Isolation and Characterization of Microbial Diversity of Soil of Dhanbad Coal Mines using Molecular Approach

 

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

School of Bioengineering and Biosciences, Lovely Professional University, Punjab-144411, India. *Corresponding Author E-mail: chirag.18298@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 identify key extremophilic species residing in the coal mines of Dhanbad plateau in Bihar, India. Dhanbad plateau soil presents a unique niche, rich in coal and minerals. Soil from the coal mines was analysed through metagenomic as well as culture-dependent approach to enlist the micro-organisms native to the place. The culturable fraction lead to the identification of Klebsiella spp, Enterobacteriaceae, Serratia marcescens, and Providencia spp

 

KEYWORDS: Microbial Diversity, Molecular techniques, 16S rRNA genes, Dhanbad Plateau, Metagenomics.

 

 


INTRODUCTION:

The existence of life on the planet Earth dates to 4.1 billion years ago with the evidences of prokaryotic life to 3.5 billion years back in time1. During this vast travel through the time life forms has improved, but from the origin of first prokaryotic cell to the domination of gigantic reptiles of Jurassic era and from the development of ganglion to the network of scintillating neurons in human mind, one thing is common and constant. They are the tiny invisible creatures sharing the journey of time with all the life forms present on the Earth with incredible potential and sustainability - ‘Microorganisms’.

 

Microorganisms are found on every inch of the planet, from the cold Arctic Circle to the dry desert of Sahara; from the rainforests of amazon to the deep world of Indian Ocean. The morphology and genetic ability to sustain in almost every environment makes bacterial population one of the most diverse population ever inhibit the planet2.

 

Per an estimation, 50% total biomass load of the planet is made up of microbial cells3. There are more number of bacteria (1029) estimated in the ocean then the stars in the Universe (1021). However, the critical point about our knowledge of microbial diversity is that till date we have information about only 1% of microbial species only round about 3,100 in an estimated number of 300,000 to 1 million microbes have been cited and documented in literature available. 99% of microbial population is still waiting for exploration4.

 

The distribution of bacterial species all over the planet contributes to the Microbial diversity. This diversity of micro contributes to the overall biodiversity of the living world and put a great impact on the ecosystem and other organism’s life. Due to different forms of bacterial cells and physiology, they can metabolize many compounds and act as decomposers in the environment2, 5.

 

Dhanbad is a district in the state of Jharkhand which is situated in the eastern part of the India at 23°37.3’ N and 24°4' N latitude and 86°6'30” E and 86°50' E longitude. Dhanbad is also situated on this plateau and the soil composition of region is highly affected by the formation of plateau 50 million years ago. Due to plateau, soil of entire Jharkhand region became rich in mineral and coal and it makes the state a 40% contributor to the total mineral resources of the Country. Major minerals found in the soil of plateau are coal, iron metal, mica, Uranium, Bauxite, copper, stone, limestone, silver, graphite, magnetite and dolomite6.

 

Exploration of Microbial diversity in the mining areas is gradually becoming interesting area of research, because the microbial diversity gives a huge impact on the biodiversity and biogeochemistry around them. Studies have been conducted to explore microbial diversity previously in mineral rich mining regions worldwide7-8Bacterial community structure and diversity could be altered during period of a decade and the tendency of the microbial population to get changed has been observed in a study of coal mines in China9

 

Different types of microbes are source of enzymes to life saving antibiotics. But, in the modern era when we are surrounded with problems like antibiotic resistant, global warming, land and water pollution, air pollution, shortage of effective drugs, it become very much important to care about those 99% population of microorganisms which has not been explored yet4,10. Analysis of microbial diversity became important in this scenario.

 

There are several techniques and methods involved in the exploration of microbial diversity, but molecular technique of characterization of microbial diversity is most effective and accurate method for the determination of microbial diversity10. Analysis of genome and conserved biomolecules like ribosomal RNA are among those modern techniques, which are in practice now days for the analysis and characterization of microbial diversity. These techniques not only provide us information about microbial population and their distribution but also equip us to identify novel bacterial strains, which can produce novel secondary metabolites useful in various aspects of human life11.

 

In this study, we used the approach of sequencing conserved 16SrRNA genes present in bacterial population of the soil and then biochemical characterization of the isolated pure cultures from the same soil. It helped us to assess different type of bacterial population present in the soil and their biochemical characteristics.

