Lignocellulosic Medium Research Articles

Isolation of Lignocellulolytic Bacteria From Erek-erek Geoforest Ijen Geopark, Indonesia

Ijen Geopark is one of the UNESCO Global Geoparks in Indonesia. The Erek-erek Geoforest (EEG) is one of the biosites in the Ijen Geopark. The EEG has an abundant biomass which is composed of lignocellulosic complex. The abundance of biomass has the potential as a source of lignocellulolytic microorganisms that can degrade lignocellulose material. The purpose of this research was to isolate lignocellulolytic bacteria from the Erek-erek Geoforest. This research method was soil sampling, physicochemical analysis of soil, isolation, and purification of bacteria, growth capability in lignocellulose media, and pathogenicity-test. The results showed that a total of 180 were isolated from EEG. There are 93 bacteria growth in lignin media and 142 bacteria growth in cellulolytic with the isolated code EIB (erek-erek Ijen Banyuwangi) under aerobic conditions which had various bacterial morphologies. To ensure the safety of using these isolates, it is necessary to do a pathogenicity test of these bacteria using a blood agar plate. Indicated pathogenic bacteria will form a clear zone around the bacterial colony on the blood media. The pathogenicity test of the highest lignocellulolytic bacteria showed that the 3 ligninolytic and 3 cellulolytic EIB bacteria isolates were non-pathogenic. Further, non-pathogenic bacteria from the soil litter of erek-erek forests can be tested for their potential as biological agents for the industry.

High-pressure microwave-assisted pretreatment of softwood, hardwood and non-wood biomass using different solvents in the production of cellulosic ethanol

BackgroundPretreatment is an indispensable stage of the preparation of lignocellulosic biomass with key significance for the effectiveness of hydrolysis and the efficiency of the production of cellulosic ethanol. A significant increase in the susceptibility of the raw material to further degradation can be attained as a result of effective delignification in high-pressure conditions. With this in mind, a method of high-pressure pretreatment using microwave radiation and various solvents (water, 40% w/v NaCS, 1% v/v H2SO4, 1% w/v NaOH or 60% v/v EtOH with an addition of 1% v/v H2SO4) was developed, enabling the acquisition of biomass with an increased susceptibility to the process of enzymatic hydrolysis. The medium obtained in this way can be used for the production of cellulosic ethanol via high-gravity technology (lignocellulosic media containing from 15 to 20% dry weight of biomass). For every type of biomass (pine chips, beech chips and wheat straw), a solvent was selected to be used during the pretreatment, guaranteeing the acquisition of a medium highly susceptible to the process of enzymatic hydrolysis.ResultsThe highest efficiency of the hydrolysis of biomass, amounting to 71.14 ± 0.97% (glucose concentration 109.26 ± 3.49 g/L) was achieved for wheat straw subjected to microwave-assisted pretreatment using 40% w/v NaCS. Fermentation of this medium produced ethanol concentration at the level of 53.84 ± 1.25 g/L. A slightly lower effectiveness of enzymatic hydrolysis (62.21 ± 0.62%) was achieved after high-pressure microwave-assisted pretreatment of beech chips using 1% w/v NaOH. The hydrolysate contained glucose in the concentration of 91.78 ± 1.91 g/L, and the acquired concentration of ethanol after fermentation amounted to 49.07 ± 2.06 g/L. In the case of pine chips, the most effective delignification was achieved using 60% v/v EtOH with the addition of 1% v/v H2SO4, but after enzymatic hydrolysis, the concentration of glucose in hydrolysate was lower than in the other raw materials and amounted to 39.15 ± 1.62 g/L (the concentration of ethanol after fermentation was ca. 19.67 ± 0.98 g/L). The presence of xylose and galactose was also determined in the obtained fermentation media. The highest initial concentration of these carbohydrates (21.39 ± 1.44 g/L) was observed in beech chips media after microwave-assisted pretreatment using NaOH. The use of wheat straw after pretreatment using EtOH with an addition of 1% v/v H2SO4 for the preparation of fermentation medium, results in the generation of the initial concentration of galactose and xylose at the level of 19.03 ± 0.38 g/L.ConclusionThe achieved results indicate a high effectiveness of the enzymatic hydrolysis of the biomass subjected to high-pressure microwave-assisted pretreatment. The final effect depends on the combined use of correctly selected solvents for the different sources of lignocellulosic biomass. On the basis of the achieved results, we can say that the presented method indicates a very high potential in the area of its use for the production of cellulosic ethanol involving high-gravity technology.

