Production of Endoglucanases by Streptomyces thermocoprophilus CP1 using Rice Straw as a Substrate

Rice straw is a major agricultural waste that can be used as an alternative substrate to expensive raw materials for endoglucanases (CMCase) production by microorganisms. This study aimed to search for a microorganism having the potential to produce endoglucanase from rice straw. From compost samples, 40 bacterial colonies were isolated on carboxymethylcellulose (CMC) agar. Among them, 16 isolates showed a hydrolysis zone on a CMC agar plate with hydrolysis (HC) values ranging from 1.15±0.02 to 4.40±0.52. Based on hydrolysis zone diameter and HC value, isolates CP1, CP2 and CP3 were further examined for their CMCase production in CMC broth. According to CMCase production and stability, isolate CP1 was selected for further study. The optimal pH and temperature for CMCase production of isolate CP1 were 5 and 45 °C, respectively. When using pre-treated rice straw as a substrate for semi-solid-state fermentation, the highest CMCase activity of 0.142 ± 0.008 U/mL was obtained in a medium containing pre-treated rice straw of 60 g/L. The sequence alignment analysis and phylogenetic analysis suggested that the isolate CP1 was likely to be Streptomyces thermocoprophilus. The microorganism obtained from this study may be not only industrially important but also beneficial to the environment.

Diversity and cellulolytic activity of culturable bacteria isolated from the gut of higher termites (Odontotermes sp.) in eastern Thailand

Boontanom P, Chantarasiri A. 2021. Diversity and cellulolytic activity of culturable bacteria isolated from the gut of higher termites (Odontotermes sp.) in eastern Thailand. Biodiversitas 22: 3349-3357. Cellulolytic bacteria are vital symbionts associated with the gut of all higher termites. Odontotermes termites are a higher termite widely found in Thailand. However, information concerning the diversity of cellulolytic bacteria in this termite gut remains inadequate. The aim of this study is to isolate and identify the culturable cellulolytic bacteria from the Odontotermes gut collected from eastern Thailand. The crude cellulases produced from the most active cellulolytic bacterium were further characterized. Thirty-two cellulolytic bacteria were isolated and subsequently classified by PCR-RFLP of the 16S rRNA gene. A total of 10 different RFLP patterns were obtained belonging to five bacterial genera, namely Acinetobacter, Bacillus, Citrobacter, Paenibacillus, and Serratia. The B. cereus strain TWV503 was considered to be the most active cellulolytic bacterium based on the CMC agar method. B. cereus strain TWV503 showed CMCase activity at 2.190 ± 0.063 U/mL of CMCase and 0.276 ± 0.031 U/mL of FPase. The optimum temperature and pH for CMCase activity were 50°C and the neutral pH ranging from 7.0 to 8.0, respectively. CMCase activity remained stable at up to 70°C and neutral pH ranging from 7.0 to 8.0 for 24 hours of incubation. This study revealed novel information related to cellulolytic bacteria isolated from the gut of Odontotermes termites collected from Thailand.

Screening Cellulolytic Fungi Isolated From Malaysia Cocoa Pod Husk and Its Culture Conditions for Cellulases Production

The aim of this study was to screen few fungal isolates from local cocoa pod husks (CPH) which able to secrete cellulases. The isolates were plated on carboxymethyl cellulose (CMC) agar plates which then incubated for two days at 28ºC. Then, these plates were stained with congo red dye for 0.5-1 h followed by destaining with 1 M NaCl solution for 15-20 minutes to observe its cellulolytic activity. One isolates which exposed the largest cellulolytic zone on CMC agar plate was selected for further study. In this study, culture conditions with respect to pH, incubation time, amount of substrate (CPH) and temperature were screened using Design expert @version 8.0 by employing two-level factorial design. The selected fungus isolate was cultured in shake flask at 37°C with agitation of 200 rpm for 5 days in incubator shaker. During fermentation period, samples were collected every day for fungal-cellulases activity of filter paper activity (FPase) and carboxymethyl cellulase (CMCase) activity. Analysis of variance (ANOVA) of this study showed that the most significant parameters that affects the production of cellulases from the selected fungi isolates were the amount of substrate (CPH) used followed by the interaction of amount of substrate with pH (p< 0.05). It showed that the cellulases activity was high when the pH 9 with more amount of substrate used. However, it was observed that less significant changes of celllulases activity when different amount of substrate was used at same pH of 3. Based on the microscopic observation of isolate, it morphology was closed to Rhizopus sp.. In conclusion, it is suggested to optimize the selected culture parameters obtained in this study in order to maximize the activity of cellulases from the selected isolates.

