Cellulase-producing Bacteria Research Articles

Production of cellulase enzymes by <i>Bacillus</i> sp and <i>Pseudomonas</i> sp isolated from anthill soil

Cellulase turns the most widespread biopolymer and biologically sustainable resource, 'cellulose,' into reducing sugar. The study aimed at producing cellulase enzymes by bacteria isolated from anthill soil. Cellulase-producing bacteria were isolated from anthill soil using Carboxymethyl cellulose (CMC) medium. The isolates were screened for cellulase production by cultural, morphological, biochemical and sugar fermentation tests. Optimization of the fermentation medium for maximum cellulase production was carried out by one factor at a time (OFAT). Data obtained were analysed with the analysis of variance (ANOVA) using SPSS 2007, version 16.0. The identified Pseudomonas sp, Staphylococcus sp, E. coli and Bacillus sp were isolated with highest potential of cellulase production. The culture conditions like pH, temperature, carbon sources and nitrogen sources were optimized. The optimum conditions found for cellulase production were 40oC at pH 8.5 with maltose as carbon source and yeast extract as nitrogen source. The highest activity and stability of cellulase enzymes between neutral to alkaline pH and high temperature.

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.

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.

Microbes on the "peachy spots" of ancient Kaihua paper: microbial community and functional analysis.

Kaihua paper is a type of precious hand-made paper in China that has been used throughout Chinese history. Due to its extraordinary whiteness and fine texture, it was adopted by the imperial palace in the reign of Emperor Kang Xi and Yong Zheng of the Qing Dynasty of China in the 17th and 18th century. It is stained by a special type of yellowish-brown spot after years of storage, which is called a "peachy spot." The formation of such spots remains unclear, although complicated physicochemical processes or microbial activities might be involved. We performed nondestructive sampling and high-throughput sequencing on peachy spot surfaces, unstained areas, and air samples in the stack room to analysis the the bacterial and fungal communities, and performed prediction of functional genes of the bacterial communities. The results showed that peachy spot formation was mainly related to bacterial communities rather than fungal communities. Significantly more potential acid- producing, acidophilic or cellulase-producing bacteria, such as, Streptococcus, Staphylococcus, and Lysinibacillus, and pigment-producing bacteria, such as Methylobacterium and Rubrobacter, were identified in the peachy spot samples. Prediction of the functional genes of the bacterial community also suggested the production of acidic substance pigments. These findings provide new insights into the pigment formation mechanism in ancient paper and open an opportunity to develop new strategies to preserve the ancient paper documents.

PHENETIC DIVERSITY OF CELLULASE-PRODUCING BACTERIA FROM WANA TIRTA KULON PROGO MANGROVE FOREST

Mangrove is an ecosystem that have important value for the environment and are a source of cellulase-producing microorganism biomass. The purpose of this study was to determine the characteristics and types, as well as the highest cellulase enzyme activity from cellulase-producing bacteria found in Wana Tirta Mangrove Forest, Jangkaran, Kulon Progo. 
 This research is a descriptive-exploratory research. Sampling in the form of litter, mud and water was carried out in the Wana Tirta Kulon Mangrove Forest which was divided into 3 plots. This sample is then isolated on selective media in the form of Carboxymethyl cellulose (CMC) In order to grow cellulolytic bacteria. The bacteria obtained are then purified and phenetic characterization. The data obtained were used to classify the bacterial isolates using MVSP identification software (Multivariate Statistical Package) 3.1 with UPGMA clustering algorithm (Unweight Pair Group Method With Arithmatic Averages) then the result is presented in the form of a dendogram.
 Result Research shows as many as 17 isolates of cellulolytic bacteria were obtained. There were 4 bacterial isolates from litter samples, 1 bacterial isolate from water samples and 12 bacterial isolates from mud samples. After being made in the form of a dendogram, 19 bacterial clusters were obtained. A total of 6 bacterial isolates had a similarity index of ≥ 72% against Bacillus pumilus namely isolates S1A 2, S2A, AL 3, AP 3, AP 6 and AP 17, 4 bacterial isolates have a similarity index of ≥ 70% to Bacillus stearothermophyllus namely AP 22, AP 24, AP 25, and AP 26 isolates. As well as 7 bacterial isolates have a similarity index of ≥ 77% against Streptomyces Sp. namely isolates S1A 1, S3B, AP 8, AP 9, AP 14, AP 20 and AP 27.The highest cellulase enzyme activity occurred in AP 14 bacterial isolate of 286.72 U / ml.

