Isolation, Identification and Characterization of Microorganisms

Home , Great Rann of Kutch, Little Rann of Kutch, Rann of Kutch, Regions of Gujarat

ACTA SCIENTIFIC MICROBIOLOGY (ISSN: 2581-3226) Volume 3 Issue 7 June 2020

Research Article

Isolation, Identification and Characterization of Microorganisms Isolated from Unexplored Saline Regions of Kutch, Gujarat, India Joshi CM1* and Trivedi NS2 Received: 1C U Shah University, Wadhwan City, Gujarat, India Published: June 09, 2020 2Gujarat University, Ahmadabad, Gujarat, India April 11, 2020 Joshi CM and *Corresponding Author: Trivedi NS. © All rights are reserved by Joshi CM, C U Shah University, Wadhwan City, Gujarat, India.

Abstract

The present investigation reveals the diversity of bacteria, fungus and archaea along the different regions of Kutch area, Gujarat, India. The samples were collected from 3 selected site of Kutch area whose temperature was between 37 - 41°C, pH 8.5, EC 1480 µs, Salinity 9.01 and 1.85 ppm TDS. 52 bacterial, 17 extreme halophilic archaea and 2 halophilic fungus strains were isolated, among these all bacterial strains were selected for further study like enzyme productivity and anti-microbial activity because the main aim of research was to isolate organism which have multiple applications. Fifteen strains produced four or more than four enzymes which have industrial application (amylase, protease, cellulose, lipase, and chitinase) and one strain of Micrococcus showed antimicrobial activity against all test cultures. Eighteen archaea and two halophilic fungi that grew at 25% salt containing medium were also Keywords: isolated. Bacteria; Fungus; Archaea; Kutch; Enzyme; Antimicrobial Activity; Salt Concentration

Introduction

fifteen isolates were isolated from the different hyper saline soil of Little Rann of Kutch, Gujarat and most of them are used as bio- Rann of Kutch is a salt marsh in the state of Gujarat, India. It can fertilizer [6]. be divided into the Great and The Little Rann of Kutch. While the Great Rann of Kutch converts to a “White Desert” in the summer Rann of Kutch has also been exploited for the isolation of Ex- with thin layer of salt crust which becomes marshy island during tremophiles. Notable studies include those involving isolation of Streptomyces rainy season, little Rann of Kutch represents salt crystallizers of In- extreme halophiles [7], Halophilic archaebacteria [8], halo tolerant dia. On the whole the ecosystem of the Rann is largely undisturbed alkaliphilic [9], moderately halophilic bacteria (Dey et al. and would be the reservoir of myriad of microorganisms ranging 2013). All these studies focus on the isolation of microbes using from normal micro flora to extreme organisms which can thrive traditional methodology. A very interesting study of Pandit., in the ever fluctuating harsh saline environments. Such places are [10] revealed a diversity of 56 to 87% Bacteria and 8 to 40% Ar- always fascinating for understanding the evolution and survival of chaea using metagenomics which opened immense possibilities of microbes in such harsh environments. further work. et al.

Lot of work has been done in terms of diversity studies in hy- In view of the work of Pandit., [10] regarding the immense Bacillus iranensis sp per saline systems. Ventosa [1] have worked on halophiles from diversity and presence of genes of industrially important enzymes, unusual habitats, . was isolated from saline this study was formulated to tap the diversity of Rann of Kutch for mud of the hypersaline lake Aran-Bidgol in Iran [2]. In the Indian isolation of industrially important microbes specially focusing on scenario hypersaline coastal regions of Gujarat, Maharashtra, Ra- those microbes which have multiple potential of bio-prospecting. jasthan and Tamil Nadu have been well studied and several Acti- nobacteria and Halophiles reported [3]. Halophilic Archaebacteria Thus, in this study undisturbed and unexplored soil samples have been reported from desert of Rajasthan (Upasani 2007). Dave were collected from three locations of Kutch (Sangur village near and Soni [4] have reported isolation of Halophilic and Alkalophilic khavda, Mandvi beach and Great Rann of Kutchh). Microbial iso- bacteria from coastal regions of Gujarat. Thirteen bacterial strains lations were performed for isolation of bacteria, archaea and acti- were isolated from Kharaghoda area of Gujarat [5]. More than nobacteria. These microbes were identified at the molecular level.

