Cellulase Production Research Articles

Investigation of the presence and enzymatic activity of fungi in stored corn (Zea mays L.) and soybean [Glycine max (L.) Merrill] grains

Soy [Glycine max (L.) Merrill] and corn (Zea mays L.) are important crops in Brazilian agribusiness. When stored improperly, these grains are susceptible to insects and microorganisms. Among the microorganisms capable of causing grain deterioration, fungi stand out. Despite the harm associated with fungi, they are biotechnologically appreciable due to their potential ability to produce hydrolytic enzymes, such as cellulases and proteases. Fungal hydrolytic enzymes have been applied in various processes in the food, textile, and detergent production industries, among others. The objective of this study was to verify the presence of fungi in samples of corn and soybean grains under storage conditions and characterize them regarding the production of cellulolytic and proteolytic enzymes. Among the results, there was a greater quantity and variety of filamentous fungi (35 morphotypes) in association with the grains evaluated, than of yeasts (three morphotypes). Representatives of the genera Aspergillus spp., Curvularia sp., and Penicillium spp. were detected, corroborating previous reports in the literature. Filamentous fungi that tested positive for cellulase production had an enzyme ratio index (ERI) varying between 1.06 and 2.57 (mean ± standard deviation = 1.37 ± 0.48), while for proteolytic activity, only the fungi isolated from soybean tested positive, with ERI (mean ± standard deviation) = 1.21 ± 0.01. Although the taxonomy of the isolates in this study has not been completely elucidated, it is possible to state that these fungi are promising for producing hydrolytic enzymes, with potential for use in the development of products and/or biotechnological processes.

Isolation and characterization of cellulase producing bacteria from forest, cow dung, Dashen brewery and agro-industrial waste.

The continuous accumulation of waste, particularly from industries, often ends up in landfills. However, this waste can be transformed into a valuable resource through innovative methods. This process not only reduces environmental pollution but also generates additional useful products. This study aims to screen novel high-efficiency cellulose-degrading bacteria from cow dung, forest soil, brewery waste, and agro-industrial waste in the Debre Berhan area for the treatment of cellulose-rich agricultural waste. The serial dilution and pour plate method was used to screen for cellulolytic bacteria and further characterized using morphological and biochemical methods. From eleven isolates cow dung 1 (CD1), cow dung 6 (CD6) and cow dung (CD3) which produced the largest cellulolytic index (3.1, 2.9 and 2.87) were selected. Samples from forest soil, and spent grain didn't form a zone of clearance, and effluent treatment and industrial waste (IW9) shows the smallest cellulolytic index. Three potential isolates were then tested for cellulolytic activity, with cow dung 1 (CD1) displaying promising cellulase activity. These bacterial isolates were then identified as Bacillus species, which were isolated from cow dung 1 (CD1) with maximum cellulase production. Cow dung waste is a rich source of cellulase-producing bacteria, which can be valuable and innovative enzymes for converting lignocellulosic waste.

Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.

Effect of medium supplementations and extraction conditions on cellulase production through solid state fermentation of oil palm empty fruit bunches

In this study, cellulase enzyme was produced through solid state fermentation (SSF), employing oil palm empty fruit bunches (EFB) as the primary substrate. Two key aspects were explored to enhance crude enzyme yield, which are medium supplementation effects during SSF and cellulase recovery. During medium supplementation, glucose and/or Tween 80 were added alongside EFB substrate and other nutrients. Enzyme yield was determined using a filter paper assay and expressed as enzyme activity. The initial addition of glucose during fermentation led to increased crude enzyme activity, as measured by the filter paper assay. The peak crude enzyme activity was observed with the addition of 3 mg of glucose, with higher amounts showing no further increase in activity. Conversely, the addition of Tween 80 did not yield any significant increase in crude enzyme activity across all concentrations tested. The extraction conditions were varied to study cellulase recovery, specifically by adjusting the solid‐to‐solvent ratio and the number of extraction stages. Higher enzyme activity was achieved with lower solid‐to‐liquid ratios, as the increased solvent volume facilitated greater enzyme extraction. However, increasing the number of extraction steps did not significantly affect the resulting cellulase activity. Overall, this research underscores the need for further process optimization for cellulase production via SSF, utilizing the widely available EFB in Indonesia.

