Accessing the Microbial Diversity of Sugarcane Fields from Gujjarwal Village, Ludhiana and their Molecular Identification

Daljeet Singh Dhanjal, Chirag Chopra, Prince Anand, Reena Singh Chopra*

School of Bioengineering and Biosciences, Lovely Professional University, Phagwara -144411, India.

*Corresponding Author E-mail: reena.19408@lpu.co.in

ABSTRACT:

Soil is the richest ecosystem there is, as far as the diversity and populations of microorganisms is concerned. It is known that there could be over ten billion microbes per gram of soil, making soil the ideal ecosystem for mining different genes, ORFs etc. Microbial Diversity forms the lifeline of an ecosystem, interfering, regulating and participating in the biogeochemical activities therein. The present study was conducted to explore and assess the microbial community by culture dependent and independent approach, of sugarcane fields of Gujjarwal Village of Ludhiana. To study microbial diversity in sugarcane fields, a total of nine different colonies were isolated and differentiated based on their cell morphology and size. Biochemical characterizations lead to the identification of Enterobacter cloacae, Proteus mirabilis and Serratia marcescens. Molecular characterization was done by isolating soil DNA (GJSF DNA) and subjected to PCR amplification for identification via 16S rRNA gene analysis. BLAST analysis revealed presence of Serratia sp., uncultured bacterium clones SS-22C07, ep138, K-15-14-19, S127B31, L1A.11C01 and uncultured Enterobacteriaceae bacterium.

KEYWORDS: Microbial Diversity, Culture Dependent, Gujjarwal, Sugarcane, Ludhiana.

INTRODUCTION:

Soil being the source of organic and inorganic nutrients, becomes the key source for the proliferation of different microbial community¹. It plays the role in degradation and recycling of the organic and inorganic material which is due to the diverse microbes present in it². Assessment of the soil has become the biggest concern for its preservation and maintenance. Where culture dependent approach provides the limited information about the microbial community as large amount of microorganism being unculturable in the in-vitro conditions³. The overall knowledge of the microbial diversity by the culture dependent is inadequate and restrained. The advancement in molecular techniques has facilitated generation of phylogenetic trees and characterization of the natural microbial community without culturing them^4-5.

Because of the substantial measure of the data that is encoded in the genome of both culturable and non-culturable microorganisms, it has inspired the scientific community to create novel molecular methods for proficient characterization. Molecular techniques are now utilized to recognize bacteria and delineate bacterial DNA differences either for individual microscopic organisms or entire microbial group. The molecular methods comprise isolation of the metagenomic DNA, amplification and sequencing with 16S rRNA gene to retrieve the information for further analysis^6-8. Molecular techniques have enabled us to access the huge diversity of unexplored microbes⁹. The 16S rRNA gene sequencing became the main attraction and useful technique to assess the novel bacterial phylum that where yet not cultured^10-11.

The aim of the present study was to explore the microbial diversity present in the sugarcane fields of Gujjarwal village, Ludhiana. The assessment of the soil revealed that phylum Enterobacteriaceae is predominant in the soil out of which Serratia marcescens, Enterobacter cloacae and Proteus mirabilis were the species that were identified by the biochemical assessment. Metagenomic DNA was isolated from the soil, amplified and 16SrRNA gene was amplified using 16S universal primers sequenced using 16S rRNA.

MATERIAL AND METHODS:

Sample Collection:

Seven soil samples were taken from different locations of the fields used for agricultural production of sugarcane from Gujjarwal village of Ludhiana located at an elevation height of 262m (30.91°N 75.85°E). The samples were collected in sterile Zip lock bags and stored at 4ºC. The soil was collected from the depth of 6-8 cm with sterile scrapper.

Isolation of Pure Cultures:

About 1 gm soil sample from Gujjarwal sugarcane field (GJSF) was placed in 250ml conical flask containing 100 ml of 2X-YT broth. The medium was incubated at 37^oC overnight in an orbital shaker (150 rpm). The inoculum prepared was then used for the spreading on the 2X-YT enrichment media by diluting it up to the 6-fold and kept 37ºC for 24 hrs. The colonies were differentiated based on their colour and morphology and the pure colonies were streaked on the 2X-YT enrichment media in a quadrant manner. The sub-cultured pure colonies were categorized based on standard gram staining protocol¹².

Biochemical Tests:

Ten biochemical tests named Indole, Methyl Red, Citrate Utilization, EMB Agar, MacConkey Agar, Triple Sugar Iron, Catalase, Nitrate Reduction and Urease test were performed per the protocol of Cappucino and Shermann (2008).

