PRODUCTION AND CHARACTERIZATION OF GREEN PLASTIC POLYMER POLYHYDROXYALKANOATES DERIVED FROM MICRO-ORGANISM USING WASTE RESOURCES

Abstract

Polyhydroxyalkanoates (PHAs) are a type of biodegradable, thermoplastic polyester produced by various microorganisms as a way to store carbon and energy inside their cells. These PHAs are made up of repeating units of hydroxyalkanoic acids, which can have different lengths and branching patterns, resulting in a wide range of PHA materials with diverse physical and chemical properties. PHAs are considered promising biopolymers that could potentially replace synthetic plastics. While the production cost of biopolymers is typically higher than that of synthetic plastics, they offer a vital solution to the problem of plastic pollution. Therefore, further research and development are needed in this area, particularly to rapidly screen and isolate bacteria capable of producing PHAs with exceptional yields at low production costs. Ultimately, polyhydroxyalkanoates are materials that can degrade naturally. PHA production can be produced using different carbon sources like sugars, lipids, and waste materials including food and agricultural waste. This approach helps reduce the environmental impact of PHA manufacturing. Moreover, PHAs can be easily degraded by microorganisms in various environments such as soil, water, and marine ecosystems, preventing their accumulation in the environment. The present research aimed to identify microorganisms capable of producing PHAs and utilize them efficiently by employing agro-industrial waste as a substrate. Chapter 1 provides an introduction to the PHA polymer and presents a review of the existing literature on the topic. It discusses the mechanism involved in the production of PHA, specifically focusing on the pathway through which it is synthesized. Additionally, this section highlights the unique features of PHA that make it suitable for a wide range of applications. The chapter also provides a brief overview of relevant studies related to PHA production using different types of waste materials and the microorganisms involved in the conversion of carbon into commercially viable polymers. Chapter 2 of the thesis focuses on a detailed study of the sample procurement process for isolating the potential PHA producer, HLI13B. This involved screening bacteria using different dyes, and the methodology and results of this screening are presented. The chapter also covers an extensive examination of the experimental setup for bacterial culture to optimize growth conditions such as pH, temperature, and glucose concentration. Polymer production using the optimized conditions, as well as the extraction of the polymer, are described in this section. Furthermore, the chapter includes a comprehensive study of the physical, chemical, and thermal characterization of the polymer. Various analyses were conducted, including FTIR, TGA, DTA, DTG, XRD, 1H NMR, GPC, SEM, SPM, Nanoindentation, and Biodegradability. These analyses aimed to determine the chemical composition, mechanical strength, and thermal stability of the polymer, thereby validating its suitability for various applications in packaging and biomedical fields. Additionally, the chapter incorporates a study of the genome analysis of the PHA producer, HLI13B, with the objective of identifying the genes responsible for PHA production within the organism. Chapter 3 focuses on the production of the PHA polymer using waste resources, specifically utilizing the isolated species HLI13B. The chapter also discusses recent studies that have utilized waste for PHA production. The primary objectives of this chapter include the production of environmentally friendly plastic, namely polyhydroxyalkanoate (PHA), from beverage industrial wastewater, as well as controlling water pollution by consuming residual sugar and reducing pH levels. PHA is a biopolymer that accumulates in various bacterial species when grown in a carbon-rich medium. The novel organism HLI13B, isolated from mess sludge, demonstrates the ability to accumulate PHA and has a strong tolerance for highly alkaline conditions. Beverage industrial wastewater, which contained residual sugar (4.65 g/L) and had a pH of 11, was aseptically collected and used as a carbon source supplemented with mineral salt media (MSM) to culture HLI13B for biomass production under optimized conditions (37 ℃ and 2% glucose). The use of wastewater as a medium significantly reduced the pH from 11 to 7.5 within 24 h and eventually to pH 6.5. The polymer yield reached up to 1.38 g/L under the optimized conditions. The accumulated polymer was extracted using solvent extraction and was found to be identical to the polymer produced using standard glucose. The agricultural applicability of the treated water and wastewater was evaluated by irrigating chickpea plants. The results indicated that the alkaline wastewater had a negative effect on plant growth. Chapter 4 focuses on the detailed study of an organism called Bacillus sp. S4, which was isolated from the roots of sugarcane plants. The chapter provides information about the screening optimization, production, and characterization of the PHA polymer produced by Bacillus sp. S4. Notably, this organism is capable of efficiently utilizing xylose, an alternative, and abundant sugar i.e., xylose. Xylose is a major component present in the leftover hydrolysate of ethanol production waste, as the yeast Saccharomyces cerevisiae primarily consumes glucose during ethanol production, leaving xylose behind. The utilization of this waste significantly reduces production costs. The chapter also delves into the characterization of the PHA polymer synthesized by Bacillus sp. S4 and explores its potential application in nanofiber synthesis. The nanofibers derived from the PHA polymer are thoroughly characterized to assess their suitability for biomedical applications. Chapter 5 has detailed the structural aspects of the enzyme involved in PHA synthesis pathways PhaC. The naturally occurring polymers PHA are incredibly useful in the medical field and tissue engineering because of their unique physical and chemical characteristics. In the PHA production pathway, PHA synthase (PhaC), is one of the significantly important enzymes for synthesizing this commercially applicable polymer. The current study concentrated on one such PhaC Class I enzyme activity using an in-silico approach. The present study initializes with the phylogenetic analysis of PHA synthase class I sequences to understand their evolution over time. Further, to investigate the structural realm, the homology modeling-based approach was used followed by Molecular Dynamics (MD) simulation. Interestingly, this enzyme was reported to have open, closed, and semi-closed conformations with an immense structural difference. Hence, the molecular dynamic simulation was performed for all three conformations and a CoA complex structure to investigate the dynamic behavior. Simulation analysis includes backbone variations, residue-wise fluctuations, and compactness of the system of the PHA synthase PhaC I. In conclusion, open conformation simulation was observed to be a more structurally moving or fluctuating structure than that of closed and semi-closed conformations. Further, CoA complexed structure attained more stable behavior over the trajectory of simulation than that of only protein (open) structure. Additionally, the regions of structural importance and indicating dynamicity are identified and their in-detailed analysis is presented. This novel study sheds light on the structural insights of PHA synthase as this enzyme has the potential target of protein engineering to facilitate it to produce a variety of biopolymers for commercial use. Chapter 6 summarizes the whole study and conclude the outcomes of the study. It also includes the future copes of the study.