Isolation, identification and screening of alkaliphilic bacteria from mangrove sediment and alkaline

154 Pages·2016·5.85 MB·English

Isolation, identification and screening of alkaliphilic bacteria from
mangrove sediment and alkaline industry - contaminated soil for
antibacterial property and chromium removal efficiency

Thesis submitted to Pondicherry University for the
Award of the degree
DOCTOR OF PHILOSOPHY IN
ECOLOGY AND ENVIRONMENTAL SCIENCES

By
Mr. R. ANANDBABU, M.Sc.,

Research Supervisor
Dr. A. YOGAMOORTHI, M.Sc., Ph.D.,
Associate professor

Department of Ecology and Environmental Sciences
School of Life Sciences
Pondicherry University
Pondicherry - 605014
India

February – 2015
Dr. A. YOGAMOORTHI, M.Sc., Ph.D.,
Associate Professor,
Department of Ecology & Environmental Sciences,
Pondicherry University,
R.V.Nagar, Kalapet,
Puducherry – 605 014, India.
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Certificate
This is to certify that the thesis entitled “Isolation, identification and screening of
Alkaliphilic bacteria from mangrove sediment and alkaline industry - contaminated
soil for antibacterial property and chromium removal efficiency” submitted by Mr.
R. ANANDBABU to the Pondicherry University for the award of the degree of Doctor of
Philosophy is the record of original research work done by the candidate during the
period 2010-2014 at Department of Ecology and Environmental Sciences, Pondicherry
University, India under my supervision and guidance and that no part thereof has been
presented for award of any Degree, Diploma, Associateship, Fellowship or any other
similar title.

Place: Puducherry (Dr. A.YOGAMOORTHI)
Date: Research Supervisor

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R. ANANDBABU
Research Scholar,
Department of Ecology and Environmental Sciences,
Pondicherry University,
Puducherry – 605 014.

Declaration

I, R. ANANDBABU hereby declare that the thesis entitled “Isolation, identification
and screening of Alkaliphilic bacteria from mangrove sediment and alkaline
industry - contaminated soil for antibacterial property and chromium removal
efficiency”, submitted to the Pondicherry University in fulfillment of the requirements
for the award of the degree of Doctor of Philosophy in Ecology and Environmental
Sciences, embodied the result of original and independent work carried out by me under
the supervision of Dr. A. YOGAMOORTHI, M.Sc., Ph.D., Associate Professor,
Department of Ecology and Environmental Sciences, Pondicherry University.
This work has not been submitted earlier, in full or part, for the award of any
other Degree, Diploma, Fellowship, Associateship or any other similar title of this
University or any other University.

Place: Puducherry (R. ANANDBABU )
Date:

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ACKNOWLEDGEMENT

It is my greatest honor to work under the guidance of Dr. A.
YOGAMOORTHI M.Sc., Ph.D., Associate Professor in Department of Ecology and
Environmental Sciences, Pondicherry University. With respect and regards, I wish to
express my sincere thanks to him for the scientific freedom, infallible guidance,
intellectual companionship, magnanimous understanding, invaluable suggestion and
endless support during my period of research.
I am highly thankful to my Doctoral Committee members Dr. C.
Thirunavukkarasu, Assistant Professor in Department of Biochemistry and
Molecular Biology, Pondicherry University and Dr. G. Poyyamoli, Associate
Professor, Department of Ecology and Environmental Sciences, Pondicherry
University for their valuable comments, suggestions during the course of my study.
I am extremely thankful to Vice Chancellor Pondicherry University, for
awarding fellowship, and I also wish to extend my heartfelt thanks to Prof. Anisa
Basheer Khan, Dean, School of Life Sciences, Prof. K. V. Devi Prasad, Head,
Department of Ecology and Environmental Sciences, and all the faculty members for
providing all the facilities and constant support and encouragement.
I wish to thank Laboratory Assistants and Non-teaching staffs of Department
of Ecology and Environmental Sciences, Pondicherry University for the help during
my study period.
My sincere thanks to Pondicherry Center for Biological Sciences, for their
timely help by extending Laboratory facilities.
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I wholeheartedly thank Mr. John Thomas, Assistant Professor, Department of
Microbiology, St. Joseph's College of Arts and Science, for encouraging me to pursue
my higher studies
I also extend my heartful thanks to my colleagues Mr. Siva Shankar R. and
Mr. T. Kaviarasan and my friends for their valuable encouragement and suggestions
– Neenadevi, Kamal, Jeeva, Pradheeps, Mani, Anand, Priya, Devanadhan,
Jayakumar, Gunaseelan, Pajanivel, Sudhakaran, Kalaimohan, Veeresh,
Parthiban, Kapil, Sadish, Afsal and Sathish.
No words can express my gratitude to my beloved family members, Mr. T.
Rangasamy and Mrs. R. Santhi Rangasamy my parents and my brothers Mr. R.
Subramani and Mr. R. Karthik and my sister-in-laws Mrs. Ranjani and Mrs.
Supriya and my niece S. Rakshitha and all the other family members without whom
this study would have ceased to be a reality.
I thank my father-in-law Mr. Padbhanaban, my mother-in-law Mrs. Rani
and my sister-in-law Ms. Sharley for their continuous support.
A special thanks to my beloved wife Mrs. Angeline Jennifer for her
understanding and love during the past few years. Her support and encouragement
was in the end what made this dissertation possible.
Last but not the least I thank the almighty for his abundant blessings and
giving me the opportunity to undergo my research in such a successful way.

