Effective Bioconversion Research Articles

In-silico discovery of bifunctional enzymes with enhanced lignocellulose hydrolysis from microbiota big data

Due to the importance of using lignocellulosic biomass, it is always important to find an effective novel enzyme or enzyme cocktail or fusion enzymes. Identification of bifunctional enzymes through a metagenomic approach is an efficient method for converting agricultural residues and a beneficial way to reduce the cost of enzyme cocktail and fusion enzyme production. In this study, a novel stable bifunctional cellulase/xylanase, PersiCelXyn1 was identified from the rumen microbiota by the multi-stage in-silico screening pipeline and computationally assisted methodology. The enzyme exhibited the optimal activity at pH 5 and 50°C. Analyzing the enzyme activity at extreme temperature, pH, long-term storage, and presence of inhibitors and metal ions, confirmed the stability of the bifunctional enzyme under harsh conditions. Hydrolysis of the rice straw by PersiCelXyn1 showed its capability to degrade both cellulose and hemicellulose polymers. Also, the enzyme improved the degradation of various biomass substrates after 168 h of hydrolysis. Our results demonstrated the power of the multi-stage in-silico screening to identify bifunctional enzymes from metagenomic big data for effective bioconversion of lignocellulosic biomass.

Polyphasic Characterization of Plant Growth Promoting Cellulose Degrading Bacteria Isolated from Organic Manures.

In the present study, twenty seven cellulose-degrading bacteria (CDB) were isolated from various organic manures and their cellulolytic activities were determined. The bacterial isolate CDB-26 showed the highest cellulolytic index, released 0.507 ± 0.025mg/ml glucose and produced 0.196 ± 0.014IU/ml cellulase enzyme under in vitro conditions. Biochemically, all the 27 isolates showed difference in the 6 biochemical tests performed. Further, all the 27 CDB isolates were subjected to various plant growth-promoting activities, and all CDB strains were positive for IAA production, GA3 production and siderophore production, whereas 19 strains were positive for ACC deaminase activity, 21 strains showed NH3 production and 19 strains were positive for HCN production. Out of 27 CDB isolates, 18 isolates were able to solubilize phosphate, 21 isolates were able to solubilize potash and 10 CDB isolates were found positive for silica solubilization. The molecular diversity among different CDB isolates was studied through ARDRA and demonstrated very high genetic diversity among these bacteria. The in vitro cellulose-degradation potential of these CDB isolates using vegetable waste as substrate were also assessed, and the 3 CDB isolates viz. Serratia surfactantfaciens (CDB-26), Stenotrophomonas rhizophila (CDB-16) and Pseudomonas fragi (CDB-5) showed the highest cellulose-degrading potential under in vitro conditions. Hence, the cellulolytic microbes isolated in the present study could be used for effective bioconversion of plant biomasses into enriched compost.

Recent progress on bio-succinic acid production from lignocellulosic biomass.

Succinic acid is a valuable bulk chemical, which has been extensively applied in food, medicine, surfactants and biodegradable plastics industries. As a substitute for chemical raw material, bio-based succinic acid production has received increasing attention due to the depletion of fossil fuels and environmental issues. Meanwhile, the effective bioconversion of lignocellulosic biomass has always been a hot spot of interest owning to the advantages of low expense, abundance and renewability. Consolidated bioprocessing (CBP) is considered to be an alternative approach with outstanding potential, as CBP can not only improve the product yield and productivity, but also reduce the equipment and operating costs. In addition, the current emerging microbial co-cultivation systems provide strong competitiveness for lignocellulose utilization through CBP. This article comprehensively discusses different strategies for the bioconversion of lignocellulose to succinic acid. Based on the principles and technical concepts of CBP, this review focuses on the progress of succinic acid production under different CBP strategies (metabolic engineering based and microbial co-cultivation based). Moreover, the main challenges faced by CBP-based succinic acid fermentation are analyzed, and the future direction of CBP production is prospected.

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications.

