Selective bio-analytical methods for specific identification and detection of toxic microcystis species

312 Pages·2013·4.59 MB·English

by  Matizirofa, Lazarus

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SELECTIVE BIO-ANALYTICAL METHODS FOR SPECIFIC IDENTIFICATION
AND DETECTION OF TOXIC MICROCYSTIS SPECIES AND MICROCYSTINS
IN WATER
_________________________________________________________________________

by
ELBERT A. MBUKWA

Thesis in fulfilment of the requirement for the degree

PHILOSOPHIAE DOCTOR
in
CHEMISTRY

in the
FACULTY OF SCIENCE
of the
UNIVERSITY OF JOHANNESBURG

SUPERVISOR: Dr. TITUS A. M. MSAGATI
CO-SUPERVISOR: Prof. BHEKIE B. MAMBA

March, 2013
AFFIDAVIT: MASTER’S AND DOCTORAL STUDENTS
TO WHOM IT MAY CONCERN

This serves to confirm that I
_______________________________________________________________________________________
Full Name(s) and Surname

ID Number
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Student number enrolled for the
___________________________________________________________________________________

Qualification
__________________________________________________________________________________________________________

In the Faculty of Science

Herewith declare that my academic work is in line with the Plagiarism Policy of the University of
Johannesburg which I am familiar.

I further declare that the work presented in the (minor
_________________________________________________________
dissertation/dissertation/thesis) is authentic and original unless clearly indicated otherwise and in
such instances full reference to the source is acknowledged and I do not pretend to receive any
credit for such acknowledged quotations, and that there is no copyright infringement in my work. I
declare that no unethical research practices were used or material gained through dishonesty. I
understand that plagiarism is a serious offence and that should I contravene the Plagiarism Policy
notwithstanding signing this affidavit, I may be found guilty of a serious criminal offence (perjury)
that would amongst other consequences compel the UJ to inform all other tertiary institutions of the
offence and to issue a corresponding certificate of reprehensible academic conduct to whomever
request such a certificate from the institution.

Signed at Johannesburg ________________ on this _________________ of______________________2013

Signature_______________________________________ Print name_______________________________

STAMP COMMISSIONER OF OATHS
Affidavit certified by a Commissioner of Oaths

This affidavit conforms with the requirements of the JUSTICES OF THE PEACE AND COMMISSIONERS OF OATHS ACT
16 OF 1963 and the applicable Regulations published in the GG GNR 1258 of 21 July 1972; GN 903 of 10 July 1998; GN
109 of 2 February 2001 as amended.
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DEDICATION

The work presented in this Thesis is dedicated to you
Gaudencia & Dyness

………………………………………………………..

Kindly remember that:
“Too much of a good thing is good thing”
(Alan Jackson)
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ACKNOWLEDGEMENTS

