Physiological study of some Bacillus spp. resistant to heavy metals and improving their ability of bioremediation

Other Title(s)

دراسة فسلجية لبعض أنواع من جنس Bacillus المقاومة للمعادن الثقيلة و تحسين قابليتها الإصحاحية

Dissertant

Mukhaifi, Iman Abub

Thesis advisor

al-Sulami, Amin Abd al-Jabbar Abd Allah

Comitee Members

Thuwayni, Qasim N. Ubayd
al-Asadi, Iqbal Jassam
Nahir, Habib S.
Mahdi, Kawthar H.

University

University of Basrah

Faculty

Science College

Department

Department of Biology

University Country

Iraq

Degree

Ph.D.

Degree Date

2014

English Abstract

In this study , a plating procedure was used for isolating Bacillus species from soil contaminated with heavy metals and from agricultural ones in Basrah governorate ,on the selective medium yeast tryptophan peptone glucose (YTPG) agar supplemented with different concentrations of heavy metals.

Isolates were identified by conventional tests .

Consequently, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis and Bacillus amyloliquefascienis were isolated from the soils of Paper factory, Abu-ALkhassib, Iron and Steel factory, Jabal Sanam and Garmat Ali soils .Bacillus cereus was isolated from petrochemical factory soil.

Biochemical tests, used to identify Bacillus species included: gelatin and starch hydrolysis, nitrate reduction, casein hydrolysis ,catalase, citrate, salinity tolerance and Gram stain.

All Bacillus isolates from soils ,appeared as Gram positive, spore forming ,rod cell, positive for casein and starch hydrolysis.

The results showed that there are specific and different degrees of resistance among wild type isolates to heavy metals in nutrient broth medium.All isolates exhibited higher resistance to heavy metals after the exposure to 6.4g/L of Acridine orange LSD=0.06900 with p<0.01 For revealing the effect of heavy metals on the growth of wild types, aliquots of 100 ml nutrient broth was placed in 250 ml conical flasks and inoculated with 1ml of 24h old broth culture of any of the Bacillus species isolates .

After 2h incubation the OD600 ,the culture starts to increase exponentially and high significant difference in growth at me 3h p=0.001,LSD=0.0334 with p<0.01 was observed .

The exponential phase, con nued un l me 17h .There were significant differences (p <0.05) of the effect of heavy metals on the growth of the Bacillus species when they were exposed to low concentra ons, 2mM and 4mM of heavy metals .

Conversely, there were highly significant differences of the heavy metals effects on the growth of the organism when exposed to higher concentra ons of 6mM and 8mM, p=0.001 with p<0.01.

For instance, the effect was so pronounced that the B.megaterium was unable to enter into the logarithmic phase of growth when it is exposed to high concentrations of Fe+2 as compared to control.

However, after the exposition to acridine orange, the B.megaterium entered into the logarithmic phase a er 8h in the presence of 12 mM of Fe+2 and 9h in the presence of 8mM of Cd+2 in case of B.cereus.

The results showed that there was high significant difference in the growth among isolates toward pH, NaCl and temperature with p<0.01 and LSD=0.28000 .Optimum temperature of the tested isolates were 30ºC in all isolates, while most of them preferd salinity of 5% and optimum pH of 7.

The results showed different uptakes of Cd+2 ,pb+2,Zn+2 ,Ni+2 , Fe+2 and Cu+2 between biomasses of mutant and wild type of Bacillus isolates in 30 ,40 and 60 mg/L of heavy metals solution.

Concentrations of Cd+2,Pb+2 and Ni+2 rapidly decreased during the first 24h in the solution with no further decrease .

While concentrations of Zn+2 ,Cu +2and Fe+2 continued decreasing until time 48h.

Percent of uptake of heavy metals decreases as the added heavy metals increases .

The results revealed that Bacillus cereus mutant has higher emulsification ac vity and biosurfactant produc on .

Using 4g/l glutamic acid ,3% glycerol, pH7,30⁰C and 3day for incuba on represent op mum condi ons for biosurfactant production.

Purified biosurfactant from the wild type and mutant B.cereus were analyzed by TLC to detect amino acids in the biosurfactant.

GC-mass results showed the presence of clear peak corresponding to Palmitic acid C16H32O2 in mutant and wild type of B.cereus.

IR-spectrometry showed the main functional groups in mutant and wild type of B.cereus .

The melting point of biosurfactant was (129 -131)⁰C and that both of them are identical.

In this study ,lipopeptide biosurfactant produced by Bacillus cereus was used for remediation of cadmium and lead from contaminated soil .

In addition to that ,the remediation was carried on by a combination of the biosurfactant and B.cereus biomass or B.licheniformis biomass.

Bioremediation is generally a slow process, addition of biosurfactant with biomass to soil enhances bioremediation of field with a significant reduction of the time required to bioremediate contaminated sites , spread and sequester of heavy metals away from contaminated soil.

Main Subjects

Biology

No. of Pages

150

Table of Contents

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Language

English

Data Type

Arab Theses

Record ID

BIM-748119