Characterization and Production of Medium-Chain-Length Polyhydroxyalkanoates (PHA) in Pseudomonas putida

Abstract

Polyhydroxyalkanoates (PHAs) are microbial bio polyesters that offer a promising biodegradable alternative to petroleum-derived plastics. This thesis focuses on the production and characterization of medium-chain-length PHAs (mcl-PHAs) by Pseudomonas putida using octanoic acid as a carbon source. The main aim was to optimize key bioprocess parameters such as temperature, pH, dissolved oxygen (DO), feeding strategy, and salt concentrations such as phosphates, magnesium and iron—to improve both PHA yield and polymer quality.

Initial optimization of growth was done in flasks, followed by fed-batch experiments conducted in 7L benchtop bioreactors. Among our tested parameters, we could identify one factor referred to hereafter as ‘X- Factor’ which significantly enhanced’ enhanced PHA accumulation and increased molecular weight of the polymer produced by this bioprocess from around, 117 kDa to 150 kDa. After that, optimized parameters were successfully scaled to fermentations in 50L, 60L and 1000L reactors, producing a similar outcome of higher molecular weights ranging between 140 to 150 kDa. This indicates the consistency, reproducibility and robustness of our findings through the different experiments across different reactors with different volume capacities and reactors.

In this study, we have successfully optimized the bioprocess conditions used for production of mcl-PHA in Bioextrax AB. leading to production of a more commercially relevant polymer with better potential in particular industrial applications.

Popular Abstract

Plastic pollution is one of the most significant environmental problems facing the world today, largely due usage of petroleum-based plastics which are non-biodegradable and accumulate in landfills and natural ecosystems, posing threats to wildlife and human health. In response, there is a urgent need to search for sustainable, biodegradable alternatives that can perform as well as traditional plastics but break down more easily in the environment. One promising solution is the use of polyhydroxyalkanoates (PHAs), which are naturally produced by certain bacteria as a way to store energy and carbon. PHAs are a diverse family of microbial polyesters, and among them, medium-chain-length PHAs (mcl-PHAs) are especially attractive because of their flexibility, elasticity, and low crystallinity. These properties make mcl-PHAs suitable for a wide range of applications, from packaging materials to medical devices. Unlike short-chain-length PHAs (scl-PHAs), which tend to be brittle, mcl-PHAs have a rubber-like texture and can be processed into soft, durable products.lThe research summarized in this thesis focuses on improving the production of mcl-PHAs using the bacterium Pseudomonas putida and octanoic acid as the primary carbon source. P. putida is a versatile, non-pathogenic bacterium capable of utilizing various carbon sources, including fatty acids and industrial waste streams. The study aimed to address key challenges in mcl-PHA production, such as inconsistent yield, variable molecular weight, and inefficient substrate consumption. To tackle these challenges, tphe research team systematically optimized several bioprocess parameters. These included temperature, pH, dissolved oxygen (DO), feeding strategies, and concentrations of essential tThe molecular weight of PHAs is crucial because it determines the mechanical strength, flexibility, and suitability of the polymer for different applications. Higher molecular weights result in stronger, more elastic materials, which are more valuable for commercial use. The study found that by optimizing the bioprocess conditions, the molecular weight of the produced mcl-PHA increased from around 117,000 Daltons to 150,000 Daltons. This improvement was consistent across different scales, from laboratory flasks to industrial bioreactors with capacities up to 1,000 liters. Such scalability is essential for commercial production, as it shows that the process is robust and reproducible. The research also employed advanced analytical techniques to characterize the produced polymers. Gel Permeation Chromatography (GPC) was used to measure molecular weight and distribution, while Thermogravimetric Analysis (TGA) assessed the thermal properties and stability of the polymers. These analyses confirmed that the optimized process consistently produced mcl-PHAs with desirable qualities for industrial applications.

In conclusion, this thesis demonstrates a successful approach to producing high-quality, biodegradable mcl-PHAs using Pseudomonas putida and octanoic acid. By carefully optimizing growth conditions and scaling up the process, the research paves the way for more sustainable plastic alternatives that could help reduce environmental pollution and dependence on fossil fuels. The findings offer hope for a future where biodegradable plastics can replace conventional materials in many everyday products, contributing to a cleaner and more sustainable world.

• author: Kumari, Kalpana
• supervisor: Klas Ingstorp; Yahia Abouelenen
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214679
• date added to LUP: 2025-10-30 10:54:34
• date last changed: 2025-10-30 10:54:34