Bacterial Diversity and Bioprospecting for enzyme among the Shola Forests of Idukki District, Kerala, India

Bacterial Diversity and Bioprospecting for enzyme among the Shola Forests of Idukki District, Kerala, India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bacterial Diversity and Bioprospecting for enzyme among the Shola Forests of Idukki District, Kerala, India Rochelle Fernandez, Prabagaran Solai Ramatchandirane This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3834802/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Shola forests are unique montane vegetations found extensively along the Southern Western Ghats of India. Samples were collected in the form of soil, slurry, and water from Pampadum, Mathikettan and Anamudi Sholas. Cultivable bacteria were isolated to explore bacterial diversity of the region. Among the total 58 morphologically divergent bacterial strains, 32 isolates showed circular configuration, 24 were irregular while 2 were punctiform. Predominance of pale white colour colonies were discernible followed by white, beige, orange, yellow, pink including transparent ones. Nearly 56.89% of the isolates were found to be Gram negative rods. Distribution of Gram positive rod and Gram positive cocci were 39.65% and 3.45% respectively. Statistical analysis (Shannon and Simpsons) was recorded. Maximum and minimum diversity was observed from the samples collected from Pampadum soil and water. Identification through 16S rRNA gene sequence of the isolates revealed predominance of 4 divisions viz., Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes , distributed in 9 genera. More genera were distributed in the phyla Proteobacteria followed by the Firmicutes . Least representations were by Actinobacteria and Bacteroidetes . The bacterial isolates had the ability to produce different enzymes. The isolate Acinetobacter dispersus (ASL31) was found to have maximum amylase activity of 62.6 ± 1.3 U/ml. The isolate Bacillus paralicheniformis (PS1) showed maximum protease activity of 49.2 ± 0.6 U/ml, and the isolate Bacillus aerius (MSL9) possessed maximum cellulase activity of about 86.2 ± 0.9 U/ml. Hence, the diverse environment of Shola forests provides adequate diversity to explore the place for various microorganisms for bioprospecting. Shola Forests Western Ghats Bacterial diversity 16S rRNA gene enzyme activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Forests engulf about 80% of terrestrial biodiversity that are essential to maintain a healthy and balanced ecosystem around the world. They are the major ecological niche that house diverse community of native and introduced organisms that serve as a significant resource for mankind. Though certain factors that determine the forest type include climate, soil type, topography, and elevation, classification of forests mainly depends upon their nature, composition, and its association with neighboring environment. Shola is one such typical tropical montane forest, ancient one with valleys and edges where there exist extremely diverse grasslands in between (Bunyan et al. 2012 ). Sholas are covered with grasslands in the mountain tops characterized by frost and fire-resistant grass species while the depressions between the mountains are covered with patches of stunted tropical forest vegetation. Together they form an extraordinary ecosystem which serve as a rich repository of rare and invaluable biodiversity (Robin and Nandini 2012 ). Such an unique ecosystem is native only to the Southern Western Ghats of India at elevations over 1,900 to 2,220 metres in the states of Karnataka, Kerala, and Tamil Nadu. Climate has a greater influence on the montane ecosystem, which gets colder as altitude rises (Perry 1994 ). Shola Forests has very high-water retention ability which means that the habitat absorbs a lot of water and lets it out through a very long period of time. Most of the South Indian rivers originate from such Shola habitat. These forests are distributed according to their elevation in western ghats where northern region is denser when compared to the southern region. Interestingly these forests house numerous endemic species due to its unique climatic conditions and high isolation. These forests are rightly called nature’s water towers that play a vital role in sustaining life of the plains. Microbial diversity plays a dominant role in the maintenance of ecosystem and deserves greater attention since extensive knowledge on microbial taxonomy and physiology will help gain better insights into the microbial world and in identifying potential novel species (Panizzon et al. 2015 ). Among the forest ecosystem, soil has a substantial role in facilitating essential nutrient and energy flow patterns (Canadell and Raupach 2008 ) where soil bacteria reside as driving force for all these ecological processes (Bardgett et al. 2008 ). Though bacteria is minimally explored in forest compared to the flora and fauna, they depict an integral part of ecological studies and microbial community. Acidobacteria , Actinobacteria , Proteobacteria , Bacteroidetes , and Firmicutes are the most abundant bacterial phyla reported in most soils (Lauber et al. 2009 ). Bacteria in forest soils actively respond to climate change (Llado et al. 2017). Many environmental factors in forest ecosystem such as carbon: nitrogen ratio, soil pH, temperature, minerals, nutrients have a substantial effect on the microbial community (Janssens et al. 2010 ). Generally, the wealth of bacterial phylum and genus varies among the soil types based on vegetation too. Even though forest ecosystem bacteria play a major part in fulfilling various essential functions like decomposition of organic matter, involvement in nitrogen cycle processes, and so on, still the complete role of bacteria in such ecosystems is to be explored and understood. Despite considerable number of studies were undertaken, exploration of total bacterial diversity in Shola Forests are very limited. Detailed studies on the microbial composition and diversity in soils of Sholas are important for a better understanding of their ecological role that can solve several environmental cues. To investigate variations in bacterial communities, three Shola National Parks in the Idukki district of Kerala, India were studied. Materials and Methods Site description and Sampling Bacterial diversity of soil, water, and slurry samples were investigated in three Shola National Parks viz., Pampadum, Mathikettan, and Anamudi of Idukki district, Kerala, India (Fig. 1 ). Pampadum shola is the smallest national park in Kerala located in the Vattavada panchayat of Devikulam Taluk covering an area of 11.75 km 2 , in the eastern part of Southern Western Ghats. The altitude ranges between 1600–2400 msl. Mathikettan shola park is spread over 12.82 km 2 in Poopara village of Udumbanchola Taluk. The altitude of the region ranges from 1200–1984 msl. Anamudi shola park comprises three shola reserve forests – Idivara, Pullarda, and Mannavan covering an area of over 42.68 km 2 . The Park is located in the Marayoor village of Devikulam Taluk and the elevation ranges from 2152 to 2305 msl. The location details of each sampling sites are listed in Table 1 . A total of 9 samples i.e., soil, water and slurry were collected from each Shola National Park. The soil samples were collected at a depth of 2–5 cm that were stored in air tight polyethylene bags, while slurry and water samples were collected in sterilized falcon tubes. All the samples were maintained at room temperature until it reached the laboratory. Table 1 Location details of the sampling sites Site Pampadum Mathikettan Anamudi Latitude (N) 10º 7’ 37.1928” 9º 59’ 5.694” 10º 12’ 7.1244” Longitude (E) 77º 15’ 29.4588” 77º 14’ 46.4568” 77º 8’ 52.8612” Elevation (msl) 1917 1314 2152 Isolation of bacteria Four different mediums viz., Nutrient agar (NA), Kings medium B base (Kings B), Tryptone soya broth (TSB) and Luria Bertani agar (LB) were selected for the cultivation of microbes. Isolation of bacteria was carried out by standard serial dilution plate technique. One gram of soil, 1ml of slurry and water samples were transferred to 9ml sterile distilled water and well shaken. Different aqueous dilutions ranging from 10 − 1 to 10 − 15 were prepared and 0.1ml of appropriate dilutions were spread plated on all four medium and plates were incubated at 37ºC for about 24 hrs. Morphological characteristics of Bacterial isolates After incubation, on the basis of colony morphology distinct microbial colonies were counted and subsequently pure cultured. The colony color, shape (punctiform, circular, filamentous, irregular, rhizoid), elevation (flat, raised, convex, pulvinate, umbonate) and margin (entire, undulate, lobate) were noted. Isolated pure cultures of bacteria were maintained in solidified agar plates at 4ºC. All the isolates were named alphanumerically and maintained in glycerol stocks. Diversity indices A range of diversity indices particularly Shannon index, the evenness indices derived from it, Simpson’s dominance index and its equitability index have been derived with bacterial communities. Where pi is the proportion of individuals of one particular species divided by the total number of individuals, ln is the natural log, S is the total number of species and ∑ is sum from species 1 to species S. Shannon equitability (E H ) is calculated by dividing Shannon diversity index by natural logarithm of species richness ln (S). Where pi 2 is the square of the proportion of individuals of one particular species divided by the total number of individuals, S is the total number of species and ∑ is sum from species 1 to species S. Simpson equitability (E D ) is calculated by the formula 1-D. Equitability takes a value between 0 and 1, with 1 being complete evenness. DNA Extraction and PCR amplification Genomic DNA was isolated from bacterial cultures grown in nutrient broth overnight, using CTAB (Cetyl trimethyl ammonium bromide) method. The 16S rRNA genes were PCR amplified with a universal primer pairs 27F and 1492R. The PCR master mix consisted of EmeraldAmp GT PCR master mix, 2µl DNA (100 ng), 1µl of each primer (10 pmol), and sterile distilled water to bring the total volume to 20µl. PCR amplification involved an initial denaturation at 96ºC for 5 min, then 35 cycles involving denaturation at 94ºC for 1 min, annealing at 48.5ºC for 1 min, extension at 72ºC for 2 min followed by a final extension at 72ºC for 10 min. Sequencing and Phylogenetic tree construction Sequence analysis was performed using an ABI PRISM Big dye terminator cycle sequencing ready reaction kit (Applied Biosystems) and an ABI 3500 Genetic Analyzer (Applied Biosystems). The sequences of the 16S rRNA gene obtained were aligned by using DNA lasergene software and were compared against the sequences available from GenBank using BLASTN program. The 16S rRNA gene sequences of those strains that represent the closest neighbour of each isolate was retrieved. The sequences were aligned using CLUSTALW and phylogenetic tree was constructed after pairwise and multiple sequence alignment using the neighbor-joining method. MEGA 7.0 software was used for the analysis. Bootstrap analysis was performed employing 1000 replicate data sets to evaluate the confidence limits of the branching. Sequence data was deposited at the National Center for Biotechnology Information (NCBI), GenBank (Supplementary Table 1). Extracellular enzyme screening The isolates were screened for the production of amylase, protease and cellulase enzyme on appropriate agar medium supplemented with respective substrates; 0.2% starch for amylase, 0.5% skim milk for protease and 1% carboxymethyl cellulose for cellulase. The plates were incubated at 37ºC for 24 hrs. The potential isolates were identified by the formation of clear zones around the colonies. The selected isolates were grown in appropriate broth conferred with respective substrates and incubated at 37ºC overnight with continuous shaking at 180 rpm. The crude extract was prepared by centrifuging the overnight culture at 10,000 rpm for 10 mins. The supernatant was filtered and was used as an enzyme source for assay. Determination of enzyme activity Qualitative assay was performed by well diffusion method. The agar medium was prepared along with respective substrates for amylase, protease and cellulase enzymes. Precisely 100 µl of the crude extracts was loaded in the wells and the plates were incubated overnight at 37ºC. Based on the diameter of the clear zone formation, potential isolates were selected for further assay. Quantitative assay was carried out spectrophotometrically in triplicates where optical densities were plotted to determine enzyme activity. Protease activity was determined by Folin’s phenol method (Tsuchida et al. 1986 ), whereas