1. M.P. Council of Science & Technology
RAPD PCR profiling of lipase producing bacterial isolates from Anhoni Hot Spring, Central India
Dr. Anita Tilwari , Senior Scientist M.Sc. Microbiology,, Ph D Biotechnology PDF Molecular Medicine
From Malaysia
CENTRE OF EXCELLENCE IN BIOTECHNOLOGY
M.P. Council of Science & Technology
(Deptt. of Science & Technology, Govt. of M.P.)
Vigyan Bhavan, Nehru Nagar, Bhopal

2. Hot springs are natural ecosystem formed by the discharge of geothermally heated ground water from the volcanic activities of the earth’s crust and cracks or joints in sedimentary rock .
In Madhya Pradesh there are many hot springs like Anhoni hot spring pipariya, Choti Anhoni Sohagpur, Fatehpur Hot spring, Dhuni pani, Babeha etc.
In Indian mythology it is a belief that the hot springs are sacred and have holy ambiance with elixir like property, which can heal people from fatal disease and also that pregnant women take bath on mahashivratri and makarsankranti to heal the impotency, to be blessed with the motherhood.

3. Anhoni hot water spring is the sulphurous spring with acidic in nature.
For several decades, the bacteria from hot springs have attracted the interest of many scientists due to their biotechnological potential and scientific curiosity.
It is very important to have increased awareness of conserving microbial diversity.
Intensive research work has already been done in India and world focusing on physiochemical properties and geological features.

4. Many scientist studied biodiversity, genotypic and phenotypic characterisation of bacteria from Tarabalo and Taptapani from Orissa India, Unkeshwar , Maharashtra, Manikaran, Himachal Pradesh, Barkeshwar, West Bengal Tatta pani from Azad Kashmir, Siloam , South Africa, Soldhar and Ringigad , Uttaranchal Himalaya, and were explored for their microbial isolation and identification and molecular characterization using 16S rRNA, fatty acid analysis and enzyme production.
But the hot springs in Madhya Pradesh were not even investigated for their physicochemical properties and microbial community diversity.

5. It is very important to study the microbial diversity in order to understand the distribution of organism, functional potential, regulation, sustainable management of disturbed biodiversity
The molecular approach to the study of microbial diversity is more effective than the conventional biochemical markers as it directly access the hereditary information and makes it easy to understand the relationship between an individual
Random amplified polymorphic DNA analysis proved to be a reliable, very simple, easy, cost effective with no prior information of DNA required and can be performed even in the remote area as no need of liquid nitrogen for DNA isolation required

6. In a large number of studies RAPD reproduced very successful results in studying the genetic diversity of bacteria from soil, water, plant, etc.
This is a first new approach towards investigation of bacterial diversity among Anhoni hot spring water microbial communities living in extreme environments.
In this study, thermophilic lipase producing bacterial isolates from Anhoni hot spring were characterized using genomic patterns obtained through RAPD.

7. Aim and Objectives
This study was conducted with the aim to evaluate diversity of lipase producing bacteria isolated from Hot spring of Madhya Pradesh. India. Different microbial and molecular techniques were used in this study.
In this context, the work focused on the following aspects:
Isolation of cultivable lipase producing bacteria
Isolation of bacteria using Morphological characterization and biochemical estimation of the isolated strains.
Molecular Characterization and evaluation of genetic diversity of the isolated strains of Bacterial strains using RAPD Analysis.
Identification of the isolates belonging to different phylogenetic groups on the basis of 16S rDNA sequencing.

8. Materials and Methods Study area and sample collection
A water sample was collected in winter season the month of January 2015 from the hot water (Temp C, pH- 3.5± 5) spring, Anhoni ( ’ 39.9” N, ’ 51.4”E) located in Hoshangabad district of Central India

9. Physicochemical analysis of Hot spring water samples
Water samples were treated and analyzed for its physiochemical properties .
Water sample is subjected to physiochemical analysis like pH
TDS
Conductivity,
Chlorine
Salinity,
Dissolved oxygen (DO)
Biological oxygen demand (BOD)
Chemical oxygen demand (COD).