R. Anandbabu

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ABBREVIATIONS

AAS : Atomic Absorption Spectrometer
APHA : American Public Health Association
BOD : Biochemical Oxygen demand
CIF : Central Instrumentation Facility

COD : Chemical Oxygen demand
Cr : Chromium
DMSO : Dimethyl sulfoxide
DNA : Deoxyribonucleic acid
DO : Dissolved Oxygen
EC : Electrical conductivity
EDX : Energy Dispersive X-ray
FTIR : Fourier Transform Infrared Spectroscopy
g : Gram
GC–MS : Gas Chromatography–Mass Spectrometry
LB : Luria Broth
mg : milligram
MIC : Minimum inhibitory concentration
ml : millilitre
mm : millimetre
MUG : Methylumbelliferyl β-D-Galactopyranoside
NaOH : Sodium hydroxide
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NCBI : National Center for Biotechnology Information
NCCLS : National Committee for Clinical Laboratory Standards
OC : Organic Carbon
OD : Optical Density
PCR : Polymerase Chain Reaction
Rpm : Rotations per minute
SEM : Scanning Electron Microscope
TEM : Transmission Electron Microscopy
TDW : Triple Distilled Water
TN : Total Nitrogen
UVI : Ultraviolet Illuminator
µg : microgram
µl : microlitre
β-Gal : Beta galactosidase