Screening and characterization of cellulolytic molds from empty fruit bunches and soils in palm oil plantation area in Indonesia

Objective This research was aimed to isolate cellulolytic molds in empty fruit bunches of oil palm (EFBOP) and soils from palm oil plantation area and identify their enzyme activities to digest EFBOP. Results A total of seven molds were successfully isolated and screened for their enzyme activities from EFBOP and the soils. The enzymes from each isolate were produced in submerged culture using Mineral Mandels and 3% of alkali pretreated pollard in triplicates. The results indicated that all of the isolates were able to hydrolyze Carboxymethyl Cellulose (CMC), Whatmann No. 1 filter paper, and also EFBOP to sugars with reducing ends that reacted to 3,5-Dinitrosalicylic acid (DNS). The CMCase activity of isolate X showed the highest while the lowest was found for isolate MT8. Filter paperase (FPase) activity of isolate X performed the highest wile the lowest were found from isolate MT3 and MT6. The saccharification activity of isolate P showed the highest while MT6 performed the lowest activity

Diversity and Activity of Aquatic Cellulolytic Bacteria Isolated from Sedimentary Water in the Littoral Zone of Tonle Sap Lake, Cambodia

Tonle Sap Lake is the largest freshwater lake in Southeast Asia, and it is regarded as one of the most biodiverse freshwater ecosystems in the world. Studies concerning aquatic cellulolytic bacteria from Tonle Sap Lake remain scarce. Cellulolytic bacteria and their cellulases play a vital role in the biogeochemical cycles of lake environments, and their application in biotechnological industries is likewise an important component of their usage. This study aimed to assess the isolation, genetic identification, bioinformatic analyses, and activity characterization of aquatic cellulolytic bacteria. The cellulolytic bacteria isolated from sedimentary water samples in the littoral zone of the lake belong to the genera Aeromonas, Bacillus, and Exiguobacterium. Several isolated aquatic bacteria were designated as rare cellulolytic microbes. Remarkably, B. mojavensis strain REP303 was initially evidenced by the aquatic cellulolytic bacterium in freshwater lake ecosystems. It was considered a highly active cellulolytic bacterium capable of creating a complete cellulase system involving endoglucanase, exoglucanase, and β-glucosidase. The encoded endoglucanase belongs to the glycosyl hydrolase family 5 (GH5), with a carboxymethylcellulase (CMCase) activity of 3.97 ± 0.05 U/mL. The optimum temperature and pH for CMCase activity were determined to be 50 °C at a pH of 7.0, with a stability range of 25–55 °C at a neutral pH of 7.0–8.0. The CMCase activity was enhanced significantly by Mn2+ and was inhibited considerably by EDTA and ethyl-acetate. In conclusion, this study is the first to report data concerning aquatic cellulolytic bacteria isolated from the littoral zone of Tonle Sap Lake. A novel strain of isolated cellulolytic B. mojavensis could be applied in various cellulose-based industries.

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C and xylanase was reduced in 53% after two hours incubation at the same temperature. CMCase and xylanase obtained by A. niger KIJH cultivated in submerged culture containing J. corumbensis as carbon source may have application in biotechnology processes that require enzymes that remain active under routine extreme conditions.

Isolation, Biochemical Characterisation and Identification of Thermotolerant and Cellulolytic Paenibacillus lactis and Bacillus licheniformis

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that, the polymer molecule will be degraded to smaller particles, used as a carbon source by microorganisms. Because of the frequently applied methods of pre-treatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C. We described their morphological features and biochemical characteristics based on CMCase activity and the api®ZYM. The growth curves, during incubation at 50 °C, were examined using the microbioreactor BioLector®. Results and conclusions. 40 bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganism. The CMCase activity was confirmed for 27 strains. However, the hydrolysis capacities (HC) were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was presented, among others, by BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. Novelty and scientific contribution. We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P.s lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of the circular bioeconomy.