amylase (Pokhrel et al. 2013 ) and cellulase activity (Miller 1959 ) was assayed by DNS (Dinitrosalicylic acid) method. Results Abundance of Bacteria in Shola samples The diversity of bacteria from soil, slurry, and water samples of the Shola forests of Kerala were enumerated through serial dilution plating technique. Four different media were employed viz., Nutrient agar, Kings medium B base, Tryptone soya broth, and Luria Bertani agar. Totally 58 strains (Supplementary Fig. S1 ) were isolated from all the 9 collected samples viz., 21 strains from Pampadum (soil: 10; slurry: 8; water: 3), 24 strains from Mathikettan (soil: 3; slurry: 9; water: 12) and 13 strains from Anamudi forest (soil: 4; slurry: 7; water: 2). The colony forming units were recorded for the dilutions from 10 − 3 to 10 − 13 . Among the soil samples, the highest bacterial population was recorded in Mathikettan (NA) where the values ranged from 132 x 10 − 13 to 311 x 10 − 9 cfu/g. Lowest density was noticed in Pampadum (LB), with values ranging from 27 x 10 − 13 to 162 x 10 − 9 cfu/g. Similarly in slurry samples, maximum population was observed in Mathikettan (LB), with values ranging from 55 x 10 − 13 to 343 x 10 − 9 cfu/ml, while the lowest population was from Anamudi (Kings B) with values ranging between 29 x 10 − 13 to 153 x 10 − 9 cfu/ml. In case of water samples, Mathikettan (TSB) exhibited higher bacterial abundance. The values ranged from 99 x 10 − 7 to 353 x 10 − 3 cfu/ml and Anamudi (LB) displayed least abundance, with values ranging from 48 x 10 − 7 to 341 x 10 − 3 cfu/ml (Fig. 2 ). Morphological characteristics of bacterial isolates Out of the 58 strains isolated, 32 isolates (55.17%) showed circular configuration and 24 (41.37%) were irregular while 2 (3.45%) exhibited punctiform configuration. Among Pampadum samples, soil and slurry were observed to be irregular compared to water which was predominantly circular. While the configuration of the Mathikettan soil isolates was circular, slurry, and water were predominantly irregular. In case of Anamudi forest, circular configuration was found to be dominating in all three sample types. Pale white colour isolates were observed mainly (Pampadum: 52.3%; Mathikettan: 33.3%; Anamudi: 23.1%), followed by white, transparent, beige, orange, yellow, and pink colour. Most of the isolates had raised colonies (47.6%; 33.3%; 23.1%) and entire margin (42.9%; 45.8%; 84.6%) in Pampadum, Mathikettan and Anamudi respectively. Distribution of Gram negative rod, Gram positive rod, and Gram positive cocci was found to be 56.89%, 39.65%, and 3.45% respectively. Shannon and Simpson diversity indices Shannon – Wiener diversity (H), its evenness (E H ), and Simpson’s index (D) and its equitability (E D ) were studied. Shannon index increases as both richness and evenness of the community increases. Simpson’s index which is a measure of dominance increases as diversity (evenness) decreases. The lesser the value of D, the more the diversity, the greater the value of E D , the more the diversity. In the present study, the H index ranged from 0.32 to 1.81 and E H ranged from 0.29 to 1.00. The D index ranged from 0.21 to 0.85 and E D ranged from 0.15 to 0.79. From the acquired results, it can be concluded that the highest and lowest values of diversity indices are recorded from soil and water samples of Pampadum respectively. Phylogenetic analysis of the 16S rRNA gene sequence The 16S rRNA gene of the selected morphologically divergent bacterial isolates was sequenced. The nearest phylogenetic neighbour of all the isolates were identified by BLAST analysis of the 16S rRNA gene sequence from GenBank database. The closest homologues sequences were retrieved and phylogenetic tree was constructed (Fig. 3 ). The study revealed 9 different genera namely Acinetobacter, Bacillus, Brevundimonas, Chryseobacterium, Enterobacter, Exiguobacterium, Pseudomonas, Rothia and Staphylococcus belonging to four divisions viz., Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria . Bacterial diversity was found to be maximum in the division of Proteobacteria in which 24 strains were among the genus of Acinetobacter, Brevundimonas, Enterobacter , and Pseudomonas. This was followed by the division Firmicutes with 17 strains spreading among the genus Bacillus, Exiguobacterium , and Staphylococcus. The remaining 2 strains each were among the division Actinobacteria and Bacteroidetes belonging to the genus Rothia and Chryseobacterium correspondingly. In Pampadum forest, the bacterial strains predominantly were among the division Firmicutes and the species include Bacillus paralicheniformis (PS1), Bacillus sp. (PS6, PSL23), Bacillus subtilis (PS15), Bacillus nakamurai (PSL25), Bacillus vallismortis (PSL30), Bacillus megaterium (PSL31), Staphylococcus sp. (PSL39) and Bacillus xiamenensis (PW54). Then comes the division Proteobacteria which comprises Pseudomonas songnenensis (PS3), Pseudomonas sp. (PS8, PSL17) Pseudomonas stutzeri (PSL27, PW52) and Pseudomonas aeruginosa (PW50). Phyla Proteobacteria was prevalent in Mathikettan forest samples and involved namely species of Pseudomonas sp. (MS1, MSL23, MW37), Pseudomonas songnenensis (MS5, MSL7, MW53), Enterobacter cloacae (MSL8), Pseudomonas stutzeri (MSL34, MW48) and Brevundimonas vesicularis (MW38). This was followed by the Phyla Firmicutes that included Bacillus aerius (MSL9), Bacillus sp. (MSL14), Bacillus xiamenensis (MSL33), Exiguobacterium acetylicum (MW35) and Exiguobacterium sp. (MW40). The Phyla Actinobacteria and Bacteroidetes were only observed in the Anamudi forest samples that comprised the species Rothia terrae (ASL30) and Chryseobacterium montanum (ASL20). The dominant Phyla in Anamudi samples were Proteobacteria with species Pseudomonas sp. (AS4), Pseudomonas songnenensis (AS8, ASL28, AW44), Pseudomonas stutzeri (AS11, ASL36, AW41) and Acinetobacter dispersus (ASL31). The Phyla Firmicutes included Staphylococcus epidermidis (AS6), Exiguobacterium sibiricum (ASL19), and Bacillus sp. (ASL23). More diverse bacterial strains were observed in the Anamudi Shola samples with four divisions ( Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria ) and 7 genera ( Pseudomonas, Acinetobacter, Staphylococcus, Exiguobacterium, Bacillus, Rothia, Chryseobacterium ). While Mathikettan and Pampadum samples showed 2 divisions ( Proteobacteria, Firmicutes ) with five ( Pseudomonas, Enterobacter, Brevundimonas, Bacillus, Exiguobacterium ) and three ( Bacillus, Staphylococcus, Pseudomonas ) genera respectively. Pseudomonas stutzeri and Pseudomonas songnenensis were found repeatedly in almost all samples of Shola Forest. Overall, the Phyla Proteobacteria was observed to be imperious in the Shola Forest samples (Fig. 4 ). Enzyme activity All the identified bacterial isolates were screened for the enzyme production viz., amylase, protease, and cellulase. Among them, 38 isolates showed positive activity for amylase, followed by 32 and 16 isolates for cellulase and protease respectively by the formation of hydrolyzed zones. While members of phylum Proteobacteria showed predominant activity for amylase and cellulase, phylum Firmicutes was good in protease activity. Nine isolates, namely Bacillus subtilis (PS15), Bacillus nakamurai (PSL25), Bacillus megaterium (PSL31), Bacillus sp. (PS6, PSL23, ASL23), Pseudomonas sp. (MW37) and Pseudomonas stutzeri (MW48, AS11) exhibited positive enzyme activity for all the three enzymes. Three isolates, specifically Enterobacter cloacae (MSL8), Staphylococcus epidermidis (AS6) and Rothia terrae (ASL30) showed negative growth for all enzymes. On the basis of the primary screening, the best isolates capable of producing the enzymes were selected for further study. The secondary screening was carried out by well diffusion method with a standard strain Pseudomonas aeruginosa (ATCC27853) (Marathe et al. 2018 ; Raju and Divakar 2013 ; Gunavathy and Boominathan 2015 ). Based on the clear hydrolyzed zones formed, potent strains namely PS1, PSL39, PW50, MSL14, MW50, ASL23 for protease, PS8, MSL14, MW35, MW40, ASL31, ASL36 for amylase and PSL25, PW54, MS5, MSL9, MSL33, ASL23 for cellulase were taken for further analysis. All the selected isolates showed almost equal and greater hydrolyzed zones than the standard (Supplementary Fig. S2). Out of the selected isolates, the strain Acinetobacter dispersus (ASL31) found to have maximum crude amylase activity (62.6 ± 1.3 U/ml). The isolate Bacillus paralicheniformis (PS1) and Bacillus aerius (MSL9) possessed maximum activity for protease (49.2 ± 0.6 U/ml) and cellulase (86.2 ± 0.9 U/ml) enzymes respectively (Fig. 5 ). Discussion The present study was carried out with an objective to report diversity of bacteria in few Shola forests of Kerala along the Western Ghats. Sahyadri hills are the lifeline of the Indian ecosystem which spans from the tip of Kanyakumari to Gujarat covering the states of Kerala, Tamil Nadu, Karnataka, Goa, Maharashtra, and Gujarat. Due to human activities over centuries, most of the pristine areas were disturbed except certain pockets like the Sholas. Among them, the remarkable ones are located in Idukki, Wayanad, Palakkad and Thrissur districts of Kerala, among which the present study mainly focused only on the Sholas of Idukki. Sampling was done in forest soil, water streams, and marshy areas from Pampadum, Mathikettan, and Anamudi Shola national park. Pampadum Shola is the smallest park covering an area of 11.75 km 2 at 1600–2000 msl, above which grasslands prevail with small forest patches distributed evenly. Mathikettan Shola houses evergreen forests, moist deciduous forests, shola grasslands, and semi-evergreens covering an area of about 12.82 km 2 at an elevation ranging between 1200–1984 msl. Anamudi forest is covered with West coast tropical evergreen, Southern hilltop tropical vegetation found at higher altitudes of about 2305 msl. The Park has one of the largest shola forest ecosystems in the country with unique habitats, covering an area of about 42.68 km 2 . Generally, such culture-based approaches underestimate the total bacterial number, as the nutritional requirements of every prokaryote present in a particular sample cannot be met, even though it contains all essential nutrients required for the physiological growth of the isolates (Orphan et al. 2000 ). Thus, these approaches are highly dependent on the medium used for isolation and incubation conditions maintained during cultivation (Jackson et al. 2013 ). Culturing in more than one medium will certainly increase the cultivation of diverse types of bacteria than screening through a single medium. Hence four different media viz., Nutrient agar, Luria Bertani agar, Tryptone soya broth, and Kings medium B base were employed to isolate bacteria. Bacterial colonies exhibit distinct characteristics like size, colour, shape, and texture, which primarily vary amid different species (Mamou et al. 2016 ). A total of 58 morphologically distinct strains were isolated. Majority of the isolates were observed to be pale white while few of them were pigmented. Circular configuration with raised colonies was predominant among the isolated strains. The bacterial load was estimated and compared among the samples, where a significantly higher population of bacteria was noticed in Mathikettan samples. The values were 311 x 10 − 9 cfu/g for soil, 343 x 10 − 9 cfu/ml for slurry and 353 x 10 − 3 cfu/ml for water. The Gram stain is a significant morphological clue among the initial characterization and classification of bacteria (Thairu et al. 2014 ). Among the total isolates, Gram negative rod (56.89%) was found to be prevalent followed by Gram positive rod (39.65%) and Gram positive cocci (3.45%) whose distribution is similar to a study conducted in the soils of grassland ecosystem of the Western Ghats that reported Gram negative bacteria to be more diverse (Rinoy 2016 ). The isolates were analyzed for richness, evenness, and diversity indices. The Shannon – Wiener (H) and its evenness (E H ) ranged from 0.32 to 1.81 and 0.29 to 1.00. The Simpsons index (D) and its equitability (E D ) ranged from 0.21 to 0.85 and 0.15 to 0.79. Maximum and minimum diversity was recorded for the samples of Pampadum soil and water. The bacterial diversity may be associated with the forest types since vegetations are known to affect soil microbial diversity and community structures. A previous study conducted in the tropical Eastern Himalaya reported that there was a fall in bacterial diversity with rising altitudes (Lyngwi et al. 2013 ). The 16S rRNA gene sequence is the most common housekeeping genetic marker employed to study bacterial phylogeny and taxonomy due to its conserved nature in almost all Eubacteria and Archaebacteria (Janda and Abbott 2007 ). The 16S rRNA gene of the isolates was sequenced and their