Isolation, Identification and Assessment of Efficient Cellulase Producing Bacteria from the Termite Guts

The present study is concerned with the screening the cellulase-producing bacteria from termite gut, assessed potential cellulase-producing bacteria and partial characterization (optimum parameters) of cellulase from isolated bacteria. The result showed that 15 out of 48 isolated strains was positive for degrading the carboxy methyl cellulose (CMC) in agar by congo-red method. After screening by DNS assay, three selected bacteria exhibited high cellulase activity that were identified as Citrobacter amalonaticus CM 1-3, Bacillus cereus CM 5-1 and Streptococcus salivarius CE 5-1 using 16S rRNA sequence analysis. All bacterial strains utilized CMC and showed the highest cellulase activity. Cellulase characterization of C. amalonaticus CM 1-3 and S. salivarius CE 5-1 was revealed optimum activity at 35°C, pH 7.0 and for 48 h. Bacillus cereus CM 5-1 represented its potential use in industrial processes due to thermostable cellulase production. The crude cellulase of this strain was purified by (NH4)2SO4 precipitation with 1.58 purification fold and 74.38% overall recovery. The optimal temperature and pH for cellulase activity of B. cereus CM 5-1 were at 40°C and pH 7.0. Thus, this study provided additional information about the diversity and partial characteristic cellulase of cellulolytic bacteria from termite gut for future industrial applications.

Diversity and cellulolytic activity of cellulase-producing bacteria isolated from the soils of two mangrove forests in Eastern Thailand

Abstract. Bamrungpanichtavorn T, Ungwiwatkul S, Boontanom P, Chantarasiri A. 2023. Diversity and cellulolytic activity of cellulase-producing bacteria isolated from the soils of two mangrove forests in Eastern Thailand. Biodiversitas 24: 3891-3902. The Southeast Asian countries hold the largest proportion of the world's mangrove area. Mangrove forests are a potential source for the isolation of economic microbial enzymes. Cellulases are a widely used microbial enzyme for cellulose degradation in various industries. Therefore, this study aimed to isolate, genetically identify, and enzymatically characterize cellulase-producing bacteria from the soils of two mangrove forests in Eastern Thailand. Twenty-six cellulase-producing bacteria were isolated and subsequently genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the 16S rRNA genes. Thirteen different RFLP patterns were obtained and genetically analyzed into six bacterial genera comprising Aeromonas, Bacillus, Chryseobacterium, Lysinibacillus, Pseudomonas, and Vibrio. The Bacillus species were the predominant cellulase-producing bacteria in the study sites. Moreover, the cellulase-producing Chryseobacterium and Lysinibacillus had hardly ever been reported. The Bacillus sp. strain RY08B was the most active cellulase-producing bacterium with 1.510±0.060 U/mL of CMCase activity. The optimum temperature and pH for the CMCase activity were determined to be 50°C at a pH of 7.0 with a thermal stability range of 25-50°C at a pH of 7.0. This bacterium could be applied in several environmentally friendly industries requiring mild conditions for their production processes.