Effect of Fomes fomentarius Cultivation Conditions on Its Adsorption Performance for Anionic and Cationic Dyes.

Lab-cultivated mycelia of Fomes fomentarius (FF), grown on a solid lignocellulose medium (FF-SM) and a liquid glucose medium (FF-LM), and naturally grown fruiting bodies (FF-FB) were studied as biosorbents for the removal of organic dyes methylene blue and Congo red (CR). Both the chemical and microstructural differences were revealed using X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, zeta potential analysis, and scanning electron microscopy, illuminating the superiority of FF-LM and FF-SM over FF-FB in dye adsorption. The adsorption process of CR on FF-LM and FF-SM is best described by the Redlich–Peterson model with β constants close to 1, that is, approaching the monolayer Langmuir model, which reach maximum adsorption capacities of 48.8 and 13.4 mg g–1, respectively, in neutral solutions. Adsorption kinetics follow the pseudo-second-order model where chemisorption is the rate-controlling step. While the desorption efficiencies were low, adsorption performances were preserved and even enhanced under simulated dye effluent conditions. The results suggest that F. fomentarius can be considered an attractive biosorbent in industrial wastewater treatment and that its cultivation conditions can be specifically tailored to tune its cell wall composition and adsorption performance.

Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes

Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.

Literature study of production dry cellulase from Trichoderma reesei, Aspergillus niger, and Bacillus subtilis

Cellulase is an enzyme that can degrade cellulose through a catalytic process. The enzyme that works synergistically to release sugar (glucose). This review aims to determine cellulase enzymes’ activation from three different microorganisms, Aspergillus niger, Bacillus subtilis, and Trichoderma reesei. Enzymes’ activation using various kinds of lignocellulosic substrates and the effect of adding carrier agents on the freeze-drying process. The cellulase enzyme from Trichoderma reesei has an activity of 3.4 IU / mL. This enzyme is produced by pre-treatment 1% H2O2 in lignocellulosic media. Aspergillus niger has a cellulase enzyme activity of 0.229 IU / mL using a sugarcane bagasse substrate that has been pre-treated by shiitake mushrooms. The cellulase enzyme from Bacillus subtilis has an activity of 0.907 IU / mL, with the addition of CMC levels of 5% as an inducer. Cellulase enzymes in liquid form are susceptible to denaturation during storage. The production of cellulase enzymes in solid form is expected to maintain the stability of the resulting cellulase enzyme activity longer. The addition of non-reducing sugar as a carrier agent in the freeze-drying process is reported to preserve the biological activity. The addition of sucrose and trehalose, each with a concentration of 300 mM, maintained the amylase enzyme activity up to 90%. The addition of sucrose with an optimum concentration of 60 mM protects both total protein stability and good lysozyme activity. It is suspected that a carrier agent with an optimal concentration can maintain protein stability in cellulase enzyme activity in this review.

PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production.