taxonomic affiliation was ascertained. The analysis revealed the coverage of four divisions namely Actinobacteria, Bacteroidetes, Firmicutes , and Proteobacteria distributed among 9 genera viz., Acinetobacter, Bacillus, Brevundimonas, Chryseobacterium, Enterobacter, Exiguobacterium, Pseudomonas, Rothia and Staphylococcus. Five phyla, mainly Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes , and Firmicutes were reported to be abundant in most soils (Lauber et al. 2009 ). The phyla Proteobacteria holds maximum bacterial diversity with 24 strains encompassing Acinetobacter, Brevundimonas, Enterobacter , and Pseudomonas which as reported in previous studies. The Proteobacteria and Acidobacteria were the most abundant phyla in the soil samples of temperate deciduous broadleaved forest and a tropical mountain rainforest of China (Wei et al. 2018 ). It has been reported that members of Proteobacteria are well distributed in almost all forest types (Zhang and Xu 2008 ). Nampoothiri et al. ( 2013 ) reported that Proteobacteria was the most abundant phylum in the water samples of the Western Ghats region, followed by Firmicutes, Actinobacteria , and Bacteroidetes. The diversity and composition of soil bacterial communities in the Forest montane region of Northeast China resulted in a high abundance of the phyla Proteobacteria (Wu et al. 2019). Environmental factors have a significant role in determining the bacterial community structure of forest soils (Xia et al. 2016 ). Soil bacterial community composition and diversity are influenced by soil properties, vegetation types, nutrient availability, and plant diversity (Liu et al. 2010 ; Naether et al. 2012 ) among which soil pH is considered an essential factor (Qiu et al. 2014 ). Generally, Shola soils are reported to be slightly acidic ranging between 5.0 and 6.4. Acidobacteria and Proteobacteria are abundant in acidic soils (Shen et al. 2013 ), while the abundances of Actinobacteria and Bacteroidetes rise with an increasing pH (Jeanbille et al. 2016 ). Proteobacteria was found to be plentiful in the acidic soils from the subtropical montane forest of Taiwan, among which β-Proteobacteria was abundant, then α-Proteobacteria and γ-Proteobacteria , while the low proportion of Acidobacteria in the study was considered to be as a result of high soil moisture and anaerobic microhabitats (Lin et al. 2010 ). Acidic soils of coniferous forests also mainly harbor Proteobacteria and Acidobacteria (Baldrian et al. 2012 ). Other predominant phyla in the present study were Firmicutes with 17 strains, which spreads among the genera Bacillus, Exiguobacterium , and Staphylococcus. This was followed by Actinobacteria and Bacteroidetes with genus Rothia and Chryseobacterium respectively. Enzymes form an important component of forest ecosystems because they play a significant part in catalyzing reactions essential for organic matter decomposition and nutrient cycling (Wang et al. 2016 ). In the present study, representative isolates were screened for their hydrolytic enzyme activity viz., amylase, protease, and cellulase. Out of 43 isolates screened, 9 strains namely Bacillus subtilis (PS15), Bacillus nakamurai (PSL25), Bacillus megaterium (PSL31), Bacillus sp. (PS6, PSL23, ASL23), Pseudomonas sp. (MW37) and Pseudomonas stutzeri (MW48, AS11) showed positive activity for all the three enzymes. Bacillus strains are considered as multifunctional microorganisms and they are one of the principal sources of enzymes from bacterial origin used in industry (Latorre et al. 2016 ). In this study, three strains specifically Enterobacter cloacae (MSL8), Staphylococcus epidermidis (AS6) and Rothia terrae (ASL30) exhibited negative results for all the enzymes, which is contrary to the earlier studies that reported Enterobacter cloacae and Staphylococcus epidermidis as enzyme producers (Lokapirnasari et al. 2015 ; Vandecandelaere et al. 2014 ). These differences in the enzyme potential may be due to the epigenetic influence of the environment. Taxonomic description of Rothia terrae , a rare soil bacterium isolated from the subtropical fields of Tainan County, Taiwan was described in 2008 by Chou et al. Later Sariturk et al. ( 2017 ), investigated it and reported that it does not produce amylase and cellulase enzyme but has a potential of beta galactosidase activity. This is the only actinobacteria identified in this entire study, which is a cream white coloured, aerobic, Gram positive bacteria that could be distinguished from other Rothia species through unique phenotypic characteristics. Enzyme assays were performed to determine their activity which provides significant details on mechanisms of enzyme catalysis and interactions with substrates. On the basis of primary and secondary screening, potential enzyme producers were selected, and quantitative assay was performed spectrophotometrically (Fersht 1999 ). The strains Acinetobacter dispersus (ASL31), Bacillus paralicheniformis (PS1), and Bacillus aerius (MSL9) possessed maximum activity for amylase (62.6 ± 1.3 U/ml), protease (49.2 ± 0.6 U/ml) and cellulase (86.2 ± 0.9 U/ml) enzymes respectively. Studies have been reported earlier, showing the potential of Acinetobacter sp. to produce amylase enzyme (Yavankar et al. 2007 ; Onishi and Hidaka 1978 ). Cellulase and protease production has been previously reported for Bacillus sp. (Oke et al. 2016 ; Suganthi et al. 2013 ; Masi et al. 2021 ). Conclusion The present study was intended to provide information on the diversity of bacteria in the Pampadum, Mathikettan, and Anamudi Sholas situated in Idukki district of Kerala, which resulted in similar reports with the earlier studies. However, vast bacterial diversity studies have been proposed in the Southern Western Ghats, but studies particularly in Shola forests are limited. Studies on microbial diversity of such unique habitats open up to vast areas of the microbial world towards such exploration. Such diversity does not restrict to microorganism per se but also extended to their metabolic activities besides their interactions with other microbes, plants, and animals. Furthermore, the results also throw light on the ability of these isolates to produce enzymes that are of great importance in various fields, as microbial enzymes are acquiring much attention with the rapid development of enzyme technology and have been extensively studied for its application in different industries and therapeutics. Declarations Acknowledgements The authors would like to acknowledge Kerala Forest Department for providing permission and required amenities for sample collection from the forest. The authors are grateful to University Grant Commission (UGC, Govt of India) for the support provided to establish infrastructure in the Department of Biotechnology grant vide UGC/SAP/No.F.3-20/2013, through Special Assistance Program (SAP) and Department of Science and technology (DST) towards Fund for Improvement of S&T Infrastructure in Universities and Higher Educational Institutions vide (FIST) and DST-PURSE phase-II vide BU/ DST-PURSE/2017/28. Authors Contributions Conceptualization: [Rochelle Fernandez] and [Solai Ramatchandirane Prabagaran]; Methodology, data analysis and interpretation: [Rochelle Fernandez]; Drafting the article: [Rochelle Fernandez]; Supervision: [Solai Ramatchandirane Prabagaran]; All the authors have read and approved the final manuscript. Funding: Not applicable Data availability Data, materials and codes can be provided upon request Competing interests No potential conflicts of interest are declared by the author(s). Ethics Approval and Consent to participate Not applicable. Human and Animal Rights No human subjects or livestock were included in this research. Consent for Publication Not applicable. References Baldrian P, Kolařík M, Stursová M, Kopecký J, Valášková V, Větrovský T, Zifčáková L, Snajdr J, Rídl J, Vlček C, Voříšková J (2012) Active and total microbial communities in forest soil are largely different and highly stratified during decomposition. ISME J 6(2):248–258. https://doi.org/10.1038/ismej.2011.95 Bardgett RD, Freeman C, Ostle NJ (2008) Microbial contributions to climate change through carbon cycle feedbacks. The ISME Journal 2(8):805–814. https://doi.org/10.1038/ismej.2008.58 Bunyan M, Bardhan S, Jose S (2012) The Shola (Tropical Montane Forest)-grassland ecosystem mosaic of Peninsular India: a review. Am J Plant Sci 3(11):1632–1639. http://dx.doi.org/10.4236/ajps.2012.311198 Canadell JG, Raupach MR (2008) Managing forests for climate change mitigation. Science 320(5882):1456–1457. https://doi.org/10.1126/science.1155458 Chou Y-J, Chou J-H, Lin K-Y, Lin M-C, Wei Y-H, Arun AB, Young C-C, Chen W-M (2008) Rothia terrae sp. nov. isolated from soil in Taiwan. Int J Syst Evol Microbiol 58(Pt 1):84–88. https://doi.org/10.1099/ijs.0.65172-0 Fersht A (1999) Structure and mechanism in protein science: A guide to enzyme catalysis and protein folding. W.H. Freeman and Company, New York Gunavathy P, Boominathan M (2015) Isolation and molecular characterization of cellulase producing bacteria from soil of sacred grove, Puducherry, India. Int J Curr Microbiol App Sci 4(12):584–590 Jackson CR, Randolph KC, Osborn SL, Tyler HL (2013) Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables. BMC Microbiol 13(1):274. https://doi.org/10.1186/1471-2180-13-274 Janda JM, Abbott SL (2007) 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45(9):2761–2764. https://doi.org/10.1128/JCM.01228-07 Janssens IA, Dieleman W, Luyssaert S, Subke J-A, Reichstein M, Ceulemans R, Ciais P, Dolman AJ, Grace J, Matteucci G, Papale D, Piao SL, Schulze E-D, Tang J, Law BE (2010) Reduction of forest soil respiration in response to nitrogen deposition. Nat Geosci 3(5):315–322. https://doi.org/10.1038/ngeo844 Jeanbille M, Buée M, Bach C, Cébron A, Frey-Klett P, Turpault MP, Uroz S (2016) Soil parameters drive the structure, diversity and metabolic potentials of the bacterial communities across temperate Beech Forest soil sequences. Microb Ecol 71(2):482–493. https://doi.org/10.1007/s00248-015-0669-5 Latorre JD, Hernandez-Velasco X, Wolfenden RE, Vicente JL, Wolfenden AD, Menconi A, Bielke LR, Hargis BM, Tellez G (2016) Evaluation and selection of Bacillus species based on enzyme production, antimicrobial activity, and biofilm synthesis as direct-fed microbial candidates for poultry. Front Vet Sci 3:95. https://doi.org/10.3389/fvets.2016.00095 Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol 75(15):5111–5120. https://doi.org/10.1128/AEM.00335-09 Lin Y-T, Huang Y-J, Tang S-L, Whitman WB, Coleman DC, Chiu C-Y (2010) Bacterial community diversity in undisturbed perhumid montane forest soils in Taiwan. Microb Ecol 59(2):369–378. https://doi.org/10.1007/s00248-009-9574-0 Liu Z, Fu B, Zheng X, Liu G (2010) Plant biomass, soil water content and soil N:P ratio regulating soil microbial functional diversity in a temperate steppe: A regional scale study. Soil Biol Biochem 42(3):445–450. https://doi.org/10.1016/j.soilbio.2009.11.027 Lladó S, López-Mondéjar R, Baldrian P (2017) Forest soil bacteria: diversity, involvement in ecosystem processes, and response to global change. Microbiol Mol Biol Rev 81(2):e00063–16. https://doi.org/10.1128/MMBR.00063-16 Lokapirnasari WP, Nazar DS, Nurhajati T, Supranianondo K, Yulianto AB (2015) Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia. Vet World 8(3):367–371. https://doi.org/10.14202/vetworld.2015.367-371 Lyngwi NA, Koijam K, Sharma D, Joshi SR (2013) Cultivable bacterial diversity along the altitudinal zonation and vegetation range of tropical Eastern Himalaya. Rev Biol Trop 61(1):467–490. https://doi.org/10.15517/rbt.v61i1.11141 Mamou G, Malli Mohan GB, Rouvinski A, Rosenberg A, Ben-Yehuda S (2016) Early developmental program shapes colony morphology in bacteria. Cell Rep 14(8):1850–1857. https://doi.org/10.1016/j.celrep.2016.01.071 Marathe SK, Vashistht MA, Prashanth A, Parveen N, Chakraborty S, Nair SS (2018) Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis , Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples. J Genet Eng Biotechnol 16(1):39–46. https://doi.org/10.1016/j.jgeb.2017.10.001 Masi C, Gemechu G, Tafesse M (2021) Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent. Ann Microbiol 71:24. https://doi.org/10.1186/s13213-021-01631-x Miller GL (1959) Use of dinitrosalycylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. http://dx.doi.org/10.1021/ac60147a030 Naether A, Foesel BU, Naegele V, Wüst PK, Weinert J, Bonkowski M, Alt F, Oelmann Y, Polle A et al (2012) Environmental factors affect Acidobacterial communities below the subgroup level in grassland and forest soils. Appl Environ Microbiol 78(20):7398–7406. https://doi.org/10.1128/AEM.01325-12 Nampoothiri KM, Ramkumar B, Pandey A (2013) Western Ghats of India: rich source of microbial