytic enzymes, cellulases and hemicellulases for effective degradation of biomass. However, the complete bioconversion of biomass is governed by the proportion and synergistic action of hydrolytic enzymes present in the cocktail. Thus, this study aims to formulate an enzyme cocktail consisting of recombinant cellulases (bifunctional cellulolytic chimeric enzyme, CtGH1-L1-CtGH5-F194A and cellobiohydrolase, CtCBH5A) and xylanases (endo-1,4-β-xylanase, CtGH11A, and β-xylosidase, BoGH43), all in crude form (unpurified enzyme present in the total cellular proteins) for producing high yield of total reducing sugars using choline chloride:acetic acid pretreated rice-straw (CApRS). D-optimal mixture design approach was used for statistically designing the optimal proportion of crude recombinant enzyme cocktail for hydrolysis of delignified rice straw. The developed enzyme cocktail comprising chimera, CtCBH5A, CtGH11 and BoGH43 in ratio 35.1:41.8:10.0:13.1 resulted in cellulolytic enzyme activity, 5.6 FPU/mL and xylanase activity, 102 U/mL. Enzymatic saccharification of 3% (w/v) delignified CApRS using developed enzyme cocktail at 187 FPU/gCApRS, pH 5.8 and 35 °C, yielded total reducing sugar of 498 mg/gbiomass, glucose yield (410 mg/g) and xylose yield (71.3 mg/g) resulting in 72% saccharification efficiency. Whereas, significantly low total reducing sugar yield of 80 mg/g and 218 mg/g were released from raw RS and partially pretreated RS, respectively, by the developed cocktail under the same hydrolytic conditions, thereby inferring the effectiveness of the enzyme cocktail against CApRS biomass. The developed enzyme cocktail was compared with a commercial enzyme cocktail and prospects its applicability in lignocellulosic biorefineries.

Biocatalytic pretreatment of rice straw by ligninolytic enzymes produced by newly isolated Micrococcus unnanensis strain B4 for downstream cellulolytic saccharification.

Rice (Oryza sativa L.) straw is an agricultural byproduct of high yield, and its disposal by burning has detrimental effect on ecosystem. It has potential as source of fermentable sugars for industrial use; however, it requires effective pretreatment to remove lignin. Bacterial enzymes based pretreatment is advantageous due to their extracellular nature, and tolerance to higher temperature, pH and oxygen limitation. We herein report screening of lignocellulose degradation environment of vermicompost for ligninolytic bacteria, and studying role of Micrococcus unnanensis strain B4 in delignification of rice straw. The bacterium was capable to degrade acid soluble and insoluble lignin; and produced lignin degrading laccase and peroxidase having maximum activity at pH 6.5 and 72 h incubation. Both enzymes exhibited alkaline pH stability, and thermal stability with retention of 100 % activity on pre-incubation at 60 ℃ for 1 h. The enzymes were used for pretreatment of rice straw using chemicals (acetic acid:hydrogen peroxide) pretreatment as reference. Scanning electron microscopy of pretreated rice straw samples showed alteration in morphology with exposure of cellulosic components. Enzymatically pretreated rice straw on saccharification by a commercial cellulase yielded about 400 mg of reducing sugar per gram, comparable to that released on chemical pretreatment. Hence, pretreatment based on M.unnanensis strain B4 and its ligninolytic enzymes can be an alternative to chemical pretreatment for saccharification of rice straw to fermentable sugars.