Lignocellulosic biomass offers a sustainable source for biofuel production that does not compete with food-based cropping systems. Importantly, two critical bottlenecks prevent economic adoption: many industrially relevant microorganisms cannot ferment pentose sugars prevalent in lignocellulosic medium, leaving a significant amount of carbon unutilized. Furthermore, chemical biomass pretreatment required to release fermentable sugars generates a variety of toxins, which inhibit microbial growth and metabolism, specifically limiting pentose utilization in engineered strains. Here we dissected genetic determinants of anaerobic xylose fermentation and stress tolerance in chemically pretreated corn stover biomass, called hydrolysate. We previously revealed that loss-of-function mutations in the stress-responsive MAP kinase HOG1 and negative regulator of the RAS/Protein Kinase A (PKA) pathway, IRA2, enhances anaerobic xylose fermentation. However, these mutations likely reduce cells’ ability to tolerate the toxins present in lignocellulosic hydrolysate, making the strain especially vulnerable to it. We tested the contributions of Hog1 and PKA signaling via IRA2 or PKA negative regulatory subunit BCY1 to metabolism, growth, and stress tolerance in corn stover hydrolysate and laboratory medium with mixed sugars. We found mutations causing upregulated PKA activity increase growth rate and glucose consumption in various media but do not have a specific impact on xylose fermentation. In contrast, mutation of HOG1 specifically increased xylose usage. We hypothesized improving stress tolerance would enhance the rate of xylose consumption in hydrolysate. Surprisingly, increasing stress tolerance did not augment xylose fermentation in lignocellulosic medium in this strain background, suggesting other mechanisms besides cellular stress limit this strain’s ability for anaerobic xylose fermentation in hydrolysate.

Homogeneous solid dispersion (HSD) system for rapid and stable production of succinic acid from lignocellulosic hydrolysate.

Continuous bio-production of succinic acid was reported in homogeneous solid dispersion (HSD) system utilizing porous coconut shell activated carbon (CSAC) as immobilization carrier. The aim of the present work was to implement the HSD system to increase the area of cell immobilization and the rate of succinic-acid production from the lignocellulosic medium. The ratio of the two enzymes (cellulase-to-hemicellulase) was initially optimized to break down the lignocellulose into its free monomers, wherein the best ratio was determined as 4:1. Succinic-acid production was evaluated in the HSD system by varying the substrate loading and dilution rate. The results showed that high productivities of succinic acid were obtained when 60g/L glucose was fed over a dilution rates ranging from 0.03 to 0.4/h. The titer of succinic acid decreased gradually with higher dilution rate, whereas the residual substrate concentration increased with it. Critical dilution rate was determined to be 0.4/h at which the best productivity of succinic acid of 6.58g/Lh and its yield of 0.66g/g were achieved using oil palm fronds (OPF) hydrolysate. This work lends evidence to the use of CSAC and lignocellulosic hydrolysate to further exploit the potential economies of scale.

Assessment of the Effect of Easily-metabolised Carbon Supplements on Xylanase Production by Newly Isolated Trichoderma asperellum USM SD4 Cultivated on Oil Palm Empty Fruit Bunches

Effect of lignocellulosic medium supplemented with selected easily-metabolised carbon sources on microbial xylanase production was assessed. A newly isolated oil-palm-waste-domesticated bio-agent-producing fungus, identified based on rRNA analysis as T. asperellum USM SD4 was used as a representative organism. The potential of T. asperellum for enhanced xylanase production was evaluated by the statistical optimization of important cultural parameters via response surface methodology (RSM). T. asperellum showed optimum xylanase activity at pH 7; temperature 27 °C; moisture content 4 mL growth medium (gm): 1 gram dried substrate (gds) and inoculum size 2 x 106 spores/mL. Xylanase activity (2,337 IU/gds) attained in this study was far higher than ever reported for T. asperellum. Using the set of optimum conditions, the mixture of supplementary sugars to the lignocellulosic medium initiated xylanase repression in a concentration-independent manner. However, the degree of repression depended on the nature and type of respectively added sugar. The repressive effect exerted by monosaccharides (xylose, glucose, and fructose) was greater than exerted by either of dimeric (cellobiose and sucrose) or polymeric (xylan) sugars. Of all added substrates, xylan exerted the least repressive effect. Using xylose as a representative sugar, mechanism of xylanase repression was decisively explained and supported with experimental data.