Isolation and molecular characterization of cellulase-producing bacteria from waste dump site

Cellulases are collections of extracellular enzymes and a complex mixture of enzyme proteins with various specificities. Cellulases work together to hydrolyze glycosidic bonds and generate monomers of glucose for fermentation. This investigation aims to isolate and molecularly characterize Bacillus species with cellulolytic ability. Bacillus species were isolated from soil samples retrieved from a dump site and screened for cellulolytic ability on carboxyl methylcellulose (CMC) agar. The percentage hydrolysis efficiency of isolates was determined and cellulase produced was quantified using CMC assay method. Biochemical identification was by Analytical Profile Index (API) Kit 50CHB/20E and API web software while molecular characterization employed 16S rRNA gene sequencing and blast search analysis. Bacillus megaterium (FSP1) and Bacillus zanthoxyli (FSP4) exhibited their cellulolytic potentials by presenting zones of clearance of about 21 ± 2.08 and 7 ± 1.00 mm on CMCA with hydrolysis efficiency of 250 and 600 % respectively. Quantification of crude cellulase revealed cellulase activity of 85 and 74μmol/ml for both bacteria species. Biochemically, the cellulolytic bacteria were identified as Bacillus megaterium (FSP1) and Bacillus zanthoxyli (FSP4) while molecularly, they were identified as Bacillus megaterium 14581 (FSP1) and Bacillus zanthoxyli 1433 (FSP4) with Reference Sequence (RefSeq) accession numbers NR_116873 and NR_164882, and showing maximum sequence similarity of 99 and 96 % respectively. Results obtained from this investigation, suggests that both bacteria species characterised, possesses good cellulolytic ability and hence can be utilized for the production of the enzyme cellulase which has a wide range of industrial application.

Lignocellulolytic Enzymes Production by Four Wild Filamentous Fungi for Olive Stones Valorization: Comparing Three Fermentation Regimens.

Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of β-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 μg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

Expression of cellobiose dehydrogenase gene in Aspergillus niger C112 and its effect on lignocellulose degrading enzymes.

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, β-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency.

Insights of Pichia kudriavzevii SVMS2019 for cellulase production and fermentation into ethanol

The use of lignocellulosic biomass to produce value-added products, such as fuel ethanol, is a current priority for global biobased economy. The quest to explore novel microbes, such as yeasts, to attain sustainable products for an ever-growing population has become highly in demand. This study might be the first to explore the rumen yeast Pichia kudriavzevii SVMS2019 for cellulase production to efficiently utilize wheat straw as biomass, which can further be converted to bioethanol. The optimization process was carried out on pre-treated wheat straw by employing one factor at a time (OFAT) and response surface methodology (RSM). Wheat straw pre-treated using NaOH alkali pretreatment was characterized with FTIR, XRD, and SEM studies. The findings revealed that the P. kudriavzevii SVMS2019 obtained maximum cellulase activity of 44.064 IU/ml at 37.5 °C, pH 3.5, 120 h, and 1.5% of NaOH pre-treated wheat straw (substrate concentration) through RSM. It exhibited 1.25-fold increased production compared to OFAT. The stability of crude cellulase was observed at 45 °C and pH 6. Further, the hydrolyzed reducing sugars produced under optimal conditions opted for fermentation, with a maximum bioethanol content of 12.13 g/l obtained at 37.5 °C in a 24 h interval.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity

BackgroundLignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.ResultsThe current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.ConclusionThe results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Studies on Cellulolytic Fungal Flora from Forests of Maharashtra, India

