Disposal of waste sludges produced in large amounts in the pulp and paper industry imposes significant environmental and economical problems. One strategy to address these issues involves revalorization of paper mill sludges by their application as substrates for microbial production of biotechnologically relevant enzymes. The application of lignocellulolytic enzymes in paper, textile and bioenergy industries is encouraged in order to decrease chemicals and energy consumptions. In the following work, deinking sludge was assessed as a substrate for production of lignocellulases. Based on the results of growth and activity screenings, Pleurotus ostreatus PLAB was chosen as the most promising candidate among 30 tested strains and its secretome was further studied by quantitative enzyme assays and mass spectrometry. While endoglucanase and xylanase activities detected in P. ostreatus secretome produced on deinking sludge were similar to activities of cultures grown on other lignocellulosic substrates, average laccase activity was significantly higher (46?000 U/kg DIS). Mass spectrometry identification of the most prominent proteins in the secretome of the target strain confirmed that significant amounts of different lignin-modifying oxidases were produced on this substrate despite its low lignin content, indicating the presence of other inducible compounds. The findings of this study suggest deinking sludge may represent a good substrate for fungal production of the aforementioned enzymes with broad biotechnological applications, including bioremediation, paper and bioenergy industries.
Bioethanol and biobutanol hold great promise as alternative biofuels, especially for transport sector, because they can be produced from lignocellulosic agro-industrial residues. From techno-economic point of view, the bioprocess for biofuels production should involve minimal processing steps. Consolidated bioprocessing (CBP), which combines various processing steps such as pretreatment, hydrolysis and fermentation in a single bioreactor, could be of great relevance for the production of bioethanol and biobutanol or solvents (acetone, butanol, ethanol), employing clostridia. For CBP, Clostridium holds best promise because it possesses multi-enzyme system involving cellulosome and xylanosome, which comprise several enzymes such as cellulases and xylanases. The aim of this article was to review the recent developments on enzyme systems of clostridia, especially xylanase and cellulase with an effort to analyse the information available on molecular approaches for the improvement of strains with ultimate aim to improve the efficiencies of hydrolysis and fermentation.
Response surface methodology (RSM) was used to optimize the enzymatic hydrolysis of corn stover (CS), an abundant agricultural residue in the USA. A five-level, three-variable central composite design (CCD) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids loadings (6.6–23.4%), and enzyme loadings (6.6?23.4 FPU g?1 DM) on glucose yield from thermo-mechanically extruded CS. The extruded CS was first hydrolyzed with the crude cellulase of Penicillium pinophilum ATCC 200401 and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance (ANOVA) showed a coefficient of determination (R2 ) of 0.82. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU g?1 DM, the enzymatic hydrolysis of pretreated CS produced a glucose yield of 57.6% of the glucose maximum yield which was an increase of 10.4% over the non-optimized controls at zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges.
Prairie cordgrass (PCG), Spartina pectinata, is considered an energy crop with potential for bioethanol production in North America. The focus of this study was to optimize enzymatic hydrolysis of PCG at higher solids loadings using a thermostable cellulase of a mutant Penicillium pinophilum ATCC 200401. A three variable, five-level central composite design of response surface methodology (RSM) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids loadings (6.6%–23.4%), and enzyme loadings (6.6–23.4 FPU/g dry matter, DM) on glucose yield from a thermo-mechanically extruded PCG. The extruded PCG was first hydrolyzed with the crude P. pinophilum cellulase and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance showed a coefficient of determination (R2) of 0.89. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU/g DM, the enzymatic hydrolysis of pretreated PCG produced a glucose yield of 76.1% from the maximum yield which represents an increase of 15% over the non-optimized controls at the zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges. Furthermore, statistical optimization of solids and enzyme loadings of enzymatic hydrolysis of biomass may have important implications for reduced capital and operating costs of ethanol production.
A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15–20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45–0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.
Switchgrass (Panicum virgatum), a perennial grass native to North America, is a promising energy crop for bioethanol production. The aim of this study was to optimize the enzymatic saccharification of thermo-mechanically pretreated switchgrass using a thermostable cellulase from Geobacillus sp. in a three-level, four-variable central composite design of response surface methodology. Different combinations of solids loadings (5 to 20%), enzyme loadings (5 to 20 FPU g-1 DM), temperature (50 to 70 oC), and time (36 to 96 h) were investigated in a total of 30 experiments to model glucose release from switchgrass. All four factors had a significant impact on the cellulose conversion yields with a high coefficient of determination of 0.96. The use of higher solids loadings (20%) and temperatures (70 oC) during enzymatic hydrolysis proved beneficial for the significant reduction of hydrolysis times (2.67-times) and enzyme loadings (4-times), with important implications for reduced capital and operating costs of ethanol production. At 20% solids, the increase of temperature of enzymatic hydrolysis from 50 oC to 70 oC increased glucose concentrations by 34%. The attained maximum glucose concentration of 23.52 g L-1 translates into a glucose recovery efficiency of 46% from the theoretical yield. Following red yeast fermentation, a maximum ethanol concentration of 11 g L-1 was obtained, accounting for a high glucose to ethanol fermentation efficiency of 92%. The overall conversion efficiency of switchgrass to ethanol was 42%.
A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600 C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600 C and 700 C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.
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).
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.
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.
Bacillus sp. was cultured in solid-state fermentation (SSF) of wheat straw to produce cellulase. The fermented biomass was harvested after 36 h of SSF at pH 8 and temperature 400C. It was filtered and centrifuged at 10,000 rpm at 4 0C and supernatant was collected as crude enzyme extract. Maximum activity of cellulase (3.775±0.13U/ml) was obtained after fermentation of wheat straw (10g) medium containing 0.2g soybean meal, 0.04g corn steep liquor (CSL), 80% moisture content (mineral salt medium, pH 8), 2-mL inoculum, and temperature 40 0C. SSF was found to be more productive than submerged fermentation (SmF) in terms of cellulase yields. The partial purification of cellulase was carried out through (NH4)2SO4 precipitation. The partially purified enzyme produced under SSF had molecular weight of 35 and 45kDa. It was active in a broad pH (4-10) and temperature range (25-550C). The optimum, pH and temperature of Bacillus cellulase were pH 5 and 450C, respectively. At 500C and 600C, the half lives of the partially purified cellulase were 194 and 163 min, respectively. All the results indicated that the Bacillus sp. had a promising application of treatment of agro-wastes and cellulase from Bacillus sp. could be potentially used in biofuel industries.
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.
Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and ?-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.