10. Isolation and pure culture development of bacteria from a hot spring
Isolation and screening of alkaline protease producing bacteria

11. Molecular Characterization:
Preparation of DNA samples for RAPD (Genomic DNA):
RAPD-PCR Analysis
Data analysis and Scoring
PIC = 2fi (1-fi), Where fi = Frequency of amplified allele (present band) and (1-fi) = frequency of the null allele (absent band) of marker (I).
Marker index was calculated according to the formula MI= Product of PIC and the number of polymorphic bands per assay unit (Powell et al., 1996).
SI= 2Nij/( Ni + NJ)

12. Overview of 16S rRNA Gene Sequencing
Cloning or
single-strand prep
DNA
16S rRNA gene
PCR
Vector + 16S rRNA
Sequencing
Database
search
Phylogenetic tree
extraction

13. PCR Amplification of 16S rRNA Gene
Genomic DNA or cell lysate
electrophoresis
PCR primers
dNTP
Taq DNA polymerase
95 C, 1 min
60 C, 1 min
72 C, 2 mins
35 cycles
1636 bp
Primer 9F
(5’-GAGTTTGATCCTGGCTCAG-3’)
Fig. Agarose gel electrophoresis of PCR-amplified 16S rRNA genes
16S rRNA gene
Primer 1542R
(5’-AGAAAGGAGGTGATCCAGCC-3’)

14. Result and Discussion:
Physiological Characterization of hot water spring:
Color Transparent Transparent
Odour Sulphorous
Flammable pH
Temperature (0C)
Molecular weight
Conductivity ms
TDS ppt 500
Salinity ppm
COD mg/L 250
Turbidity NTU 200
DO mg/L 7
BOD µg/ml 30

16. Total no. of polymorphic bands (b) Total no. of monomorphic bands (b)
Molecular Characterization of Bacterial isolates from Hot water spring
Primer
Accessions
Total no. of bands (a)
Total no. of polymorphic bands (b)
Total no. of monomorphic bands (b)
Polymorphism (b/a × 100)
Fragment size
(bp)
RBa-1
AM911690 10
100%
RBa-2
AM773311 8
RBa-3
AM773772 7
RBa-4
AM911679 5 3 2
60%
RBa-5
AM911680 9 1
88.8%
RBa-6
AM773778
RBa-7
AM773318 13
RBa-8
AM911681
RBa-9
AM911682
RBa-10
AM911683 4
Total
10 Primers 71 68 03
94.8
Average
7.1
6.8
0.3
94.9

17. Jaccard’s similarity coefficient among different bacterial isolates through RAPD.

18. HSM 1
HSM3
HSM 4
HSM5
HSM6A
HSM6 B
HSM7
HSM8
HSM9
HSMC A-1
CA-2
CA-4
HSMC A-5
CA-6
CA-7
HSM-1 1
HSM-3
0.339
HSM-4
0.298
0.318
HSM-5
0.220
0.328
0.382
HSM- 6A
0.299
0.393
0.333
HSM- 6B
0.305
0.268
0.35
0.197
HSM-7
0.231
0.208
0.235
0.188
0.355
0.338
HSM-8
0.246
0.141
0.27
0.238
0.379
0.537
0.344
HSM-9
0.184
0.212
0.175
0.211
0.253
0.347
0.391
HSM- CA1
0.157
0.161
0.169
0.25
0.213
0.329
HSM- CA2
0.192
0.191
0.131
0.276
0.316
0.256
0.378
0.387
HSM- CA4
0.239
0.2
0.145
0.181
0.24
0.156
0.198
0.413
HSM- CA5
0.214
0.324
0.187
0.261
0.203
0.281
0.28
0.403
0.508
HSM- CA6
0.225
0.266
0.247
0.282
0.321
0.21
0.341
0.351
0.457
HSM- CA7
0.16
0.271
0.182
0.19
0.128
0.134
0.274
0.552

19. Paired group Jaccard coefficient matrix of Bacterial isolates based on RAPD marker.

20. >gi| 123456|CA - 1 HSM | 16S ribosomal RNA gene, partial Sequence
Total sequence: 1472bp
>gi| |CA - 1 HSM | 16S ribosomal RNA gene, partial Sequence
TGATGtTAgcGGcGGACGGGTGAgTAaCACGTGGgTAACCTGCCTGTaAGACTGGGATAACTCCGGGAACCGGGGCTAaTACCGGATGcTTGTTTATGGTTCAAACATAAAAGGTGGCTTCGGCTACCCTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCaACGATGCGAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTGCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCgCGGTGAATaCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCaCGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTA

21. >gi|559795282|ref|NR_104873. 1| Bacillus subtilis subsp
>gi| |ref|NR_ | Bacillus subtilis subsp. inaquosorum strain BGSC 3A28 16S ribosomal RNA gene, partial sequence
TTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTT

22. >gi | 123456|M-4HSM| 16S ribosomal RNA gene, partial sequence
CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGTCTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATCCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAgtcGGtGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATtGGGGTGAAGTC
Total sequence: 672bp

23. Results of BLAST in NCBI Database

24. Results of BLAST in NCBI Database

25. Phylogenetic analysis of 16S rRNA gene sequences from isolate bacterial of hot. Phylogenetic trees were constructed using sequences from the representative bacterial. The dendrograms show the result from analysis using Megha 6 and neighbor-joining.

26. Phylogenetic tree (Bacillus)

27. THANK YOU