I would like to express my sincere and heartfelt thanks to my supervisors, Dr T.A.M.
Msagati and Prof B.B. Mamba. Dear my supervisors, your efforts go beyond the list of
things that can be mentioned here to express how much I am indebted to you. However, to
mention the least, this work would not have been made possible if it were not for the
invaluable and kind guidance, support, contributions, directions, courage, etc, that I had
received from you during the course of my program. At times, when I was so depressed
and pressed down your support lifted me up and led me to a right direction. I am indeed
grateful, thank you so very much. I am also so grateful to Prof. V. Wepener for his
sincerity, help and above all his support during sampling and direct involvement in a
molecular study component that has contributed much to my PhD degree. Dear Prof.
Wepener, your support for a research visit to Israel, has been an unforgettable event that
has enriched my academic life. From Israel, I am indebted to Prof. S. Boussiba for his
willingness to accept my research visit and for his supervision and support during my stay
in his laboratory. Dear Drs. L. Stefan, K. Yuval your supervision and guidance I received
are highly appreciated. Thank you very much for your invaluable contributions. Dear
academic staff, colleagues, technical staff and S. Illana from the Landau Family-
Microalgal Biotechnology Laboratory (MBL), you were so great and very supportive and
made my research work an exciting event in Israel. May you receive the blessings from the
Lord God of Israel! Tehla (Israel post office) I thank you for connecting me with my
family.
I acknowledge all technical and administrative staff members from the Departments of
Applied Chemistry (at DFC) and Zoology (at APK) for your kind support during my
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program at the laboratories housed in the above Departments, University of Johannesburg.
Almighty Lord God bless you all.
A day could not pass through in a laboratory without interacting or seeking assistance or
help from a colleague in the Laboratory no matter on what scale the asking could be
measured. Therefore, I would like to appreciate the companionship I enjoyed from you all
dear ones while working at the Department of Applied Chemistry at Doornfontein
Campus. Administrative staff (Faculty of science, DFC/APK), thank you very much.
Great are days you stay with your family, the opposite is very difficult to compare to say
the least. I therefore would like to gratefully express my sincere thanks to you my wife
Gaudencia, our daughter Dyness and the family at large for your endurance, support and
continued prayer, and encouragement to me. Guess what? I love you. Yes, Dyness Dad has
now completed his home work! So he will be home and available for the evening walks,
prayer and Fridays. Also acknowledged in this list are all family members that have been
so supportive to my family every now and then during my absence. Pastor Joseph and the
church family please receive and share God’s blessing with your families.
I would like to acknowledge the World Bank-Science, Technology and Higher Education
Project (WB-STHEP) through the Dar es Salaam University College of Education, DUCE
(University of Dar es Salaam) for supporting my PhD program. I am thankful to DUCE
(University of Dar es Salaam) for generously granting me a study leave with special thanks
going to the offices of the Principal, DPA, STHEP-DUCE (DP-PFA), Dean (Sci) & HoD
(Chem). It is very difficult to mention every one by name or otherwise. Therefore from
whomever I received a hand, word, wish or prayer of any kind; please receive my sincere
appreciation.
All glory is in the hands of the Almighty God. “He alone stretches out the heavens and
treads on the waves of the sea” Job 9:8 (NIV). Amen.
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PUBLICATIONS AND CONFERENCES

PUBLICATIONS
The work presented in this Thesis is based on a number of papers, some of which have
already been published. These papers are listed below and will be referred in text using
respective Roman numbers. Conference presentation titles are also included in this section.

PAPER I (Chapter 4)

Mbukwa, E., Msagati, TAM., Mamba, BB., Boussiba, S., Wepener, V., Leu, S., Kaye, Y.
Toxic Microcystis novacekii T20-3 from Phakalane ponds, Botswana: PCR amplifications
of microcystin synthetase (mcy) genes, extraction and LC-ESI-MS identification of
microcystins (2012), (Manuscript in Preparation).

PAPER II (Chapter 5)
Elbert A. Mbukwa, Sammy Boussiba, Victor Wepener, Stefan Leu, Yuval Kaye, Titus A.
M. Msagati and Bhekie B. Mamba. PCR amplification and DNA sequence of mcyA gene, a
case of the distribution profile of a toxigenic Microcystis aeruginosa in the Hartbeespoort
Dam, South Africa. Journal of Water and Health (2012) (Submitted Manuscript).

PAPER III (Chapter 6)
Elbert A. Mbukwa, Sammy Boussiba, Victor Wepener, Stefan Leu, Yuval Kaye, Titus
A. M. Msagati and Bhekie B. Mamba. Potential use of dissolved cyanobacterial DNA for
monitoring toxic Microcystis cyanobacteria in filtered water. J. Phys. Chem. Earth (2012)
(Submitted Manuscript).

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Publications and Conferences
PAPER IV (Chapter 7)
Mbukwa, E.A., Msagati, T.A.M., Mamba, B.B. Quantitative variations of intracellular
microcystin-LR, -RR and -YR in samples collected from four locations in Hartbeespoort
Dam in North-West Province (South Africa) during the 2010/2011 summer season. Int. J.
Environ. Res. Public Health. (2012), 9: 3484-3505.
PAPER V (Chapter 8)
Mbukwa, E.A., Msagati, T.A.M, Mamba, B.B. 2012a. Supported liquid membrane-liquid
chromatography–mass spectrometry analysis of cyanobacterial toxins in fresh water
systems. J. Phys. Chem. Earth. (2012), 50–52: 84–91.

