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Microbiology and Biotechnology Letters

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Environmental Microbiology (EM)  |  Microbial Ecology and Diversity

Microbiol. Biotechnol. Lett. 2022; 50(2): 245-254

https://doi.org/10.48022/mbl.2201.01006

Received: January 17, 2022; Revised: May 4, 2022; Accepted: May 19, 2022

Evaluation, Characterization and Molecular Analysis of Cellulolytic Bacteria from Soil in Peshawar, Pakistan

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,     hinaali.ibs@suit.edu.pk

Abstract

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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

Graphical Abstract


Introduction

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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 [1]. In plant biomass, Cellulose accounts for 40 billion tons per year, which makes it the most abundant and primary product [2]. Plant biomass is also present in less amount in hemicelluloses and least of all lignin [3]. 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 [4]. 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 [5]. 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 [6]. 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 [7].

Bacteria can be used to degrade cellulose into simple sugars because they can adapt to environmental conditions and biochemical changes such as Pseudomonas aeruginosa, Serretiamarcescens, Nocardia, Arthrobacter, Micrococcus etc. Bacterial studies are of great importance due to their cellulolytic enzyme production and biochemical flexibility [8]. Keeping in view the potential applications of cellulase enzyme in different industrial processes, it is necessary to isolate, identify and characterize cellulase producing bacteria from soil. In this study, three bacterial strains were isolated from soil of bagasse of sugar mill and sawdust from carpenter shop and were screened for their cellulolytic activity. These bacterial strains were identified by 16S rRNA gene sequencing and different culture parameters have been examined for enzyme production such as time, temperature, pH, and nitrogen and carbon sources. Furthermore, also the operational stability has been investigated to full fill the requirement of different industrial processes.

Materials and Methods

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Isolation of cellulolytic bacteria from soil samples

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.

Identification of selected isolates

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 [9].

Molecular identification of bacterial isolates

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 [10]. 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 [11].

Secondary screening for production of cellulase enzyme

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 [12]. 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 [3].

Enzyme assay

Enzyme assay was carried out according to the method described by Karmakar et al. [13]. 100 μl of crude enzyme supernatant along with 100 μl of 1% CMC as a substrate and cellulase activity was measured by estimation of glucose by DNSA method. Standard graph prepared by concentration of standard glucose solution. 175 μl distilled H2O was added to 125 μl of NaPO4 buffer with pH 7, incubated for 20 min at 50℃ in a water bath, and then 500 μl of DNS solution was added. The test tubes were heated for 10 min at 100℃ in water bath tubes. It was then allowed to cool to room temperature and absorbance taken at 570 nm. The color intensity of the solution was detected by measuring the (OD) using a spectrophotometer for measurement of enzyme assay at 540 nm. One unit of cellulase activity is defined as “the amount of cellulase required to catalyze the formation of reducing sugar which is equal to 1 mole of D glucose per minute under assay conditions” [14, 15].

Enzyme activity (U/ml) = (Consumed substrate) (μmol/ ml) × Total Reaction Volume (ml)/(Reaction time (min)) × (Enzyme volume(ml)).

Enzyme production under optimized conditions

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.

Results

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Screening and identification of cellulolytic bacteria

Three cellulase positive isolates were identified from soil samples Khazana Sugar Mills, Peshawar. They include: B. cerus, B. subtilis and B. stratosphericus. The cultural, biochemical, and morphological characteristics of these isolates are shown in Table 1. These three isolates were named as HB2, HS5 and HS9. These isolates showed clear zones (hydrolysis) on CMC nutrient agar medium supplemented with Congo red (Fig. 1).

Table 1 . Biochemical characteristics of the bacterial isolates.

Sample IDCitrate TestEosin Methylene BlueMackonkeyBlood AgarMSA MediaCoagulase TestOxidase TestUrease TestCatalase TestIndole TestIdentified Genus
HB2NegativeNegativePink growthGreenish growthNegativeNegativeNegativeNegative (yellowish color)PositiveNegativeBacillus
HS5NegativePurple growthPink growthGreenish GrowthYellow growthNegativePositiveMedium positivePositiveNegativeBacillus
HS9NegativeNegativeNegativeGreenish growthYellow growthPositivePositiveSlow positivePositiveNegativeBacillus


Figure 1.Bacterial isolates show clear zones on carboxymethyl cellulose medium (CMC).

Morphological and biochemical characteristics

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).

Molecular Identification of cellulolytic bacteria

Further confirmation was done by molecular identification. Pure culture of selected isolates was sent to Macrogen Korea for 16S rRNA gene sequencing [18]. 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 B. cerus, B. subtilis and B. stratosphericus using 16S rRNA sequencing. All the three bacterial sequences were present in NCBI GenBank, and the best sequence alignment results were noted. The phylogenetic tree was constructed through MEGA 6.0 to understand evolutionary relationship (Figs. 2−4) [11].

Figure 2.Phylogenetic analysis of Bacillus Cereus by maximum likelihood.

Figure 3.Phylogenetic analysis of Bacillus subtilis by Maximum Likelihood method.

Figure 4.Phylogenetic analysis of Bacillus stratosphericus by maximum likelihood method.

Effect of temperature and incubation time on enzyme production

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).

Figure 5.Effect of incubation temperature and time on enzyme activity and growth of (A) HB2 (B) HS5 and (C) HS9.

Effect of pH on enzyme production

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).

Figure 6.Effect of pH on cellulase enzyme production and growth of (A) HB2 (B) HS5 and (C) HS9.

Effect of carbon source on enzyme production

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).

Figure 7.Effect of carbon sources on enzyme activity and growth of (A) HB2 (B) HS5 and (C) HS9. Error bars shows standard deviation.

Effect of nitrogen sources on enzyme production

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).

Figure 8.Effect of nitrogen sources on cellulase enzyme activity and growth of (A) HB2 (B) HB5 and (C) HB9. Error bar shows standard deviation.

Discussion

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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 B. cerus, B. subtilis and B. stratosphericus by using 16S rRNA gene sequencing.

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. B. cerus and B. subtilis are the most common organism found in soil. Previous studies shows that different cellulose producing bacteria were isolated from different sources including B. stratosphericus which was reported from high attitudes [19]. Later, the same bacteria were reported from spoilage and soft rot of vegetables and fruits in Egypt [20]. This study is of its kind to report the cellulase producing bacteria from saw dust which is a by product of wood working areas. Furthermore, using 16S rRNA confirmed that one of the isolates is B. stratosphericus, which is for the first time reported from sawdust having better cellulolytic activity.

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 [23]. 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 Bacillus strains [24].

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, B. subtilis and B. stratosphericus) were reported from sawdust in Peshawar region. Different growth parameters (Temperature, pH, Incubation time, Nitrogen and Carbon sources) were optimized in lab for maximum enzyme production. Briefly, we found that when lactose was used as a carbon source, meat extract as a nitrogen source, the selected isolates showed maximum enzyme activity at pH 7 and at 40℃. Moreover, B. stratosphericus which was previously isolated from high altitudes, we reported it from a carpenter shop in Peshawar, Khyber Pakhtunkhwa, Pakistan. Peshawar has many pharmaceutical and food industries where they purchase cellulase enzyme for different process. Findings of the current study may be used to use these bacteria for industrial scales production of cellulase using the same parameters to get maximum production.

Acknowledgments

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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.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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