Isolation and Amplification of Metagenomic DNA:

1gm of the soil (GJSF) was inoculated in 100 mL of 2X-YT broth and incubated overnight in an orbital shaker. After the overnight incubation, contaminant free high molecular weight DNA was isolated from the soil as reported earlier¹³. The purity of the DNA sample was determined spectrophotometrically at a wavelength of 230, 260, and 280 nm and concentration of the DNA was calculated by the formula, Concentration (µg/ml) = Absorbance at 260nm × 50 (µg/ml) × Dilution factor¹⁴.

Molecular characterization of the isolated DNA (GJSF) was done by amplifying, sequencing and analysing the 16S rRNA gene. PCR amplification of 16S rRNA gene was accomplished by mixing 7µL of molecular grade water (Himedia), 2.5µL of the Taq Polymerase 10X buffer, 5µL of the template DNA, 2.5µL of the dNTP mix (10mM), 1µL of each 452F(5’-GACTGGGGTGAAGTCGTAAC-3’) and 452R (5’-TGGCTGGGTTGCCCCATTCGG-3’) universal primers (10pmol) and 1U/µL of Taq polymerase. A negative control (sterile deionized water) and a positive control (Escherichia coli genomic DNA) were included in the experiment to confirm validity of results. The reaction mixture was incubated in a DNA Thermal Cycler (Eppendorf) with the following cycling conditions: denaturation step of 2 min at 95ºC, followed by 35 cycles (denaturation at 95ºC for 30 secs, annealing at 50ºC for 50 secs, and extension at 72ºC for 1 min 45 secs) and final extension at 72ºC for 8 min. The amplified product (10μL) was electrophoresed in 1% agarose gel, in 1XTAE buffer, ethidium bromide solution 10 mg/mL, at 100 V for 1 h. The gel was visualized under UV light and photographed and the amplicon was send for sequencing analysis at Bioserve Biotechnologies, Hyderabad.

Table 1: Biochemical assessment of the isolated bacterial colonies (+ve – Positive, -ve – Negative, Lac⁺- Lactose Fermenting, Lac^- - Lactose Non-fermenting, G- gas, K/A - Glucose Fermenter and A/A – Glucose and Sucrose Fermenter)

Samp-le

Gram Stain-ing

Indole Test

Methyl

Red Test

Voges- Proskauer

Test

Citrate Utiliza

tion Test

EMB Agar Test

MacConkey Agar Test

Triple Sugar

Iron Test

(Slant/Butt)

Catalase Test

Nitrate Reduction Test

Ure-ase Test

Identified Bacteria

References

LdhD1

-ve

+ve

Lac⁺

Lac⁺+ G

K/A + G

+ve

Serratia marcescens

Mahendra., (2015)

LdhD2

-ve

+ve

Lac⁺

Lac⁺+ G

A/A + G

+ve

Enterobacter cloacae

Patil et al., (2015).

LdhD3

-ve

+ve

Lac⁺

Lac⁺+ G

A/A + G

+ve

Enterobacter cloacae

Patil et al., (2015).

LdhD4

-ve

+ve

Lac⁺

Lac⁺+ G

K/A + G

+ve

-ve

Serratia marcescens

Mahendra., (2015)

LdhD5

-ve

+ve

-ve

Lac^-

Lac^-+ G

K/A + G

+ve

Proteus mirabilis

Jasuja et al., (2013)

LdhD6

-ve

+ve

Lac⁺

Lac⁺+ G

K/A + G

+ve

Serratia marcescens

Mahendra., (2015)

LdhD7

-ve

+ve

Lac⁺

Lac⁺+ G

A/A + G

+ve

Enterobacter cloacae

Patil et al., (2015).

LdhD8

-ve

+ve

Lac⁺

Lac⁺+ G

A/A + G

+ve

Enterobacter cloacae

Patil et al., (2015).

LdhD9

-ve

+ve

Lac⁺

Lac⁺+ G

A/A + G

+ve

Enterobacter cloacae

Patil et al., (2015).

RESULTS:

Isolation of Pure Cultures:

The isolation criteria involved serially diluting the soil to 10^-5 and 10^-6-fold and spread-plated on the 2X-YT media. Screening of the different CFUs was done based on size and morphology, for sub culturing and isolating the pure cultures. Nine pure colonies were isolated from GJSF soil after dilution by 10^-5 and 10^-6-fold. Analysis of all colonies was done for biochemical test-based identification. On performing the gram staining all isolates were found to be gram negative and rod shaped. They were then identified based on morphology, biochemical analysis such as indole, MR-VP, Citrate, EMB, MacConkey, TSI, Catalase, Nitrate reduction and Urease test by referring Bergey’s Manual of Systematic Bacteriology.

