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Environmental Microbiology (EM) | Microbial Ecology and Diversity
Microbiol. Biotechnol. Lett. 2022; 50(2): 245-254
Hira Ikram1, Hamid Ali Khan2*, Hina Ali1*, Yanhui Liu3, Jawairia Kiran1, Amin Ullah5, Yaseen Ahmad1, Sadia Sardar1, and Alia Gul4
1Institute of Biological Sciences, Sarhad University of Science and Information Technology, Peshawar 25000, Pakistan
2Director Office of Reseaerch, Innovation and Commercialization, Sarhad University of Sciecnce and IT, Peshawar 25000, Pakistan
3Center for Genomics and Biotechnology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, P.R. China
4Department of Botany Hazara University, Mansehra, Mansehra 21300, Pakistan
5Department of Health and Biological Sciences, Abasyn University Peshawar, KPK 25000, Pakistan
Correspondence to :
Hina ali, email@example.com
Cellulases are a group of biocatalyst enzymes that are capable of degrading cellulosic biomass present in the natural environment and produced by a large number of microorganisms, including bacteria and fungi, etc. In the current study, we isolated, screened and characterized cellulase-producing bacteria from soil. Three cellulose-degrading species were isolated based on clear zone using Congo red stain on carboxymethyl cellulose (CMC) agar plates. These bacterial isolates, named as HB2, HS5 and HS9, were subsequently characterized by morphological and biochemical tests as well as 16S rRNA gene sequencing. Based on 16S rRNA analysis, the bacterial isolates were identified as Bacillus cerus, Bacillus subtilis and Bacillus stratosphericus. Moreover, for maximum cellulase production, different growth parameters were optimized. Maximum optical density for growth was also noted at pH 7.0 for 48 h for all three isolates. Optical density was high for all three isolates using meat extract as a nitrogen source for 48 h. The pH profile of all three strains was quite similar but the maximum enzyme activity was observed at pH 7.0. Maximum cellulase production by all three bacterial isolates was noted when using lactose as a carbon rather than nitrogen and peptone. Further studies are needed for identification of new isolates in this region having maximum cellulolytic activity. Our findings indicate that this enzyme has various potential industrial applications.
Keywords: Cellulose, cellulase, enzyme assay, carboxymethyl cellulose
Most of the biomass present on earth is in the form of cellulose which is major constituent of cell wall and accounts 50% for dry weight . In plant biomass, Cellulose accounts for 40 billion tons per year, which makes it the most abundant and primary product . Plant biomass is also present in less amount in hemicelluloses and least of all lignin . Microorganisms that are found in nature like bacteria and fungi, both could degrade different types of cellulose which can be soluble (amorphous) and insoluble (crystalline) by the action of cellulases and hemicelluloses . In plant biomass, structural polysaccharides can be degraded by combined effects of enzymes such as cellulases, hemicellulases, and glycosyl hydrolases. Cellulase play important role in the breakdown of complex structure into simple monomeric form which are industrially applicable . Cellulose found in the cell wall of plant is commonly degraded by the combined action of a multi-component enzymatic system of cellulase which play main role in bioconversion of cellulosic material . Many bacterial strains have been discovered from soil that has ability to degrade cellulosic biomass. The hydrolysis of cellulosic biomass is performed by synergistic action of three main enzymes. These enzymes are cellobiohydrolase or exoglucanase, endoglucanase or carboxymethyl cellulase and cellobiase or b-glucosidase .
Bacteria can be used to degrade cellulose into simple sugars because they can adapt to environmental conditions and biochemical changes such as
Cellulolytic bacteria were isolated from different soil samples collected aseptically near Khazana Sugar Mills, Peshawar. The Soil samples were collected below the sawdust and Bagasse surface at approximately 10 cm depth with the help of sterile spatula followed by thoroughly mixing in the bag. Isolation of cellulolytic bacteria was done by serial dilution technique up to 10-6. Samples were spread on CMC Nutrient agar supplemented with Congo red. Clear zones appeared indicating cellulose hydrolysis. The positive isolates were subsequently sub-cultured. These isolates were further identified through morphological and biochemical tests.
