Exploration of halotolerant bacteria for its potential in degradation of dissolved ammonia from Aquacuture.

1. Exploring the potential of halotolerant bacteria in the degradation of
dissolved ammonia from aquaculture farm
Hari Mohan Jha
MB0515
Under supervision of
Prof. Savita Kerkar
Dept. of Biotechnology, Goa university

2. Dissertation Stage - II
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3. Aim
• EXPLORING THE POTENTIAL OF HALOTOLERANT BACTERIA IN THE DEGRADATION OF DISSOLVED
AMMONIA FROM AQUACULTURE FARM.
3

4. Objectives
• Isolation of ammonia utilizing bacteria from aquaculture pond.
• Screening of bacteria for ammonia oxidizing activity.
• Determining the effects of the Ammonia Oxidizing Bacteria (AOB) on the concentration
of inorganic nutrients and physico-chemical parameters.
• Partial Identification of the AOB.
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5. Results
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6. Bacteria isolation
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 Sample collected from Site 1 (S1A, S1B,
S1C) and Site 4 (S4A, S4B, S4C)
• Water sample was collected from Dr. Rivonkar Aquaculture
Farm (15.4602° N,73.8950° E)

7. Bacterial growth analysis
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• Figure showing bacterial culture growth for
SK0116 to SK1016 (SKCC).
• No significant growth observed in SK0116,
SK0216, SK0316, SK0416, SK0516.
• SK0616, SK0716, SK0916, SK0816 showed
maximum growth within allotted period of
time.
• Figure showing bacterial growth for SK1116, SK1216,
SK1316, SK1416, SK1716, ASK9, ASK35, ASK11,
ASK29, ASK186
0
0.2
0.4
0.6
0.8
SK1116 SK1216 SK1316 SK1416 SK1716 ASK9 ASK35 ASK11 ASK29 ASK186
Opticaldensity
Bacterial culture
Bacterial Growth
2 Days 4 Days 6 Days
]o

8. 8
• Figure showing bacterial growth for S1A, S1B,
S1C, S4A, S4B, and S4C ( From aquaculture farm)
• Every bacterial culture showed significant growth
in provided conditions.

9. Analysis for Ammonium degradation
• Figure showing ammonium concentration at
interval of 2days for first 10 bacterial culture
from SKCC.
• SK0916 showed only ammonium degradation
i.e. 41% , whereas SK1016, SK0816 showed
increased then degradation in ammonium
concentration.
9
0
20
40
60
80
100
120
140
160
180
200
Control SK0116 SK0216 SK0316 SK0416 SK0516 SK0616 SK0716 SK0916 SK1016 SK0816
Conc(mg/L)
Culture
Day 2 Day 4 Day 6

10. •
10
• Figure showing ammonium concentration at
interval of 2days for next 10 culture.
• ASK29 showed only ammonium degradation
0
20
40
60
80
100
120
SK1116 SK1216 SK1316 SK1416 SK1716 ASK9 ASK35 ASK11 ASK29 ASK186
AmmoniumConc.(mg/L)
Bacterial culture
2Days 4Days 6Days
• Figure showing ammonium concentration at interval
of 2days for aquaculture isolates.
• S1A showed significant ammonium degradation i.e.
42% in six days interval.

11. Nitrite estimation
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0
1
2
3
4
5
6
7
SK09 ASK29 S1A
Conc.(uM)
• Figure showing nitrite production by SK09, ASK 29 and S1A

12. Nitrate estimation
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• Figure showing nitrate production by SK09 and S1A

13. Overall study on Bacterial strain
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• Figure showing the correlation in different physico
chemical character for S1A

14. 14
• Figure showing the correlation in different physico
chemical character for SK09

15. Comparison between bacterial culture on the basis observed traits
SK09 (SKCC)
• less
• 41%
• 1uM
• 1.3uM
• 3.02mg/L
S1A (Aquaculture farm)
• more
• 42%
• 5.7uM
• 2.82uM
• 0.63mg/L
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• Characterstics
• Growth
• Ammonium degradation
• Nitrite production
• Nitrate production
• Oxygen consumption

16. Bacterial identification
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• Gram staining for S1A under 100X magnification oil immersion

17. Biochemical Test
Carbohydrates S1A Carbohydrates S1A Carbohydrates S1A
Lactose + Salicin + Sodium gluconate -
Xylose + Dulcitol - Glycerol +
Maltose + Inositol - alpha-methy-D-mannoside -
Fructose + Sorbitol + Xylitol -
Dextrose + Mannitol + ONPG +
Galactose - Adonitol - Esculine hudrolysis +
Raffinose + Arabitol - D- Arabinose -
trehalose + Erythritol - Citrate utilization -
Melibiose + alpha-methy-D-glucose - malonate utilization -
Sucrose + Rhamnose - Sorbose -
L-Arabinose + Cellobiose + Melezitose -
Mannose + Inulin +
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18. Scanning electron microscopy
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• Figure showing SEM image of S1A at different magnification i.e.5000X and 7000X respectively
• The size of bacteria found to be 2.640um.

19. 16S rRNA sequencing
• >S1A
AACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATT
GAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCA
ACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA
GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAAC
TCTGTTGTTAGGGAAGAACAAGTATGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTAC
GTGCCAGCAGCCGCGGTAATACGTAGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTA
AGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGC
GGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACT
GACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA
GTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAA
ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCA
GGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCG
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGC
ACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCT
ACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCG
GATTGTAGCCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGG
GCCTTGTACACACCGCCCGTCACACCACGAGAGTTT GTAACACCC
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20. Evolutionary relativeness.
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22. • Closest neighbours to S1A
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23. Summary
• The project work focused on the potential application of halotolerant bacteria for degradation of
dissolved ammonia.
• Halotolerant Bacteria from an aquaculture farm were isolated and hypersaline bacteria from SKCC
were used for the study.
• The farm and saltern bacteria were screened for Ammonium oxidization activity.
• Ammonia Oxidising Bacteria (AOB) were further analysed for its effect on different
physiochemical parameters and nutrients.
• The best ammonia oxidising bacteria were selected based on its potential to degrade dissolved
ammonia and oxygen utilisation.
• The bacteria S1A showed similarity with Bacillus aryabhattai (99.85%), Bacillus
megaterium (99.7%) and Bacillus flexus (98.86%) based on 16S rRNA sequencing, phylogenetic
analysis, morphological and biochemical characteristics.
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24. Conclusion
• The halotolerant bacteria S1A isolated from aquaculture farm showed ammonium oxidizing
activity which could be explored further as a bioremidiator of Ammonia from aquaculture farm.
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25. Acknowledgement
I would like to thank
• Department of Biotechnology , New Delhi
• HOD Dept. of Biotechnology and My Guide Prof. Savita kerkar
• All faculty members Usha Ma’am, Barros Ma’am, Ghadi Sir, Abhishek Sir, Gautam Sir
• Samantha Ma’am and all Research scholar of our Dept.
• Non teaching staff
• My classmates , my juniors and our seniors .
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28. Thank you
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