Isolation and Amplification of DNA:

In the present study, the extracted metagenomic DNA (GJSF DNA) was of good quality, concentration and was of high molecular weight as analysed on 0.8% (w/v) agarose gel (Fig1). The isolated high molecular weight DNA was checked for yield and purity based on the absorbance ratios at 260⁄230nm (DNA⁄ Humic acids) and 260⁄280 nm (DNA⁄ protein) spectrophotometrically¹⁵. 16S r-DNA gene fragment was amplified from metagenomic (GJSF) DNA with annealing conditions based on the Tm of the 16S primers. The sample showed a single amplicon at 1.5 kb (Fig 1).

Fig.1: Showing Agarose Gel Electrophoresis of metagenomic DNA (GJSF DNA) and PCR-amplified 16S rRNA gene. A (from left to right): 0.7% Agarose gel showing one well containing GJSF metagenomic DNA; B: 1% Agarose gel showing 16S rRNA gene amplified from metagenomic DNA of GJSF (right), using 16S Universal Primers.

Sequencing Analysis:

The sequence obtained was analysed through multiple sequence alignment as well as BLAST tool of NCBI. Upon BLAST analysis, it was found that the sequence matched with Serratia sp. AH-7-3, Uncultured bacterium clone SS-22C07, Uncultured bacterium clone ep138, Uncultured Serratia sp. clone K-15-14-19, Uncultured Enterobacteriaceae bacterium, Uncultured bacterium clone S127B31, Uncultured soil bacterium clone L1A.11C01 and Uncultured bacterium clone d5-34 to the extent of coverage of 85% with each subject sequence. To understand the extent of diversity, a phylogenetic tree was constructed using Clustal Omega(Fig 2).

DISCUSSION:

The results of 10 tests represented in table 1 were essential to establish a system for assessment of Enterobacteriaceae spp. The Enterobacteriaceae spp. majorly belongs to category of gram -ve bacteria. The IMViC test showed the results --++ for the LdhD1, LdhD2, LdhD3, LdhD4, LdhD6, LdhD7, LdhD8, LdhD9; and ++-- for LdhD5 respectively. (Table 1). The synthesis of tryptophanase, ability to create acidic environment, inability to catabolize the citrate and to synthesize acetyl methyl carbinol by the LdhD5 for Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization Tests respectively, differentiate it from the other colonies which showed similar results to each other and results were found similar with findings of previously documented literature¹⁶. All colonies could ferment lactose except LdhD5 which was confirmed by culturing the isolated colonies on EMB and MacConkey Agar. Additionally, gas production was seen for all the colonies. The Triple Sugar Iron Test was performed to assess the ability of the colonies to ferment three-different sugars and produce H₂S. Thus, on evaluation it was found that LdhD1, LdhD4, LdhD5, LdhD6 were glucose fermenting bacteria as they were alkaline on the slant and acidic on the butt whereas LdhD2, LdhD3, LdhD7, LdhD8, LdhD9 could ferment all the three sugars as they changed the colour from red to yellow on both slant as well by reducing the pH of the media. The Positive result by all bacterial colonies for catalase, nitrate reduction and Urease test confirmed the synthesis of the catalase, nitrate reductase and urease enzyme by the isolated bacterial colonies. Thus, on evaluating these biochemical tests and by interpreting the result by following the previous documented literature and Bergey’s Manual, the bacterial samples LdhD1, LdhD4, LdhD6 were identified as Serratia marcescens, LdhD2, LdhD3, LdhD7, LdhD8, LdhD9 (Table 1) were identified as Enterobacter cloacae and LdhD5 was identified as Proteus mirabilis^17-18,16.

Molecular analysis was carried out by amplifying and sequencing the 16S rRNA gene from the metagenomic DNA using conserved universal primers. The PCR amplicon of approximately 1.5kb was sequenced at Bioserve Biotechnologies, Hyderabad. The species that could be identified through sequencing followed by BLAST analysis included Serratia species and unculturable clones form various libraries. To have an idea about the origin of these strains, a phylogenetic tree was also developed, which showed organisms of diverse lineages.

ACKNOWLEDGMENT:

The authors thank the senior administration of Lovely Professional University for providing the seed grant for initiating the project.

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Received on 25.05.2017 Modified on 19.06.2016

Research J. Pharm. and Tech 2017; 10(10):3439-3442.

DOI: 10.5958/0974-360X.2017.00612.6