biodiversity. J Sci Ind Res 72:617–623 Oke MA, Annuar MSM, Simarani K (2016) Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic substrates. BioRes 11(3):5854–5869 Onishi H, Hidaka O (1978) Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp. Can J Microbiol 24(9):1017–1023. https://doi.org/10.1139/m78-169 Orphan VJ, Taylor LT, Hafenbradl D, Delong EF (2000) Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Appl Environ Microbiol 66(2):700–711. https://doi.org/10.1128/AEM.66.2.700-711.2000 Panizzon JP, Pilz Júnior HL, Knaak N, Ramos RC, Ziegler DR, Fiuza LM (2015) Microbial diversity: relevance and relationship between environmental conservation and human health. Braz Arch Biol Technol 58(1):137–145. https://doi.org/10.1590/S1516-8913201502821 Perry DA (1994) Forest ecosystems. Johns Hopkins University Press, London Pokhrel B, Wanjare P, Singh S, Purushotham B, Kumara SM (2013) Isolation, screening and characterization of promising-amylase producing bacteria from sewage enriched soil. International Journal of Advanced Biotechnology Research (4):286–290 Qiu S-L, Wang L-M, Huang D-F, Lin X-J (2014) Effects of fertilization regimes on tea yields, soil fertility, and soil microbial diversity. Chil J Agric Res 74(3):333–339. https://doi.org/10.4067/S0718-58392014000300012 Raju EVN, Divakar G (2013) Production of amylase by using Pseudomonas aeruginosa isolated from garden soil. International Journal of Advances in Pharmacy, Biology and Chemistry 2(1):50–56 Rinoy Varghese (2016) Diversity, distribution and physiological capabilities of culturable heterotrophic bacteria from grassland, shola, Myristica swamp and evergreen forest soils of Southern Western Ghats, PhD thesis, Mahatma Gandhi University. http://hdl.handle.net/10603/194974 Robin VV, Nandini R (2012) Shola habitats on sky islands: Status of research on montane forests and grasslands in southern India. Curr Sci 103(12):1427–1437 Sariturk S, Aygan A, Ekiz DO, Oruc O (2017) Production and partial characterization of β-Galactosidase from Rothia terrae J9. Journal of Molecular Biology and Biotechnology 1(2):24–31 Shen C, Xiong J, Zhang H, Feng Y, Lin X, Li X, Liang W, Chu H (2013) Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain. Soil Biol Biochem 57:204–211. http://dx.doi.org/10.1016/j.soilbio.2012.07.013 Suganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam KM (2013) Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. Journal of Genetic Engineering and Biotechnology 11(1):47–52. https://doi.org/10.1016/j.jgeb.2013.02.002 Thairu Y, Nasir IA, Usman Y (2014) Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan African Journal of Medicine 1(4):168–174. https://doi.org/10.4103/2384-5147.144725 Tsuchida O, Yamagota Y, Ishizuka J, Arai J, Yamada J, Takeuchi M, Ichishima E (1986) An alkaline protease of an alkalophilic Bacillus sp. Curr Microbiol 14:7–12. https://doi.org/10.1007/BF01568094 Vandecandelaere I, Depuydt P, Nelis HJ, Coenye T (2014) Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms. Pathog Dis 70(3):321–331. https://doi.org/10.1111/2049-632X.12133 Wang W, Page-Dumroese D, Lv R, Xiao C, Li G, Liu Y (2016) Soil enzyme activities in Pinus tabuliformis (Carriere) plantations in northern China. Forests 7(6):112. https://doi.org/10.3390/f7060112 Wei H, Peng C, Yang B, Song H, Li Q, Jiang L, Wei G, Wang K, Wang H, Liu S, Liu X, Chen D, Li Y, Wang M (2018) Contrasting soil bacterial community, diversity, and function in two forests in China. Front Microbiol 9:1693. https://doi.org/10.3389/fmicb.2018.01693 Wu S-J, Deng J-J, Yin Y, Qin S-J, Zhu W-X, Zhou Y-B, Wang B, Ruan H, Jin L (2020) Bacterial community changes associated with land use type in the forest montane region of Northeast China. Forests 11(1):40. https://doi.org/10.3390/f11010040 Xia Z, Bai E, Wang Q, Gao D, Zhou J, Jiang P, Wu J (2016) Biogeographic distribution patterns of bacteria in typical Chinese forest soils. Front Microbiol 7:1106. https://doi.org/10.3389/fmicb.2016.01106 Yavankar SP, Pardesi KR, Chopade BA (2007) Species distribution and physiological characterization of Acinetobacter genospecies from healthy human skin of tribal population in India. Indian J Med Microbiol 25(4):336–345. https://doi.org/10.4103/0255-0857.37335 Zhang L, Xu Z (2008) Assessing bacterial diversity in soil: a brief review. Journal of Soils and Sediments 8(6):379–388 Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3834802","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266140191,"identity":"461bff20-3dcb-4a15-9f16-50bbba16863d","order_by":0,"name":"Rochelle Fernandez","email":"","orcid":"","institution":"Bharathiar University","correspondingAuthor":false,"prefix":"","firstName":"Rochelle","middleName":"","lastName":"Fernandez","suffix":""},{"id":266140192,"identity":"056f72f8-5c67-478e-8e9d-b056811f4ca5","order_by":1,"name":"Prabagaran Solai Ramatchandirane","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYHACNijNfAxCHwAjorSwpZGshccMrgUvMJc+/OzBj192+fyze7495qlhkOO7kcB4uACPFsu+NHPD3r5kyxl3zm435jnGYCx5I4Hh8Aw8WgzOMJhJ8PYwGzDcyN0mzdvAkLgBpIUHrxb2b5J/e+oN5G/kPANpqSdCC4+ZNM+PwwYGN3LYQFoSDAhpsezhKTeWbThuYHgD6Kk5xyQMZ5552IBXizkP+7aHb/5UG8jdSH724E2NjTzf8eTDn/E6DEQwtsH5EiBuAx4NUC0Mf/CqGQWjYBSMgpEOAJYDTljoVEdxAAAAAElFTkSuQmCC","orcid":"","institution":"Bharathiar University","correspondingAuthor":true,"prefix":"","firstName":"Prabagaran","middleName":"Solai","lastName":"Ramatchandirane","suffix":""}],"badges":[],"createdAt":"2024-01-04 14:33:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3834802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3834802/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49450579,"identity":"738985ad-9c75-4a62-bb04-b5cbd8e703b5","added_by":"auto","created_at":"2024-01-11 04:37:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":381637,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the study area\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/732ff8e2a28fc45a468b0db7.png"},{"id":49450581,"identity":"86121715-d508-4336-b2af-a50b2854fa40","added_by":"auto","created_at":"2024-01-11 04:37:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47546,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing the abundance of bacteria in different Shola samples\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/25849e6494bc6189080e3b8e.png"},{"id":49451224,"identity":"9d583a20-1261-40eb-91f2-d08b1c958a0a","added_by":"auto","created_at":"2024-01-11 04:45:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146234,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed based on the partial 16S rRNA gene sequence from the bacterial isolates of Shola Forest using neighbor-joining method with the aid of MEGA 7.0 software. Bootstrap values (expressed as percentage of 1000 replications) greater than 50 % are given at nodes. \u003cem\u003eMethanothermococcus thermolithotrophicus \u003c/em\u003ewas taken as an out group. The scale bar represents 0.05 substitutions per alignment position in the tree\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/15c59101e0892bc3b7f45d7a.png"},{"id":49450583,"identity":"3f5697c1-1fdf-4320-a229-9a3e8e00ea50","added_by":"auto","created_at":"2024-01-11 04:37:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":242747,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of different species among the sampling regions of Shola Forest\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/b80739c16f4f4bda54e8a244.png"},{"id":49450580,"identity":"9f3fe476-88fd-4861-b068-f43ebded8aa7","added_by":"auto","created_at":"2024-01-11 04:37:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124574,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative assay for the determination of enzyme activity. The values are represented as mean ± SE (n = 3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/7eba623670cc93eda050ab36.png"},{"id":50575347,"identity":"ee4f4e7f-7a59-46a4-962d-f43b43febc7a","added_by":"auto","created_at":"2024-02-02 17:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1288959,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/f1061fed-6413-43af-800a-75577b329e4d.pdf"},{"id":49450584,"identity":"4bc142df-6c9f-4ecc-b2f3-406cef308b9e","added_by":"auto","created_at":"2024-01-11 04:37:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3770653,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-3834802/v1/839975d9c539ccb097bf6f33.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacterial Diversity and Bioprospecting for enzyme among the Shola Forests of Idukki District, Kerala, India","fulltext":[{"header":"Introduction","content":"\u003cp\u003eForests engulf about 80% of terrestrial biodiversity that are essential to maintain a healthy and balanced ecosystem around the world. They are the major ecological niche that house diverse community of native and introduced organisms that serve as a significant resource for mankind. Though certain factors that determine the forest type include climate, soil type, topography, and elevation, classification of forests mainly depends upon their nature, composition, and its association with neighboring environment. Shola is one such typical tropical montane forest, ancient one with valleys and edges where there exist extremely diverse grasslands in between (Bunyan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSholas are covered with grasslands in the mountain tops characterized by frost and fire-resistant grass species while the depressions between the mountains are covered with patches of stunted tropical forest vegetation. Together they form an extraordinary ecosystem which serve as a rich repository of rare and invaluable biodiversity (Robin and Nandini \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Such an unique ecosystem is native only to the Southern Western Ghats of India at elevations over 1,900 to 2,220 metres in the states of Karnataka, Kerala, and Tamil Nadu. Climate has a greater influence on the montane ecosystem, which gets colder as altitude rises (Perry \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Shola Forests has very high-water retention ability which means that the habitat absorbs a lot of water and lets it out through a very long period of time. Most of the South Indian rivers originate from such Shola habitat. These forests are distributed according to their elevation in western ghats where northern region is denser when compared to the southern region. Interestingly these forests house numerous endemic species due to its unique climatic conditions and high isolation. These forests are rightly called nature\u0026rsquo;s water towers that play a vital role in sustaining life of the plains.\u003c/p\u003e \u003cp\u003eMicrobial diversity plays a dominant role in the maintenance of ecosystem and deserves greater attention since extensive knowledge on microbial taxonomy and physiology will help gain better insights into the microbial world and in identifying potential novel species (Panizzon et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among the forest ecosystem, soil has a substantial role in facilitating essential nutrient and energy flow patterns (Canadell and Raupach \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) where soil bacteria reside as driving force for all these ecological processes (Bardgett et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Though bacteria is minimally explored in forest compared to the flora and fauna, they depict an integral part of ecological studies and microbial community. \u003cem\u003eAcidobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e are the most abundant bacterial phyla reported in most soils (Lauber et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Bacteria in forest soils actively respond to climate change (Llado et al. 2017). Many environmental factors in forest ecosystem such as carbon: nitrogen ratio, soil pH, temperature, minerals, nutrients have a substantial effect on the microbial community (Janssens et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Generally, the wealth of bacterial phylum and genus varies among the soil types based on vegetation too. Even though forest ecosystem bacteria play a major part in fulfilling various essential functions like decomposition of organic matter, involvement in nitrogen cycle processes, and so on, still the complete role of bacteria in such ecosystems is to be explored and understood.\u003c/p\u003e \u003cp\u003eDespite considerable number of studies were undertaken, exploration of total bacterial diversity in Shola Forests are very limited. Detailed studies on the microbial composition and diversity in soils of Sholas are important for a better understanding of their ecological role that can solve several environmental cues. To investigate variations in bacterial communities, three Shola National Parks in the Idukki district of Kerala, India were studied.