Isolation, characterization and identification of cellulase (Endo-β-1,4-glucanase) producing bacteria from diverse locations


 The objective of the study was to isolate cellulase producing bacteria from natural sources and identify the bacterial isolates that produce thermostable enzyme. Bacteria were isolated from samples collected from different locations like mango leaf litter, termite gut, composting yard (municipal solid waste, compost, and soil), soil from decaying paper waste, decaying wood, and saline coastal belt soil by enrichment in broth containing 1% cellulose and subsequent culturing on plates and the purified cultures stored in slants of the solidified agar medium of the same composition as enrichment medium. Primary screening of the isolates for cellulase (Endo β-1,4-glucanase) production was done by growing them on Carboxy Methyl Cellulose (CMC) agar medium and screening using indicator dye Congo red. Secondary screening of the positive isolates showing cellulolytic Index > 4 was done by crude cellulase enzyme extraction and assay using 1% CMC in 1N citrate buffer (pH 5.0) at different pH values (4.8- 7.0) and temperatures (35- 70℃). Cellulase activity (EA) in U/mL was calculated based on the absorbance change per min. which depends on the amount of reducing sugar produced by the enzyme catalysed conversion of CMC to reducing sugar. Isolates were characterized morphologically and biochemically and six isolates (Gram -ve: T1, MSW, C1, S2 and Gram +ve: W2, P) having significantly higher EA were selected for DNA extraction. DNA was quantified and the DNA of four isolates (MSW, S2, W2, P) were selected for sequence analysis. The 16S rDNA sequencing was carried out through PCR amplification and Blast using NCBI blast similarity search tool resulted in the identification of MSW, S2, W2 as E.coli, Sulfitobacter pontiacus, Bacillus subtilis respectively and P as Bacillus cereus and Rhodococcus erythropolis strain e1. Isolates showed significantly higher cellulase activity (EA) at pH 6.2 and at varying temperatures. Bacterial isolates P and C2 showed significantly higher activity at 40℃, MSW at 60℃ and others at 50℃ (EA ranging from 0.0054 – 0.0604 U/mL). Isolates W2, P, S2 had EA ranging from 0.006 - 0.03 U/mL at 70℃. This study showed that the cellulase producers could be grown at different temperatures (40 - 70℃) on cellulose based substrate. In this study the substrate specificity indicates that the crude enzyme is an endo- β-1,4-glucanase that plays a prominent role in initiating and sustaining the hydrolytic process and randomly cleaves cellulose into glucose and olygomeric polysaccharides. Hence by selecting samples with cellulosic substance from different locations with extreme environmental conditions like high temperature, high salinity it is possible to isolate cellulolytic bacteria producing cellulase enzymes that are stable at higher processing temperatures and other harsh conditions which is of importance in industrial applications.
 Keywords: Endo-β-1,4-glucanase, Cellulolytic index, Cellulase activity, Thermostable

Gut Microbiota of Freshwater Gastropod (Bellamya aeruginosa) Assist the Adaptation of Host to Toxic Cyanobacterial Stress

Gut microbes play a critical role in helping hosts adapt to external environmental changes and are becoming an important phenotype for evaluating the response of aquatic animals to environmental stresses. However, few studies have reported the role that gut microbes play after the exposure of gastropods to bloom-forming cyanobacteria and toxins. In this study, we investigated the response pattern and potential role of intestinal flora in freshwater gastropod Bellamya aeruginosa when exposed to toxic and non-toxic strains of Microcystis aeruginosa, respectively. Results showed that the composition of the intestinal flora of the toxin-producing cyanobacteria group (T group) changed significantly over time. The concentration of microcystins (MCs) in hepatopancreas tissue decreased from 2.41 ± 0.12 on day 7 to 1.43 ± 0.10 μg·g-1 dry weight on day 14 in the T group. The abundance of cellulase-producing bacteria (Acinetobacter) was significantly higher in the non-toxic cyanobacteria group (NT group) than that in the T group on day 14, whereas the relative abundance of MC-degrading bacteria (Pseudomonas and Ralstonia) was significantly higher in the T group than that in the NT group on day 14. In addition, the co-occurrence networks in the T group were more complex than that in the NT group at day 7 and day 14. Some genera identified as key nodes, such as Acinetobacter, Pseudomonas, and Ralstonia, showed different patterns of variation in the co-occurrence network. Network nodes clustered to Acinetobacter increased in the NT group from day 7 to day 14, whereas the interactions between Pseudomonas and Ralstonia and other bacteria almost changed from positive correlations in the D7T group to negative correlations in the D14T group. These results suggested that these bacteria not only have the ability to improve host resistance to toxic cyanobacterial stress by themselves, but they can also further assist host adaptation to environmental stress by regulating the interaction patterns within the community. This study provides useful information for understanding the role of freshwater gastropod gut flora in response to toxic cyanobacteria and reveals the underlying tolerance mechanisms of B. aeruginosa to toxic cyanobacteria.