The selected bacterial isolate that showed the highest cellulase activity was identified based on the morphological i.e., Colony morphology and zone of clearance of cellulase enzyme, followed by biochemical parameters i.e., gram staining, Eosin methylene blue, Mackonkey, Blood agar, MSA media, Coagulase test, Urease test, Indole, Citrate Utilization Test, Catalase test and Oxidase test .
Molecular identification based on 16S RNA was carried out for the positive bacterial isolates to confirm their identity. Pure culture of positive bacterial isolates was sent to Macrogen Korea Sequencing Company for DNA extraction and subsequent 16S rRNA gene sequencing. Universal Primers (27F: AGAGTTTGATCTGGCTCAG) (1492R: GGTTACCTTGTTACG ACTT) were used for 16S rRNA sequencing . The amplified 16S rRNA PCR product was sequenced and trimmed. The unknown organism was identified using the maximum aligned 16S rRNA sequences available in the GenBank of NCBI through BLAST search. MEGA 6.0 software was used for the construction of phylogenetic tree to understand the evolutionary relationship .
The isolates that showed maximum cellulolytic activity during screening were subjected to enzyme production. A single colony from pure cultures were inoculated in nutrient broth and incubated at 37℃ for 24 h. 100 μl of the above overnight culture was used as inoculum source for the production medium . In 50 ml of Erlenmeyer flasks, 25 ml of nutrient broth containing 1% carboxy methyl cellulose (CMC) was prepared for each of the selected isolate’s strains including a control and autoclaved at 121℃ for 15 min. Broth media was inoculated with 250 μl of bacterial culture and incubated overnight at 37℃ for 150 rpm to obtain clear supernatant. The fermented broth was centrifuged at 10,000 rpm for 10 min and supernatant was used for enzyme activity assay .
Enzyme assay was carried out according to the method described by Karmakar
Enzyme activity (U/ml) = (Consumed substrate) (μmol/ ml) × Total Reaction Volume (ml)/(Reaction time (min)) × (Enzyme volume(ml)).
Optimization of the fermentation medium for maximum cellulase production was carried out. The effect of various factors affecting cellulase production was determined by measuring enzyme activity at different pH value (5−9) and temperature (35−50℃) [16, 17]. The effect of various carbon sources such as maltose, lactose, and fructose concentration were examined in the production medium. Various nitrogen sources like yeast extract, peptone and meat extracts were examined for their effect on enzyme production and growth of bacterial isolates.
Three cellulase positive isolates were identified from soil samples Khazana Sugar Mills, Peshawar. They include:
The selected bacterial isolates were further identified through morphological and biochemical tests. The cultural, colony morphology and characteristics of these isolates were observed (Table S1). All three strains were identified as gram-positive rods (Table 1).
Further confirmation was done by molecular identification. Pure culture of selected isolates was sent to Macrogen Korea for 16S rRNA gene sequencing . Universal Primers were used for sequencing (Table S7) The amplified 16S rRNA PCR product was sequenced. The result of molecular identification for selected isolates revealed to be
HB2 showed maximum cellulose production and optical density activity for 72 h at 40℃. (Fig. 5A). The optimum cellulose activity of HS5 was observed at 40℃ for 24 h. The maximum activity of HS9 at 40℃ for 48 h and the maximum optical density was at peak at 40℃ for 48 h measured at 600 nm (Figs. 5B and 5C).
