ARSENIC BIOREMEDIATION

Prepared by : Students of Microbiology (AS114) , UiTM

Nurshahira Bt Ruslan Nurhasniza Bt Tajuddin Nur Azila Suhana Bt Mat Yunos Nurul Ain Najihah Bt Norazizan Muhammad Azizi Hazim B. Ruszaidin

Title of journal chosen: Arsenic mediated modifications in Bacillus aryabhattai and

their biotechnological applications for arsenic bioremediation

Authors’ name : Namrata Singh, Sunil Gupta, Naina Marwa, Vivek Pandey, Praveen C.Verma,

Sushma Rathaur, Nandita Singh

Editor : Martin Leemakers

Available online : 10 September 2016

History of Bioremediation600 B.C.- Bioremediation dicovered by Romans who utilized the microorganisms to treat the waste water

1960 - George M Robinson invented bioremediation during experimentation with dirty jars

1975- Ananda Mohan Chakrabraty developed the oil-eating superbug that able to degrade some component in crude oil

1989- The term bioremediaton began to widespread after The Exxon Valdez oil spill in Prince William Sound, Alaska

1990- Derek Lovley and coworkers was the first proposed to cleanup of uranium comtamination in groundwater

Problems* 2 most toxic forms of arsenic : Arsenate [As(V)] & Arsenite [As(III)] that exist in environment ( water and agriculture land)* Arsenate is more toxic than arsenite * This toxic come from human activities such as pesticide, wood preservative, mining and smelting operation.

Biotechnology solutions :* Bacillus aryabhattai show capability for uptake and volatilization of arsenic * It can be use in arsenic bioremediation* In this bacteria, there are arsenic operon that play important role to reduce arsenic toxicity* 7 defferentially expressed protein had been found to be up-regulated in bacterial cell upon As exposure which may have role in reducing As toxicity in bacterial cell .

METHODS

Bacterial strain and growth condition

Arsenic accumulation capacity of bacterial cells from liquid media

ars Gene determination

Electrophoresis of total bacterial protein

Sample preparation for SDS-PAGE

Protein identification

MS and MS/MS analysis

Scanning electron microscope coupled with energy dispersive X-ray (SEM-EDS) spectroscopy

Fourier-transform infrared (FTIR) spectroscopy

1) Bacterial strain & growth condition

* Obtain strain from the rice rhizosphere.* Streak strain on plates that was added with sodium arsenate and sodium arsenite separately, then incubated.* Inoculated in broths that was added arsenate and arsenite separately, then incubated for 24 hours by shaking (200rpm) at 32°C. * The absorbance in broths was measured at 600nm.

Rice Rhizosphere

2) Arsenic accumulation capacity of bacterial cell in liquid media

* The strain grown in nutrient agar that was added with arsenate

* The sample then centrifuged at 5000rpm for 10 minutes.

* The pellet that formed was let to dried, digested with HNO3, then determined using ICP-MS spectroscopy

* The ability of strain to volatilize arsenic was calculated.

ICP-MS SPECTROSCOPY

3) Ars Genes Determination

* Specific primers designed: arsR, arsD, arsA, arsB, arsC, arsH and arsAB.

* Perform PCR (involves template [genomic DNA] and primers [arsR, arsA, arsD, arsB,arsC,arsH and arsAB] )

* ars gene amplification was analysed by gel electrophoresis.

4)Electrophoresis of total bacterial protein

* Grown bacteria in nutrient broth (with & without arsenate)

* The bacteria then centrifuged, wash with NaCl2, resuspend in Tris-HCL, disruptured by sonication, centrifuged again and the sample collected

5) SDS-PAGE

* Sample from above step was taken for further the SDS-PAGE

* The protein bands was resulted from the SDS-PAGE process.

6) Protein Identification

* To extract peptide

* Protein bands that yield from the SDS-PAGE are used in this process

* The gel was washed with ammonium bicarbonate, de-stained, dried. Immersed in ammonium bicarbonate

* The sample digested

* Then the peptide were extracted from this sample

* The peptide desalted

7) MS & MS/MS Analysis* Identify peptide sample using MS/MS machine

8) SEM-EDS Spectroscopy* To observe surface structure & metal distribution of strain culture in Arsenate.

9) FTIR Spectroscopy* Grow bacteria in broth, incubate, centrifuge, washed, and dried for 24 hours at 40°C

* This dried sample analysed using FTIR spectrophotometer.