The search for novel renewable products over synthetics hallmarked this decade and those of the recent past. Most economies that are prospecting on biodiversity for improved bio-economy favor renewable resources over synthetics for the potential opportunity they hold. However, this field is still nascent as the bulk of the available resources are non-renewable based. Microbial metabolites, emphasis on secondary metabolites, are viable alternatives; nonetheless, vast microbial resources remain under-exploited; thus, the need for a continuum in the search for new products or bio-modifying existing products for novel functions through an efficient approach. Environmental distress syndrome has been identified as a factor that influences the emergence of genetic diversity in prokaryotes. Still, the process of how the change comes about is poorly understood. The emergence of new traits may present a high prospect for the industrially viable organism. Microbial enzymes have prominence in the bio-economic space, and proteases account for about sixty percent of all enzyme market. Microbial keratinases are versatile proteases which are continuously gaining momentum in biotechnology owing to their effective bio-conversion of recalcitrant keratin-rich wastes and sustainable implementation of cleaner production. Keratinase-assisted biodegradation of keratinous materials has revitalized the prospects for the utilization of cost-effective agro-industrial wastes, as readily available substrates, for the production of high-value products including amino acids and bioactive peptides. This review presented an overview of keratin structural complexity, the potential mechanism of keratin biodegradation, and the environmental impact of keratinous wastes. Equally, it discussed microbial keratinase; vis-à-vis sources, production, and functional properties with considerable emphasis on the ecological implication of microbial producers and catalytic tendency improvement strategies. Keratinase applications and prospective high-end use, including animal hide processing, detergent formulation, cosmetics, livestock feed, and organic fertilizer production, were also articulated.

Assessment of the Functional and Technological Properties of Zooprotein Based on Fly Larvae (Lucilia Caesar) As Feed Composition

Recently, biotechnologies are gaining large scale as promising areas of science that are exploring the possibilities of using living organisms, systems or products of their vital functions for solving technological problems. The object of present research is the high-protein preparation Zooprotein based on the larvae of the fly species Lucilia Caesar. The insect gained wide popularity due to the highly effective bioconversion of various solid organic waste, as well as the high nutritional value of the larvae with the possibility of using fly larvae in farm animals and aquaculture feeding. The high-protein preparation Zooprotein based on the larvae of Lucilia Caesar can be considered as a promising functional ingredient for feeding diets for various animal species due to unique chemical composition. Present study provides data on the antimicrobial properties of Lucilia Caesar flies from the point of view of the safety in use of the high-protein feed ingredient Zooprotein as feed composition. In addition, the functional and technological properties of Zooprotein were evaluated in comparison with other commonly used ingredients in the feed industry, in particular fish and meat and bone meal. Also, the main basis for its application is the ability to bind moisture and fat, create a specific structure for the finished product, and, finally, the harmlessness of using this product. The main purpose of present research is to study the functional and technological properties of high-protein preparation Zooprotein in comparison with other commonly used feed ingredients.

Development of a new algorithm for obtaining highly effective bio-fertilizer from mixtures of peat and bird droppings

VNIIMZ has long and fruitfully been working on the creation of bio-fertilizers and multifunctional bio drugs produced by the method of directional bioconversion. This article reflects the innovative elements of a fundamentally new technology for producing promising bio-fertilizers. The development of a new bioconversion algorithm for peat and bird droppings mixtures was aimed at creating a competitive bio-fertilizer with favorable acidity, a high number of agronomically beneficial microflora, the presence of nutrients in a form available for plants and microflora, and was done by choosing the most effective bioconversion process. Compared 3 different process parameters, laid three times. Their assessment was carried out according to the content in the initial peat and bird droppings mixtures and in the final products (variants of the biological fertilizer) ammonifying and amylolytic microorganisms, sanitary indicative microflora (enterobacteria), fungi, actinomycetes, on the activity of oxidoreductases, on the level of agrochemical parameters. The algorithm for conducting the fermentation process includes acid hydrolysis of the initial mixture and two temperature stages. At the first stage, proceeding at 55-60С within 24 hours, the necessary ecological bio-fertilizer is achieved, characterized by a minimum of sanitary and representative microflora - enterobacteria and low content of spoilage microorganisms - fungal flora. At the second stage of bioconversion, occurring at a temperature of 36-39 С for 96 hours, the microflora actively develops, participating when using this fertilizer in the formation of soil structure, a high level of its effective fertility. During the development process, the choice was made of the option of conducting a fermentation process, which differs from the others by a higher content of agronomically beneficial microflora, a minimum of microorganisms of the genus Enterobacter and a high content of key nutrients (nitrogen, phosphorus, potassium) for plant growth.

