1. 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
2. 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
3.
4. History of Bioremediation
600 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
5. 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.
6. 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.
7. 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
8. Protein identification
MS and MS/MS analysis
Scanning electron microscope coupled with energy
dispersive X-ray (SEM-EDS) spectroscopy
Fourier-transform infrared (FTIR) spectroscopy
9. 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.
11. 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.
13. 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.
14. 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.
15.
16. 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
17. 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.
21. 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.
22. 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.
23.
24. * 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.
25. 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.
26.
27. 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.
28. * 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.
29.
30. 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.
31. * 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.
32. 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).
33.
34. conclusion
The stain Bacillus aryabhattai
(NBRI014) was capable of removing
considerable amount of arsenic (As)
by expressing ars genes.
Elemental composition and relative
distribution of arsenic (As) in bacterial
cell, revealed that functional groups
involved in arsenic (As) binding and it was
confirmed by FTIR analysis.
The strain NBRI014 also be able to
remove 7 new unregulated proteins
which may increase bacterial tolerance
under arsenic stress condition.