MS & MS/MS ANALYSIS

Result and discussion :

3.1 Arsenic accumulation capacity of bacterial cells from broth

* Concentration of As(V) in the bacterial biomass increases, concentration of As(V) in liquid medium decreases.

* Amount of bioaccumulation and biovolatilization increases, with increasing time period.

* Control sample (not exposed to NBRI014), no changes observed in As(V) concentration.

* NBRI014 has capability of removing As(V) from liquid medium by biovolatilization.

3.2 ars Gene profiling of selected As tolerant strain

* Multiple ars (arsenic resistant) genes had been evaluted for their presence by using gene specific primers. Eg : arsAB, arsD, arsR, arsH, arsC, arsA and arsB.

i) arsC gene – act as a cytoplasmic As(V) reductase and reduce arsenate to arsenite.

i) arsH gene – confer high levels of arsenite resistance.

iii) arsD gene – control the expression of the As operon.

iv) arsR gene – works as an As inducible which regulates ars operon in the presence of As(III).

v) arsB gene – pump As(III) out of the cells using proton motive force.

vi) arsA gene – is an As(III)-activated ATPase.

vii) arsAB gene – functions in the reduction mechanism of the arsA ATPase.

* As resistant gene (arsRDABC)

- helps in As detoxification and reduces As(V) into As(III).

- its mechanism were well established in As resistant bacteria. Eg : Klebsiella, Escherichia, Pseudomonas and Serptococcus.

- volatilization rate affected by the expression of ars operon in bacterial strain.

- could play a useful ecological role in remediation of As toxicity and mobility in As-contaminated sites.

3.3 SEM-EDS analysis of bacterial strain

SEM micrographs analysis* NBRI014 grown without As(V) – circular, rod shaped, smooth surface.

* NBRI014 grown with As(V) – irregular, enlarged with rough and wrinkled appearances.

* arsenate may be located in the vacuole of bacterial cells.

Energy dispersive X-ray spectroscopy(EDS) analysis* As(V) treated cells – arsenic peak was observed.

* control –no such peak was observed.

3.4 FTIR spectroscopy in bacterial biomass

* Without As(V) - Infrared absorption frequencies of each peak and the corresponding functional group were displayed.

* With As(V) - Clear spectrum changes were observed in range of 1000-500 per cm and 3500-3000 per cm

* It is may be due to the metal chelating where the metal bound to functional group ligands on the bacterial cell surface.

* There was a metal binding process proceeding on the surface of the bacterial cells with functional group.

* Involved in the interaction with metal ions.

* Broad absorption peak within 1000-500 per cm – indicates the existence of C-Br, N-H, and the C-H bond.(amino group)

* Strong band within 3500-3000 per cm – due to N-H and O-H bond.(alcohol group)

* These indicates the biosorption activity.

* With As(V)- negatively-charged group present in the bacterial cell wall absorb metal cations through various mechanism, eg : electrostatic interaction, van der waals forces, covalent bonding.

* As was suggested to be complexes with polarizable functional group on the cell surfaces of Bacillus aryabhattai.

3.5 MALDI TOF analysis of bacterial protein

SDS-PAGE and Image Quant TL 7.0 analysis * Showed 17 clear bands in Bacillus aryabhattai.

* The band were cut and trypsin digested for identification.

* It show out of 17 protein in Bacillus spp. , 43% were up-regulated while 57% have no change in expression pattern.

* Up-regulated protein related to the energy metabolism, proline synthesis and membrane protein.

* Eg, an enzyme (L-amino acid amidase) relate to proline biosynthesis was up-regulated.

* High proline level confers tolerance to bacteria against high As stress.

* Elongation Factor Tu (EF-Tu) -a protein that perform multiple function.

* Induction of EF-Tu in presence of As showed the adaptation bacteria toward the given stress condition.

* It show the interplay of proteins in coping with high As stress/accumulation.

MS & MS/MS Analysis* The expressed protein databases with peptide mass fingerprints obtained by MALLDI-TOP MS- indicates the proteome level on microbe response mechanism in stressful condition.

* It reveals the accumulation potential of NBRI014 in As polluted sites.

* This strain prove to be responsible for detoxification As due to the presence of ars operon system(own defence mechanism for As resistance).

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