The growth medium pH is one of the most important physical parameters which played an important role in enzyme secretion. The fermentation was carried out with CMC in shaking incubator for 72 h at pH ranged 3−5 to determine optimum pH for enzyme production. The best optimum cellulase production was noted at pH 7 for 48 h for HB2 and optimum growth density was at peak at pH for 48 h (Fig. 6A). The maximum cellulase production and optical growth density of HS5 at pH 7 for 48 h are presented in Figure 6B. Maximum enzyme production for HS9 was observed at pH 7 for 24 h with maximum optical growth density at pH 7 for 48 h time interval (Fig. 6C).
The cellulolytic bacteria were treated with different carbon sources (fructose, maltose, and lactose) at 1% concentration at different incubation time interval (24− 72 h). The results were obtained using photo spectrometer. The maximum enzyme activity and optical density of growth was observed for HB2 using lactose as a carbon source for 48 h time interval (Fig. 7A). Whereas HS5 also showed maximum production of enzyme and optical density for 24 h using lactose as a carbon source. Lactose was found to be the best source of carbon for the bacterial isolate HS9 in the production of cellulase enzyme for 48 h time interval, while the optical density for growth of HS9 was also on peak for fructose as a carbon source for 48 h interval period. (Figs. 7B and 7C).
Nitrogen is an essential element that play important role in the growth of microorganism and in the production of enzymes. Different nitrogen sources were used (Meat extract, Yeast extract, and Peptone) at concentration of 1%. The bacterial strain HB2 showed optimum enzyme production while optical density was at peak using meat extract as a nitrogen source at 48 h interval (Fig. 8A). The maximum cellulase production was noted by HS5 for 24 h but the optical density was at peak for HS9 at 48 h interval time using meat extract as a nitrogen source (Fig. 8B). Maximum enzyme activity was also noted by bacterial strain for 72 h using peptone as a nitrogen source. Although, HS9 revealed maximum optical density for growth at 48 h interval when peptone was used as a nitrogen source (Fig. 8C).
The natural environment is full of biological waste that can be used by microorganism for their survival and ultimately changing waste material to less toxic or useful products. Some of these microorganisms having the ability to degrade cellulose to reducing sugars that can be used in many industrial applications. In current study, cellulolytic bacteria were isolated from natural environment. In present study, three bacterial isolates were isolated from natural environment having the potential to produce cellulase enzyme that convert cellulose to reducing sugar. These isolates were later confirmed as
Cellulases producing bacterial isolates were identified on their ability to produce clear zone on CMC agar plate indicating their ability to degrade cellulose. For further optimization and maximum production of cellulase, different growth parameters such as pH, temperature, incubation period, substrate concentration and carbon sources were optimized.
Soil is one of the main reservoirs which provide nutrient and energy for supporting the growth of microorganisms.
Studies reported that temperature greatly affect growth of microbes and ultimately extra cellular enzyme production [21, 22]. Keeping in view, different conditions based on variation in temperature range was provided to the selected strains. A significant increase in the amount of enzyme production was observed at 40℃ with 1% CMC concentration . All three isolates were evaluated for different pH as previous studies shows that besides temperature, and carbon, pH also affect enzyme production. Interestingly, maximum activity was observed at pH 7 among all the selected isolates (Fig. 6). Current findings are in correlation with the finding of others for different
Different microbes prefer different sources of carbon for their growth. In current study, maximum growth and enzyme activity was observed for HB2 using lactose as a carbon source for 48 h. Moreover, HS5 showed maximum activity when incubated for 48 h using meat extract and peptone as nitrogen source. In conclusion, all the three bacterial isolates showed maximum cellulase production when grown on meat extract as nitrogen source and lactose as carbon source (Figs. 7 and 8).
Current study is the only study of its kind in which three different cellulolytic bacteria (B. cereus,
We are thankful to Dr. Hina Ali for her assistance in Bioinformatics and the organization of manuscript and Mr. Shafiq for his helping in experimental lab work and all the authors for contribution in this manuscript. This study was partially supported by Sarhad University of Science and IT, Peshawar, Pakistan.
The authors have no financial conflicts of interest to declare.
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