• Feedstocks Analysed at Celignis
    Straw

Background on Straw

Straw is a general term that can cover most solid plant residues from crops. It can include oilseed rape, rye, barley, wheat, oats, beans, and peas. In Ireland, the cereal straws are the most abundant. When wheat or barley is threshed, straw is a byproduct that is typically laid back on the field during the combining process. Some may be collected and used for bedding, feed supplement, or mushroom compost production. However, much is left to rot on the field.

The yield of straw per unit mass of grain will vary according to the plant type and the local environment. However, the average yield of wheat straw is 1.3 to 1.4 kg per kg of grain.

Analysis of Straw at Celignis



Celignis Analytical can determine the following properties of Straw samples:



Lignocellulosic Properties of Straw

Cellulose Content of Straw

The chemical compositions of straws will be dependent on the relative proportions of the components of the plant (e.g. nodes, internodes etc.) and the chemical compositions of these components. The harvesting procedure is also important since it determines how well the different components are collected.

The specific cultivar of the species may also be important as the relative proportions of leaves and internodes may differ between varieties and different cultivars may also lose differing quantities of leaves in the harvesting process, due to variations in the brittleness of leaves.

Celignis founder Daniel Hayes has extensive experience in the collection, preparation, and chemical/infrared analysis of straw samples. He has carried out research projects, funded by the Irish Environmental Protection Agency and the Irish Department of Agriculture, that involved the analysis of a variety of straw types.

Cellulose is the principal component in straw samples. The cellulose content can vary between different varieties of the same plant species. There is greater variability in cellulose content between straws from different plant species.

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Hemicellulose Content of Straw

Hemicellulose is the second most abundant constituent in straws, with xylose being the principal hemicellulos sugar, followed by arabinose. Galactose is present in concentrations that are typically around three times less than arabinose, whilst rhamnose and mannose are minor components.

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Lignin Content of Straw

Lignin is the third most abundant polymer in most straws (after cellulose and hemicellulose). The lignin content can vary between different varieties of the same plant species. There is greater variability in lignin content between straws from different plant species.

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Starch Content of Straw

The starch contents of straws are typically quite low but can vary according to the maturity of the plant.

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Uronic Acid Content of Straw

Uronic acids can be present as side chains attached to the main backbone of hemicelluloses in straws. They concentrations of uronic acids tends to be greatest in the nodes, lower in the internodes, and at intermediate levels in the leaves.

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Enzymatic Hydrolysis of Straw

We can undertake tests involving the enzymatic hydrolysis of Straw. In these experiments we can either use a commercial enzyme mix or you can supply your own enzymes. We also offer analysis packages that compare the enzymatic hydrolysis of a pre-treated sample with that of the native original material.

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Bioenergy Properties of Straw

Ash Content of Straw

The ash content of agricultural straws is comparable to grassy energy crops but higher than most woody feedstocks.

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Heating (Calorific) Value of Straw

Straws have good heating values, meaning that they are suitable for utilisation in boilers for the production of heat and/or electricity. However the effective heating value will depend greatly on the moisture content of the biomass.

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Ash Melting Behaviour of Straw

Ash melting, also known as ash fusion and ash softening, can lead to slagging, fouling and corrosion in boilers which may reduce conversion efficiency. We can determine the ash melting behaviour of Straw using our Carbolite CAF G5 BIO ash melting furnace. It can record the following temperatures:

Ash Shrinkage Starting Temperature (SST) - This occurs when the area of the test piece of Straw ash falls below 95% of the original test piece area.

Ash Deformation Temperature (DT) - The temperature at which the first signs of rounding of the edges of the test piece occurs due to melting.

Ash Hemisphere Temperature (HT) - When the test piece of Straw ash forms a hemisphere (i.e. the height becomes equal to half the base diameter).

Ash Flow Temperature (FT) - The temperature at which the Straw ash is spread out over the supporting tile in a layer, the height of which is half of the test piece at the hemisphere temperature.



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Major and Minor Elements in Straw

Examples of major elements that may be present in Straw include potassium and sodium which are present in biomass ash in the forms of oxides. These can lead to fouling, ash deposition in the convective section of the boiler. Alkali chlorides can also lead to slagging, the fusion and sintering of ash particles which can lead to deposits on boiler tubes and walls.

