ABSTRACT
Polyhydroxybutyrate
(PHB) are biodegradable materials, which are accumulated to store carbon and
energy in various microorganisms.
Soil
samples used for the study were collected from groundnut farm garden. The
isolates were screened for PHB production using sudan III stain as well as
submerged fermentation. Four (4) of the best PHB producing bacteria were
selected for further study. A total of twenty one (21) bacteria were isolated
form the soil sample in which Bacillus
sp had the highest percentage frequency of occurrence (54.5%). Follow in order
by Enterococcus sp (13.63%), Veillonella
sp (9.1%), Micrococcus
sp. (4.5 %), Corynebacterium
sp.(4.5%) Yersinia Pestis (4.5%), Lactobacillus fermenti
(4.5%) and Citrobacter sp. (4.5%).
The fermentation pattern of PHB production
was studied using Nitrogen source. NH4Cl was found to be the best nitrogen
source (1.560a) for growth of PHB producing bacteria (Citrobacter
sp) while maximum PHB production (1.899a)
was observed in Citrobacter sp when
KNO3 was used as nitrogen source.
The study carried out shown that
nitrogen sources significantly influence the production of PHB .
TABLE
OF CONTENT
Title page
Certification
Dedication
Acknowledgement
Abstract
Table of content
CHAPTER
ONE
1.0 Introduction
1.1 Aims and objectives
1.2 Statement of problem
1.3 Justification
CHAPTER
TWO
2.0 Literature review
CHAPTER
THREE
3.0 Materials and method
3.1 Preparation of media
3.2 Isolation of bacterial from soil samples
3.3 Maintenance of culture
3.4 Identification of bacterial isolates
3.4.1 Morphological characterization
3.4.2 Biochemical characterization
3.5 Screening o isolate for PHB production using
staining techniques.
3.6 PHB production
3.7 PHB extraction
CHAPTER FOUR
4.0 Result and discussion
CHAPTER FIVE
Conclusion
and appendix
CHAPTER ONE
1.0 INTRODUCTION
Polyhydroxybutyrate
(PHB) is one of the polyhydroxyalkanoates (PHAs) which has biodegradable and
biocompatible properties. They are adopted in the biomedical field, in for
example, medical implants and drug delivery carriers (Keshavarz and Roy, 2010).
Polyhydroxybutyrate
was discovered and identified as a granular component in bacterial cells. PHB
can grow in a wide variety of natural environment and is the reserve polymer
(intracellular granules) found in many types of bacteria in nature e.g. in
soil, sea water, sewage sludge or compost.
Polyhydroxyalkanoates
(PHAs) can be classified into two groups depending on the number of carbon
atoms in the monomer unit: short-chain-length. (SCL-PHA) 3-5 carbon atom containing
monomer and medium chain-length (MCL-PHAs), 6-14 carbon atom containing
monomers (Steinbuchel and Valentin, 1995).
The
discovery of a polyester by Smet et al.
(1983) was the first report of accumulation of medium-chain-length (MCl-PHAs),
having constituents of 6-14 carbon atoms in axenic culture (Smet et al., 1983).
Currently,
more than 140 hydroxyalkanoic acid have been identified as constituent of
polyhydroxyalkanoates (PHAs) representing a versatile class of microbial
polymer (Steinbuchel and Valentin, 1995). Besides linear and branched 3-4-,5- and
6-hydroxyalkanoates, various functionalized PHAs constituents such as
polyhydroxyalkanoates (PHAs) containing halogenated or aromatic side chains
have been described (Abe et al.,
1990).
The
physical properties of the homopolymer of hydroxybutyrate (PHB) are similar to
those of poly propylene, for example regarding melting point, crystallinity,
glass transition temperatures etc, and represent a stiff and brittle material
(Hocking and Marchessault, 1994). The properties are improved in copolymers
containing up to 25 mol% 3-hydroxyvalerate (3HV), where toughness and
flexibility are increased and the decrease in crystallinity and melting point
advance the melt-processing of the polymer without being degraded(Marchessault
and Yu, 2002).
Therefore,
the poly (3HB-CO-3HV) copolymer gained industrial interest and in the late
1980s, commercialization under the trade name Biopol was initiated (Holmer et al., 1984). Biopol can be processed
to useful materials by various processes such, as extrusion, injection molding,
fiber spinning, coating or foaming. Because of the versatile applications as
thermoplastic biopolymers, polyhydroxyalkanoates (PHAs) can also be used for
natural fiber composites or as binder in paints and for various application in
medicine and pharmacy such as tissue engineering (Kessler et al., 1999)
Polyhydroxylbutyrate(PHB)
is a commonly found substance and readily biodegradable are aerobically and
anaerobically. Microbes can use polyhydroxylbutyrate (PHB) exists in the
cytoplasmic fluid in the form of crystalline granule about 0.5μm in diameter.