The present investigation was conducted to isolate effective cellulolytic fungal flora from soil samples collected from various forests in Maharashtra (India). The study aimed to determine hydrolytic zones and relative enzyme activity for selecting most efficient fungal isolates. Further, the isolates were identified using morphological identification technique. Altogether 137 fungal isolates were obtained in the present study, collected from forests of Lonavala, Chandrapur, Mahabaleshwar, Gadchiroli and Bhandardara. The screening of these fungal isolates for cellulase production revealed that 41 isolates had varying degrees of cellulolytic activity. However, isolate C77 produced the largest clearance zone (4.8 cm) on carboxymethyl cellulose (CMC) medium. The relative enzyme activity, expressed as the cellulolytic index, ranged from 1.1 to 3.5 among the 41 fungal isolates. Notably, C65 displayed the highest cellulolytic index, indicating superior enzyme activity, while other isolates like G5X, B24 and M145 also exhibited significant cellulolytic activity. From the present investigation it is concluded that Aspergillus terreus Thom, Trichoderma spp. Aff. T. harzianum Rifai, Aspergillus fumigatus, and Aspergillus spp. Aff. A. flavus Link isolated from the forests of Chandrapur, Gadchiroli, Bhandardara and Mahabaleshwar, respectively, were the most efficient cellulose degrading fungi.

Enhancement of cellulase production by cellulolytic bacteria SB125 in submerged fermentation medium and biochemical characterization of the enzyme

Microbial diversity from indigenous cultures has the potential to accelerate lignocellulose degradation through enzymes and make composting economically feasible. Therefore, this study is designed to boost cellulase output from a bacterial strain obtained from soil using a one-variable-at-a-time approach and response surface methodology. The bacteria recognized as Bacillus tequilensis (ON754229) produced the maximum cellulase at a temperature of 37 °C, pH -7.0, and incubation time of 72 h. A major contribution was anticipated by glucose (17 %) and ammonium sulfate (11 %) with cellulase activity of 0.56 U/mL in the optimized medium. The enzyme possessed activity of CMCase, FPase, and amylase of 0.589 μmol/min, 1.22 μmol/min, and 0.92 μmol/min respectively. SDS-PAGE showed a 65 kDa molecular weight of the enzyme capable of degrading cellulose, as confirmed by zymogram analysis. The enzyme showed relatively moderate thermo-stability towards neutral pH conditions possessing optimum conditions at pH 6.5 and temperature of 50 °C. The Km and Vmax values were 11.44 mM and 0.643 μmol/min respectively. The presence of MgSO4, ZnSO4, and Triton X- 100 increased the enzymatic reaction however AgNO3, EDTA, and HgCl2 altered the activation process. These results showed cellulase from B. tequilensis SB125 would be suitable for conventional industrial processes that convert biomass into biofuels.

Use of an inexpensive carbon source for the production of a cellulase enzyme complex from Penicillium ucsense S1M29 and enzymatic hydrolysis optimization

AbstractThe high cost of cellulolytic enzyme complexes (CECs) has been a significant impediment to the commercial production of bioproducts from lignocellulose biomass. This study aimed to develop a cost‐effective CEC derived from Penicillium ucsense (former Penicillium echinulatum), utilizing diverse forms of pretreated sugarcane bagasse as the primary carbon/inductor source. Among the different pretreatments used, the hydrothermal pretreatment followed by NaOH delignification (BHD) produced higher FPase and xylanase activities (4.5 FPU mL–1 and 120 IU mL–1) in bioreactor experiments at 20 g BHD L–1 initial concentration. A batch‐mode assay conducted across a range of initial carbon source (5 to 60 g L–1) confirmed the highest FPase activity (4.0 to 5.0 FPU mL–1 at 120 h), in the range of 20–40 g BHD L–1. During these assays the agitation rate, controlled by dissolved O2, tended to stabilize at lower levels, indicating substrate limitation. Conversely, higher initial carbon source concentrations led to an excess of glucose, likely triggering carbon catabolite repression and inhibiting cellulase production. This insight prompted the development of a controlled pulsed fed‐batch strategy, resulting in FPase activity of 11 FPU mL–1 at 220 h using 90 g L–1 BHD controlled fed into the bioreactor. An enzymatic hydrolysis procedure using the generated CEC was also optimized using a central composite rotational design (CCRD). The optimized enzyme hydrolysis conditions achieved a reducing sugar concentration of 80.9 g L–1 in 48 h using 170 g L–1 of BHD as the substrate at a ratio of 15 FPU of enzyme substrate per g of BHD. A preliminary economic assessment demonstrated that, for a first‐ and second‐generation (1G + 2G) ethanol biorefinery, the cost contribution of enzymes would be about US$0.2/L of biofuel. In conclusion, an efficient and highly productive procedure was developed successfully for the production of a CEC. It was particularly effective for the enzymatic hydrolysis of pretreated sugarcane bagasse.