Citation: . Acta Scientific Microbiology Joshi CM and Trivedi NS “Isolation, Identification and Characterization of Microorganisms Isolated from Unexplored Saline Regions of Kutch, Gujarat, India". 3.7 (2020): 03-09. Isolation, Identification and Characterization of Microorganisms Isolated from Unexplored Saline Regions of Kutch, Gujarat, India

They were then tested for the antimicrobial potential and the abil- analysis was performed according to neighbour joining methods ity to produce enzymes including Amylase, Protease, Cellulase, Li- using MEGA version 7.0. Enzymatic study Materialspase, and Chitinase and Methods [11-28]. Collection of soil samples The isolates were screened for production of different enzymes using their respective media as mentioned in table 1. For this pur- Soil samples were collected from different locations on the ba- pose, cultures were spot inoculated on assay plates and incubated sis of differences in their physical parameters and to avoid recur- for 4 - 7 days. Name of rence of identical cultures. The collection sites includes Mandvi Media Method Enzymes beach (N 22. 82513 E 69.33753), and Great Rann of Kutchh (N 23. 82763 E 69.52002) in Kutch area of Gujarat from the depth of 15 cm in sterile polythene bags and air tight container with the help Amylase Minimal medium Clearing aroundsolution the growth with starch after flooding with iodine of sterile spatula and were transported to laboratory for further medium processing. Enrichment and isolation Protease Casein skim milk Clearing around the growth

Cellulase Carboxymethyl Clearing aroundsolution the growth At first, the soil slurry was made by suspending 1g of the col- Cellulose medium after flooding with iodine lected dry soil in 10 ml distilled water. The slurry was mixed by media Lipase Tributyrin Agar Clearing around the growth vortexing for 1 minute, the coarse particles-8 were allowed to settle and serial dilutions were made till 10 . 100 µl aliquots of dilutions -2 -4 -6 -8 Chitinase Chitin Agar mediaTable 1Clearing around the growth 10 , 10 , 10 and 10 were spread on Starch casein Agar, Nutrient Agar and HV-YPC media in triplicates (A, B and C). All spread plates were incubated at 30°C for a period of 14 days. On day 14, colo- Experiment was performed in triplicates. After incubation, nies formed were observed and morphologically different colonies plates were observed for zone of clearance due to enzyme activity. were subculture on Starch Casein Agar and HV-YPC media for fur- Antibiogram analysis therDNA characterization amplification, sequencing and studies. and phylogenetic analysis This screening was done for 52 bacterial isolates. Analysis was Genomic DNA was isolated from the isolates using Qiacube fol- done using crude extract obtained by spinning the culture broth et al. lowing the protocol given by the manufacturer. Lysis of cultures at 5000 rpm for 2 minutes. The assay was conducted by agar well and chloroform wash were done manually and then the superna- diffusion method (Perez., 1990). Test cultures i.e. two gram tant was subjected to column purification step, washing and elu- positive and two gram negative were first spread on nutrient agar ° tion in Qiacube. The eluted DNA was directly subjected to PCR plates. Wells were punched using cup-borer and filled with 50 μl amplification. Amplification of bacterial isolates was done using culture supernatant. Plates were incubated at 30 C for 24 hours. ° 16s universal primers 8F (5’3’) and 1492R using touchdown PCR. Sterile nutrient broth was taken as control. The plates were incu- Similarly, for amplification of Archaea, 21f and 958R were used bated over night at 30 C in incubator. The antimicrobial activity of and fungal DNA was amplified by ITS 1 and ITS 4. The PCR product Resultsmicrobes was analyzed by presence of inhibition zones. was detected by agarose gel electrophoresis and was visualized by Physico-chemical properties of the samples ultraviolet (UV) fluorescence. PCR products were purified using EXOSAP. Cycle sequencing of purified PCR products was performed Both soil and water samples were collected from the hypersa- using BIGDYE employing same primers except for bacteria, where line environment of Kutch. The color of water sample was light red 704F and 907R primers were used. Cycle sequencing products to deep red indicating archaeal population. And the muddy soil were purified by using X-terminator and Sam solution. Sequenc- with dark brown color was collected. The concentration of NaCl in ing was done using the 3500 genetic analyser from Applied Bio- majority of the water samples collected from hypersaline environ- systems. Compilation of the individual sequences obtained was ments of the Kutch was near saturation, indicating the extremity done using BIOEDIT software. Sequence homology was done us- of the environment. The pH of the samples varied from location to ing NCBI BLAST and the organisms were identified. Phylogenetic

05 Name of Organism NCBI Accession number location and ranged from 8.70 to 13.8. The total dissolved solids Micrococcus luteus