 

MATERIAL AND METHODS:

Collection of Soil:

The collection of soil sample was done under aseptic conditions. Soil was taken after digging for two feet with the help of sterilized tools, transferred into the aseptic zip-lock polybag, and sealed. In the laboratory, the sample was kept at a dry place inside a marked airtight plastic container at 4oC. Analysis of the soil sample was done for gathering the information of physical and chemical properties of the soil. Separate tests were done for assessing these properties including test for pH, Bulk density and Specific gravity of the soil12.

 

Metagenomic DNA Extraction:

Metagenomic DNA was also extracted directly from soil by modifying the previously reported method12-13.

 

Validation and quality determination of the isolated DNA:

Spectrophotometric analysis of the DNA was done for quality determination of the obtained DNA. Ratio of absorbance at 260nm to 280nm and 260nm to 230nm were used as qualitative parameters.

 

Amplification of the 16S rRNA genes from isolated metagenomic DNA:

Due to the conserved nature of 16S rRNA gene universal bacterial 16S rRNA primers were utilized for the Polymerase Chain Reaction amplification of the gene from isolated DNA14-16. The protocol for the amplification of 16S rRNA gene was standardized per the quality of isolated DNA and conditions. Universal primers 452F (5’-GACTGGGGTG AAGTCGTAAC-3’) and Reverse Primer- 452R (5’-TGGCTGGGTTGCCCCATTCGG-3’) were used for amplifying the 16S rRNA gene form the DNA samples. Sequencing of the amplified 16S rRNA genes was performed at Bioserve, Hyderabad.

 

Isolation of culturable microbes from the soil sample:

For characterization of culturable microbial strains present in the soil sample biodiversity analysis through enrichment culture methods were used as an alternative approach. This had allowed to analysis of morphological and biochemical properties of the bacterial strains present. 1 gram of the soil sample was used to inoculate 100 ml 2X-YT broth and kept at 37oC for 24 hours in incubator shaker. Next, the cultures were serially diluted up to 106 dilutions in sterile test tubes and were plated on 2X-YT Agar. Pure cultures were isolated by sub-culturing of the identified colonies.

 

Biochemical testing of the isolated pure cultures:

Biochemical characterization was done by performing 10 biochemical tests for each isolated pure culture with standard method described previously11. These tests included Indole test, Methyl red test, Voges Proskauer Test, Citrate Utilization Test, EMB Agar test, Mackonkey Agar test, TSI Agar Test, Nitrate Reduction Test, Urease Test, and Catalase Test.

 

Identification of unknown isolates:

Based on the results of biochemical tests, Bergey’s Manual of Systemic Bacteriology used to identify the genus and species of the pure cultures.

 

RESULTS:

Metagenomic DNA Extraction and PCR Amplification of 16S rRNA Gene:

Metagenomic DNA was analysed by electrophoresis on 0.7% agarose gel. High Molecular weight DNA was observed in DBSM (Dhanbad Soil Metagenomic) DNA (Fig. 1A). The spectrophotometric analysis of the extracted DNA was performed. The spectrophotometric analysis of the isolated DNA, showed the A260/280 and A260/230 ratio to be 1.92 and 2.14 respectively, showing that the isolated DNA was of good quality. The concentration of the DNA sample was calculated to be 5226µg/mL.

 

The amplified product was analysed on 1% agarose gel electrophoresis. The sample showed an amplification of about 1.5kb and was sequenced at Bioserve Biotechnologies, Hyderabad, India. The sequence revealed presence of two extremophilic organisms viz. Geobacillus thermooleovorans and Bacillus halodurans. BLAST analysis showed 29% coverage and 74% identity with Bacillus halodurans C-125 sequence (Accession No. BA000004.3).

 

Identification of Culturable Microbes:

Sub-culturing was done by streaking the agar plates with the cultures obtained from soil followed by incubation at 37oC for 24 hrs. The colonies of different morphology and size were observed on the master plate of 105 and 106 dilution. Out of the eleven colonies obtained (four from 105 dilutions and seven from 106 dilution) having different size and morphology were marked on the plates for further isolation and preparation of the pure cultures.

 

The pure cultures were designation for convenience in biochemical testing for the identification of strains.

 

 

Fig. 1: (A) 0.8% agarose gel showing high-molecular weight DBSM DNA; B: 1% agarose gel showing 16S rRNA gene amplified from DBSM DNA.