Isolation, screening and optimization of cellulase production by a novel bacterial isolate Enterococcus durans

Cellulose is an abundant plant biomass and a renewable source of energy in the ecosphere. The breakdown of cellulose occurs via the cellulase enzyme, which is commonly produced by microbes. This study aimed to optimize the fermentation parameters for enhanced cellulase production. Standardized parameters include isolation and screening of cellulase-producing bacteria (CPB), production of an enzyme, biochemical and molecular identification of bacterial isolate, optimization of cultural parameters, and application in wash performance. A total of 581 bacterial strains were isolated from soil samples, of which 16 isolates formed zones of hydrolysis on carboxymethylcellulose (CMC) agar media and were categorized as CPB. Based on maximum hydrolysis zone formation, three isolates, Krishi Vigyan Kendra-5 (KVK-5), Greenhouse-4 (GA-4), and Medicinal Garden-5 (MG-5) were chosen for bacterial cellulase production (BCP), with the isolate MG-5 proving to be the best cellulase producer (1.75 ± 0.01 U ml-1). Based on 16S rRNA gene sequencing the isolate MG-5 was identified as Enterococcus durans. The optimized parameters for the production of the cellulolytic enzyme were an incubation period of 48 h, CMC (carbon source), and yeast extract (nitrogen source) at a concentration of 1.5% w/v, pH 7, 45 °C, 1.5% v/v inoculum size and 100 rpm. Optimum conditions resulted in a 1.92-fold increase (3.36 U ml-1) in cellulase activity. Cellulase enzyme when used with detergent (Surf Excel), resulted in more efficient removal of chocolate stains on cotton fabric. This is the first report of Enterococcus durans producing cellulolytic enzymes. The analysis of cellulase in stain removal provides valuable evidence regarding the application of this enzyme in laundry cleaning.

Screening of bioflocculant and cellulase-producing bacteria strains for biofloc culture systems with fiber-rich carbon source.

The biofloc technology (BFT) system has been widely applied in the shrimp and fish culture industry for its advantages in water-saving, growth improvement, and water quality purification. However, The BFT system usually takes a long time to establish, and the extra carbon source input increases the maintenance cost of the system. In this study, we aimed to develop a low-cost and high-efficient BFT system for Litopenaeus vannamei by applying bacteria that could promote the formation of BFT and utilize cheap carbon sources. Three bioflocculant-producing bacteria strains (M13, M15, and M17) have been screened from a cellulolytic strain collection. All three strains have been identified as Bacillus spp. and can use sugarcane bagasse (SB) as a carbon source, which is a cheap byproduct of the sucrose industry in the tropic area of China. Compared to sucrose, the addition of SB and the three strains could improve the biofloc formation rate, biofloc size distribution, ammonia removal rate, and the growth performance of the shrimps. These results suggest that the bioflocculant and cellulase-producing bacteria strains could promote the biofloc formation and the growth of shrimps by using SB as an economic substitute carbon source in the BFT shrimp culture system.

Cross-habitat distribution pattern of Bacillus communities and their capacities of producing industrial hydrolytic enzymes in Paracel Islands: Habitat-dependent differential contributions of the environment

Bacillus as a predominant genus of enzyme-producing bacteria presents desirable features to fulfill the vast demand of specific industries, whereas the knowledge of the Bacillus communities and their capacities of producing industrial hydrolytic enzymes across the microhabitats of the Paracel Islands is limited. Herein, a total of 193 culturable Bacillus strains belonging to 19 species were isolated across the microhabitats of seawater, sediment, coral and seagrass, covering 39 stations of the Paracel Islands. Each microhabitat displayed its unique species, while the species of Bacillus paramycoides besides being the dominant species with an abundance of 54.94% also was the only species shared by all microhabitats of the Paracel Islands. Of the Bacillus communities, 97.41% of the isolates exhibited the capacity of producing one-or-more types of enzymes with comparatively higher and broader ranges of enzyme activities, including 163 protease-, 27 cellulase-, 118 alginate lyase-, 140 K-carrageenase- and 158 agarose-producing strains. By the correlation analyses of “Bacillus-environmental factors” and “Enzyme-producing Bacillus-environmental factors”, the cross-habitat distribution and enzyme-producing capacity pattern of the Bacillus communities were strongly driven by habitat type, and the environmental factors made habitat-dependent differential contributions to that in the Paracel Islands. It's worth noting that the cellulase-producing strain wasn't detected in seagrass due to its survival strategy to prevent cellulose degradation by inhibiting cellulase-producing bacteria, while coral contained more stable microbial metabolic functions to protect against environmental fluctuations. These findings besides providing large quantities of promising enzyme-producing candidates for specific industrial desires, also facilitate the development and utilization of marine microbial resources and the environmental policy- and/or law-making according to environmental features across the microhabitats of the Paracel Islands.

Antibiotic Treatment Reduced the Gut Microbiota Diversity, Prolonged the Larval Development Period and Lessened Adult Fecundity of Grapholita molesta (Lepidoptera: Tortricidae).

Simple SummaryThe gut microbiota, which has an important influence on insect physiology and ecology, can be affected by many factors, such as antibiotics, temperature, diet, and species. However, herein we demonstrate the effects of antibiotics on the gut microbiota and on Grapholita molesta development and fecundity. Five antibiotics, ciprofloxacin, streptomycin, chloramphenicol, ampicillin, and rifampicin, were screened by the drug-sensitive disc method. Then, the effects of antibiotics on oriental fruit moth growth, reproduction, and gut microbiota structure were investigated by comparison of biological parameters and 16S amplicon sequencing technology. The results illustrated ciprofloxacin, showing the largest inhibition zone diameter, to be the most suitable antibiotic to inhibit the gut microbiota of G. molesta a minimum inhibitory concentration of 1 μg/mL. After feeding ciprofloxacin, the relative abundance of Actinobacteria and Cyanobacteria significantly decreased, while that of Bacteroidetes and Firmicutes increased. Finally, ciprofloxacin feeding affected larval growth, development, and reproduction significantly, resulting in prolonged larval development duration, shortened adult longevity, and significantly decreased single female oviposition and egg hatchability. In addition, we isolated and purified some culturable bacteria belonging to Proteobacteria, Firmicutes, Actinobacteria, and cellulase-producing bacteria from the G. molesta midgut.Grapholita molesta, the oriental fruit moth, is a serious pest of fruit trees with host transfer characteristics worldwide. The gut microbiota, which plays a crucial part in insect physiology and ecology, can be influenced by many elements, such as antibiotics, temperature, diet, and species. However, the effects of antibiotics on G. molesta gut microbiota are still unclear. In this study, we selected five common antibiotic agents to test the inhibition of G. molesta gut microbiota, and found ciprofloxacin shown the best antibacterial activity. After feeding 1 μg/mL of ciprofloxacin, the relative abundance of Actinobacteria and Cyanobacteria decreased significantly, while that of Firmicutes and Bacteroidetes increased. PICRUSt2 analysis indicated that most functional prediction categories were enriched in the G. molesta gut, including amino acid transport and metabolism, translation, ribosomal structure and biogenesis, carbohydrate transport and metabolism, transcription, cell wall/membrane/envelope biogenesis, and energy production and conversion. Finally, ciprofloxacin feeding significantly affected larval growth, development, and reproduction, resulting in prolonged larval development duration, shortened adult longevity, and significantly decreased single female oviposition and egg hatchability. In addition, we isolated and purified some culturable bacteria belonging to Proteobacteria, Firmicutes, Actinobacteria, and cellulase-producing bacteria from the G. molesta midgut. In brief, our results demonstrate that antibiotics can have an impact on G. molesta gut bacterial communities, which is beneficial for host growth and development, as well as helping female adults produce more fertile eggs. These results will thus provide a theoretical reference for developing new green control technology for G. molesta.

Molecular characterization of cellulase genes in Pseudomonas stutzeri

BackgroundCellulose is one of the most abundant natural sources of carbon. In biofuel manufacturing, cellulase is used as an enzyme to hydrolyze cellulose into a fermentable product (glucose). Pseudomonas stutzeri is one of the microorganisms found in cattle rumen. The microbiome of the rumen is heterogeneous and known for its potentiality to efficiently hydrolyze cellulose. Recent studies have identified, cloned, and crystallized one of the cellulase genes present in P. stutzeri, the A1501 cellulase gene (PST_2494 gene). ResultsThis study describes the isolation of cellulase-producing bacteria from sheep's rumen. The highest cellulase-producing isolate was identified as P. stutzeri by 16s rDNA sequencing. qRT-PCR was used to measure the cellulase gene expression levels, revealing a higher gene expression of the PST_1459 gene (4 folds) compared to PST_2494 genes. Moreover, cellulase productivity was enhanced by UV irradiation mutagenesis. ConclusionsSheep's rumen bacterial isolates were tested for their cellulase productivity, and the highest was identified as P. stutzeri. An investigation of the cellulase genes of P. stutzeri revealed the presence of an unidentified cellulase gene (PST_1459). A qRT-PCR reaction was carried out to validate and measure the expression levels of different cellulase genes, revealing a higher gene expression of the PST_1459 gene than PST_2494 genes. Moreover, UV irradiation mutagenesis was performed to enhance cellulase productivity. The gene expression tested by qRT-PCR confirmed the enhancement of cellulase productivity in some of the mutants obtained.How to cite: Al Makishah NH, Elfarash AE. Molecular characterization of cellulase genes in Pseudomonas stutzeri. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.004.

Screening and Determination of Potential Cellulolytic Bacteria from Mangrove Ecosystem

The mangrove ecosystem is a coastal wetland resource that has a diversity of biodiversities such as flora, fauna, and microorganisms. Microbes are an important component in the mangrove environment not only play a role in creating and maintaining boosters but also work as a source of biotechnology products. Cellulolytic bacteria are a group of cellulase-producing bacteria capable of breaking down cellulose into glucose monomers, and cellulose as a source of carbon and energy. This study aims to screen and determine the diversity of cellulolytic bacteria from the mangrove ecosystem. The research method was experimentally by exploring the diversity of cellulolytic bacteria from soil and sediment from the mangrove ecosystem. Three sampling locations were selected, such as litter soil, soil in the root area, and soil in the tidal area. Screening of potential bacteria producing cellulase enzymes was carried out by growing bacteria on Carboxy Methyl Cellulose (CMC) agar medium which was cultured for 48 hours at 37°C. The bacteria that produced the greatest cellulolytic activity were characterized by their morphological and physiological characteristics. The results of bacterial characterization were then adjusted according to Bergey's Manual of Determinative Bacteriology. Based on the analysis showed three bacterial isolates were obtained that had the largest cellulolytic activity index, namely MS06 (9.73), MS08 (5.41), and MS02 (5.07). The results showed that three isolates had the same characteristics as the bacterial genera Bacillus, Cellulomonas, and Micrococcus.

Harnessing the potential use of cellulolytic Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG) isolated from the guts of termites (Isoptera)

BackgroundFor many years, denim-heavy quality cotton twill colored with indigo colors and with a well-worn/faded look has held a lot of appeal. Machine damage, drainage system blockage, and other issues come with the conventional usage of pumice stones for “stone-washing” denims. In view of the abovementioned information, a range of works has been done to investigate the economic prospects of bacterial cellulase enzymes for use in industrial processes, including biopolishing in the textile sector. Ethiopia has excellent termite diversity to isolate bacterial gut-associated cellulose enzymes for biostoning applications. The main purpose of this study was, therfore, to decipher how to isolate and characterize cellulase enzymes from termite (Isoptera) gut bacteria with the intention of employing it for biostoning of textiles.PurposeTo use cellulolytic enzymes of Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG) isolated from termite guts in biostoning of textiles and improving garment quality.MethodsCellulase enzyme-producing bacteria were isolated and screened from the guts of worker termites sampled from Meki and Zeway termite mounds in the Central Rift Valley region of Ethiopia. Bacterial screening, biochemical, morphological, and 16S rRNA sequence identification techniques were employed to characterize the bacterial strains. In addition, the production, optimization, and purification of the associated cellulase enzymes were employed, and the potential application of the enzymes for biostoning of a textile was demonstrated.ResultThe isolated M21WG was found to be 99% identical to the Klebsiella oxytoca (MT104573.1) strain, while the isolated Z6WG showed 97.3% identity to the Klebsiella sp. strain (MN629242.1). At an ideal pH of 7, a temperature of 37 °C, a 72-h incubation time, and a substrate concentration of 1.5% carboxymethylcellulose sodium, the maximum activity of the crude cellulase extract from these bacteria was assessed. These bacteria produced cellulase enzymes that were moderately efficient. Consequently, it was determined that the cellulase enzymes were effective for biostoning of denim cloth.ConclusionIt was determined that Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG) could be used as a doorway to better understand harnessing the use of these cellulase-producing bacteria from termite (Isoptera) guts. In this study, it was also attempted to assess the effectiveness of the two bacterial isolates in biostoning in anticipation of their potential application in the textile realm.

Screening of cellulase-producing bacteria and their effect on the chemical composition and aroma quality improvement of cigar wrapper leaves

A high yield cellulase production strain was screened from the surface of cigar wrapper leaves and added to cigar wrapper leaves for fermentation in the present study. The enzyme-producing strain was identified as Bacillus velezensis, and its maximum carboxymethyl cellulase activity could reach 44.3 U/mL. Making the cellulose degradation rate of cigar wrapper leaves as the index, the fermentation temperature, inoculum concentration, and carbon and nitrogen source addition concentration were optimized by response surface methodology. The results showed that under the conditions of inoculation concentration of 20%, fermentation temperature of 37 °C, addition of 4.5 ‰ glucose and 2.25 ‰ glutamate, the highest degradation rate of tobacco cellulose was 31.7%. Significance analysis results showed that the order of factors affecting the degradation rate of cigar wrapper cellulose was: inoculation concentration > carbon and nitrogen source addition concentration > temperature. The analysis of aroma substances under the optimal conditions found that the total amount of aroma substances of cigar wrapper increased by 26.1% compared with that before fermentation, in which β-damascenone, megastigmatrienone, farnesyl acetone, and solanone were significantly improved. The results were conducive to improving the aroma quality and concentration of cigar wrapper leaves.

Screening and identification of cellulose-degrading bacteria from soil and leaves at Kerman province, Iran.

Cellulosic biomass is considered one of the most promising sources for the production of alternative renewable bioenergy and other valuable products. Identification and optimization of strains with high enzymatic activity that can overcome constraints imposed by the cellulosic structure is an essential step in the development of new biotechnologies. The aim of this study was to isolate and identify thermophilic (50°C) and mesophilic (37°C) cellulolytic bacteria from soil and leaves samples at Kerman, Iran. Degrader bacteria were isolated using serial dilution and pour plate method. Media contained carboxymethylcellulose (CMC), and filter paper was used as sources of carbon. Totally 22 mesophilic and 17 thermophilic bacterial strains which produced clear zones were further identified by morphological and biochemical tests. Screening of purified bacteria was performed to identify cellulase-producing bacteria by Congo red test. These bacteria were compared to each other based on cellulase activity, the percentage of growth, and extracellular protein amounts. The strains with the highest enzymatic activity were determined by the DNS method. The isolated US5 and US7 grew rapidly, and produced cellulase. The US5 created the largest clear zones (7mm). Besides, these strains were selected for analysis of 16S rRNA sequence. The results showed that selected bacteria strains belong to Brevibacillus borstelensis. The B. borstelensis strains isolated in this study showed a suitable cellulase enzyme activity.

Screening and identification of thermophilic cellulolytic bacteria isolated from sawdust compost

Composting process mainly depends on the metabolic pathways of the microorganism and involves the activity of different enzymes. Thermophilic cellulase-producing bacteria isolated from sawdust compost were tested for formation of a visible zone around the colonies on the agar plates medium containing carboxymethyl cellulose at 50ºC. Screening of carboxymethyl cellulase producing isolates was further realized on the basis in liquid medium by DNS method. Among 29 isolates investigated, V1 and V11 strains exhibited maximum enzyme activity of 1.9 and 2.3 U/mL, respectively. These isolates were selected for morphological, physiological and biochemical studies and 16S rRNA gene analysis. They were found a Gram-positive, rod-shaped spore forming cells, which were identified as Bacillus megaterium (V1) and Bacillus subtilis (V11) based on cell morphology, nucleotide homology and phylogenetic analysis. The optimal temperature for activity of endoglucanases (CMCase) ranged from 35–45°C (strain V1) and 40– 50ºC (strain V11). Our findings showed that Bacillus megaterium (V1) and Bacillus subtilis (V11) cellulase demonstrate thermophilic characteristics within wide range of temperature and meets the requirements for commercial enzymes.

Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia

Abstract. Fitri L, Bessania MA, Septi N, Suhartono S. 2021. Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia. Biodiversitas 22: 5169-5180. Cellulolytic actinobacteria are cellulase-producing bacteria capable of degrading cellulose. This study aimed to isolate, characterize, evaluate the cellulolytic ability, and to determine physiological characterization of soil cellulolytic actinobacteria isolated from the Ujung Pancu area, Aceh Besar. Isolation of actinobacteria from soil samples was performed using serial dilution method on Yeast Malt Agar (YMA) medium. Morphological characterization was carried out by growing isolates on YMA, Oatmeal Agar (OA), and Yeast Starch Agar (YSA) media. Cellulolytic ability was determined by calculating the cellulolytic index (IS) on 1% carboxymethyl cellulose (CMC) medium after adding 0.1% congo red solution. Physiological characterization of cellulolytic actinobacteria tested in this study was salinity, pH, and carbon source in liquid Yeast Malt (liquid YM), and the growth was measured at a wavelength of 581nm. The results showed that a total of nine isolates of actinobacteria were isolated, which belonged to the genus Streptomyces. Cellulolytic test results showed that eight isolates had the ability to degrade cellulose. Isolates AUP-04, AUP-03, and AUP-01 had the highest cellulolytic index value. Physiological characterization results revealed that three isolates had different tolerances for salinity levels, pH, and types of carbon sources. AUP-03 isolate grew well at 10% salinity with an OD value of 0.88, isolate AUP-01 grew at 5% salinity with an OD value of 0.49, whereas isolate AUP-04 grew well on media that did not contain salinity. All three isolates grew well at pH 6 with OD values of 0.93, 1.12, and 1.27. AUP-03 and AUP-01 isolates grew well on media containing dextrose as carbon source with OD values of 0.154 and 0.17, respectively, while isolate AUP-04 grew well on glucose-containing media with an OD value of 0.22.