INFLUENCE OF DIFFERENT NITROGEN SOURCES ON GROWTH AND PHB PRODUCTION OF BACTERIAL ISOLATES

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 (1.560^a) for growth of PHB producing bacteria  (Citrobacter sp) while maximum PHB production  (1.899^a) 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 .

CHAPTER ONE

1. 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 (P^H, 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).