PAPER VI (Chapter 9)
Elbert A. Mbukwa, Titus A.M. Msagati and Bhekie B. Mamba. Preparation of
guanidinium terminus-molecularly imprinted polymers for selective recognition and solid-
phase extraction (SPE) of [arginine]-microcystins. Anal Bioanal Chem (2013) (In Press).
Research paper presentations at international and/or local conferences
1. Elbert Mbukwa, Titus A.M. Msagati and Bhekie Mamba. Solid-phase extraction
and high performance liquid chromatography-mass spectrometric determination of
microcystins from algal blooms. Poster presentation at the international conference
held in April, 2011 in Brazil.
2. Elbert A. Mbukwa, Titus A.M. Msagati and Bhekie B. Mamba. Supported liquid
membrane-liquid chromatography-mass spectrometry analysis of cyanobacterial
toxins in fresh water systems. Poster presentation at the 12th
WaterNet/WARFSA/GWP-SA international symposium held from 26-28th October,
2011 in Maputo, Mozambique.
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Publications and Conferences
3. Elbert A. Mbukwa, Titus A.M. Msagati and Bhekie B. Mamba. Synthesis of
guanidinium terminus-molecularly imprinted polymers and their applications for
selective removal of highly toxic microcystins in water. Poster presentation at the
5th DST/MINTEK annual NIC workshop "Building a healthy nation through
nanotechnology", held from 21-22nd September, 2012 in Cape Town, South Africa.
4. Elbert A. Mbukwa, Sammy Boussiba, Victor Wepener, Stefan Leu, Yuval Kaye,
Titus A. M. Msagati and Bhekie B. Mamba. Investigation of a PCR method for
early detection of toxic Microcystis sp in water using dissolved cyanobacterial
DNA templates. Poster presentation at the international UNESCO/DAAD-Alumni
conference on “Water in Africa”. Sustainable water management in African
countries, held from 1st-3rd October, 2012 in Kisumu, Kenya.
5. Elbert A. Mbukwa, Sammy Boussiba, Victor Wepener, Stefan Leu, Yuval Kaye,
Titus A. M. Msagati and Bhekie B. Mamba. Potential use of dissolved
cyanobacterial DNA for monitoring toxic Microcystis cyanobacteria in filtered
water. Oral presentation at the 13th WaterNet/WARFSA/GWP-SA International
symposium held from 31st October – 2nd November, 2012 in Johannesburg, South
Africa.

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ABSTRACT

The aim of this study was to investigate the toxigenicity of Microcystis spp from the water
environment using a multi-disciplinary approach involving molecular-based methods for
early detection of toxigenic species, chromatographic separation method development and
organic synthesis of polymers for selective extraction, separation, structural
characterization and quantification of microcystins. The occurrence and potential for
production of MCs from Microcystis spp growing in the Hartbeespoort Dam (South Africa)
and Phakalane pond effluents (Botswana) were determined using polymerase Chain
Reaction (PCR) method after DNA extraction. Purified DNA extracts from algal/water
samples were confirmed to be of cyanobacterial origin by PCR amplification of
cyanobacterial specific 16S-rRNA gene and separated by agarose gel electrophoresis and
products visualized after ethidium bromide staining. A product size pertaining to 16S-
rRNA gene PCR product was 500 bp based on the PCR primer pair used in this study. The
investigated DNA samples showed the presence of six microcystin synthetase (mcy) genes,
namely mcyA, -B, -C, -D, -E & -G that are known to be directly involved in the
biosynthesis of MCs in toxigenic Microcystis spp. The above genes were amplified using
gene specific PCR primers followed by agarose gel electrophoresis and visualized after
ethidium bromide staining. Respective PCR product sizes (bp) were confirmed based on
theoretical calculations and literature information. In this study four (4) new and highly
conserved PCR primers were designed and used for consistent and successful
amplifications of mcyA and mcyB genes from the Hartbeespoort Dam samples. These new
primers were also shown to successfully amplify mcyA & mcyB genes from Phakalane
pond Microcystis isolates (samples). Results from DNA sequencing using new mcyA
primers and subsequent BLASTN sequence alignment showed that the DNA samples from
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