Carbon nanotubes molybdenum disulfide 3D nanocomposite as novel nanoscaffolds to immobilize Lens culinaris β-galactosidase (Lsbgal): Robust stability, reusability, and effective bioconversion of lactose in whey

Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris β-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.

Enhanced Enzymatic Hydrolysis of Pennisetum alopecuroides by Dilute Acid, Alkaline and Ferric Chloride Pretreatments.

In this study, effects of different pretreatment methods on the enzymatic digestibility of Pennisetum alopecuroides, a ubiquitous wild grass in China, were investigated to evaluate its potential as a feedstock for biofuel production. The stalk samples were separately pretreated with H2SO4, NaOH and FeCl3 solutions of different concentrations at 120 °C for 30 min, after which enzymatic hydrolysis was conducted to measure the digestibility of pretreated samples. Results demonstrated that different pretreatments were effective at removing hemicellulose, among which ferric chloride pretreatment (FCP) gave the highest soluble sugar recovery (200.2 mg/g raw stalk) from the pretreatment stage. In comparison with FCP and dilute acid pretreatment (DAP), dilute alkaline pretreatment (DALP) induced much higher delignification and stronger morphological changes of the biomass, making it more accessible to hydrolysis enzymes. As a result, DALP using 1.2% NaOH showed the highest total soluble sugar yield through the whole process from pretreatment to enzymatic hydrolysis (508.5 mg/g raw stalk). The present work indicates that DALP and FCP have the potential to enhance the effective bioconversion of lignocellulosic biomass like P. alopecuroides, hence making this material a valuable and promising energy plant.

Bioconversion of agricultural waste and its efficient utilization in the hilly ecosystem of Northeast India

PurposeA suitable strategy for bioconversion of agricultural waste into quality organic manure and its efficient utilization was assessed in the hilly ecosystem of Northeast India.MethodsPits were dug in terrace lands and filled with chopped plant biomass (rice straw, maize stover and mixed weed biomass) with alternate layers of cow dung at 3:2 ratio. The treatment consisting of control (plant biomass + cow dung), plant biomass + cow dung + earthworm (Eisenia fetida) (with or without poly lining in the pit) and plant biomass + cow dung + earthworm + cellulose-degrading microorganism (Pseudomonas sp.) (with or without poly lining in the pit) was used for the bioconversion process.ResultsCombined inoculation of E. fetida and Pseudomonas sp. with poly lining in the pit significantly enhanced compost recovery (29.8%), cellulase activity (55.2%) and the C:N ratio (1.9%) as compared to control. Significant achievement in yield components and nutrient status in soil was observed after combined application of compost with 50% recommended dose of fertilizer.ConclusionThe current investigation established dual inoculation effect of E. fetida and Pseudomonas sp. with poly lining in the pit as a suitable technology for faster decomposition and effective bioconversion of agricultural waste into quality organic manure. A treatment imposed with such organic manure will be supportive in enhancing crop production as well as improving soil health.

A Demonstration of the Consistency of Maize Stover Pretreatment by Soaking in Aqueous Ammonia from Bench to Pilot-Scale

Soaking in aqueous ammonia (SAA) is a means of pretreating biomass at moderate temperatures and ammonia concentrations (15% w/w). To establish process consistency and scalability, sieved maize stover was pretreated at 50-ml, 300-ml, and 100-l scales. Each scale was carried out through different methods. Sealed reactor tubes were used for 50-ml pretreatment. Fabric dyeing apparatus was used for the 300-ml pretreatment and a commercial Littleford DVT reactor was used for 100-l pretreatment. For each scale, biomass washing and solid-liquid separations were scaled appropriately. Washed pretreated solids were analyzed for composition and recovery of dry biomass and carbohydrates calculated. Nearly 100% of the glucan content was recovered in pretreated solids at all three scales, indicating the viability of SAA pretreatment. Pretreated solids (15% w/w) were hydrolyzed in a 1-l twin Sigma blade mixer using Cellic CTec2 (15 FPU.g-glucan−1) followed by fermentation in shake flasks. Hydrolytic yields ranged 65–70% across scale treatments. In comparison, fermentative yields averaged 95% across scale treatments, indicating saccharification to be a rate-limiting step in effective bioconversion of lignocellulose.