We can also determine the levels of 13 different minor elements (such as arsenic, copper, and zinc) that may be present in Straw.

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Analysis of Straw for Anaerobic Digestion



Biomethane potential (BMP) of Straw

Straw can be somewhat recalcitrant to degradation in anaerobic digestion processes due to the predominance of lignocellulosic material. However, it can be used as a co-feed with more labile feedstocks (e.g. manures). Alternatively, pre-treatment processes that reduce the recalcitrance of the lignocellulosic matrix can be employed.

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Physical Properties of Straw



Bulk Density of Straw

At Celignis we can determine the bulk density of biomass samples, including Straw, according to ISO standard 17828 (2015). This method requires the biomass to be in an appropriate form (chips or powder) for density determination.



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Particle Size of Straw

Straw can be somewhat recalcitrant to degradation in anaerobic digestion processes due to the predominance of lignocellulosic material. However, it can be used as a co-feed with more labile feedstocks (e.g. manures). Alternatively, pre-treatment processes that reduce the recalcitrance of the lignocellulosic matrix can be employed.

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Publications on Straw By The Celignis Team

Haigh K.F, Petersen A.M, Gottumukkala, L, Mandegari M, Naleli, K, Gorgens J.F (2018) Simulation and comparison of processes for biobutanol production from lignocellulose via ABE fermentation, Biofuels, Bioproducts and Biorefining 12(6): 1023-1036

Six conceptual process scenarios for the production of biobutanol from lignocellulosic biomass through acetone?butanol?ethanol (ABE) fermentation, using reported data on process performances, were developed with ASPEN Plus® V8.2 software. The six scenarios covered three fermentation strategies, i.e. batch separate hydrolysis and fermentation (SHF), continuous SHF, and batch simultaneous saccharification and fermentation (SSF) integrated with gas stripping (GS). The two downstream processing options considered were double?effect distillation (DD) and liquid?liquid extraction and distillation (LLE&D). It was found that the SSF?GS/DD scenario was the most energy efficient with a liquid fuel efficiency of 24% and an overall efficiency of 31%. This was also the scenario with the best economic outcome, with an internal rate of return (IRR) of 15% and net present value (NPV) of US$387 million. The SSF?GS/DD scenario was compared to a similar molasses process, based on the product flow rates, and it was found that the molasses process was more energy efficient with a gross energy value (GEV) of 23?MJ?kg1 butanol compared to ?117?MJ?kg1 butanol for the lignocellulosic process. In addition, the molasses?based process was more profitable with an IRR of 36% compared to 21%. However, the energy requirements for the molasses process were supplied from fossil fuels, whereas for the lignocellulose processes a portion of the feedstock was diverted to provide process energy. Improved environmental performance is therefore associated with the lignocellulosic process.

Gottumukkala L.D. Gorgens J.F (2016) Biobutanol production from lignocellulosics, Biofuels Production and future perspectives, Taylor & Francis group

Next-generation biofuels from renewable sources have gained interest among research investigators, industrialists, and governments due to major concerns on the volatility of oil prices, climate change, and depletion of oil reserves. Biobutanol has drawn signicant attention as an alternative transportation fuel due to its superior fuel properties over ethanol. e advantages of butanol are its high energy content, better blending with gasoline, less hydroscopic nature, lower volatility, direct use in convention engines, low corrosiveness, etc. Butanol production through (acetone, butanol, and ethanol) ABE fermentation is a well-established process, but it has several drawbacks like feedstock cost, strain degeneration, product toxicity, and low product concentrations. Lignocellulosic biomass is considered as the most abundant, renewable, low-cost feedstock for biofuels. Production of butanol from lignocellulosic biomass is more complicated due to the recalcitrance of feedstock and inhibitors generated during the pretreatment and hydrolysis process. Advanced fermentation and product recovery techniques are being researched to make biobutanol industrially viable.