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSite description and Sampling\u003c/h2\u003e\n \u003cp\u003eBacterial diversity of soil, water, and slurry samples were investigated in three Shola National Parks viz., Pampadum, Mathikettan, and Anamudi of Idukki district, Kerala, India (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Pampadum shola is the smallest national park in Kerala located in the Vattavada panchayat of Devikulam Taluk covering an area of 11.75 km\u003csup\u003e2\u003c/sup\u003e, in the eastern part of Southern Western Ghats. The altitude ranges between 1600–2400 msl. Mathikettan shola park is spread over 12.82 km\u003csup\u003e2\u003c/sup\u003e in Poopara village of Udumbanchola Taluk. The altitude of the region ranges from 1200–1984 msl. Anamudi shola park comprises three shola reserve forests – Idivara, Pullarda, and Mannavan covering an area of over 42.68 km\u003csup\u003e2\u003c/sup\u003e. The Park is located in the Marayoor village of Devikulam Taluk and the elevation ranges from 2152 to 2305 msl. The location details of each sampling sites are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eA total of 9 samples i.e., soil, water and slurry were collected from each Shola National Park. The soil samples were collected at a depth of 2–5 cm that were stored in air tight polyethylene bags, while slurry and water samples were collected in sterilized falcon tubes. All the samples were maintained at room temperature until it reached the laboratory.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLocation details of the sampling sites\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePampadum\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMathikettan\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAnamudi\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLatitude (N)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10º 7’ 37.1928”\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9º 59’ 5.694”\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10º 12’ 7.1244”\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLongitude (E)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77º 15’ 29.4588”\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77º 14’ 46.4568”\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77º 8’ 52.8612”\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eElevation (msl)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2152\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eIsolation of bacteria\u003c/h2\u003e\n \u003cp\u003eFour different mediums viz., Nutrient agar (NA), Kings medium B base (Kings B), Tryptone soya broth (TSB) and Luria Bertani agar (LB) were selected for the cultivation of microbes. Isolation of bacteria was carried out by standard serial dilution plate technique. One gram of soil, 1ml of slurry and water samples were transferred to 9ml sterile distilled water and well shaken. Different aqueous dilutions ranging from 10\u003csup\u003e− 1\u003c/sup\u003e to 10\u003csup\u003e− 15\u003c/sup\u003e were prepared and 0.1ml of appropriate dilutions were spread plated on all four medium and plates were incubated at 37ºC for about 24 hrs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphological characteristics of Bacterial isolates\u003c/h2\u003e\n \u003cp\u003eAfter incubation, on the basis of colony morphology distinct microbial colonies were counted and subsequently pure cultured. The colony color, shape (punctiform, circular, filamentous, irregular, rhizoid), elevation (flat, raised, convex, pulvinate, umbonate) and margin (entire, undulate, lobate) were noted. Isolated pure cultures of bacteria were maintained in solidified agar plates at 4ºC. All the isolates were named alphanumerically and maintained in glycerol stocks.\u003c/p\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003eDiversity indices\u003c/h2\u003e\n \u003cp\u003eA range of diversity indices particularly Shannon index, the evenness indices derived from it, Simpson’s dominance index and its equitability index have been derived with bacterial communities.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"435\" height=\"98\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere \u003cstrong\u003epi\u003c/strong\u003e is the proportion of individuals of one particular species divided by the total number of individuals, \u003cstrong\u003eln\u003c/strong\u003e is the natural log, \u003cstrong\u003eS\u003c/strong\u003e is the total number of species and ∑ is sum from species 1 to species S. Shannon equitability (E\u003csub\u003eH\u003c/sub\u003e) is calculated by dividing Shannon diversity index by natural logarithm of species richness ln (S).\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"352\" height=\"94\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eWhere \u003cstrong\u003epi\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e is the square of the proportion of individuals of one particular species divided by the total number of individuals, \u003cstrong\u003eS\u003c/strong\u003e is the total number of species and ∑ is sum from species 1 to species S. Simpson equitability (E\u003csub\u003eD\u003c/sub\u003e) is calculated by the formula 1-D. Equitability takes a value between 0 and 1, with 1 being complete evenness.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDNA Extraction and PCR amplification\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eGenomic DNA was isolated from bacterial cultures grown in nutrient broth overnight, using CTAB (Cetyl trimethyl ammonium bromide) method. The 16S rRNA genes were PCR amplified with a universal primer pairs 27F and 1492R. The PCR master mix consisted of EmeraldAmp GT PCR master mix, 2µl DNA (100 ng), 1µl of each primer (10 pmol), and sterile distilled water to bring the total volume to 20µl. PCR amplification involved an initial denaturation at 96ºC for 5 min, then 35 cycles involving denaturation at 94ºC for 1 min, annealing at 48.5ºC for 1 min, extension at 72ºC for 2 min followed by a final extension at 72ºC for 10 min.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eSequencing and Phylogenetic tree construction\u003c/h2\u003e\n \u003cp\u003eSequence analysis was performed using an ABI PRISM Big dye terminator cycle sequencing ready reaction kit (Applied Biosystems) and an ABI 3500 Genetic Analyzer (Applied Biosystems). The sequences of the 16S rRNA gene obtained were aligned by using DNA lasergene software and were compared against the sequences available from GenBank using BLASTN program. The 16S rRNA gene sequences of those strains that represent the closest neighbour of each isolate was retrieved. The sequences were aligned using CLUSTALW and phylogenetic tree was constructed after pairwise and multiple sequence alignment using the neighbor-joining method. MEGA 7.0 software was used for the analysis. Bootstrap analysis was performed employing 1000 replicate data sets to evaluate the confidence limits of the branching. Sequence data was deposited at the National Center for Biotechnology Information (NCBI), GenBank (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eExtracellular enzyme screening\u003c/h2\u003e\n \u003cp\u003eThe isolates were screened for the production of amylase, protease and cellulase enzyme on appropriate agar medium supplemented with respective substrates; 0.2% starch for amylase, 0.5% skim milk for protease and 1% carboxymethyl cellulose for cellulase. The plates were incubated at 37ºC for 24 hrs. The potential isolates were identified by the formation of clear zones around the colonies. The selected isolates were grown in appropriate broth conferred with respective substrates and incubated at 37ºC overnight with continuous shaking at 180 rpm. The crude extract was prepared by centrifuging the overnight culture at 10,000 rpm for 10 mins. The supernatant was filtered and was used as an enzyme source for assay.\u003c/p\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003eDetermination of enzyme activity\u003c/h2\u003e\n \u003cp\u003eQualitative assay was performed by well diffusion method. The agar medium was prepared along with respective substrates for amylase, protease and cellulase enzymes. Precisely 100 µl of the crude extracts was loaded in the wells and the plates were incubated overnight at 37ºC. Based on the diameter of the clear zone formation, potential isolates were selected for further assay. Quantitative assay was carried out spectrophotometrically in triplicates where optical densities were plotted to determine enzyme activity. Protease activity was determined by Folin’s phenol method (Tsuchida et al. \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e), whereas amylase (Pokhrel et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) and cellulase activity (Miller \u003cspan class=\"CitationRef\"\u003e1959\u003c/span\u003e) was assayed by DNS (Dinitrosalicylic acid) method.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAbundance of Bacteria in Shola samples\u003c/h2\u003e \u003cp\u003eThe diversity of bacteria from soil, slurry, and water samples of the Shola forests of Kerala were enumerated through serial dilution plating technique. Four different media were employed viz., Nutrient agar, Kings medium B base, Tryptone soya broth, and Luria Bertani agar. Totally 58 strains (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were isolated from all the 9 collected samples viz., 21 strains from Pampadum (soil: 10; slurry: 8; water: 3), 24 strains from Mathikettan (soil: 3; slurry: 9; water: 12) and 13 strains from Anamudi forest (soil: 4; slurry: 7; water: 2).\u003c/p\u003e \u003cp\u003eThe colony forming units were recorded for the dilutions from 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e. Among the soil samples, the highest bacterial population was recorded in Mathikettan (NA) where the values ranged from 132 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e to 311 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/g. Lowest density was noticed in Pampadum (LB), with values ranging from 27 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e to 162 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/g. Similarly in slurry samples, maximum population was observed in Mathikettan (LB), with values ranging from 55 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e to 343 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/ml, while the lowest population was from Anamudi (Kings B) with values ranging between 29 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e to 153 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/ml. In case of water samples, Mathikettan (TSB) exhibited higher bacterial abundance. The values ranged from 99 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e to 353 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e cfu/ml and Anamudi (LB) displayed least abundance, with values ranging from 48 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e to 341 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e cfu/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMorphological characteristics of bacterial isolates\u003c/h2\u003e \u003cp\u003eOut of the 58 strains isolated, 32 isolates (55.17%) showed circular configuration and 24 (41.37%) were irregular while 2 (3.45%) exhibited punctiform configuration. Among Pampadum samples, soil and slurry were observed to be irregular compared to water which was predominantly circular. While the configuration of the Mathikettan soil isolates was circular, slurry, and water were predominantly irregular. In case of Anamudi forest, circular configuration was found to be dominating in all three sample types. Pale white colour isolates were observed mainly (Pampadum: 52.3%; Mathikettan: 33.3%; Anamudi: 23.1%), followed by white, transparent, beige, orange, yellow, and pink colour. Most of the isolates had raised colonies (47.6%; 33.3%; 23.1%) and entire margin (42.9%; 45.8%; 84.6%) in Pampadum, Mathikettan and Anamudi respectively. Distribution of Gram negative rod, Gram positive rod, and Gram positive cocci was found to be 56.89%, 39.65%, and 3.45% respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eShannon and Simpson diversity indices\u003c/h2\u003e \u003cp\u003eShannon \u0026ndash; Wiener diversity (H), its evenness (E\u003csub\u003eH\u003c/sub\u003e), and Simpson\u0026rsquo;s index (D) and its equitability (E\u003csub\u003eD\u003c/sub\u003e) were studied. Shannon index increases as both richness and evenness of the community increases. Simpson\u0026rsquo;s index which is a measure of dominance increases as diversity (evenness) decreases. The lesser the value of D, the more the diversity, the greater the value of E\u003csub\u003eD\u003c/sub\u003e, the more the diversity. In the present study, the H index ranged from 0.32 to 1.81 and E\u003csub\u003eH\u003c/sub\u003e ranged from 0.29 to 1.00. The D index ranged from 0.21 to 0.85 and E\u003csub\u003eD\u003c/sub\u003e ranged from 0.15 to 0.79. From the acquired results, it can be concluded that the highest and lowest values of diversity indices are recorded from soil and water samples of Pampadum respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of the 16S rRNA gene sequence\u003c/h2\u003e \u003cp\u003eThe 16S rRNA gene of the selected morphologically divergent bacterial isolates was sequenced. The nearest phylogenetic neighbour of all the isolates were identified by BLAST analysis of the 16S rRNA gene sequence from GenBank database. The closest homologues sequences were retrieved and phylogenetic tree was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The study revealed 9 different genera namely \u003cem\u003eAcinetobacter, Bacillus, Brevundimonas, Chryseobacterium, Enterobacter, Exiguobacterium, Pseudomonas, Rothia\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e belonging to four divisions viz., \u003cem\u003eActinobacteria, Bacteroidetes, Firmicutes, Proteobacteria\u003c/em\u003e. Bacterial diversity was found to be maximum in the division of \u003cem\u003eProteobacteria\u003c/em\u003e in which 24 strains were among the genus of \u003cem\u003eAcinetobacter, Brevundimonas, Enterobacter\u003c/em\u003e, and \u003cem\u003ePseudomonas.