(TDS) of brine samples varied from 1.10 to 1.85 ppm, electrical Virgibacillus chiguensis -1 KY681702 conductivity (EC) of water sample varied from-1 was 148 mS cm Kokuria palustris ° KY681703 and that of soil or mud sample was 118 mS cm . The temperature Bacillus sonorensis KY681704 of samples ranged from 20 to 26 C. The properties of the both sam- Bacillus infantis KY681705 ples is mentioned in the table 2. Bacillus cereus Sample No. Sample 1 Sample 2 KY681706 Bacillus anthracis KY681707 Haloferax alexendrinus KY681708 Latitude N 23. 82763 N 22.82513 Pseudomonas putida KY681709 Longitude E 69.52002 E 69.33753 Bacillus foraminis KY681710 pH 13.8 8.70 Micrococcus aloeverae KY681711 Salinity 10.2 8.01 -1 -1 Staphylococcus hominis KY681712 EC 148.0mS cm 118.7 mS cm Bacillus licheniformis KY681713 TDS 1.85ppm° 1.14 ppm° Bacillus firmus KY681714 Temperature 26.6TableC 2 20.0 C Bacillus anthracis KY681715 Staphylococcus epidermis Isolation of the microorganisms KY681716 Bacillus licheniformis KY681717 Bacillus oceanisediminis There were numerous presences of microbes but the organisms KY681718 Virgibacillus with different cultural characteristics were selected. In case of ex- KY681719 Bacillus firmus treme halophiles only colored colonies were counted. The preva- KY681720 Bacillus siamensis lence of hard, sticky colonies was also observed which varied from KY681721 Bacillus velenzensis light pink to cream. Two fungal cultures were also isolated which KY681722 were found to be grown on the media. Bacillus licheniformis KY681723 Bacillus cereus A total of 71 isolates have been obtained in this study including KY681724 Staphylococcus epidermis bacteria, halophilic bacteria and archaea. Halotolerant Fungi have KY681725 Bacillus endophyticus also been isolated which could grow on media containing 25% salt KY681726 Bacillus endophyticus (Table 3). KY681727 Bacillus anthracis Sr. No. Microorganisms Number of Isolates KY681728 Virgibacillus pantothenticus KY681729 2 Providencia stuartii 1 Bacteria 48 KY681730 3 Bacillus subtilis Archea 13 KY681731 02 Staphylococcus epidermis Halophilic bacteria 08 KY681732 Bacillus licheniformis 4 Halotolerant fungiTable 3 KY681733 Bacillus licheniformis KY681734 Bacillus licheniformis Identification of isolates based on 16S rRNA sequencing KY681735 Bacillus flexus KY681736 Bacillus thuringiensis Identification of isolates on the basis of 16S rRNA sequencing KY681737 Bacillus amyloliquifaciens revealed the presence of many different groups of microbes rang- KY681738 Bacillus, Micrococcus, Staphylococcus, Providencia, Brachybacte- Bacillus subtilis ing from Bacteria to Archea. Including some common generas like KY681739 rium, Virgibacillus, Halobacillus, Haloarcula, Chromohalobacter, Brachybacterium paraconglom KY681740 Heloferax, Halobacterium, Aspergillus Micrococcus luteus KY681741 . Table 4 Shows the name of Micrococcus endophyticus KY681742 organisms along with their NCBI accession numbers. KY681743

Bacillus subtilis Number of organisms showing Positive Sr. No. Enzymes Bacillus lichenifromis Result KY681744 Bacillus licheniformis 36 KY681745 Staphylococcus sp. 2 KY681746 1 Amylase Haloferax sp. 3 33 KY681747 Protease 40 Haloferax sp. KY681748 Lipase Haloferax volcanii KY681749 4 Cellulase 38 Halobacterium salinarum KY681750 5 Chitinase Table 5 19 Halomonas elongate KY681751 Chromohalobacter salexigens KY681752 Halobacterium salinarum KY681753 Haloferax alexendrinus KY681754 Halobacterium salinarum KY681755 Haloferax lucentense KY681756 Halomonas elongate KY681757 Halomonas elongata KY681758 Halobacterium salinarum KY681759 Haloarcula amylolytica KY681760 Paracoccus sp. KY681761 Bacillus korlensis Graph 1: KY681762 Bacillus sp. KY681763 Shows organisms showing maximum amylase activity. Bacillus amyloliquifaciens KY681764 Arthrobacter sp. KY681765 Halobacillus sp. KY681766 Arthrobacter sp. KY681767 Haloferax alexandrinus KY681768 Halobacterium salinarum KY681769 Aspergillus versicolor KY681770 Aspergillus parasiticus KY681771 Table 4 KY681772