Beta-hydroxybutyrate is connected by ester linkage and from polyhydroxybutyrate
(Andreson and Dawes, 1990). This can be extracted from the cells as native
granule or by solvent extraction (Doi and lee, 1990) and processed in the way as
polypropylene.
These
biopolymers were also found to increase the resistance of bacteria (Tombolini
and Nuti, 1989). Lemoigne, Grelet and Croson (1950) draw attention to the
different amount of poly-beta – hydroxybutyrate obtained by Bacterim megaterium on different media
and Maraca and Wilkinson (1958) showed that more of the substance was formed as
the glucose concentration of the growth medium was increased; the subsequent
depletion of the product during the later stage of growth suggested a storage
function. They also showed that organism rich in poly-beta-hydroxybutyrate had
a slower rate of autolysis than organisms poor in poly-beta-hydroxybutyrate. It
is probable; therefore, that poly-beta-hydroxybutyrate act as a reserve carbon
and energy source. Tinelli (1955) found that the major part of the material was
metabolized at sporulation and deduced that the two processes were intimately
connected (Kato et al., 1992)
The
bacterium capable of producing polyhydroxybutyrate has been identified in more
than twenty (20) bacterial genera, including Azotobacter, Bacillus,
Beijernickia, Pseudomonas. Alcaligenes, Rhizobium
and Rhodospirillum (Sudesh et al., 2000). Many researchers have
explained that soil bacteria generally produce polyhydroxybutyrate. Polyhydroxybutyrate
PHB) production increases if ambient conditions (PH, temperature,
nutrients) are made available (Hanzlikova et
al., 1985).
Polyhydroxyalkanoate
is one such biodegradable microbial polymer which is accumulated in bacteria as
intracellular storage granules in the presence of excess carbon sources and
limited nitrogen source (Anderson and Dawes, 1990).
The
polymer is known to occur as intracellular granules in several genera of micro-organisms.
The granules are synthesized by prokaryotes using fatty acids, sugars and other carbon sources
(Atkins and kennedy, 1985). Polyhydroxybutyrate is insoluble in water,
resistant to ultraviolet radiation and is impermeable to oxygen and is very
much suitable for use as food packaging material. This polymer is readily
degraded in the soil and sewage and can be processed using the extrusion
technology that is currently used in making polyethylene or polypropylene films
(Byrom, 1987). The polyhydroxyalkanoate content and its composition are
influenced mainly by the strain of the micro-organisms (Halami, 2008). To
achieve a cost effective polyhydroxyalkanoate production, the availability of
an efficient bacterial strain is a prerequisite and is a focus of interest for
many investigation (Green span et al.,
1985).
The
polyhydroxybutyrate production using inexpensive carbon sources in the form of
starch by the indigenous strain can be advantageous as the complex starch
substrates can be used directly without involvement of any hydrolysis step (Hahn
et al., 1995).
Polyhydroxybutyrate
(PHB) is used in food packaging, plastic films, surgical sutures (Stitching of
the edges of a wound or incision), controlled drug deliver e.t.c (kim et al., 1994). This bioplastic has many
applications in bone plates, nails, screws and in the treatment of
osteomyclitis (Hanzlikova et al.,
1985).
1.1 AIMS AND OBJECTIVES OF THE
RESEARCH
1.
Isolation and biochemical identification of polyhydroxybutyrate (PHB) producing
bacteria from soil.
2. Screening of
polyhydroxybutyrate (PHB) producing bacteria isolated from soil.
3. Polyhydrobutyrate (PHB)
Production.
4. Determination of effect of
different nitrogen sources on growth and polyhydroxybutyrate (PHB) production.
1.2 STATEMENT OF PROBLEM
The accumulation of these non-degradable
plastic in the environment is a menacing (threating) draw back increasing day
by day. Oil based polymers take many years to degrade which poses an
environment problem in some areas, and causes deleterious effects to wild life.
To overcome this, production of environmental friendly plastic have been
discovered.
1.3 JUSTIFICATION
The continuous exhaustion of fossil
fuels led to the research for the production of bio-degradable plastic from
renewable sources the production of biodegradable polymers from renewable
resources is the need of the hour, in the face of these ecological facts.
Login To Comment