Characterization of a novel manganese peroxidase from white-rot fungus Echinodontium taxodii 2538, and its use for the degradation of lignin-related compounds

A novel manganese peroxidase (MnP) was isolated and characterized in this study. The MnP was produced by a new selective linguini-degrading white-rot fungus Echinodontium taxodii 2538 (E. taxodii 2538) on natural lignocellulose medium of moso bamboo. The purified MnP had an estimated molecular mass of 53.4kDa, showing a single band on sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE), and composed of an amino acid sequence of GTTPSNGVVVP at N-terminal. The enzyme showed maximum activity when incubated at pH 3.5 or 55°C and could maintain a high enzymatic activity after 24h incubation under a broad range of pH (2.0–6.0) and temperature (below 45°C). The kinetic parameters revealed that the MnP had the highest affinity toward MnSO4 (Km values was 0.35μM) among all the substrates. Degradation of different types of lignin model compounds by MnP was investigated. It revealed that the MnP could oxidize both phenolic and nonphenolic lignin units. In addition, the MnP combined with laccase (Lac) from E. taxodii 2538 could degrade lignin more efficiently. In summary, this study provided a potential enzyme and a promising path for more efficient lignin modification.

Synergistic growth in bacteria depends on substrate complexity.

Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (μ). Bacterial synergism also promoted the production of β-1,4-glucosidase but not the production of cellobiohydrolase or β-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment.

Selection and molecular characterization of cellulolytic–xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites

Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass are a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences from the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120h incubation was 121.08units/L/h and 348units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83units/L/h and 359units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation.

Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules.

BackgroundTwo major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients.ResultsIn silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the encapsulated yeast, and glucose, mannose, galactose and xylose were utilised in parallel from the beginning of the cultivation.ConclusionsEncapsulation of xylose-fermenting S. cerevisiae leads to improved simultaneous and efficient utilisation of several sugars, which are utilised sequentially by suspended cells. The greatest improvement is obtained in inhibitory media. These findings show that encapsulation is a promising option for production of second-generation bioethanol.

Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity.

The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3% compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident.

Comparison of the growth of filamentous fungi and yeasts in lignocellulose-derived media

Abstract Five microorganisms of potential interest for on-site enzyme production in lignocellulosic biorefineries were evaluated with regard to inhibitor tolerance and nutrient utilization. Prehydrolysate and hydrolysate of Norway spruce were used in concentrations of 25%, 50%, 75% and 100% and compared to reference media without fermentation inhibitors. Monosaccharide sugars, oligosaccharides, and small aliphatic acids were monitored to investigate nutrient utilization in the lignocellulosic media. The microorganisms studied were the filamentous fungi Aspergillus niger and Trichoderma reesei and the three yeasts Pichia pastoris, Saccharomyces cerevisiae, and Yarrowia lipolytica. All five fungi had the ability to grow in media with 25% prehydrolysate or 25% hydrolysate. The S. cerevisiae strain had the highest inhibitor tolerance of the microorganisms studied and grew in media with 50% prehydrolysate or 75% hydrolysate. In medium with 25% prehydrolysate A. niger and Y. lipolytica gave high biomass yields of 0.46 and 0.32 g/g on initial carbon source, which corresponded to 0.46 and 0.43 g/g on consumed carbon source, respectively. The ethanol yield on consumed carbon source in 50% hydrolysate was 0.29 g/g for P. pastoris and 0.33 g/g for S. cerevisiae. The capability of A. niger to utilize a broad range of nutrients appears especially useful for enzyme production using residual streams. The high tolerance against inhibitors exhibited by S. cerevisiae could be particularly useful in a consolidated bio-process where the fermenting microorganism contributes also by producing enzymes.

Ligninolytic peroxidase gene expression by Pleurotus ostreatus: Differential regulation in lignocellulose medium and effect of temperature and pH

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.

