DEPARTMENT OF BIOTECHNOLOGY

COURSE CODE-1156BT701
MAJOR PROJECT
WINTER SEMESTER(2023-2024)
VIVA VOCE

ASSESSMENT OF THE ZINC SOLUBILIZATION POTENTIAL OF

BACTERIAL ISOLATES FROM AGRICULTURAL SOILS

SUPERVISED BY
Dr. V. SUGUMARI, Assistant professor,
Department of Biotechnology
PRESENTED BY
1. Prejesh Samuel Sofalas (VTUNo: 16032)
(Reg. No: 20UEBT0025)
2. Prince Preetham Raj (VTUNo: 12015)
(Reg. No: 20UEBT0026)

30-04-2024 1
OUTLINE

• INTRODUCTION
• RESEARCH GAP
• OBJECTIVE
• RESEARCH PLAN
• RESULTS
• REFERENCE

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INTRODUCTION

Role of Zinc in plants

▪ Zinc (Zn) is a vital element for the growth and development of plants. It is classified as
one among the eight key trace elements required for crop growth and output.
▪ Zinc has a significant part in consumption and metabolic processes of both eukaryotic and
prokaryotic organisms and act as a cofactor or metal activator in numerous enzyme
systems.
▪ Zinc is also essential for the synthesis of proteins and the utilization of carbohydrates in
plants, which serve as the basis and energy for growth.
▪ Zinc is required for the formation of chlorophyll, a green pigment that is crucial for
photosynthesis and the overall vitality of plants. It acts as an important cofactor in the
synthesis of chlorophyll.
▪ Plants require adequate zinc to develop to the fullest extent possible and to be healthy
overall.

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INTRODUCTION (Cont...)

Image source- https://agrovet.farmerstrend.co.ke/online-agrovet-shop-in-kenya/the-role-of-

zinc-in-plant-development-its-deficiency-toxicity-symptoms/
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INTRODUCTION (Cont...)

Zinc Solubilizing Bacteria

▪ Zinc solubilizing microorganisms solubilize zinc through various mechanisms, one of

which is acidification.

▪ These bacteria produce organic acids in soil which sequester the zinc cations and decrease
the pH of the nearby soil. The anions can also chelate zinc and enhance zinc solubility.

▪ These bacteria can also produce plant growth-promoting substances such as

phytohormones and siderophores which aid in enhancing plant growth, development, and
stress tolerance.

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REVIEW OF LITERATURE
Journal title Journal BACTERIA Year AUTHOR
Name

Contribution of Zinc Solubilizing Frontiers in Pseudomonas fragi 2017 Sana Kamrana,

Bacteria in Growth Promotion and Microbiology Izzah Shahid,
Zinc Content of Wheat Deeba N. Baig,
Muhammad
Rizwan, Kauser A.
Malik, Samina
Mehnaz

Zinc solubilizing bacteria (Bacillus Journal of Bacillus megaterium 2020 Kalpana Bhatt &
megaterium) with multifarious plant Animal Dinesh Kumar
growth promoting activities alleviates Science and Maheshwari
growth in Capsicum annuum L Biotechnology

Zinc solubilizing bacteria from the Rhizosphere Pseudomonas 2017 Iti Gontia-Mishra,
rhizosphere of rice as prospective aeruginosa Swapnil Sapre,
modulator of zinc biofortification in Sharad Tiwari
rice

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REVIEW OF LITERATURE(Cont...)
Journal title Journal BACTERIA Year AUTHOR
Name

Isolation and Characterization of Zinc International B. aryabhatta 2016 Vidyashree N.D,

Solubilizing Bacteria From Stone Journal of Muthuraju R.,
Quarry Dust Powder Agriculture Panneerselvam P.,
Sciences Saritha B.,
Ganeshamurthy
N.A.

Isolation and Identifcation of Insoluble Journal of Soil Enterobacter cloacae 2021 Fatemeh
Zinc‑Solubilising Bacteria Science and Hashemnejad,
and Evaluation of Their Ability to Plant Nutrition Mohsen Barin,
Solubilise Various Zinc Minerals Maryam Khezri,
Youbert Ghoosta,
Edith C. Hammer
Isolation, characterization, and effect Saudi Journal Pseudomonas 2019 Ahmad Zaheer,
of phosphate-zinc-solubilizing of Biological guariconensis Arif Malik,
bacterial strains on chickpea (Cicer Sciences Ahmad Sher,
arietinum L.) growth Muther Mansoor
Qaisrani, Asim
Mehmood

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RESEARCH GAP

▪ Despite extensive studies on zinc solubilizing bacteria (ZSB), there is

still a need to explore and identify novel bacterial strains capable of
efficient zinc solubilization.