Gottumukkala L.D, Sukumaran R.K. Mohan S.V. Valappil S.K. Sarkar O, Pandey A (2015) Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium sporogenes BE01: bio-electrochemical analysis of the electron transport mediators involved, Green chemistry 17(5): 3047-3058

Clostridium sporogenes BE01, a non-acetone forming butanol producer, can produce hydrogen and volatile fatty acids (VFAs) during butanol fermentation from rice straw hydrolysate. Bio-electrochemical analysis revealed the changes that occurred in the redox microenvironment and electron transport mediators during fermentation at different pH and CaCO3 concentrations. CaCO3 played a very important role in enhancing the production of hydrogen, volatile fatty acids and solvents by stimulating the changes in the electron transport system. The electron transport system mediated by NAD/NADH, flavins, Fe–S clusters, protein bound FAD, and cytochrome complex in C. sporogenes BE01 was analysed by cyclic voltammetry (CV). Electrokinetic analysis revealed that the favorability for redox reactions increased with an increase in pH, and the polarization resistance reduced significantly with CaCO3 supplementation.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Pandey A (2014) Growth and butanol production by Clostridium sporogenes BE01 in rice straw hydrolysate: kinetics of inhibition by organic acids and the strategies for their removal, Biomass Conversion and Biorefinery 4(3): 277-283

Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resins—XAD 4, XAD 7, XAD 16, and an anion exchange resin—Seralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.

Gottumukkala, L. D, Valappi, S. K. (2013) Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01, Bioresource Technology 145: 182-187

Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Mathiyazhakan, K (2013) Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01, Bioresource technology 145: 182-187

Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

Hayes, D. J. M. (2012) Review of Biomass Feedstocks and Guidelines of Best Practice, DIBANET WP2 Report150 pages

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This document is the result of the evaluation of biomass feedstocks, from Europe and Latin America, that took place as part of the DIBANET project. That project is co-financed from the 7 th Framework Programme for Research and Technological Demonstration of the European Union. (Title: Enhancing international cooperation between the EU and Latin America in the field of biofuels; Grant Agreement No: 227248-2).

The work in Task 2.1 of Work Package 2 (WP2) at DIBANET partners UL, CTC, and UNICAMP involved evaluating, on a number of levels, potential feedstocks for utilisation in the DIBANET acid-hydrolysis process (WP3). In the early stage of the project a wide number of feedstocks were examined and relevant secondary compositional data were sought from the literature. Selected feedstocks were analysed at the laboratories of UL, CTC, and UNICAMP and, from these, a limited number of feedstocks were subjected to more in-depth analysis/evaluation.

Work at UL focused on Miscanthus, cereal straws, and waste papers. The wet-chemical and spectroscopic analysis that was carried out on a wide number of Miscanthus samples have allowed for in-depth understandings to be reached regarding the changes in lignocellulosic composition, and potential biomass/biofuel yields that could be realised over the harvest window. Straws present much less chemical variation but have enough structural carbohydrates to warrant their processing in the DIBANET technology. Waste papers can have amongst the highest total carbohydrate contents of any of the feedstocks studied.

Work at CTC focused on the residues of the sugarcane industry - sugarcane bagasse and sugarcane trash (field residues from harvesting). A large number of samples were collected from a variety of sugar mills and plantations. It has been seen that there can be a significant variation in the composition of different bagasse samples, particularly with regards to the ash content. Sugarcane trash has lower total carbohydrates contents than bagasse but is still a suitable feedstock for DIBANET.

Work at UNICAMP focused on the evaluation of residues from the banana, coffee, and coconut industries. It was found that these also have potential for utilisation in the DIBANET process, however the value of the residues for this end-use is dependent on which part of the plant is utilised. For instance, coffee husks have sufficient structural carbohydrates to allow for decent yields of levulinic acid, formic acid, and furfural in DIBANET, however the leaves of the coffee plant do not. Leaves from the banana plant are also of less value for DIBANET than the other parts of the plant (e.g. stem).

A major output of this Deliverable is the downloadable electronic database that contains all of the WP2 analytical data obtained during the course of the project. It contains analytical data and predicted biorefining yields for a total of 1,281 samples. It can be obtained, free of charge, from the DIBANET website and will be a valuable tool for stakeholders in biorefining projects.

This document presents the data and evaluations that were made regarding biomass feedstocks, and also puts forward guidelines of best practice in terms of making the best use of these resources. A shortened version of this document can also be downloaded from the DIBANET website.