Response surface optimized free radical grafting of methyl methacrylate on de-lignified rice straw for evaluating its application potential as flame retardant roofing material

The main objective of this investigation is to convert chemically bi-step pre-treated rice straw encompassing delignification and inorganic materials loading into a low cost roofing material by graft copolymerization of methyl methacrylate (MMA) via free radical mechanism. Central composite design (CCD) process utilizing response surface methodology (RSM) approach was adopted for the optimization of time duration, concentration of methyl methacrylate and reaction temperature (three-independent factors) at five levels. Optimization of the procedure was done by an estimated regression model (quadratic) in combination with an analysis of variance (ANOVA) depicting how the independent test variables have a comparative impact. Contour plots (2D) as well as response surface (3D) plots helped in investigating the cumulative interactions between the variables critical to optimization as well as in deciphering under which conditions optimal grafting can occur. Percentage of grafting (%G) was the response variable, which was a dependent output variable. A quadratic response surface model, according to estimated values could then be developed to relate every independent variable to percentage of grafting (%G). Optimal conditions projected by RSM were 3.99 cc amount of monomer, 119.85 min duration of time and 62.89 °C grafting temperature. At this combination, the highest percentage of grafting of 50.24% was predicted, with desirability = 1.00, the observed result indicates perfect reproducibility of the experiment. This theoretical value was compared with experimental result and the observed result is 50.68% which is very close to the software predicted value. The response surface quadratic model adequacy was satisfactory with the coefficient of regression of 0.9815. According to the Design-Expert software this predicted value of regression coefficient confirmed the accuracy of model fitness with the observed experimental data.

Profitable biomethane production from delignified rice straw biomass: the effect of lignin, energy and economic analysis

A cost and time saving strategy for the recovery of biomethane from rice straw using a novel phase-separated pretreatment.

Bioconversion of rice straw by synergistic effect of in-house produced ligno-hemicellulolytic enzymes for enhanced bioethanol production

In the present work attempts have been made to mimic the natural system of biomass delignification using laccase from Myrothecium verrucaria ITCC-8447 and synergistic effect of cellulase and xylanase from Schizophyllum commune NAIMCC-F-03379 and Aspergillus oryzae ITCC-8571 respectively for enhanced saccharification and subsequent bioethanol production. The laccase mediated pretreatment resulted in 6.7% delignification of rice straw (RS). The structural analysis of enzymatic pretreated RS using XRD and FTIR suggested high crystallinity index (CrI) and improved accessibility of cellulose and hemicelluloses as compared to untreated RS. The single step simultaneous delignification, saccharification and fermentation (SDSF) of RS using partially purified in-house enzyme cocktail with Saccharomyces cerevisiae MTCC-173 resulted in sugar yield of 26.7 ± 0.2 g/L with an experimental ethanol yield of 6.47 ± 0.16 g/L which is comparable to maximum sugar and ethanol yield of 31.2 ± 0.7 g/L and 7.34 ± 0.39 g/L respectively using commercial enzyme cocktail with S. cerevisiae MTCC-173. The SDSF was obtained as a suitable single step process for the ethanol production from RS.

Utilization of rice straw and used paper for the recycle papermaking

Rice Straw is one of the most important materials that has been used for pulp and paper production due to its abundance and cost-effectiveness. However, the pulping and papermaking characteristics of the mixture of rice straw and used paper have rarely been investigated. In this experiment, delignified rice straw was mixed with used paper in order to make recycle papers with acceptable properties. Soda pulping process was carried out to remove out of lignin. The delignification was designed to measure the effects in terms of sodium hydroxide concentration and temperature, on the cellulose and lignin content of rice straw, and its tensile strength and water absorption. The mass ratio of used paper and rice straw was varied and its physical properties was observed and compared to paper from natural rice straw. The delignification was conducted using NaOH concentrations (4-10% w/w) and temperatures (60-90°C) for 1 hour. The ratio of rice straw to used paper was varied (1:1-1:9). An optimum condition was obtained from rice straw delignification at NaOH concentration of 8% and temperature of 90°C. The results of the optimum condition obtained the highest amount of cellulose content and tensile strength while having the lowest amount of lignin content and water absorption.