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Sawdust from four types of woods (Milicia excelsa, Gmelina arborea, Afzelia africana and Khaya senegalensis) was used for cultivation of Pleurotus pulmonarius. Growth and yield of fruiting bodies were monitored and biological efficiency (BE) determined. Proximate composition of fruiting bodies was determined; total phenol content (TPC) and total flavonoid content (TFC) of the extract from the mushrooms were investigated. Results obtained show significant yield (P<0.05) of 361.30±4.55 g/kg substrate (BE = 36.13%) on G. arborea followed by K. senegalensis. The highest crude protein content (19.69±0.09%) was observed in mushrooms harvested on A. africana. Crude fibre was significantly (P<0.05) higher in mushrooms harvested from A. africana. Magnessium, sodium, potassium and calcium content of all the mushrooms were significantly different but all had significantly (P<0.05) higher potassium content (3.52±0.11 to 7.09±0.004 mg/kg). The highest TPC and TFC were recorded in mushrooms harvested from M. excelsa and the least TPC was observed for mushrooms harvested from K. senegalensis while the least TFC was recorded in G. arborea and A. africana. These findings indicate effective economic bioconversion of sawdust with varying effects on yield, nutritional composition, TPC and TFC of P. pulmonarius. G. arborea gave a better yield and can be further exploited in various capacities. Key words: Sawdust, biological efficiency, Pleurotus pulmonarius, proximate composition.

Coal methanogenesis: a review of the need of complex microbial consortia and culture conditions for the effective bioconversion of coal into methane

Microbial biodegradation of coal into low-molecular-weight compounds such as methane has been extensively researched in the last two decades because of the underlying environmental and industrial applications of this technique as compared to the chemical and physical methods of coal conversions. However, the irregular structure of coal and the need for complex microbial consortia under specific culture conditions do not make this biotransformation an ideal process for the development of anaerobic bioreactors. The most abundant species in a methanogenic culture are acetoclastic and hydrogenotrophic methanogens which utilize acetate and H2+CO2, respectively. Medium- to low-rank coals such as high-volatile bituminous, sub-bituminous and lignite are more promising in this bioconversion as compared to semi- and meta-anthracite coals. While covering the details of the ideal culture conditions, this review enlightens the need of research setups to explore the complex microbial consortia and culture conditions for maximum methane production through coal methanogenesis.

Efficient Enzymatic Digestion of Alkali Treated Maize Stover Holocellulose by Developing Balanced Cocktail of Cellulolytic and Hemicellulolytic Enzymes

Enzymatic hydrolysis of lignocellulosic biomass is a crucial step for bioethanol production. Highly balanced composition of cellulases and hemicellulases are required for effective bioconversion of lignocellulose into fermentable sugars. As commercial cellulases are often poor in certain hemicellulolytic activities, supplementation of such cellulases with accessory hemicellulolytic enzymes can yield high concentration of fermentable sugars. In the present investigation an attempt was made to develop a balanced cocktail for enzymatic hydrolysis of maize stover. Mild alkali treatment (2% NaOH) of maize stover was found to be beneficial as substrate loss was minimum and holocellulose content was unaffected. Response surface methodology was used for optimization of enzymatic saccharification process using three variables viz. level of crude β-xylosidase, level of commercial cellulases and time. Maximum 601.7 mg/g reducing sugars were obtained with 78.33% saccharification yield using optimum parameters viz 13.0 FPU/g commercial cellulase (MAPs 450), 74.42 U/g crude β-xylosidase and 36 h. Further, the role of accessory hemicellulolytic enzymes was proved by replacing crude β-xylosidase with partially purified β-xylosidase. As compared to crude β-xylosidase, supplementation of partially purified β-xylosidase at the same level, yield of reducing sugars was 536 mg/g which is only 11% lesser than crude enzyme. Moreover, developed enzyme cocktail was equally efficient at low temperature (30 °C) and at high substrate loading (30%). Enzymatic hydrolysate obtained after saccharification was efficiently fermented by ethanologenes.