Hayes, D. J. M. (2011) Analysis of Lignocellulosic Feedstocks for Biorefineries with a Focus on The Development of Near Infrared Spectroscopy as a Primary Analytical Tool, PhD Thesis832 pages (over 2 volumes)

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The processing of lignocellulosic materials in modern biorefineries will allow for the production of transport fuels and platform chemicals that could replace petroleum-derived products. However, there is a critical lack of relevant detailed compositional information regarding feedstocks relevant to Ireland and Irish conditions. This research has involved the collection, preparation, and the analysis, with a high level of precision and accuracy, of a large number of biomass samples from the waste and agricultural sectors. Not all of the waste materials analysed are considered suitable for biorefining; for example the total sugar contents of spent mushroom composts are too low. However, the waste paper/cardboard that is currently exported from Ireland has a chemical composition that could result in high biorefinery yields and so could make a significant contribution to Ireland’s biofuel demands.

Miscanthus was focussed on as a major agricultural feedstock. A large number of plants have been sampled over the course of the harvest window (October to April) from several sites. These have been separated into their anatomical fractions and analysed. This has allowed observations to be made regarding the compositional trends observed within plants, between plants, and between harvest dates. Projections are made regarding the extents to which potential chemical yields may vary. For the DIBANET hydrolysis process that is being developed at the University of Limerick, per hectare yields of levulinic acid from Miscanthus could be 20% greater when harvested early compared with a late harvest.

The wet-chemical analysis of biomass is time-consuming. Near infrared spectroscopy (NIRS) has been developed as a rapid primary analytical tool with separate quantitative models developed for the important constituents of Miscanthus, peat, and (Australian) sugarcane bagasse. The work has demonstrated that accurate models are possible, not only for dry homogenous samples, but also for wet heterogeneous samples. For glucose (cellulose) the root mean square error of prediction (RMSEP) for wet samples is 1.24% and the R2 for the validation set ( ) is 0.931. High accuracies are even possible for minor analytes; e.g. for the rhamnose content of wet Miscanthus samples the RMSEP is 0.03% and the is 0.845. Accurate models have also been developed for pre-treated Miscanthus samples and are discussed. In addition, qualitative models have been developed. These allow for samples to be discriminated for on the basis of plant fraction, plant variety (giganteus/non-giganteus), harvest-period (early/late), and stand-age (one-year/older).

Quantitative NIRS models have also been developed for peat, although the heterogeneity of this feedstock means that the accuracies tend to be lower than for Miscanthus. The development of models for sugarcane bagasse has been hindered, in some cases, by the limited chemical variability between the samples in the calibration set. Good models are possible for the glucose and total sugars content, but the accuracy of other models is poorer. NIRS spectra of Brazilian bagasse samples have been projected onto these models, and onto those developed for Miscanthus, and the Miscanthus models appear to provide a better fit than the Australian bagasse models.

Vasudeo Zambare (2011) Optimization of amylase production from Bacillus sp. using statistics based experimental design, Emirates Journal of Food and Agriculture 23(1): 37-47

Production of amylase under submerged fermentation Bacillus sp. was investigated using wheat bran, soybean meal and CaCO3 (WSC) medium. Response surface methodology (RSM) was used to evaluate the effect of the main variables, i.e., pH (11.35), temperature (35.16°C) and inoculum size (2.95%) on amylase production by applying a full factorial central composite design (CCD). The mutual interaction between these variables resulted into 4.64 fold increase in amylase activity as compared to the non-optimized environmental factors in the basal medium.

V.P. Zambare, Lew P. Christopher (2011) Statistical analysis of cellulase production in Bacillus amyloliquefaciens UNPDV-22, Extreme Life, Biospeology & Astrobiology 3(1): 38-45

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The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of fermentation medium components (wheat bran, soybean meal, and malt dextrin) on cellulase activity. Results suggested that wheat bran, soybean meal, and malt dextrin all have significant impact on cellulase production. The use of RSM resulted in a 70% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 11.23 U/mL was obtained in a fermentation medium containing wheat bran (1.03%, w/v), soybean meal (2.43%, w/v), and malt dextrin (2.95%, w/v).