Adsorption of Ammonia Nitrogen in Aqueous Solution Using Zeolite A

The capacity of alternative adsorbents including rice straw, delignified rice straw and zeolite A for ammonia nitrogen removal from aqueous solution was investigated. The adsorption experiments were carried out in batch system with initial ammonia nitrogen concentration of 0.5 mg-N/l, 1000 ml ammonia nitrogen solution for 1 g of adsorbent. Experimental data indicated that rice straw and delignified rice straw cannot adsorb ammonia nitrogen. However, zeolite A provided rapidly adsorption capacity for ammonia nitrogen removal. The amount of ammonia nitrogen adsorbed by zeolite A was 0.256 mg/g at 1 minute after the experiment was initially conducted and slightly stable at 0.275 mg/g after 10 minutes. The adsorption experiments were carried out at 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg-N/l of ammonia nitrogen concentration. The results indicated that zeolite A had the adsorption capacity of 0.102, 0.294, 0.551, 1.153, 2.303, and 3.897 mg/g respectively at equilibrium time. The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms. Based on R2, Freundlich model was more promising to represent the adsorption data than Langmuir model.

Dissecting the effect of polyethylene glycol on the enzymatic hydrolysis of diverse lignocellulose

Natural lignocellulose is used as raw material to produce chemicals through biological transformation. The accessibility of cellulase to substrate was also one of the limiting factors of industrial production. Polyethylene glycol (PEG) can be used as additive in enzymatic hydrolysis of lignocellulose. In this study, enzymatic activity on simultaneous or non-simultaneous addition of PEG 4000 was investigated, and the partly delignified rice straw, the rice straw and filter paper were used as substrates, respectively. Enzyme activity was characterized by reducing sugar concentration in supernatant which was quantified through 3,5-dinitrosalicylic acid (DNS) method. Addition of PEG has been proven to facilitate enzymatic hydrolysis of lignocellulosic materials. Furthermore, PEG had the positive effect on hydrolytic enzyme activity of pure cellulose materials without lignin. Changes in lignocellulose materials have been observed by inverted microscope and Scanning electron microscope (SEM), and no chemical changes were shown by Fourier transform infrared spectroscopy (FTIR). The promotion of PEG on enzymatic hydrolysis of pure cellulose materials may be due to its loose physical structure and similar phenomenon in natural lignin materials. PEG loosens the physical structure of lignocellulose, thus facilitating enzymatic hydrolysis. This may be a new idea to optimize the lignocellulosic enzymatic hydrolysis process.

Optimizing acrylonitrile grafting onto delignified rice straw via response surface methodology towards its flame retardancy and durability intensification

The current research emphasizes on grafting of acrylonitrile onto pretreated rice straw for making low cost roofing material. The technique was improved by means of optimization for analyzing the impact of some parameters such as polymer dose, time and temperature of grafting. The optimization process applied in this research work was response surface methodology (RSM). The process was optimized through quadratic model of regression using ANOVA study (analysis of variance). 3-dimensional (3D) surface plots of the response and 2-dimensional (2D) contour analyses were adopted for further investigation of the collective synergies amidst the functional variables and to find out the optimal values for the percentage of grafting. The maximum grafting percentage was found at a grafting time of 97.59 min, grafting temperature of 77.52 oC and monomer concentration of 16.82 cubic centimeters (cc). At this condition, the highest percentage of polymer grafting from predictions according to the employed model was 60.07% which showed desirability equal to 1. The validation of these predictions was performed experimentally, with the observed result being 59.97% which is nearly equal to the theoretical values predicted by the model. Therefore, the model was able to deliver an optimized condition appropriate for polymer grafting onto rice straw for intensifying its durability.

Biological pretreatment of rice straw using an alkalophilic fungus MVI.2011 for enhanced enzymatic hydrolysis yield

Ethanol from agricultural residues is a sustainable energy source, but involves complex steps like pretreatment for the removal of lignin. Conventional pretreatment methods make use of costly chemicals or energy intensive machines. Alternatively, biological pretreatment methods are sustainable, energy and cost efficient, but take at least two weeks to bring about delignification. An alkalophilic dimorphic dueteromycete coded MVI.2011, has been known to synthesize large quantities of ligninases in a short span of time; ligninases being the key enzymes in the biological delignification of lignocelluloses. Therefore, in this study, the effectiveness of this fungus in the delignification of rice straw is explored, with a view to reduce the pretreatment time. Solid state cultivation of rice straw with MVI.2011 for two weeks and submerged cultivation for one week showed 54% and 74.2% improvement in percentage of saccharification after enzymatic hydrolysis. The results of fungal pretreatment in 7.5 l bioreactor are also promising and faster than existing biological methods. Percentage of saccharification can be improved further, if submerged cultivation conditions are optimized to minimize the cellulolytic and hemicellulolytic activities of the fungus. Hence pretreatment with MVI.2011 is a potential alternative to the prevailing time-consuming fungal pretreatment methods for sustainable ethanol production.