An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.

The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA-sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA-sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. The tRNA-gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum

BackgroundLow-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain.ResultsWe began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum.ConclusionsOur study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

Culture of Trichoderma sp. with biochar to produce high-activity cellulase in a laboratory

Biochar (BC) was used in Trichoderma sp. culture to produce high-activity cellulase on a laboratory scale. The biochar was added into the flask before being applied to the fermenter to identify the enhancement effect and to determine the best amount of addition and the most suitable incubation period. Cellulase production was performed with a working volume of 4 L, and enzymatic hydrolysis was conducted to evaluate the saccharification ability of the enzyme. During incubation, the activities of three enzymes (Endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH)) were measured for three days, and the cellulase activity was determined using a filter paper unit (FPU). In flask scale, EG, BGL, and CBH activities were increased by 1.4, 2.1, and 1.8 folds, respectively, and the incubation period was shortened by adding BC. In the fermenter scale, EG, BGL, and CBH activities were noticeably enhanced by 12.1, 5.8, and 7.2 folds, respectively, and FPU was 42.1 (9.8 folds). Additionally, the conversion rates of cellulose and steam exploded softwood and hardwood were 109.4%, 75.4%, and 87.3%, which were similar to a commercial enzyme (Cellic CTecⅡ). This study demonstrated that biochar could be used to produce high-activity cellulase in a shorter period and suggests a novel method for effective cellulase production.

Extraction of Cellulases Produced through Solid-State Fermentation by Trichoderma reesei CCT-2768 Using Green Coconut Fibers Pretreated by Steam Explosion Combined with Alkali

The industrial processing of coconut to produce valuable foods, such as water and milk, generates large volumes of waste, especially the fruit shell. Despite this, material can be used in bioprocess applications, e.g., the production of enzymes, its recalcitrance hinders the cultivation of microorganisms, and low productivity is usually achieved. In this study, the production of cellulolytic enzymes through solid-state fermentation (SSF) and their extraction was investigated using the green coconut fiber pretreated by steam explosion, followed by alkali. The fungus Trichoderma reesei CCT-2768 was cultivated, using an experimental design, to study the effect of the water activity and the amount of biomass in the reactor. The combination of the pretreatment strategies yielded more porous biomass, with less hemicellulose (5.38%, compared to 10.15% of the raw biomass) and more cellulose (47.77% and 33.96% in the pretreated and raw biomasses, respectively). The water activity significantly affected the production of cellulases, with maximum activity yielded at the highest investigated value (0.995). Lastly, the extraction of the enzymes from the cultivation medium was studied, and a 9 g/L NaCl solution recovered the highest CMCase and FPase activities (5.19 and 1.19 U/g, respectively). This study provides an important contribution to the valorization of the coconut residue through (i) the application of the steam explosion technology to optimize the production of cellulases using the SSF technology and (ii) their extraction using different solvents.

Isolation, screening and optimization of alkaliphilic cellulolytic fungi for production of cellulase

Abstract This study concerns with the production and partial characterization of alkaline cellulase from alkaliphilic cellulolytic (AC) fungi isolated fromsoil in Perlis, Malaysia. The best fungi strain was selected on the basis of producing the highest cellulase at high pH conditions. Cellulase from the selected fungi strain was further characterized under saccharification but varies in operating parameters. Finally, the kinetic model describing the growth of the AC fungi strain was studied by employing the logistic model. Among the tested fungi strains, Basidiomycetes strain (BK1) showed high potentiality for the production of maximum alkaline cellulase production at pH 9 after 72 h of incubation at 30°C containing 6 g·L−1 carboxyl methyl cellulose. The saccharification process showed that the enzyme favour high alkaline condition and proves thermotolerant properties, while 15% (v/v) enzyme loading and 1% substrate concentration recorded the highest glucose production at about 1.2–1.3 mg·mL−1. The novelty of the study is to identify and optimize a unique indigenous fungi that emit alkaliphilic cellulase as alternative usage in biotechnology industries due to its capacity to adapt to the extreme conditions of specific industrial processes. There are revolutionary options for use in biotechnological businesses that involve high pH and therefore have substantial biotechnological promise.