V.P. Zambare, Lew P. Christopher (2011) Optimization of culture conditions for cellulase production from thermophilic Bacillus strain, Journal of Chemistry and Chemical Engineering 5(7): 521-527

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The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of culture conditions (temperature, pH, and inoculum) on cellulase activity. Results suggested that temperature and pH all have significant impact on cellulase production. The use of RSM resulted in a 96% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 13 U/mL was obtained at a temperature of 42.24 °C, pH of 5.25, and inoculum size of 4.95% (v/v) in a fermentation medium containing wheat bran, soybean meal and malt dextrin as major nutritional factors.

Singhania R.R, Sukumaran R.K, Rajasree K.P, Mathew A, Gottumukkala L.D, Pandey A (2011) Properties of a major ?-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources, Process Biochemistry 46(7): 1521-1524

Aspergillus niger NII-08121/MTCC 7956 exhibited differences in expression of ?-glucosidase (BGL) in response to carbon sources provided in the medium. Activity staining with methyl umbelliferyl ?-d-glucopyranoside (MUG) indicated that four different isoforms of BGL were expressed when A. niger was grown under submerged fermentation with either lactose or cellulose, whereas only two were expressed when wheat bran or rice straw was used as the carbon source. Among the four isoforms of BGL expressed during lactose supplementation, two were found to retain 92% and 82% activity respectively in presence of 250 mM glucose in the MUG assay. The major ?-glucosidase (BGL1) was purified to homogeneity by electro elution from a Native PAGE gel. The purified 120 kDa protein was active at 50 °C and was stable for 48 h without any loss of activity. The optimum pH and temperature were 4.8 and 70 °C respectively.

V. P. Zambare, Lew P. Christopher (2010) Solid state fermentation and characterization of a cellulase enzyme system from Aspergillus niger SB-2, International Journal of Biological Sciences and Technology 2(3): 22-29

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The focus of this study was on the solid state fermentation (SSF) of cellulase enzymes produced by Aspergillus niger SB-2 utilizing lignocellulosic agricultural waste as carbon and energy source. Optimization of the SSF media and parameters resulted in a 32% increase in the cellulase activity. Maximum enzyme production of 1,325±7.1 IU/g dry fermented substrate was observed on wheat bran and rice bran supplemented with malt dextrin and soybean meal at pH 6 and 300C after incubation for 120 h. The cellulase activities presented here appear to be among the highest reported in literature for A. niger to date. The A. niger SB-2 cellulase was partially purified and characterized. Zymogram analysis of the sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed two bands of cellulase activity with molecular weights of 30 and 45 kDa. To the best of our knowledge, a 45 kDa cellulase from A. niger has not been previously described in literature. The enzyme was active in a broad pH (4-7) and temperature (30-550C) range with a pH optimum of 6 and a temperature optimum of 450C. At 50 and 600C, the cellulase half life was 12.4 and 4.1 h, respectively. Dithiothreitol, iodoacetamide and Mg+2 acted as activators of cellulase activity. Kinetics studies indicated that the substrate specificity of A. niger SB-2 cellulase was 18% higher on insoluble cellulose than on soluble cellulose. Therefore, the cellulase complex of A. niger SB-2 would be useful in bioprocessing applications where efficient saccharification of lignocellulosic biomass is required.

Parameswaran, B, Raveendran S, Singhania, R.R, Surender V, L Devi, Nagalakshmi S, Kurien N, Sukumaran R.K, Pandey A. (2010) Bioethanol production from rice straw: an overview, Bioresource technology 101(13): 4767-4774

Rice straw is an attractive lignocellulosic material for bioethanol production since it is one of the most abundant renewable resources. It has several characteristics, such as high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars. But there occur several challenges and limitations in the process of converting rice straw to ethanol. The presence of high ash and silica content in rice straw makes it an inferior feedstock for ethanol production. One of the major challenges in developing technology for bioethanol production from rice straw is selection of an appropriate pretreatment technique. The choice of pretreatment methods plays an important role to increase the efficiency of enzymatic saccharification thereby making the whole process economically viable. The present review discusses the available technologies for bioethanol production using rice straw.





Examples of Other Feedstocks Analysed at Celignis



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