\u003c/em\u003e This was followed by the division \u003cem\u003eFirmicutes\u003c/em\u003e with 17 strains spreading among the genus \u003cem\u003eBacillus, Exiguobacterium\u003c/em\u003e, and \u003cem\u003eStaphylococcus.\u003c/em\u003e The remaining 2 strains each were among the division \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e belonging to the genus \u003cem\u003eRothia\u003c/em\u003e and \u003cem\u003eChryseobacterium\u003c/em\u003e correspondingly.\u003c/p\u003e \u003cp\u003eIn Pampadum forest, the bacterial strains predominantly were among the division \u003cem\u003eFirmicutes\u003c/em\u003e and the species include \u003cem\u003eBacillus paralicheniformis\u003c/em\u003e (PS1), \u003cem\u003eBacillus\u003c/em\u003e sp. (PS6, PSL23), \u003cem\u003eBacillus subtilis\u003c/em\u003e (PS15), \u003cem\u003eBacillus nakamurai\u003c/em\u003e (PSL25), \u003cem\u003eBacillus vallismortis\u003c/em\u003e (PSL30), \u003cem\u003eBacillus megaterium\u003c/em\u003e (PSL31), \u003cem\u003eStaphylococcus\u003c/em\u003e sp. (PSL39) and \u003cem\u003eBacillus xiamenensis\u003c/em\u003e (PW54). Then comes the division \u003cem\u003eProteobacteria\u003c/em\u003e which comprises \u003cem\u003ePseudomonas songnenensis\u003c/em\u003e (PS3), \u003cem\u003ePseudomonas\u003c/em\u003e sp. (PS8, PSL17) \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (PSL27, PW52) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PW50). Phyla \u003cem\u003eProteobacteria\u003c/em\u003e was prevalent in Mathikettan forest samples and involved namely species of \u003cem\u003ePseudomonas\u003c/em\u003e sp. (MS1, MSL23, MW37), \u003cem\u003ePseudomonas songnenensis\u003c/em\u003e (MS5, MSL7, MW53), \u003cem\u003eEnterobacter cloacae\u003c/em\u003e (MSL8), \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (MSL34, MW48) and \u003cem\u003eBrevundimonas vesicularis\u003c/em\u003e (MW38). This was followed by the Phyla \u003cem\u003eFirmicutes\u003c/em\u003e that included \u003cem\u003eBacillus aerius\u003c/em\u003e (MSL9), \u003cem\u003eBacillus\u003c/em\u003e sp. (MSL14), \u003cem\u003eBacillus xiamenensis\u003c/em\u003e (MSL33), \u003cem\u003eExiguobacterium acetylicum\u003c/em\u003e (MW35) and \u003cem\u003eExiguobacterium\u003c/em\u003e sp. (MW40). The Phyla \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e were only observed in the Anamudi forest samples that comprised the species \u003cem\u003eRothia terrae\u003c/em\u003e (ASL30) and \u003cem\u003eChryseobacterium montanum\u003c/em\u003e (ASL20).\u003c/p\u003e \u003cp\u003eThe dominant Phyla in Anamudi samples were \u003cem\u003eProteobacteria\u003c/em\u003e with species \u003cem\u003ePseudomonas\u003c/em\u003e sp. (AS4), \u003cem\u003ePseudomonas songnenensis\u003c/em\u003e (AS8, ASL28, AW44), \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (AS11, ASL36, AW41) and \u003cem\u003eAcinetobacter dispersus\u003c/em\u003e (ASL31). The Phyla \u003cem\u003eFirmicutes\u003c/em\u003e included \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (AS6), \u003cem\u003eExiguobacterium sibiricum\u003c/em\u003e (ASL19), and \u003cem\u003eBacillus\u003c/em\u003e sp. (ASL23). More diverse bacterial strains were observed in the Anamudi Shola samples with four divisions (\u003cem\u003eProteobacteria, Firmicutes, Bacteroidetes, Actinobacteria\u003c/em\u003e) and 7 genera (\u003cem\u003ePseudomonas, Acinetobacter, Staphylococcus, Exiguobacterium, Bacillus, Rothia, Chryseobacterium\u003c/em\u003e). While Mathikettan and Pampadum samples showed 2 divisions (\u003cem\u003eProteobacteria, Firmicutes\u003c/em\u003e) with five (\u003cem\u003ePseudomonas, Enterobacter, Brevundimonas, Bacillus, Exiguobacterium\u003c/em\u003e) and three (\u003cem\u003eBacillus, Staphylococcus, Pseudomonas\u003c/em\u003e) genera respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e and \u003cem\u003ePseudomonas songnenensis\u003c/em\u003e were found repeatedly in almost all samples of Shola Forest. Overall, the Phyla \u003cem\u003eProteobacteria\u003c/em\u003e was observed to be imperious in the Shola Forest samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme activity\u003c/h2\u003e \u003cp\u003eAll the identified bacterial isolates were screened for the enzyme production viz., amylase, protease, and cellulase. Among them, 38 isolates showed positive activity for amylase, followed by 32 and 16 isolates for cellulase and protease respectively by the formation of hydrolyzed zones. While members of phylum \u003cem\u003eProteobacteria\u003c/em\u003e showed predominant activity for amylase and cellulase, phylum \u003cem\u003eFirmicutes\u003c/em\u003e was good in protease activity. Nine isolates, namely \u003cem\u003eBacillus subtilis\u003c/em\u003e (PS15), \u003cem\u003eBacillus nakamurai\u003c/em\u003e (PSL25), \u003cem\u003eBacillus megaterium\u003c/em\u003e (PSL31), \u003cem\u003eBacillus\u003c/em\u003e sp. (PS6, PSL23, ASL23), \u003cem\u003ePseudomonas\u003c/em\u003e sp. (MW37) and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (MW48, AS11) exhibited positive enzyme activity for all the three enzymes. Three isolates, specifically \u003cem\u003eEnterobacter cloacae\u003c/em\u003e (MSL8), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (AS6) and \u003cem\u003eRothia terrae\u003c/em\u003e (ASL30) showed negative growth for all enzymes. On the basis of the primary screening, the best isolates capable of producing the enzymes were selected for further study.\u003c/p\u003e \u003cp\u003eThe secondary screening was carried out by well diffusion method with a standard strain \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC27853) (Marathe et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Raju and Divakar \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gunavathy and Boominathan \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Based on the clear hydrolyzed zones formed, potent strains namely PS1, PSL39, PW50, MSL14, MW50, ASL23 for protease, PS8, MSL14, MW35, MW40, ASL31, ASL36 for amylase and PSL25, PW54, MS5, MSL9, MSL33, ASL23 for cellulase were taken for further analysis. All the selected isolates showed almost equal and greater hydrolyzed zones than the standard (Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003eOut of the selected isolates, the strain \u003cem\u003eAcinetobacter dispersus\u003c/em\u003e (ASL31) found to have maximum crude amylase activity (62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 U/ml). The isolate \u003cem\u003eBacillus paralicheniformis\u003c/em\u003e (PS1) and \u003cem\u003eBacillus aerius\u003c/em\u003e (MSL9) possessed maximum activity for protease (49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 U/ml) and cellulase (86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 U/ml) enzymes respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study was carried out with an objective to report diversity of bacteria in few Shola forests of Kerala along the Western Ghats. Sahyadri hills are the lifeline of the Indian ecosystem which spans from the tip of Kanyakumari to Gujarat covering the states of Kerala, Tamil Nadu, Karnataka, Goa, Maharashtra, and Gujarat. Due to human activities over centuries, most of the pristine areas were disturbed except certain pockets like the Sholas. Among them, the remarkable ones are located in Idukki, Wayanad, Palakkad and Thrissur districts of Kerala, among which the present study mainly focused only on the Sholas of Idukki.\u003c/p\u003e \u003cp\u003eSampling was done in forest soil, water streams, and marshy areas from Pampadum, Mathikettan, and Anamudi Shola national park. Pampadum Shola is the smallest park covering an area of 11.75 km\u003csup\u003e2\u003c/sup\u003e at 1600\u0026ndash;2000 msl, above which grasslands prevail with small forest patches distributed evenly. Mathikettan Shola houses evergreen forests, moist deciduous forests, shola grasslands, and semi-evergreens covering an area of about 12.82 km\u003csup\u003e2\u003c/sup\u003e at an elevation ranging between 1200\u0026ndash;1984 msl. Anamudi forest is covered with West coast tropical evergreen, Southern hilltop tropical vegetation found at higher altitudes of about 2305 msl. The Park has one of the largest shola forest ecosystems in the country with unique habitats, covering an area of about 42.68 km\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenerally, such culture-based approaches underestimate the total bacterial number, as the nutritional requirements of every prokaryote present in a particular sample cannot be met, even though it contains all essential nutrients required for the physiological growth of the isolates (Orphan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Thus, these approaches are highly dependent on the medium used for isolation and incubation conditions maintained during cultivation (Jackson et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Culturing in more than one medium will certainly increase the cultivation of diverse types of bacteria than screening through a single medium. Hence four different media viz., Nutrient agar, Luria Bertani agar, Tryptone soya broth, and Kings medium B base were employed to isolate bacteria.\u003c/p\u003e \u003cp\u003eBacterial colonies exhibit distinct characteristics like size, colour, shape, and texture, which primarily vary amid different species (Mamou et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A total of 58 morphologically distinct strains were isolated. Majority of the isolates were observed to be pale white while few of them were pigmented. Circular configuration with raised colonies was predominant among the isolated strains. The bacterial load was estimated and compared among the samples, where a significantly higher population of bacteria was noticed in Mathikettan samples. The values were 311 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/g for soil, 343 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e cfu/ml for slurry and 353 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e cfu/ml for water.\u003c/p\u003e \u003cp\u003eThe Gram stain is a significant morphological clue among the initial characterization and classification of bacteria (Thairu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among the total isolates, Gram negative rod (56.89%) was found to be prevalent followed by Gram positive rod (39.65%) and Gram positive cocci (3.45%) whose distribution is similar to a study conducted in the soils of grassland ecosystem of the Western Ghats that reported Gram negative bacteria to be more diverse (Rinoy \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The isolates were analyzed for richness, evenness, and diversity indices. The Shannon \u0026ndash; Wiener (H) and its evenness (E\u003csub\u003eH\u003c/sub\u003e) ranged from 0.32 to 1.81 and 0.29 to 1.00. The Simpsons index (D) and its equitability (E\u003csub\u003eD\u003c/sub\u003e) ranged from 0.21 to 0.85 and 0.15 to 0.79. Maximum and minimum diversity was recorded for the samples of Pampadum soil and water. The bacterial diversity may be associated with the forest types since vegetations are known to affect soil microbial diversity and community structures. A previous study conducted in the tropical Eastern Himalaya reported that there was a fall in bacterial diversity with rising altitudes (Lyngwi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 16S rRNA gene sequence is the most common housekeeping genetic marker employed to study bacterial phylogeny and taxonomy due to its conserved nature in almost all Eubacteria and Archaebacteria (Janda and Abbott \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The 16S rRNA gene of the isolates was sequenced and their taxonomic affiliation was ascertained. The analysis revealed the coverage of four divisions namely \u003cem\u003eActinobacteria, Bacteroidetes, Firmicutes\u003c/em\u003e, and \u003cem\u003eProteobacteria\u003c/em\u003e distributed among 9 genera viz., \u003cem\u003eAcinetobacter, Bacillus, Brevundimonas, Chryseobacterium, Enterobacter, Exiguobacterium, Pseudomonas, Rothia\u003c/em\u003e and \u003cem\u003eStaphylococcus.