A New Approach to Using Dried Hybrid Poplar as a Potential Commodity Feedstock for Sugar Production

Use of a commodity feedstock with uniform physical and chemical characteristics can maximize the efficiency of sugar production in a lignocellulosic biorefinery. However, efficient handling and storage necessitates dry biomass, while effective bioconversion is maximized with high moisture biomass. To bridge this gap, the effect of moisture content on hybrid poplar bioconversion was investigated. Fresh hybrid poplar chips were both oven- and air-dried and rehydrated by soaking in water prior to SO2-catalyzed steam explosion. Following enzymatic hydrolysis, pretreated solids derived from air-dried and oven-dried chips (10% and 0% moisture content) were 11–24% less digestible than never-dried chips at 50% moisture content. Upon soaking the same poplar chips in water prior to SO2 impregnation, pretreatment, and hydrolysis, there was a dramatic improvement in digestibility of up to 44%, thought to be due to improved SO2 uptake and subsequent hemicellulose hydrolysis in the dried chips. Monomeric glucose and xy...

Highly efficient bioconversion of methane to methanol using a novel type I Methylomonas sp. DH‐1 newly isolated from brewery waste sludge

AbstractBackgroundMethane is the major component of natural and shale gas. Methane can be converted into methanol via a bioprocess using methanotrophs, and methanol is a valuable chemical feedstock for the production of value‐added chemicals. This work demonstrates highly effective bioconversion of methane to methanol using a newly isolated novel methanotroph, Methylomonas sp. DH‐1.ResultsA novel methanotroph strain was isolated from activated sludge from a brewery plant and characterized using phylogenetic analysis, electron microscopy and chemotaxonomic analysis. This aerobic, Gram‐negative, non‐motile rod‐shaped type I methanotroph was designated as Methylomonas sp. DH‐1. The growth condition of Methylomonas sp. DH‐1 and batch methane‐to‐methanol bioconversion conditions such as methane concentration, pH, biocatalyst loading, concentration of formate and MDH inhibitor were analyzed and optimized. Methanol was produced from methane with a 1.340 g L−1 titer, a 0.332 g L−1 h−1 volumetric conversion rate and a 0.0752 g g−1 cell h−1 specific methanol conversion rate.ConclusionIt was demonstrated that isolation and application of a new methanotroph strain is a practical way of improving bioconversion efficiency in the conversion of methane to methanol. Moreover, one promising feature of Methylomonas sp. DH‐1 for methanol production was its extremely high tolerance to methanol up to 7%(v/v), which is advantageous for high‐titer methanol production. © 2016 Society of Chemical Industry

Modulating the thermostability of Endoglucanase I from Trichoderma reesei using computational approaches.

In the last decades, effective cellulose degradation became a major point of interest due to the properties of cellulose as a renewable energy source and the widespread application of cellulases (the cellulose degrading enzymes) in many industrial processes. Effective bioconversion of lignocellulosic biomass into soluble sugars for ethanol production requires use of thermostable and highly active cellulases. The library of current cellulases includes enzymes that can work at acidic and neutral pH in a wide temperature range. However, only few cellulases are reported to be thermostable. In order to alleviate this, we have performed a hybrid approach for the thermostabilization of a key cellulase, Endoglucanase I (EGI) from Trichoderma reesei. We combined in silico and in vitro experiments to modulate the thermostability of EGI. Four different predictive algorithms were used to set up a library of mutations. Three thermostabilizer mutations (Q126F, K272F, Q274V) were selected and molecular dynamics simulations at room temperature and high temperatures were performed to analyze the effect of the mutations on enzyme structure and stability. The mutations were then introduced into the endoglucanase 1 gene, using site-directed mutagenesis, and the effect of the mutations on enzyme structure and stability were determined. MD simulations supported the fact that Q126F, K272F and Q274V mutations have a thermostabilizing effect on the protein structure. Experimental studies validated that all of the mutants exhibited higher thermostability compared with native EGI albeit with a decrease in specific activity.

Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst.