Parametric optimization of delignification of rice straw through central composite design approach towards application in grafting

The present investigation deals with process optimisation of delignification of rice straw towards its micro-porous structural enhancement for its utilization in polymer grafting. The individual effect of influential parameters viz. sodium hydroxide concentration (1–12%, w/v), reaction time (30–126 min), and temperature (20–150 °C) on delignification were studied in a single mode batch process. The process parameters were further optimized with Central composite design (CCD) approach of response surface methodology in Design expert software. Delignification of rice straws was observed to follow quadratic equation. Analysis of variance (ANOVA) study suggested the equation to be significant for the process with major impact of sodium hydroxide concentration on the delignification process than reaction time and temperature. The optimized parametric conditions of delignification are: alkali concentration 7.59%, reaction time 75.11 min, and reaction temperature 40 °C. The software predicted lignin extraction concentration to be 72.4 mg/g, which upon experimentation was found to be 70.03 mg/g. Instrumental analysis of the delignified rice straw demonstrated porous structure and change in surface chemistry due to lignin removal. Therefore, the delignified rice straw obtained under optimized conditions were found to be appropriate for grafting of polymers which improved its resilience for variable usages.

Hyper-production of butyric acid from delignified rice straw by a novel consolidated bioprocess

A novel consolidated bioprocess for hyper-production of butyric acid from delignified rice straw without exogenous enzymes involved was developed by co-fermentation of Clostridium thermocellum ATCC 27405 and C. thermobutyricum ATCC 49875. Feasibility of the consolidated bioprocess was approved by batch fermentations, with the optimum pH of 6.5. Fed-batch fermentation with a constant pH of 6.5 at 55 °C could enhance the butyric acid yield to a remarkable 33.9 g/L with a selectivity as high as 78%. Metabolic analysis of the co-culture indicated that sugars liberated by C. thermocellum ATCC 27405 were effectively converted to butyric acid by C. thermobutyricum ATCC 49875. Secondary metabolism of C. thermobutyricum ATCC 49875 also contributed to the hyper-production of butyric acid, resulting in the re-assimilation of by-products such as acetic acid and ethanol. This work provides a more effective fermentation process for butyric acid production from lignocellulosic biomass for future applications.

Saccharification of delignified rice straw by Fusarium oxysporum for ethanol production by Saccharomyces cerevisiae

In the present work, a preliminary test proved that there was no contradiction between using Saccharomyces cerevisiae as single culture or mixed with Fusarium oxysporum in fermenting glucose to ethanol. Rice Straw was subjected to delignification by alkaline/hypochlorite method. Delignified rice straw (RS) and carboxy methyl cellulose (CMC) individually were used in bioconversion process for ethanol production. Fusarium oxysporum performed fermentation with and without aeration, achieving ethanol production of 0.8µl/ml and 2.9µl/ml, respectively, proving the importance of air absence in this process. The CMC and RS were applied in a submerged culture to give maximum cellulases activity of 0.444U and 1.474U after 8 days, respectively. In order to apply consolidated bioprocessing, saccharification of either CMC or RS by F. oxysporum was applied in a single submerged culture giving maximum saccharification efficiency of 16.8% and 16% after 7 and 11 days, respectively. In the same submerged culture, Saccharomyces cerevisiae was used to ferment the released sugar under no aeration achieving ethanol production of 6.1% and 3.878% (v/v) corresponding to fermentation efficiency of 42.4% and 34.4%, respectively.