▪ Investigating such microbial diversity could lead to the discovery of

novel strains that could be more effective in enhancing zinc availability
for plants.

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OBJECTIVES

▪ To collect samples from different sources.

▪ To isolate bacteria which is capable of solubilizing zinc and do further

screening and characterization.

▪ To analyze the effect of zinc solubilizing bacteria on plant growth.

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RESEARCH PLAN

Serial dilution and spread Sub-culturing of different

Collection of soil sample
plating the sample strains on a fresh medium

Culturing of strains on Bunt

16srRNA Sequencing of Identification of zinc
and Rovira medium with
bacteria solubilizing bacteria
zinc compound

In vitro zinc solubilization

Plant experiment.
using plate assay

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 METHODOLOGY
COLLECTION SOIL SAMPLE

• Soil samples were collected from two different fields where different plant
species were grown.

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METHODOLOGY (Cont...)

SERIAL DILUTION

• The soil samples were serially diluted in test tubes till 10 ⁻⁵.
• Then dilutions 10⁻³, 10⁻⁴, and, 10⁻⁵ were spread plated in an agar
medium, Inorder to isolate colonies. (Incubated for 16 hours)

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 METHODOLOGY (Cont...)

SUBCULTURING OF DIFFERENT STRAINS IN FRESH MEDIUM

• From the spread plate, the different looking colonies are then
subcultured in a new different petri dish agar medium.

• The colonies were isolated for identification purposes.

• The incubation period for patched strains is 48 hours.

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METHODOLOGY (Cont...)
SUBCULTURING IN BUNT AND ROVIRA MEDIUM

• The media was altered for the target type bacteria (ZSB) and
patched.
• The subcultured strains were then cultured again along with zinc
compounds such as; ZnCO3 & ZnO in Bunt and Rovira medium.
• This time alone the incubation period extends upto 3 days as the
growth of the bacteria in the Bunt and Rovira medium is quite
progressive.
• The Zone of clearance can be clearly perceivable in the Bunt and
Rovira medium.
• Thus, the ZSB bacteria were isolated, and grown seperately in
individual petri dishes.

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METHODOLOGY (Cont...)
BUNT AND ROVIRA MEDIUM COMPOSITION
Chemical compound Amount per litre of vol
(in grams)
Glucose 20
Ammonium Sulfate ((NH₄)₂SO₄) 0.5
Peptone 1
Potassium Hydrogen Phosphate (K₂HPO₄) 0.4
Yeast Extract 1
Tetrachloroferrate (FeCl₄) 0.01
Magnesium Chloride (MgCl₂) 0.1
Agar 20
Zinc Compound 1%

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METHODOLOGY (Cont...)

NEXT-GENERATION SEQUENCING OF THE BACTERIA

• The isolated zinc solubilising bacteria was given for sequencing and
based on BLAST analysis the bacteria was identified

Quadrant streaking

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16
METHODOLOGY (Cont...)

IN VITRO ZINC SOLUBILIZATION USING PLATE ASSAY

• The overnight grown colonies of the bacterial strain were transferred

aseptically on zinc medium plates with varying zinc compound
concentrations.

• The plates were incubated at 37°C for 3 days.

• The ZSB strain produced clear zones around colonies.

• The diameter of these zones were recorded.

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17
METHODOLOGY (Cont...)
PLANT EXPERIMENT
• Green gram was selected and zinc oxide was selected as the zinc
source.
• Three treatments were carried out:
1. Control (without zinc source + without bacteria)
2. Control + ZnO (without bacteria)
3. ZnO + Bacillus niacini
• ZSB were individually inoculated in 60 ml nutrient broth and grown in
shaking incubator at 120 rpm, 37°C.
• After 24 h, the Cells were harvested from culture at 6,000 rpm for 20
min.
• Seedlings were transferred in pots and bacterial inoculum was given to
each respective pot
• Plants were harvested after 7 days and root and shoot lengths were
recorded

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18
RESULTS

SERIAL DILUTION

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RESULTS (Cont...)

SUBCULTURING OF DIFFERENT STRAINS IN FRESH MEDIUM

From Cluster bean soil From Lady’s finger soil

sample. sample.
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RESULTS (Cont...)

SUBCULTURING IN BUNT AND ROVIRA MEDIUM

with ZnCO3 with ZnO

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 RESULTS (Cont...)
NEXT-GENERATION SEQUENCING OF THE BACTERIA

• Based on 16s rRNA sequence analysis, the isolate was identified as

Bacillus niacini with 99.88% homology.
• Sequence length was found to be 815bp.