Enzymatic activity Graph 2:

Shows organisms showing maximum chitinase activity. The bacterial isolates were screened for enzymatic and antimicrobial activities. Enzyme screening included industrially important enzymes like Amylase, Lipase, Protease, Cellulase and Chi- tinase. Maximum isolates were positive for protease activity and minimum number was for Chitinase enzyme (Table 5). The organisms showing maximum enzyme activity was analyzed (Graph 1 to 5). Antimicrobial activity

Total 48 bacterial cultures were screened for the antimicrobial activity. The activity was observed against two Gram negative and two Gram Positive test organisms. Maximum number of organisms Graph 3:

Shows organisms showing maximum lipase activity.

Fungi have also been isolated which could grow on media containing 25% salt.

The bacterial isolates were screened for enzymatic and antimicrobial activities. Enzyme screening included industrially important enzymes like Amylase, Lipase, Protease, Cellulase and Chi- tinase. Maximum isolates were positive for protease activity and minimum number was for Chitinase enzyme. The results of Amy- lase has wide range of industrial applications such as food, fermen- tation, and pharmaceutical industry due to its role in hydrolysis Graph 4: of starch molecules into glucose polymers. Protease is also one of Shows organisms showing maximum cellulase activity. the industrially important enzymes which are mainly used in De- tergent, Tannery, Food, Metal recovery and wastewater treatment like industries. Lipase has industrial role in Pharmaceutical, Food, Detergent, Textile, Leather, and Cosmetic like industries. Cellulase is mainly used in pulp and paper, Textile, Bioethanol, Wine and Brewery, Food processing, Agricultural, and animal feed industries. Chitinase has mainly agricultural use chitinase producing organisms are used as bio-fertilizers. As the given organisms have been isolated from the hypersaline soil so they can be used as effective bio-fertilizers in saline regions.

Some of the isolates show the ability to produce multiple enzymes out of 52 bacterial isolates 10 organisms had shown the Graph 5: ability to produce all five enzymes. And 21 isolates had shown the Shows organisms showing maximum protease activity. ability to produce four enzymes. Bacillus subtilis and Antimicrobial agents are widely used as pharmaceutical prod- were able to show positive activity against ucts and they have also been used in bio- pesticides. Many isolates Enterobacter cloacae Minimum number of organisms were able to show Positive activity have even shown the antimicrobial activity against both gram nega- Bacillus subtilis, against . tive and gram positive test organisms. Out of 52 bacterial isolates Sr No. Name of Test No. of org. showing Staphylococcus aureus, 6 Organisms Activity 51 organisms have shown positive activity against Pseudomonas aeruginosa and 11 have shown positive activity against Staphylococcus aureus Enterobacter cloacae have shown positive activity against 2 Bacillus subtilis 1 10 1 have shown positive activity agent . 3 Pseudomonas aeruginosa 06 47 Enterobacter cloacae From these organisms one organism have also been reported 4 01 which has ability to produce all 5 enzymes as well as it has shown Table 6: Micrococcus aloeverae antimicrobial activity against all sensitive test cultures. That organ- Shows number of organisms showing antimicrobial ism has been identified as (KY681712). With activity against two gram positive and two gram negative the use of this multiple ability organism many of the industrial que- pathogenic strains. Discussion and Conclusion ries can be resolved as many benefits can be obtained from a single organism having both economical and industrial benefits.

In this study, we provide a preliminary description of a complex Kutch metagenomic studies have shown the presence of wide community of Microorganisms associated with saline soil of Kutch range of microbes from which few have been isolated and identified Gujarat. A total of 71 isolates have been obtained in this study in the present study it also reveals the presence different enzymes including bacteria, halophilic bacteria and archaea. Halotolerant so those organisms which are still uncultured can be cultured and many different industrially important enzymes can be obtained. Citation: . Acta Scientific Microbiology Joshi CM and Trivedi NS “Isolation, Identification and Characterization of Microorganisms Isolated from Unexplored Saline Regions of Kutch, Gujarat, India". 3.7 (2020): 03-09. Isolation, Identification and Characterization of Microorganisms Isolated from Unexplored Saline Regions of Kutch, Gujarat, India

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