Identification of Locally Isolated Cellulolytic Stenotrophomonas maltophilia from Rice Straw and Optimization of its Cellulase Activity

A highly cellulolytic bacterium was locally isolated from rice straw and identified as Stenotrophomonas maltophilia. Identification of the isolate based on the morphological, cultural and biochemical characteristics was confirmed with 16S rDNA analysis. The bacterium showed the highest level of reducing sugar and extracellular protein production when incubated for 3 days (348.75 μg/ml and 288.5 μg/ml respectively) at 40°C temperature (463.0 μg/ ml and 333.0 μg/ml respectively) and pH 6.5 (360.0 μg/ml and 349.0 μg/ml respectively) in Winstead’s broth having 1.5% CMC and 0.2% Yeast Extract as carbon and nitrogen source respectively. Crude cellulase enzymes produced by the bacterium showed the highest CMCase activity rather than FPase, Avicelase and ²-Glucosidase activities. Cellulase activity of the crude enzyme was also determined using the same parameters. The crude cellulase enzyme showed the highest CMCase activity when incubated for 60 minutes (232.5 U/ml), at pH 6.5 (105.0 U/ml), 35°C temperature (69.75 U/ml) using CMC and Peptone as carbon and nitrogen source respectively. Crude cellulase showed the highest activity in presence of mercury and SDS as metal and detergent respectively. Substrate specificity and SDS-PAGE analysis reveals that the cellulase may be an endo-1,4-glucanase. Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 15-22

Protective effects of five surfactants on cellulase in the saccharification of corn stover based on the impeded Michaelis-Menten model

Protective effects of five surfactants were investigated relative to the saccharification of lignocellulose using the impeded Michaelis-Menten model (IMM). The yield of total reducing sugar (Ytrs) and cellulase activity were indexed as the effect of surfactant. The IMM was used to fit the correlation between Ytrs and reaction time to obtain the index (Kobs,0) reflecting the accessibility between cellulose and lignocellulose and the comprehensive index (Ki) reflecting cellulase inactivation and non-specific site adsorption. Results showed that the strongest protective effect was found from polyoxyethylene (80) sorbitan monooleate, followed by rhamnolipid. The surfactants protected cellulase from inactivation and nonspecific site adsorption of lignocellulose in the saccharification, leading to enhanced cellulase activity, especially with respect to carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase) activities. The maximum Ytrs was obtained when the CMCase activity was 136.2 U/mL, while the FPase and β-glucosidase activities should be as high and low as possible, respectively, under the optimized condition. These findings lay the foundation for improving the saccharification efficiency of cellulase and reducing the cost of saccharification of biomass cellulose.

Synergy between recombinant endoglucanase a and exoglucanase s secreted by bacillus subtilis WB800N

An approach for dertemining the synergistic activity of cellulosomal catalytic units in culture media is among essential steps in the research of artificial cellulosomes. Endoglucanase A (CelA) and exoglucanase S (CelS) are two abundant cellulosomal cellulases secreted by anaerobic thermophilic bacterium Clostridium thermocellum and mostly responsible for the cellulolytic activities. They are the well-known candidates of catalytic units for artificial mini-cellulosome. Their synergistic activities play important roles in cellulolytic degradation. CelA cleaves inside the cellulose chains and produces more chain ends for the next step controlling by CelS. While endoglucanase demonstrates distinct activity on carboxymethyl cellulose, it still lacks the specific substrate for quantifying exoglucanase activity. In this research, we introduced an approach for measuring the synergistic activity for these endo and exoglucanase. We designed two recombinant proteins CelA and CelS secreted by the host-cell Bacillus subtilis WB800N in order to investigate their synergistic activity in culture media. The secretory expression was confirmed by tandem mass spectrometry. A modified dinitrosalicylic acid assay was performed on 96 well-plate for quantifying cellulolytic activities of the secreted cellulases in culture media. When adding CelA into CelS, CMCase activities were enhanced and higher than the total of their individual CMCase activities at some cases. When mixing 3 of CelA with 5 of CelS, the CMCase activity was enhanced about 35.5% of the total activities from individual ones. This indicated the synergistic activity of the endo and exoglucanase could degrade cellulose more efficiently than their individual activities. The research also provides the essential materials and methods for further research on designing mini-cellulosome secreted by B. subtilis.