 


 

Table 1: Biochemical Characterization of the isolates and identification of the species (+ve means positive; -ve means negative, Lac+ means lactose ferming, Lac- means lactose non-fermenting, A/A means glucose and sucrose fermting)


Strain

Test

DbdP1

DbdP2

DbdP3

DbdP4

DbdP4

DbdP5

DbdP6

DbdP7

DbdP8

DbdP9

DbdP10

DbdP11

References

Indole

-ve

-ve

+ ve

- ve

- ve

+ ve

- ve

- ve

- ve

- ve

+ ve

+ ve

Alexander and Strete, 2001

Methyl Red

-ve

- ve

+ ve

+ve

- ve

+ ve

+ ve

- ve

+ ve

- ve

+ ve

- ve

Brenner et al., 1999

Voges Proskeur

+ve

- ve

- ve

- ve

- ve

- ve

- ve

- ve

- ve

- ve

- ve

+ ve

Brenner et al., 1999

Citrate Agar

+ ve

+ ve

+ ve

- ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

- ve

+ ve

Bachoon and Wendy, 2008

EMB Agar

Lac+

Lac+

Lac-

Lac+

Lac+

Lac-

Lac+

Lac+

Lac+

Lac-

Lac+

Lac-

Swapna et al., 2015

Mac Conkey Agar

Lac+

Lac+

Lac-

Lac+

Lac+

Lac-

Lac+

Lac+

Lac+

Lac-

Lac+ Gas

Lac-

Swapna et al., 2015

TSI Agar

A/A

- ve

A/A

- ve

A/A

A/A

- ve

A/A

A/A

A/A

A/A Gas

A/A

Brenner et al., 1999

Catalase Test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

Brenner et al., 1999

Nitrate Test

+ ve

+ ve

+ ve

+ ve

+ ve

+ ve

- ve

+ ve

+ ve

+ ve

+ ve

+ ve

Brenner et al., 1999

 


DISCUSSION:

IMViC tests were helpful in differentiation of Enterobacteriaceae present in the soil sample, as negative Indole test indicated the inability of organism to produce tryptophanase enzyme and breakdown of tryptophan present in the medium. A negative methyl red test indicated the lack of production of mixed acids during metabolism due to inability to utilize pyruvate as a substrate in mixed acid fermentation pathway (BM). We determined that our 3 unidentified strains DbdP1, DbdP7 and DbdP11 had characteristic features of Klebsiella including the ability to ferment lactose. But, some of the member of the Klebsiella   has shown a positive indole test, such as Klebsiella pneumonia and Klebsiella oxytoca having the ability to utilize tryptophan and produce indole. Hence, this feature helped to differentiate them from other Klebsiella species16. Genus Escherichia can do mixed acid fermentation in anaerobic conditions and utilization of tryptophan due to production of tryptophanase enzyme. Sample DbdP4 and DbdP10 displayed the biochemical characteristics of the genus Escherichia, as generally Escherichia members lack in utilization of citrate present in medium depicting negative citrate test. It has its characteristic ability to produce gas during the fermentation of lactose and other sugars21. A positive indole test, utilization of the citrate, reduction of nitrate and production of acid from utilization of lactose, glucose and maltose are characteristic features of Serratia species. But some of the member of species also shows positive Voges-proskaeur test and characteristic production of red pigment lead to the identification of Serratia marcescens. These features were displayed by the samples DbdP2 and DbdP9. Providencia species are non-fermenter of sucrose and do not can produce gas from the glucose, but they show positive indole and methyl red tests indicating that they have the characteristics of the production of tryptophanase enzyme and ability to do the mixed acid fermentation. Providencia rettgeri have all the characteristics of the genus including positive Voges-proskaeur test indicating fermentation of glucose and production of acetoin and negative test for production of urease enzyme22. These similar biochemical characteristics were found in the samples coded DbdP3 and DbdP5 suggesting them to be Providencia species and particularly Providencia rettgeri. DbdP6 and DbdP8 samples had shown negative indole test, positive methyl red and negative Voges-Proskaeur test, apart from that DbdP8 also ferment lactose and reduce nitrate Therefore, showing all these characteristics, both the samples utilized citrate as carbon source present in medium, indicating that they might be the member of the Citrobacer species.

 

ACKNOWLEDGEMENT:

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

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest

 

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Received on 23.09.2018         Modified on 06.11.2018

Accepted on 04.12.2018      © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1137-1140.

DOI: 10.5958/0974-360X.2019.00187.2