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CHAPTER – 1

1. Introduction
Alkaliphilic microorganisms attract increased attention during the last decades
in the context of their great potential for biotechnological applications and research of
ecological diversity (Rossi et al., 2003). Several alkaliphilic bacteria have been
isolated from different environments for example deserts, soda lakes, and arid soils (Li et
al., 2002). Natural alkaline environments, in particular soda lakes, which are
distributed worldwide, contain large numbers of alkaliphiles (Jones et al., 1998;
Martins et al., 2001). Most intense and extensive intervals of organisms have been
observed generally in “moderate” environments. It would also have been known to
have an “extreme” environment on Earth are considered to avoid the endurance of
existence. Habitats in these ecological environments, for instance pH, temperature and
salinity concentrations are exceedingly elevated or low. The extreme environment is
populated by a group of organisms that have been particularly modified to these
specific circumstances and these types of intense microbes are frequently referred to
as alkaliphiles, halophiles, thermophiles and acidophiles, and reflects an exacting type
extreme environment in which they inhabitat (Horikoshi. 1991).
Naturally stirring environments with excessive pH values that hold up the
growth of microorganisms are broadly scattered. Frequently organisms that thrive in
these environments were exposed to a neutral pH value than the normal value of their
environment owing to the nature of their microenvironment. Soda lakes may be most
effective in an environment that naturally occurs in the world with an average major
productivity on standard are at slightest an order of magnitude beyond that of a
regular aquatic environment. This is producing significant results from the assumption
that the surface temperature is quite high (30-45 °C), elevated light intensity and
infinite backup of HCO3 in photosynthesis. These lakes sustain large standing crops
in a different variety of micro-organisms that are certainly prevalent in origin
(Grant.1992, 1993)
One of the largely prominent characters of several alkaline, saline lakes is the
coloration of the waters. Depending on a diversity of circumstances associated with
water chemistry, the population density of microorganisms could colour the lakes
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green, orange, purple or red. In several cases it has been potential to illustrate a clear
suggestion of microbiological activities only because of algae blooms cyanobacteria,
eubacteria or archaebacteria (Tindall, 1988).
Looking on the whole physiological diversity, it can be said that natural soda
lake environment that supports the growth of microorganisms hold both aerobic and
anaerobic extremophiles, in other cases, such as growth, aerobic alkaliphiles, high pH
have been exposed to grow in higher pH environmental circumstances. However
studies focus, mainly on the isolation of anaerobic species that grow under most pH
environmental situations are still in improvement. Therefore, extra widespread
isolation and characterization efforts are necessary. In analysis of the variety of
physiological groups are established in soda lakes, one can predicted that alkaliphilic
phenotypes may evolved several times. Biodiversity is currently assuming alkaliphiles
have existed since Archaean times, allowing the evolution of the community
independent of alkaliphiles early in the history of the world (Jones et al., 1998).
In addition, a variety of industrial wastewaters harbour an extensive choice of
alkaline microorganisms isolated from many environments. The outlets of some
chemical industries, such as cement production shows high alkalinity, but are it seems
that devoid of life as no organisms seem to be alive and has the ability to stand
extremely high pH (> 12) and lethal compounds. However, the developments of
microbial diversity in fewer alkaline industrial effluents are developed by the lye
treatment of indigo and potato fermentation. So far, several alkaliphilic
microorganisms have been isolated from diverse environments, such as Bacillus
vedderi from bauxite waste, Exiguobacterium auriantiacum from potato waste
processing and Bacillus alcaliphilus from indigo fermentation process (Agnew et al.,
1995 and Gee et al., 1980)
Alkaliphilic bacteria are considered as a subgrouping of extremophile
microorganism, enzymes from these organisms are extremely imperative for various
biotechnological and industrial processes under very inconsiderate alkaline
conditions. The word "alkaliphile" is used for microbes that grow best in a pH beyond
9, however cannot grow or grow gradually at pH near-neutral. Alkaliphiles include
prokaryotes, eukaryotes, and archaea (Horikoshi, 1996, 1999, 2004).
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Alkaliphiles have made a huge impact in the industrial applications. Studies on
these organisms have led to the unearthing of various types of enzymes that reveal
attractive features. Biological detergents have alkaline enzymes, for example alkaline
cellulases, proteases as well as lipase, etc. are produced from alkaliphiles (Fukumori
et al., 1985; Fujiwara and Yamamoto 1987; Fujiwara et al., 1991). Microorganisms
have great expertise in their potential to cause various types of bioactive compounds.
Astronomically of antibiotics that have been shown to be caused by various types of
bacteria, such as actinomycetes. Screening of bacteria from alkaline environments or
those who grew up under extreme circumstances remain an area of cultural and cost
effective for the survey. Some antibiotics initially engendered by certain bacteria
when an alkaline medium with a high pH (pH 9-10.5) were used (Sato et al., 1983).
The earliest statement of antibiotic production in alkaline medium by a team
of Sato (1980) who establishes that 2 antifungal peptides produced by Paecilomyces
lilacinus 1907 at 10.5 pH. Afterwards Nocardiopsis dassonvillei OPC-15, was
isolated, that suggests the production of the antibiotic phenazine at pH 10.0 (Tsujibo
et al,. 1988). (Dietera et al., 2003) identified Pyrocoll as one of the substances
produced by alkaliphilic Streptomyces sp. Pyrocoll exhibited activity against fungi,
protozoa, and even different human cancer cell lines. Lawton et al, (2007) foremost
reported the production of a 2-peptide lantibiotic (lanthionine – consist of peptide
antibiotics) by an alkaliphilic Bacillus namely B. halodurans C-125.
High proportion of antimicrobial compounds in the production of a species
possibly connected among natural role, playing a suspicious accomplishment to
strains into a conventional microbial community (El-Banna, 2003).
Bacteriocin are antimicrobial peptides widespread produced among bacteria
(Cotter, et al., 2005), In spite of intensive work on bacteriocin produced by lactic acid
bacteria, Bacillus species, comprising a diversity of industrially significant species
and has a history of secure use in food and pharmaceutical industry (Vaucher et al.,
2010), Alkaliphilic microorganisms particularly Bacillus species have fascinated
much attention due to its ability to produce the extracellular metabolites that has been
used and are constant at high pH. The abnormal properties of these compounds have
the potential chance for their exploitation in processes challenging such extreme
conditions (Takami et al., 1999). The development of resistance to several drugs is a
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