BackgroundCYP106A2 from Bacillus megaterium ATCC 13368 was first identified as a regio- and stereoselective 15β-hydroxylase of 3-oxo-∆4-steroids. Recently, it was shown that besides 3-oxo-∆4-steroids, 3-hydroxy-∆5-steroids as well as di- and triterpenes can also serve as substrates for this biocatalyst. It is highly selective towards the 15β position, but the 6β, 7α/β, 9α, 11α and 15α positions have also been described as targets for hydroxylation. Based on the broad substrate spectrum and hydroxylating capacity, it is an excellent candidate for the production of human drug metabolites and drug precursors.ResultsIn this work, we demonstrate the conversion of a synthetic testosterone derivative, cyproterone acetate, by CYP106A2 under in vitro and in vivo conditions. Using a Bacillus megaterium whole-cell system overexpressing CYP106A2, sufficient amounts of product for structure elucidation by nuclear magnetic resonance spectroscopy were obtained. The product was characterized as 15β-hydroxycyproterone acetate, the main human metabolite. Since the product is of pharmaceutical interest, our aim was to intensify the process by increasing the substrate concentration and to scale-up the reaction from shake flasks to bioreactors to demonstrate an efficient, yet green and cost-effective production. Using a bench-top bioreactor and the recombinant Bacillus megaterium system, both a fermentation and a transformation process were successfully implemented. To improve the yield and product titers for future industrial application, the main bottlenecks of the reaction were addressed. Using 2-hydroxypropyl-β-cyclodextrin, an effective bioconversion of 98% was achieved using 1 mM substrate concentration, corresponding to a product formation of 0.43 g/L, at a 400 mL scale.ConclusionsHere we describe the successful scale-up of cyproterone acetate conversion from shake flasks to bioreactors, using the CYP106A2 enzyme in a whole-cell system. The substrate was converted to its main human metabolite, 15β-hydroxycyproterone acetate, a highly interesting drug candidate, due to its retained antiandrogen activity but significantly lower progestogen properties than the mother compound. Optimization of the process led to an improvement from 55% to 98% overall conversion, with a product formation of 0.43 g/L, approaching industrial process requirements and a future large-scale application.

Effective bioconversion of sophoricoside to genistein from Fructus sophorae using immobilized Aspergillus niger and Yeast.

In this study, sophoricoside from Fructus sophorae was highly bioconversed to genistein by co-immobilized Aspergillus niger and Yeast. Bioconversion conditions for genistein were optimized with single-factor experiments. The optimal conditions were as follows: microbial concentration 1.5×10(7)cells/mL, wet weight of microorganisms beads 10.0g/g material, pH 5, ratio of liquid to solid 25:1 (mL/g), temperature 32°C and time 24h. Under these conditions, a 34.45-fold increase in production of genistein was observed with a bioreactor. Moreover, the antioxidant activities of the extracts from the fermented and untreated F. sophorae were 0.287±0.11, 0.384±0.08mg/mL (IC50) and 1.84±0.13, 1.28±0.25mmol Fe(II)/g, according to the DPPH test and FRAP assay, respectively. The results indicated that the method described in the current work were valuable procedure for the production of genistein, which is of most importance for industrial scale applications as well as food industry.

Using low temperature to balance enzymatic saccharification and furan formation during SPORL pretreatment of Douglas-fir

Comparing analytical results for Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL) of Douglas-fir (Pseudotsuga menziesii) at two different temperatures shows that the apparent activation energy of sugar degradation is higher than that of hemicellulose hydrolysis, approximately 161kJ/mole versus 100kJ/mole. Thus, one can balance the production of degradation products against hemicellulose hydrolysis and therefore the enzymatic saccharification efficiency of the resultant substrate by changing pretreatment temperature and duration. Specifically, pretreatment at 165°C for 75min significantly reduced furan formation compared with the pretreatment at 180°C for 30min while maintaining the same pretreatment severity and therefore the same substrate enzymatic digestibility (SED). Obtaining high SED with Douglas-fir is also limited by lignin content. Fortunately, the bisulfite in SPORL provides delignification activity. By combining kinetic models for hemicelluloses hydrolysis, sugar degradation, and delignification, the performance of pretreatment can be optimized with respect to temperature, duration, acid, and bisulfite loading. The kinetic approach taken in this study is effective to design viable low temperature pretreatment processes for effective bioconversion of lignocelluloses.

Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose

BackgroundPlant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis.ResultsA novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay.ConclusionWe present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass.