Carboxymethylcellulose Agar Medium Research Articles

Isolation and Screening of Cellulolytic Bacteria from Yak (Bos grunniens) Excreta from Lahaul Valley Region for Pre-treatment of Paddy Straw in Himachal Pradesh

The present study was conducted during August, 2021 at Department of Agricultural Engineering, College of Agriculture, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur to isolate and identify the cellulolytic bacteria from yak dung, yak dung manure and soil samples from spiti region in Lahaul-Spiti district of Himachal Pradesh. The bacterial isolates were identified by using morphological and biochemical standard methods, and identification based on Bergey’s Manual of Determinative Bacteriology. Cellulolytic bacteria from mentioned samples were isolated and cultured on CMC (Carboxymethyl cellulose) agar medium. The activity of cellulolytic bacterial was conducted based on halo area and cellulolytic index on CMC agar medium. Among 24 isolates of bacteria, 08 isolates were identified as cellulolytic bacteria. Furthermore, our isolates with higher cellulolytic index were identified as the Bacillus and Pseudomonas genus. Out of eight efficient cellulolytic isolates were identified as Bacillus spp. (06) three from yak dung manure, two from yak dung and one from soil sample and Pseudomonas spp. (02) one from yak dung manure and one from soil sample. Most Efficient cellulose degrading isolates of Bacillus spp. and Pseudomonas spp. were employed for biological pretreatment of paddy straw for two weeks and then run in laboratory biogas digester along with control digester. These digester sets were under observation from the second day of run upto three weeks and it was observed that the there was 10% hike in biogas production as compare to control digester on 22nd days at laboratory condition.

Characterization of Cellulose-Degrading Bacteria Isolated from Soil and the Optimization of Their Culture Conditions for Cellulase Production

The characterization of bacteria with hydrolytic potential significantly contributes to the industries. Six cellulose-degrading bacteria were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus by Congo red method using carboxymethyl cellulose agar medium and identified as Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Their cellulase production was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, substrate concentration, nitrogen, and carbon sources using the dinitrosalicylic acid and response surface methods. Except for Paenarthrobacter sp. MKAL1, all strains are motile. Only Bacillus sp. MKAL6 was non-salt-tolerant and showed gelatinase activity. Sucrose enhanced higher cellulase activity of 78.87 ± 4.71 to 190.30 ± 6.42 U/mL in these strains at their optimum pH (5-6) and temperature (35-40°C). The molecular weights of these cellulases were about 25kDa. These bacterial strains could be promising biocatalysts for converting cellulose into glucose for industrial purposes.

Isolation and characterization of Rhodococcus qingshengii, a cellulolytic bacteria from Cnaphalocrocis medinalis (Lepidoptera: Crambidae) gut

Herbivorous Lepidopteran insects depend on microbial cellulases for cellulose digestion. The present study aimed to investigate the herbivorous Lepidoptera Cnaphalocrocis medinalis gut for isolation of cellulose degrading bacteria. A Gram positive rod, non-motile, mesophilic, neutrophile bacterial strain TCI11 was isolated from C. medinalis (Lepidoptera: Crambidae) 5th instar larvae gut. The isolate was found to be a potent cellulose degrader, which was isolated on carboxymethylcellulose agar medium using carboxymethylcellulose as a sole carbon source and screened by Congo red and Gram’s iodine dye staining method. The cellulose degrading strain was characterized on the basis of Gram staining, morphological and biochemical properties. Based on 16S rRNA gene analysis, the strain was found to be most closely related to Rhodococcus baikonurensis, Rhodococcus erythropolis, Rhodococcus globerulus (99.77, 99.35 and 99.06% similarity, respectively) and was identified as Rhodococcus qingshengii. R. qingshengii is being reported for the first time as a potent cellulose degrader. The source of isolation of this particular bacterial strain is also novel.

PRODUCTION OF AMYLOCELLULOLYTIC ENZYMES AND THEIR VIABILITY ON CARRIER MEDIA BY Bacillus sp. U4 AND Pseudomonas sp. U3

Amylase and cellulase are enzymes that are amyocellulolytic. Both are extracellular enzymes that can degrade organic materials, namely starch and cellulose. The purpose of this study was to determine whether Bacillus sp. U4 and Pseudomonas sp. U3 could produce amylocellulolytic enzymes and determine their viability after being incubated for 2 hours on the husk, peat, and sawdust carrier media. Screening for the presence of amylase was carried out on a selective medium, namely nutrient agar-amylum 0.5 %, and cellulase on carboxymethyl cellulose-agar medium. Both isolates were inoculated into sawdust, husk, peat carrier media and incubated for 2 hours. Viability was observed using the total plate count method. Bacillus sp. U4 and Pseudomonas sp. U3 can produce amylase and cellulase. The best isolate viability after incubation for 2 hours was found in husk carrier media with 2.13 x 105 CFU/gr of husk media.

Isolation and Identification Cellulolytic Bacteria from Termite Gut Obtained from Indralaya Peatland area

The production of second-generation bioethanol as renewable energy has developed very rapidly and has become a promising alternative energy source. Bioethanol production using biomass can be obtained alternatively from cellulose in wood, sawdust, organic waste, and agricultural waste. This research used termites obtained from Indralaya peatland area as organisms that can decompose cellulose into glucose with the cellulase enzymes produced by bacteria in their digestive tract. Cellulases are enzymes capable of hydrolyzing lignocellulose into glucose. The study aimed to isolate and identify of cellulolytic bacteria from termite gut obtained from Indralaya peatland area. The bacterial isolates were classified by using morphological and biochemical standard methods, and identification based on Bergey’s Manual of Determinative Bacteriology. Cellulolytic bacteria of termite gut were isolated and cultured on CMC (Carboxymethyl cellulose) agar medium. The activity of cellulolytic bacterial was conducted based on halo area and cellulolytic index on CMC agar medium. Among 64 isolates of bacteria, 24 isolates were identified as cellulolytic bacteria. Futhermore, our isolates with higher cellulolytic index were identified as the Staphylococcus, Microbacterium, Bacillus, and Brevibacterium genus.

Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia

Abstract. Setyaningsih PP, Ningsih F, Rachmania MK, Syafitri WA, Sari DCAF, Yabe S, Yokota A, Oetari A, Sjamsuridzal W. 2019. Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia. Biodiversitas 20: 3134-3141. This study investigated 17 thermophilic Actinobacteria isolated from the soil of geysers in the Cisolok geothermal area, West Java, as potential producers of cellulase. Screening for cellulase was performed on minimal (Mm) agar medium with and without the addition of 1% (w/v) carboxymethylcellulose (CMC) and microcrystalline cellulose (MCC), then incubated at 45, 50, 55 and 60°C for up to 7 days. Formation of clear zones around colonies indicated cellulose hydrolysis. The results showed that 15, 14, 4, and 3 isolates showed cellulolytic activity on CMC agar medium at 45, 50, 55, and 60°C, respectively, after 7 days of incubation. Three potential isolates showed cellulolytic activity on MCC agar medium after being incubated for 7 days at 45°C. Molecular identification based on the 16S rRNA gene was performed for three isolates with positive cellulolytic activity at 60°C. The results showed that the three isolates are closely related to Actinomadura keratinilytica WCC-2265T with 99.93-100% sequence similarities. A phylogenetic tree based on 16S rRNA gene sequences confirmed that the three isolates were clustered together with Actinomadura keratinilytica WCC-2265T with 100% bootstrap value. The tree also showed that cellulase producers and non-cellulase producers in Thermomonosporaceae are grouped into different clades.

Isolation, Screening and Characterization of Cellulase Producing Bacterial Isolates from Municipal Solid Wastes and Rice Straw Wastes

Cellulolytic bacteria were isolated and screened from municipal solid wastes and rice straw wastes using Carboxy Methyl Cellulose (CMC) agar medium as a selective medium. Production of clear zones by the bacterial isolates on CMC agar medium supplemented with 1% CMC was considered as indicative of extracellular cellulase activity. The size of transparent zone diameter was considered as proportional to the level of cellulase production. These bacterial isolates were identified as Bacillus sp., Pseudomonas sp. and Serratia sp. based on morphological, cultural and biochemical characteristics. A basal medium containing CMC, KH2PO4, K2HPO4, MgSO4, (NH4)2SO4, CaCl2 and FeSO4 at pH 7.0 was used for cellulase production. The assay of cellulase in term of CMCase was performed by measuring the release of reducing sugar. Different physicochemical parameters were optimized for cellulase production at shake flask fermentation. Time course study revealed that maximum level of cellulase was produced by Bacillus and Serratia isolates after 24 h of cultivation and by Pseudomonas isolates after 42 h of cultivation. Optimum level of cellulase was produced by Pseudomonas and Bacillus isolates at 37°C, and that by Serratia isolate was at 35°C. Optimum pH for cellulase production by these bacterial isolates was 7.0. Optimum temperature and pH for the activity of cellulase from these isolates were 40°C and 7.0, respectively. The cellulase from these isolates was found almost stable up to 55°C and at pH 7.0 for 1 h. The crude cellulase could liberate reducing sugar from filter paper through hydrolysis. Results showed that bacterial isolates produced significant level of cellulase with cellulose degrading capability.

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.

Microbodies and lipid bodies in the hyphal tips of Sclerotinia sclerotiorum

Hyphal tips of Sclerotinia sclerotiorum grown on a carboxymethylcellulose agar medium were studied with interference contrast, phase contrast, and dark-field optics and with the electron microscope. Microbodies with hexagonal crystalline inclusions were most abundant in a zone 80–160 μ from the hyphal apex. It is suggested that these bodies are involved in synthetic and storage functions. Lipid bodies were identified by their fluorescence with Nile blue and by their refractivity using dark field optics. They were most abundant in the first 80 μ of the hyphal tip and are thought to originate in this zone. Nuclei were randomly distributed throughout the hyphal tip cell.