\u003c/em\u003e Five phyla, mainly \u003cem\u003eAcidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e were reported to be abundant in most soils (Lauber et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The phyla \u003cem\u003eProteobacteria\u003c/em\u003e holds maximum bacterial diversity with 24 strains encompassing \u003cem\u003eAcinetobacter, Brevundimonas, Enterobacter\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e which as reported in previous studies. The \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eAcidobacteria\u003c/em\u003e were the most abundant phyla in the soil samples of temperate deciduous broadleaved forest and a tropical mountain rainforest of China (Wei et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It has been reported that members of \u003cem\u003eProteobacteria\u003c/em\u003e are well distributed in almost all forest types (Zhang and Xu \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Nampoothiri et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that \u003cem\u003eProteobacteria\u003c/em\u003e was the most abundant phylum in the water samples of the Western Ghats region, followed by \u003cem\u003eFirmicutes, Actinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes.\u003c/em\u003e The diversity and composition of soil bacterial communities in the Forest montane region of Northeast China resulted in a high abundance of the phyla \u003cem\u003eProteobacteria\u003c/em\u003e (Wu et al. 2019).\u003c/p\u003e \u003cp\u003eEnvironmental factors have a significant role in determining the bacterial community structure of forest soils (Xia et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Soil bacterial community composition and diversity are influenced by soil properties, vegetation types, nutrient availability, and plant diversity (Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Naether et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) among which soil pH is considered an essential factor (Qiu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Generally, Shola soils are reported to be slightly acidic ranging between 5.0 and 6.4. \u003cem\u003eAcidobacteria\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e are abundant in acidic soils (Shen et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), while the abundances of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e rise with an increasing pH (Jeanbille et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eProteobacteria\u003c/em\u003e was found to be plentiful in the acidic soils from the subtropical montane forest of Taiwan, among which \u003cem\u003eβ-Proteobacteria\u003c/em\u003e was abundant, then \u003cem\u003eα-Proteobacteria\u003c/em\u003e and \u003cem\u003eγ-Proteobacteria\u003c/em\u003e, while the low proportion of \u003cem\u003eAcidobacteria\u003c/em\u003e in the study was considered to be as a result of high soil moisture and anaerobic microhabitats (Lin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Acidic soils of coniferous forests also mainly harbor \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eAcidobacteria\u003c/em\u003e (Baldrian et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Other predominant phyla in the present study were \u003cem\u003eFirmicutes\u003c/em\u003e with 17 strains, which spreads among the genera \u003cem\u003eBacillus, Exiguobacterium\u003c/em\u003e, and \u003cem\u003eStaphylococcus.\u003c/em\u003e This was followed by \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e with genus \u003cem\u003eRothia\u003c/em\u003e and \u003cem\u003eChryseobacterium\u003c/em\u003e respectively.\u003c/p\u003e \u003cp\u003eEnzymes form an important component of forest ecosystems because they play a significant part in catalyzing reactions essential for organic matter decomposition and nutrient cycling (Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the present study, representative isolates were screened for their hydrolytic enzyme activity viz., amylase, protease, and cellulase. Out of 43 isolates screened, 9 strains namely \u003cem\u003eBacillus subtilis\u003c/em\u003e (PS15), \u003cem\u003eBacillus nakamurai\u003c/em\u003e (PSL25), \u003cem\u003eBacillus megaterium\u003c/em\u003e (PSL31), \u003cem\u003eBacillus\u003c/em\u003e sp. (PS6, PSL23, ASL23), \u003cem\u003ePseudomonas\u003c/em\u003e sp. (MW37) and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (MW48, AS11) showed positive activity for all the three enzymes. \u003cem\u003eBacillus\u003c/em\u003e strains are considered as multifunctional microorganisms and they are one of the principal sources of enzymes from bacterial origin used in industry (Latorre et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, three strains specifically \u003cem\u003eEnterobacter cloacae\u003c/em\u003e (MSL8), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (AS6) and \u003cem\u003eRothia terrae\u003c/em\u003e (ASL30) exhibited negative results for all the enzymes, which is contrary to the earlier studies that reported \u003cem\u003eEnterobacter cloacae\u003c/em\u003e and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e as enzyme producers (Lokapirnasari et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vandecandelaere et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These differences in the enzyme potential may be due to the epigenetic influence of the environment. Taxonomic description of \u003cem\u003eRothia terrae\u003c/em\u003e, a rare soil bacterium isolated from the subtropical fields of Tainan County, Taiwan was described in 2008 by Chou et al. Later Sariturk et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), investigated it and reported that it does not produce amylase and cellulase enzyme but has a potential of beta galactosidase activity. This is the only actinobacteria identified in this entire study, which is a cream white coloured, aerobic, Gram positive bacteria that could be distinguished from other \u003cem\u003eRothia\u003c/em\u003e species through unique phenotypic characteristics.\u003c/p\u003e \u003cp\u003eEnzyme assays were performed to determine their activity which provides significant details on mechanisms of enzyme catalysis and interactions with substrates. On the basis of primary and secondary screening, potential enzyme producers were selected, and quantitative assay was performed spectrophotometrically (Fersht \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The strains \u003cem\u003eAcinetobacter dispersus\u003c/em\u003e (ASL31), \u003cem\u003eBacillus paralicheniformis\u003c/em\u003e (PS1), and \u003cem\u003eBacillus aerius\u003c/em\u003e (MSL9) possessed maximum activity for amylase (62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 U/ml), protease (49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 U/ml) and cellulase (86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 U/ml) enzymes respectively. Studies have been reported earlier, showing the potential of \u003cem\u003eAcinetobacter\u003c/em\u003e sp. to produce amylase enzyme (Yavankar et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Onishi and Hidaka \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Cellulase and protease production has been previously reported for \u003cem\u003eBacillus\u003c/em\u003e sp. (Oke et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Suganthi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Masi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study was intended to provide information on the diversity of bacteria in the Pampadum, Mathikettan, and Anamudi Sholas situated in Idukki district of Kerala, which resulted in similar reports with the earlier studies. However, vast bacterial diversity studies have been proposed in the Southern Western Ghats, but studies particularly in Shola forests are limited. Studies on microbial diversity of such unique habitats open up to vast areas of the microbial world towards such exploration. Such diversity does not restrict to microorganism per se but also extended to their metabolic activities besides their interactions with other microbes, plants, and animals. Furthermore, the results also throw light on the ability of these isolates to produce enzymes that are of great importance in various fields, as microbial enzymes are acquiring much attention with the rapid development of enzyme technology and have been extensively studied for its application in different industries and therapeutics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe authors would like to acknowledge Kerala Forest Department for providing permission and required amenities for sample collection from the forest. The authors are grateful to University Grant Commission (UGC, Govt of India) for the support provided to establish infrastructure in the Department of Biotechnology grant vide UGC/SAP/No.F.3-20/2013, through Special Assistance Program (SAP) and Department of Science and technology (DST) towards Fund for Improvement of S\u0026amp;T Infrastructure in Universities and Higher Educational Institutions vide (FIST) and DST-PURSE phase-II vide BU/ DST-PURSE/2017/28.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConceptualization: [Rochelle Fernandez] and [Solai Ramatchandirane Prabagaran]; Methodology, data analysis and interpretation: [Rochelle Fernandez]; Drafting the article: [Rochelle Fernandez]; Supervision: [Solai Ramatchandirane Prabagaran]; All the authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data, materials and codes can be provided upon request\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eNo potential conflicts of interest are declared by the author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and Animal Rights\u0026nbsp;\u003c/strong\u003eNo human subjects or livestock were included in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaldrian P, Kolař\u0026iacute;k M, Stursov\u0026aacute; M, Kopeck\u0026yacute; J, Val\u0026aacute;\u0026scaron;kov\u0026aacute; V, Větrovsk\u0026yacute; T, Zifč\u0026aacute;kov\u0026aacute; L, Snajdr J, R\u0026iacute;dl J, Vlček C, Voř\u0026iacute;\u0026scaron;kov\u0026aacute; J (2012) Active and total microbial communities in forest soil are largely different and highly stratified during decomposition. ISME J 6(2):248\u0026ndash;258. https://doi.org/10.1038/ismej.2011.95 \u003c/li\u003e\n\u003cli\u003eBardgett RD, Freeman C, Ostle NJ (2008) Microbial contributions to climate change through carbon cycle feedbacks. The ISME Journal 2(8):805\u0026ndash;814. https://doi.org/10.1038/ismej.2008.58 \u003c/li\u003e\n\u003cli\u003eBunyan M, Bardhan S, Jose S (2012) The Shola (Tropical Montane Forest)-grassland ecosystem mosaic of Peninsular India: a review. Am J Plant Sci 3(11):1632\u0026ndash;1639. http://dx.doi.org/10.4236/ajps.2012.311198\u003c/li\u003e\n\u003cli\u003eCanadell JG, Raupach MR (2008) Managing forests for climate change mitigation. Science\u003cem\u003e \u003c/em\u003e320(5882):1456\u0026ndash;1457. https://doi.org/10.1126/science.1155458 \u003c/li\u003e\n\u003cli\u003eChou Y-J, Chou J-H, Lin K-Y, Lin M-C, Wei Y-H, Arun AB, Young C-C, Chen W-M (2008) \u003cem\u003eRothia terrae\u003c/em\u003e sp. nov. isolated from soil in Taiwan. Int J Syst Evol Microbiol 58(Pt 1):84\u0026ndash;88. https://doi.org/10.1099/ijs.0.65172-0 \u003c/li\u003e\n\u003cli\u003eFersht A (1999) Structure and mechanism in protein science: A guide to enzyme catalysis and protein folding. W.H. Freeman and Company, New York\u003c/li\u003e\n\u003cli\u003eGunavathy P, Boominathan M (2015) Isolation and molecular characterization of cellulase producing bacteria from soil of sacred grove, Puducherry, India. Int J Curr Microbiol App Sci 4(12):584\u0026ndash;590\u003c/li\u003e\n\u003cli\u003eJackson CR, Randolph KC, Osborn SL, Tyler HL (2013) Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables. BMC Microbiol 13(1):274. https://doi.org/10.1186/1471-2180-13-274 \u003c/li\u003e\n\u003cli\u003eJanda JM, Abbott SL (2007) 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45(9):2761\u0026ndash;2764. https://doi.org/10.1128/JCM.01228-07 \u003c/li\u003e\n\u003cli\u003eJanssens