3-90-2 リグニン含有率の異なるイナワラの水熱処理(Session 3 バイオマス等,ポスター発表)

3-90-2 リグニン含有率の異なるイナワラの水熱処理(Session 3 バイオマス等,ポスター発表)

Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass.

The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified. Cellulosome complexes were reconstituted from 3, 12, 30, and 40 components based on their contents in the native cellulosome. The activity of the enzyme-saturated complex indicated that greater enzymatic variety generated more synergy for the degradation of crystalline cellulose and delignified rice straw. Surprisingly, a less complete enzyme complex displaying fewer than nine enzyme molecules was more efficient for the degradation of delignified rice straw than the enzyme-saturated complex, despite the fact that the enzyme-saturated complex exhibited maximum synergy for the degradation of crystalline cellulose. These results suggest that greater enzymatic diversity of the cellulosome is crucial for the degradation of crystalline cellulose and plant biomass, and that efficient degradation of different substrates by the cellulosome requires not only a different enzymatic composition, but also different cellulosome structures.

Butanol production from alkali-pretreated rice straw by co-culture of Clostridium thermocellum and Clostridium saccharoperbutylacetonicum

The co-culture of cellulolytic Clostridium thermocellum NBRC 103400 and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4 produced 5.5g/L of butanol from 40g/L of delignified rice straw pretreated with 1% (wt/vol) NaOH. The addition of cellulase (100U/gbiomass) in a co-culture system significantly increased butanol production to 6.9g/L using 40g/L of delignified rice straw. Compared to the control, this increase in butanol production was attributed to the enhancement of exoglucanase activity on lignocellulose degradation in experimental samples. The results showed that the co-culture system in conjunction with enhanced exoglucanase activity resulted in cost-effective butanol production from delignified rice straw.

Identification of Xylanase Signal Peptide in Culture Supernatant of Clostridium saccharoperbutylacetonicum Strain N1-4 Cultured on Delignified Rice Straw

The butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4 abundantly secreted xylanase into the culture supernatant when cultured on delignified rice straw. The xylanase signal peptide was identified based on its N-terminal amino acid sequence and was presumed to be secreted by the Sec-system. The presence of xylanase in the medium indicated that the strain was capable of utilizing xylan as a substrate. Also, butanol production obtained using a medium supplemented with xylan was comparable to that obtained using the same strain cultured on a medium supplemented with glucose. The signal peptide facilitated the secretion of exogenous cellulases expressed by butanol-producing strains and promoted butanol production from delignified rice straw.

Production, purification and characterization of a novel GH 12 family endoglucanase from Aspergillus terreus and its application in enzymatic degradation of delignified rice straw

Endoglucanase production was carried out using in-house isolate Aspergillus terreus on rice straw under solid state fermentation. An increase of 1.25-fold endoglucanase production was obtained under optimized conditions using response surface methodology. The enzyme was purified to homogeneity by gel filtration chromatography. Its molecular weight was determined as 28.18 kDa by gel filtration and 29.13 kDa on SDS-PAGE. The enzyme displayed maximum activity at 50 °C and pH 4.8. It was stable for 240 min at 50 °C and 120 min at 60 °C but rapidly inactivated at 70 °C. The purified enzyme was specific towards carboxymethyl-cellulose but showed no activity for cellobiose or xylan. Maximum velocity (Vmax) and KM were 16.15 μmol min−1 mg−1 and 12.01 mg ml−1, respectively. AgNO3, KCl, NaCl, and MnSO4 were found to inhibit enzyme activity while CaCl2 and ZnSO4 activated the enzyme. Internal peptide mass fingerprinting analysis identified that the protein belongs to GH12 superfamily endoglucanases. External supplementation of the purified enzyme to the crude cellulase showed 38.7% increase in saccharification efficiency of the delignified rice straw compared to the crude cellulase alone. The results demonstrated that the addition of GH 12 family purified endoglucanase to the crude cellulase can efficiently convert lignocellulosic biomass to fermentable sugars.