Application of Trametes pubescens for dye removal and biological pretreatment of sugarcane bagasse

AbstractSugarcane bagasse (SB) is an abundantly available agrowaste that is used extensively as a substrate for the production of different industrial enzymes. It is a renewable resource, rich in fermentable sugars. However, the presence of lignin makes it difficult for microbes to degrade it and so it is difficult to utilize it. Keeping in mind the role of white rot fungi in lignin degradation, Trametes pubescens MB89 was used in this study for the delignification of SB with the simultaneous production of laccase. The fungus was also used for the degradation of malachite green dye. A Box–Behnken design (BBD) was used to optimize variables affecting solid state fermentation of SB for laccase production. The leftover SB after fermentation was used for cellulase production by the thermophilic bacterium Neobacillus sedimentimangrovi UE25. The BBD depicted optimized conditions as 60% moisture, particle size 100 μ, 10 days’ incubation period, 450 μM CuSO4, and a temperature of 30 °C. The laccase titers of 1271 IU mL−1 were obtained under optimized conditions. Neobacillus sedimentimangrovi UE25 produced 4.574 IU mL−1 of endoglucanase and 1.812 IU mL−1 of β‐glucosidase by utilizing fungal pretreated SB. Scanning electron microscopy and Fourier transform infrared spectroscopy confirmed the impact of fungal cultivation on SB. Pores were observed after microbial pretreatment and there were changes in the morphological characteristics such as the pith and fiber of SB. To test the ability of laccase to break down aromatic dyes, we grew the fungus T. pubescens MB89 in a nutrient‐rich medium supplemented with malachite green dye. The fungus was able to remove 73.68% of the malachite green dye from the medium in 24 h. This shows that T. pubescens MB89 has a range of potential applications for environmental and industrial purposes.

Screening and characterization of thermostable enzyme-producing bacteria from selected hot springs of Ethiopia.

Thermostable microbial enzymes play an important role in industries due to their stability under harsh environmental conditions, including extreme temperatures. Despite their huge application in different industries, however, the thermostable enzymes of thermophilic microorganism origin have not yet been fully explored in Ethiopia. Here, we explored thermophilic bacteria and their enzymes from selected hot spring water and sediment samples. Accordingly, thermophilic bacteria were isolated and screened for the production of extracellular hydrolytic enzymes. Promising numbers of isolates were found as producers of the enzymes. The potent enzyme producers were further identified using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry analysis and 16S rRNA gene sequencing. The findings revealed the presence of potential hydrolytic enzyme-producing thermophilic bacteria in hot springs of Ethiopia and necessitate further comprehensive study involving other extreme environments. Our findings also revealed the potential of Ethiopian hot springs in the production of thermostable enzymes of significant application in different industries, including food industries.

Characterization and Identification of Thermophilic Bacteria Isolated from Different Sites Located in Al-Jouf Region, Saudi Arabia

This study aims to investigate the morphological and biochemical characteristics of thermophilic bacteria isolated from thermophilic biotopes in the Al-Jouf region of Saudi Arabia for the first time. Additionally, the production of thermostable bacterial enzymes (proteases, amylases, cellulases, gelatinases, and lipases) was investigated. Six main bacterial species were identified via 16S rRNA sequencing and phylogenetic analysis. Twenty water and soil samples were collected from several thermophilic sites in the Dumat Al-Jandal and Al-Qurayyat cities. In total, 28 bacterial strains were isolated and biochemically characterized. Most isolated strains showed high protease and amylase production. 46 and 28% of isolated strains showed the production of cellulase and gelatinase, respectively. However, lipase production was not observed in any isolated bacteria. Four Bacillus licheniformis and two Bacillus subtilis strains exhibited high GC content (55%). Our findings suggest thermophilic bacteria as a source of thermostable enzymes for pharmaceutical and industrial applications.