IA, Dieleman W, Luyssaert S, Subke J-A, Reichstein M, Ceulemans R, Ciais P, Dolman AJ, Grace J, Matteucci G, Papale D, Piao SL, Schulze E-D, Tang J, Law BE (2010) Reduction of forest soil respiration in response to nitrogen deposition. Nat Geosci 3(5):315\u0026ndash;322. https://doi.org/10.1038/ngeo844\u003c/li\u003e\n\u003cli\u003eJeanbille M, Bu\u0026eacute;e M, Bach C, C\u0026eacute;bron A, Frey-Klett P, Turpault MP, Uroz S (2016) Soil parameters drive the structure, diversity and metabolic potentials of the bacterial communities across temperate Beech Forest soil sequences. Microb Ecol 71(2):482\u0026ndash;493. https://doi.org/10.1007/s00248-015-0669-5 \u003c/li\u003e\n\u003cli\u003eLatorre JD, Hernandez-Velasco X, Wolfenden RE, Vicente JL, Wolfenden AD, Menconi A, Bielke LR, Hargis BM, Tellez G (2016) Evaluation and selection of \u003cem\u003eBacillus\u003c/em\u003e species based on enzyme production, antimicrobial activity, and biofilm synthesis as direct-fed microbial candidates for poultry. Front Vet Sci 3:95. https://doi.org/10.3389/fvets.2016.00095 \u003c/li\u003e\n\u003cli\u003eLauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol 75(15):5111\u0026ndash;5120. https://doi.org/10.1128/AEM.00335-09 \u003c/li\u003e\n\u003cli\u003eLin Y-T, Huang Y-J, Tang S-L, Whitman WB, Coleman DC, Chiu C-Y (2010) Bacterial community diversity in undisturbed perhumid montane forest soils in Taiwan. Microb Ecol 59(2):369\u0026ndash;378. https://doi.org/10.1007/s00248-009-9574-0 \u003c/li\u003e\n\u003cli\u003eLiu Z, Fu B, Zheng X, Liu G (2010) Plant biomass, soil water content and soil N:P ratio regulating soil microbial functional diversity in a temperate steppe: A regional scale study. Soil Biol Biochem 42(3):445\u0026ndash;450. https://doi.org/10.1016/j.soilbio.2009.11.027 \u003c/li\u003e\n\u003cli\u003eLlad\u0026oacute; S, L\u0026oacute;pez-Mond\u0026eacute;jar R, Baldrian P (2017) Forest soil bacteria: diversity, involvement in ecosystem processes, and response to global change. Microbiol Mol Biol Rev 81(2):e00063\u0026ndash;16. https://doi.org/10.1128/MMBR.00063-16 \u003c/li\u003e\n\u003cli\u003eLokapirnasari WP, Nazar DS, Nurhajati T, Supranianondo K, Yulianto AB (2015) Production and assay of cellulolytic enzyme activity of \u003cem\u003eEnterobacter cloacae\u003c/em\u003e WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia. Vet World 8(3):367\u0026ndash;371. https://doi.org/10.14202/vetworld.2015.367-371 \u003c/li\u003e\n\u003cli\u003eLyngwi NA, Koijam K, Sharma D, Joshi SR (2013) Cultivable bacterial diversity along the altitudinal zonation and vegetation range of tropical Eastern Himalaya. Rev Biol Trop 61(1):467\u0026ndash;490. https://doi.org/10.15517/rbt.v61i1.11141 \u003c/li\u003e\n\u003cli\u003eMamou G, Malli Mohan GB, Rouvinski A, Rosenberg A, Ben-Yehuda S (2016) Early developmental program shapes colony morphology in bacteria. Cell Rep 14(8):1850\u0026ndash;1857. https://doi.org/10.1016/j.celrep.2016.01.071 \u003c/li\u003e\n\u003cli\u003eMarathe SK, Vashistht MA, Prashanth A, Parveen N, Chakraborty S, Nair SS (2018) Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e obtained from sea water samples. J Genet Eng Biotechnol 16(1):39\u0026ndash;46. https://doi.org/10.1016/j.jgeb.2017.10.001 \u003c/li\u003e\n\u003cli\u003eMasi C, Gemechu G, Tafesse M (2021) Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent. Ann Microbiol 71:24. https://doi.org/10.1186/s13213-021-01631-x \u003c/li\u003e\n\u003cli\u003eMiller GL (1959) Use of dinitrosalycylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426\u0026ndash;428. http://dx.doi.org/10.1021/ac60147a030 \u003c/li\u003e\n\u003cli\u003eNaether A, Foesel BU, Naegele V, W\u0026uuml;st PK, Weinert J, Bonkowski M, Alt F, Oelmann Y, Polle A et al (2012) Environmental factors affect Acidobacterial communities below the subgroup level in grassland and forest soils. Appl Environ Microbiol 78(20):7398\u0026ndash;7406. https://doi.org/10.1128/AEM.01325-12 \u003c/li\u003e\n\u003cli\u003eNampoothiri KM, Ramkumar B, Pandey A (2013) Western Ghats of India: rich source of microbial biodiversity. J Sci Ind Res\u003cem\u003e \u003c/em\u003e72:617\u0026ndash;623\u003c/li\u003e\n\u003cli\u003eOke MA, Annuar MSM, Simarani K (2016) Enhanced endoglucanase production by \u003cem\u003eBacillus aerius\u003c/em\u003e on mixed lignocellulosic substrates. BioRes 11(3):5854\u0026ndash;5869\u003c/li\u003e\n\u003cli\u003eOnishi H, Hidaka O (1978) Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp. Can J Microbiol 24(9):1017\u0026ndash;1023. https://doi.org/10.1139/m78-169 \u003c/li\u003e\n\u003cli\u003eOrphan VJ, Taylor LT, Hafenbradl D, Delong EF (2000) Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Appl Environ Microbiol 66(2):700\u0026ndash;711. https://doi.org/10.1128/AEM.66.2.700-711.2000 \u003c/li\u003e\n\u003cli\u003ePanizzon JP, Pilz J\u0026uacute;nior HL, Knaak N, Ramos RC, Ziegler DR, Fiuza LM (2015) Microbial diversity: relevance and relationship between environmental conservation and human health. Braz Arch Biol Technol 58(1):137\u0026ndash;145. https://doi.org/10.1590/S1516-8913201502821 \u003c/li\u003e\n\u003cli\u003ePerry DA (1994) Forest ecosystems. Johns Hopkins University Press, London\u003c/li\u003e\n\u003cli\u003ePokhrel B, Wanjare P, Singh S, Purushotham B, Kumara SM (2013) Isolation, screening and characterization of promising-amylase producing bacteria from sewage enriched soil. International Journal of Advanced Biotechnology Research (4):286\u0026ndash;290\u003c/li\u003e\n\u003cli\u003eQiu S-L, Wang L-M, Huang D-F, Lin X-J (2014) Effects of fertilization regimes on tea yields, soil fertility, and soil microbial diversity. Chil J Agric Res 74(3):333\u0026ndash;339. https://doi.org/10.4067/S0718-58392014000300012 \u003c/li\u003e\n\u003cli\u003eRaju EVN, Divakar G (2013) Production of amylase by using \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolated from garden soil. International Journal of Advances in Pharmacy, Biology and Chemistry 2(1):50\u0026ndash;56\u003c/li\u003e\n\u003cli\u003eRinoy Varghese (2016) Diversity, distribution and physiological capabilities of culturable heterotrophic bacteria from grassland, shola, Myristica swamp and evergreen forest soils of Southern Western Ghats,\u003cem\u003e \u003c/em\u003ePhD thesis, Mahatma Gandhi University. http://hdl.handle.net/10603/194974 \u003c/li\u003e\n\u003cli\u003eRobin VV, Nandini R (2012) Shola habitats on sky islands: Status of research on montane forests and grasslands in southern India. Curr Sci 103(12):1427\u0026ndash;1437\u003c/li\u003e\n\u003cli\u003eSariturk S, Aygan A, Ekiz DO, Oruc O (2017) Production and partial characterization of \u0026beta;-Galactosidase from \u003cem\u003eRothia terrae\u003c/em\u003e J9. Journal of Molecular Biology and Biotechnology 1(2):24\u0026ndash;31\u003c/li\u003e\n\u003cli\u003eShen C, Xiong J, Zhang H, Feng Y, Lin X, Li X, Liang W, Chu H (2013) Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain. Soil Biol Biochem 57:204\u0026ndash;211. http://dx.doi.org/10.1016/j.soilbio.2012.07.013 \u003c/li\u003e\n\u003cli\u003eSuganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam KM (2013) Screening and optimization of protease production from a halotolerant \u003cem\u003eBacillus licheniformis\u003c/em\u003e isolated from saltern sediments. Journal of Genetic Engineering and Biotechnology 11(1):47\u0026ndash;52. https://doi.org/10.1016/j.jgeb.2013.02.002\u003c/li\u003e\n\u003cli\u003eThairu Y, Nasir IA, Usman Y (2014) Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan African Journal of Medicine 1(4):168\u0026ndash;174. https://doi.org/10.4103/2384-5147.144725 \u003c/li\u003e\n\u003cli\u003eTsuchida O, Yamagota Y, Ishizuka J, Arai J, Yamada J, Takeuchi M, Ichishima E (1986) An alkaline protease of an alkalophilic \u003cem\u003eBacillus\u003c/em\u003e sp. Curr Microbiol 14:7\u0026ndash;12. https://doi.org/10.1007/BF01568094\u003c/li\u003e\n\u003cli\u003eVandecandelaere I, Depuydt P, Nelis HJ, Coenye T (2014) Protease production by \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e and its effect on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e biofilms. Pathog Dis 70(3):321\u0026ndash;331. https://doi.org/10.1111/2049-632X.12133 \u003c/li\u003e\n\u003cli\u003eWang W, Page-Dumroese D, Lv R, Xiao C, Li G, Liu Y (2016) Soil enzyme activities in \u003cem\u003ePinus tabuliformis\u003c/em\u003e (Carriere) plantations in northern China. Forests 7(6):112. https://doi.org/10.3390/f7060112 \u003c/li\u003e\n\u003cli\u003eWei H, Peng C, Yang B, Song H, Li Q, Jiang L, Wei G, Wang K, Wang H, Liu S, Liu X, Chen D, Li Y, Wang M (2018) Contrasting soil bacterial community, diversity, and function in two forests in China. Front Microbiol 9:1693. https://doi.org/10.3389/fmicb.2018.01693 \u003c/li\u003e\n\u003cli\u003eWu S-J, Deng J-J, Yin Y, Qin S-J, Zhu W-X, Zhou Y-B, Wang B, Ruan H, Jin L (2020) Bacterial community changes associated with land use type in the forest montane region of Northeast China. Forests 11(1):40. https://doi.org/10.3390/f11010040 \u003c/li\u003e\n\u003cli\u003eXia Z, Bai E, Wang Q, Gao D, Zhou J, Jiang P, Wu J (2016) Biogeographic distribution patterns of bacteria in typical Chinese forest soils. Front Microbiol 7:1106. https://doi.org/10.3389/fmicb.2016.01106 \u003c/li\u003e\n\u003cli\u003eYavankar SP, Pardesi KR, Chopade BA (2007) Species distribution and physiological characterization of Acinetobacter genospecies from healthy human skin of tribal population in India. Indian J Med Microbiol 25(4):336\u0026ndash;345. https://doi.org/10.4103/0255-0857.37335 \u003c/li\u003e\n\u003cli\u003eZhang L, Xu Z (2008) Assessing bacterial diversity in soil: a brief review. Journal of Soils and Sediments\u003cem\u003e \u003c/em\u003e8(6):379\u0026ndash;388\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Shola Forests, Western Ghats, Bacterial diversity, 16S rRNA gene, enzyme activity","lastPublishedDoi":"10.21203/rs.3.rs-3834802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3834802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eShola forests are unique montane vegetations found extensively along the Southern Western Ghats of India. Samples were collected in the form of soil, slurry, and water from Pampadum, Mathikettan and Anamudi Sholas. Cultivable bacteria were isolated to explore bacterial diversity of the region. Among the total 58 morphologically divergent bacterial strains, 32 isolates showed circular configuration, 24 were irregular while 2 were punctiform. Predominance of pale white colour colonies were discernible followed by white, beige, orange, yellow, pink including transparent ones. Nearly 56.89% of the isolates were found to be Gram negative rods. Distribution of Gram positive rod and Gram positive cocci were 39.65% and 3.45% respectively. Statistical analysis (Shannon and Simpsons) was recorded. Maximum and minimum diversity was observed from the samples collected from Pampadum soil and water. Identification through 16S rRNA gene sequence of the isolates revealed predominance of 4 divisions viz., \u003cem\u003eProteobacteria, Firmicutes, Actinobacteria, Bacteroidetes\u003c/em\u003e, distributed in 9 genera. More genera were distributed in the phyla \u003cem\u003eProteobacteria\u003c/em\u003e followed by the \u003cem\u003eFirmicutes\u003c/em\u003e. Least representations were by \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e. The bacterial isolates had the ability to produce different enzymes. The isolate \u003cem\u003eAcinetobacter dispersus\u003c/em\u003e (ASL31) was found to have maximum amylase activity of 62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 U/ml. The isolate \u003cem\u003eBacillus paralicheniformis\u003c/em\u003e (PS1) showed maximum protease activity of 49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 U/ml, and the isolate \u003cem\u003eBacillus aerius\u003c/em\u003e (MSL9) possessed maximum cellulase activity of about 86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 U/ml. Hence, the diverse environment of Shola forests provides adequate diversity to explore the place for various microorganisms for bioprospecting.\u003c/p\u003e","manuscriptTitle":"Bacterial Diversity and Bioprospecting for enzyme among the Shola Forests of Idukki District, Kerala, India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-11 04:37:43","doi":"10.21203/rs.3.rs-3834802/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ee3cbb55-c375-47fc-8e3d-b6d942b8afbe","owner":[],"postedDate":"January 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-02T17:14:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-11 04:37:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3834802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3834802","identity":"rs-3834802","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}