47 Screening, Characterization and Quantification of PHB Producing Bacillus flexus Strain Isolated from Majha Region of Punjab Madhuri Girdhar 1 , Saidu M Bashir 1 , Ajay Kumar Sharma 2 , Hasibur Rehman 3 , Anand Mohan 1 * 1 Department of Biotechnology & Biosciences, Lovely Professional University, Punjab 144411, India 2 Department of Chemistry, Lovely Professional University, Punjab 144411, India 3 University of Tabuk, Tabuk, Saudi Arabia *corresponding author: Dr. Anand Mohan, Email: anandmohan77@gmail.com, Telephone/Mobile No. +919357721332 Abstract Microbial Bioplastic production is a fascinating area of research in the field of polymer science. Present work investigates the screening of PHB (Polyhydroxybutyrate) producing bacterial strain isolated from the agriculture soil of Majha region (district - Gurdaspur) of Punjab. PHB is an intracellular granule, which is a carbon and energy reservoir for the bacteria under starvation or stress conditions. Five bacterial isolates were screened through Sudan black staining out of which two colonies were observed as accumulator of PHB granule. Further studies for optimized growth of bacterial isolates were performed and carried by spectrophotometric analysis. Extraction of PHB after optimized growth was performed by solvent extraction method. The extracted sample was characterized by FTIR and thin layer chromatography (TLC) and was compared with standard PHB (Sigma-Aldrich) for confirmation of quality. Production level of PHB was observed to be in the range of 10.65% to 15.68% in two different strains with an incubation period of 72 hrs. Biochemical characterization was performed to identify the bacterial isolates at initial level. The high PHB producing strain M1 was further characterized as Bacillus flexus by 16S rRNA sequencing technique [Acession no. KJ939621]. Keywords Bioplastic, Bacillus strain, Optimization, Starvation, Polyhydroxybutyrate etc. 1. Introduction Over the past century of industrial growth and anthropogenic development human dependency on plastics and plastic based products is increasing continuously. There are a lot of ways in which plastics are utilized by human population viz., packaging material, building and construction, transportation, medical and health, electrical and electronics, agriculture, sports and leisure products [1, 2]. Both production and after life remediation of plastic generates enormous amount of waste which is difficult to decompose and also generates green house gases along with other kind of health hazard problems in humans [3]. The molecular size of plastic is up to 1,50,000 dalton which makes it resistant to degradation naturally. Consumption of petrochemical-based plastics is linked directly with diminishing oil resources. Bioplastic can act as potential material, to displace this ever-increasing waste of plastic and equally can be utilized in all applications. Merit of Bioplastic over synthetic plastic, is its degradability with time and in certain cases non immunogenic nature which can be utilized for designing of medical implants and devices. Bioplastics consists of thermostable polyesters and are an important class of advanced biomaterial. Biopolymers have an advantage for being degradable under normal environmental conditions and their physio-chemical properties are similar to that of synthetic polymers. Currently, biodegradable polymers such as PLA (Polylactic acid), PHA (Polyhydroxy alkanoates), PHB and cellophane have created lot of interest for scientists and researchers. Bacteria have the ability to accumulate bioplastics under stress conditions in the form of polyhydroxyalkanoates (PHAs) which can be purified from them [4]. PHAs are 3-hydroxy fatty acid monomers that form linear, head-to-tail polyesters. PHA is usually produced as a polymer of 103–104 monomers, which accumulates in the bacterial inclusions of size 0.2–0.5 μm in diameter. Poly-3-hydroxybutyrate (PHB) is a linear polyester of D (-)-3 hydroxybutyric acid which was first discovered in bacteria by Lemoigne in 1925. PHB is member of PHA family and most widely used for production at industrial level. Polyhydroxyalkanoates (PHAs) are polyesters which are synthesized by various microorganisms such as Pseudomonas putida, Alcaligenes latus, Aeromonas hydrophila, Bacillus spp. and Ralstonia eutropha undergoes nutrient imbalance conditions, such as higher carbon ratio with reduced phosphorus, nitrogen and oxygen [5]. PHA consists of different classes depending upon the variance of groups and their position on the main chain. PHAs can be found as homopolymer or as co-polymer and have approximately 150 different constituents hydroxyalkanoate (HA), 3-hydroxybutyrate (3HB), 4- hydroxybutyrate (4HB), 3-hydroxyvalerate (3HV), 3- hydroxyhexanoate (3HHx), 3- hydroxydecanoate (3HD), short chain-length (SCL), and medium chain-length (MCL) [6]. Fig 1: General Formula of PHB Current study involved screening of PHB producing bacteria and characterization of PHB produced for its quality. 2. Material and methods 2.1: Collection of Samples Soil samples were collected from different sites of agriculture area situated at Gurdaspur (Majha Region of Punjab). The samples were stored in sterile plastic bags at 4 o C and were transferred to laboratory. 2.2: Isolation of PHB Producing Bacteria 1 gram soil sample was used for the serial dilution. Six sterile test tubes were taken and serial dilution was performed. Spreading of samples at dilution 10 -3 , 10 -4 and 10 -5 were done on nutrient agar medium using L shaped spreader. All the petriplates were incubated at 37°C for 24-48 hours. 2.3: Morphological Analysis of isolated bacteria Pure bacterial colonies were characterized for colour, form, elevation, margin etc. Bacterial isolates were morphologically analyzed on the basis of simple staining and cell morphology using microscope.