Adaptation of Dekkera bruxellensis to lignocellulose‐based substrate

Adaptation of Dekkera bruxellensis to lignocellulose hydrolysate was investigated. Cells of D. bruxellensis were grown for 72 and 192H in batch and continuous culture, respectively (adapted cells). Cultivations in semisynthetic medium were run as controls (nonadapted cells). To test the adaptation, cells from these cultures were reinoculated in the lignocellulose medium, and growth and ethanol production characteristics were monitored. Cells adapted to lignocellulose hydrolysate had a shorter lag phase, grew faster, and produced a higher ethanol concentration as compared with nonadapted cells. A stability test showed that after cultivation in rich medium, cells partially lost the adapted phenotype but still showed faster growth and higher ethanol production as compared with nonadapted cells. Because alcohol dehydrogenase genes have been described to be involved in the adaptation to furfural in Saccharomyces cerevisiae, an analogous mechanism of adaptation to lignocelluloses hydrolysate of D. bruxellensis was hypothesized. However, gene expression analysis showed that genes homologous to S. cerevisiae ADH1 were not involved in the adaptation to lignocelluloses hydrolysate in D. bruxellensis.

Production and partial purification of cellulase from a novel fungus, Aspergillus flavus BS1

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.

Stimulatory Effect of Different Lignocellulosic Materials for Phenolic Compound Production and Antioxidant Activity from Inonotus obliquus in Submerged Fermentation

White-rot fungus Inonotus obliquus grown in submerged culture produces antioxidative phenolic compounds. In this study, addition of lignocellulosic materials into the liquid culture increased the production and antioxidant activity of extra- and intra-cellular phenolic compounds (EPC and IPC, respectively). The production of EPC and IPC was significantly enhanced by wheat straw (by 151.2 and 45.3 %), sugarcane bagasse (by 106.9 and 26.1 %), and rice straw (by 67.6 and 38.9 %). Both of the EPC and IPC extracts from the three substrates showed a higher hydroxyl and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity than those from the control medium. The highly active polyphenols such as tea catechins of epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG), and phelligridin G in the EPC extracts increased by 113.1, 75.0, and 86.3 % in the sugarcane bagasse medium. Davallialactone and inoscavin B in the EPC extracts were generated in large amounts in the lignocellulose media but not found in the control medium. The IPC extract from the wheat straw medium had the highest production of EGCG and ECG (17.6 and 18.1 mg/l). The different enhancement among the materials was attributed to the content and degradation rate of cellulose, hemicellulose, and lignin. The different antioxidant activity of the EPC and IPC extracts was related to their phenolic compositions.

Selective suppression of bacterial contaminants by process conditions during lignocellulose based yeast fermentations

BackgroundContamination of bacteria in large-scale yeast fermentations is a serious problem and a threat to the development of successful biofuel production plants. Huge research efforts have been spent in order to solve this problem, but additional ways must still be found to keep bacterial contaminants from thriving in these environments. The aim of this project was to develop process conditions that would inhibit bacterial growth while giving yeast a competitive advantage.ResultsLactic acid bacteria are usually considered to be the most common contaminants in industrial yeast fermentations. Our observations support this view but also suggest that acetic acid bacteria, although not so numerous, could be a much more problematic obstacle to overcome. Acetic acid bacteria showed a capacity to drastically reduce the viability of yeast. In addition, they consumed the previously formed ethanol. Lactic acid bacteria did not show this detrimental effect on yeast viability. It was possible to combat both types of bacteria by a combined addition of NaCl and ethanol to the wood hydrolysate medium used. As a result of NaCl + ethanol additions the amount of viable bacteria decreased and yeast viability was enhanced concomitantly with an increase in ethanol concentration. The successful result obtained via addition of NaCl and ethanol was also confirmed in a real industrial ethanol production plant with its natural inherent yeast/bacterial community.ConclusionsIt is possible to reduce the number of bacteria and offer a selective advantage to yeast by a combined addition of NaCl and ethanol when cultivated in lignocellulosic medium such as wood hydrolysate. However, for optimal results, the concentrations of NaCl + ethanol must be adjusted to suit the challenges offered by each hydrolysate.

Stress‐related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.