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 RESULTS (Cont...)
PHYLOGENETIC-TREE

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 RESULTS (Cont...)
IN VITRO ZINC SOLUBILIZATION USING PLATE ASSAY
• Zinc solubilizing ability of the bacterial strains was evaluated by determining zone diameter
after incubation for 3 days.
• A zone of 0.3cm was observed when a medium with ZnO concentration of 50mg/100ml was
used.
• A zone of 0.5cm was observed when a medium with ZnO concentration of 100mg/100ml was
used.
• A zone of 0.7cm was observed when a medium with ZnO concentration of 150mg/100ml was
used.

Medium with 50mg/100ml Medium with 100mg/100ml Medium with 150mg/100ml

ZnO concentration ZnO concentration ZnO concentration
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 RESULTS (Cont...)
IN VITRO ZINC SOLUBILIZATION USING PLATE ASSAY

• No clearance zone was formed when a medium with ZnO concentration of 200mg/100ml was
used.
• No clearance zone was formed when a medium with ZnO concentration of 250mg/100ml was
used.
• This indicates that the zinc supply to the bacteria has reached its peak, and concentrations
exceeding 150mg/100ml result in zinc toxicity or inhibition.

Medium with 200mg/100ml Medium with 250mg/100ml

ZnO concentration ZnO concentration
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 RESULTS (Cont...)
PLANT EXPERIMENT

• There was an increase in the shoot and root lengths of plants treated with
ZnO and zinc solubilizing bacteria.
• There was a decrease in the shoot and root lengths of plants treated with
only ZnO.

TREATMENT SHOOT LENGTH ROOT LENGTH

Control 7.7cm 2.9cm

Control + ZnO 5cm 1.8cm

ZnO + Bacillus niacini 9.5cm 4.2cm

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 RESULTS (Cont...)
PLANT EXPERIMENT

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REFERENCES

1. Kamran, S., Shahid, I., Baig, D. N., Rizwan, M., Malik, K. A., & Mehnaz, S. (2017).
Contribution of zinc solubilizing bacteria in growth promotion and zinc content of wheat.
Frontiers in microbiology, 8, 322772.
2. Bhatt, K., & Maheshwari, D. K. (2020). Zinc solubilizing bacteria (Bacillus megaterium) with
multifarious plant growth promoting activities alleviates growth in Capsicum annuum L. 3
Biotech, 10(2), 36.
3. Gontia-Mishra, I., Sapre, S., & Tiwari, S. (2017). Zinc solubilizing bacteria from the rhizosphere
of rice as prospective modulator of zinc biofortification in rice. Rhizosphere, 3, 185-190.
4. Vidyashree, N. D. (2016). Isolation and characterization of zinc solubilizing bacteria from stone
quarry dust powder. Intl J Agri Sci ISSN, 0975-3710.
5. Hashemnejad, F., Barin, M., Khezri, M., Ghoosta, Y., & Hammer, E. C. (2021). Isolation and
identification of insoluble zinc-solubilising bacteria and evaluation of their ability to solubilise
various zinc minerals. Journal of soil science and plant nutrition, 21, 2501-2509.
6. Zaheer, A., Malik, A., Sher, A., Qaisrani, M. M., Mehmood, A., Khan, S. U., ... & Rasool, M.
(2019). Isolation, characterization, and effect of phosphate-zinc-solubilizing bacterial strains on
chickpea (Cicer arietinum L.) growth. Saudi journal of biological sciences, 26(5), 1061-1067.

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REFERENCES

7. Upadhyay, H., Gangola, S., Sharma, A., Singh, A., Maithani, D., & Joshi, S. (2021). Contribution
of zinc solubilizing bacterial isolates on enhanced zinc uptake and growth promotion of maize
(Zea mays L.). Folia microbiologica, 66, 543-553.
8. Naz, I., Ahmad, H., Khokhar, S. N., Khan, K., & Shah, A. H. (2016). Impact of zinc solubilizing
bacteria on zinc contents of wheat. Am. Euras. J. Agric. Environ. Sci, 16, 449-454.
9. Yadav, R. C., Sharma, S. K., Varma, A., Rajawat, M. V. S., Khan, M. S., Sharma, P. K., ... &
Saxena, A. K. (2022). Modulation in biofertilization and biofortification of wheat crop by
inoculation of zinc-solubilizing rhizobacteria. Frontiers in Plant Science, 13, 777771.
10. Sirohi, G., Upadhyay, A., Srivastava, P. S., & Srivastava, S. (2015). PGPR mediated Zinc
biofertilization of soil and its impact on growth and productivity of wheat. Journal of soil science
and plant nutrition, 15(1), 202-216.

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 THANK YOU

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