Abstract
The strategy of waste valorisation reduces the bulk of waste load as well as restricts the possibility of potential contamination to the environment. Different types of wastes can be used as a substrate for the biosynthesis of value-added products. For instance, waste streams from agro-based and food industries serve as promising primary feedstock sources for the biosynthesis of ethanol, succinic acid, levulinic acid, biopolymers, and other valuable by-products. Among the above-mentioned value-added products, biopolymers have attracted enormous interest from environmental and economic standpoints. Biopolymers are widely recognised for their potential medical applications; and, by definition, are high molecular weight polymers that are degraded naturally over time with the help of microorganisms and enzymes. An easily accessible natural biopolymer is cellulose. Compared to other natural biopolymers, cellulose is an inexpensive, ubiquitous, and abundant polysaccharide extracted from plant waste. Cellulose composites, viz. cellulose nanofibres (CNFs) and cellulose nanocrystals (CNCs) are biocompatible with excellent crosslinking abilities to serve as an efficient entrapment agent for microbes. Recent studies have reported that cellulose hydrogels derived from plant or bacterial-based cellulose nano-fibres are promising agents for immobilizing probiotics. These features of cellulose hydrogels enable the construction of a bioreactor that simulates the human intestinal ecosystem, having varying porosity, pH, and oxygen gradient. Alongside this, the cross-linking density of these hydrogels can be altered to achieve spatial control over nutrient levels.In the first project, this quality of cellulose hydrogels is explored, and a concept of growing multiple probiotics in a single reactor is developed. The study focused on cultivating ten selected probiotics in a single-vessel fermenter to minimise the time and cost of production of multi-strain probiotic products. Previous studies on the production of probiotics essential for human health have been fixated on the Bacteroides and Lactobacillus strains. In addition, the selected strains are difficult to culture due to being strict anaerobes, limited biomass yield and restricted growth rates. As a result, the fermentation profile for the selected strains has not been reported so far. In order to counter these research gaps, cellulose hydrogels were deployed as an immobilization matrix to encapsulate the probiotics. First, the strains were cultivated individually to define their growth profiles and evaluate cellulose biopolymers' efficiency as an immobilization agent. Later, multiple strains were grown together in a single-vessel reactor to determine the efficiency of isFBB fermentation strategy using cellulose hydrogels as an immobilization matrix. Precisely, CNF-alginate hydrogels fared best amongst all other combinations (CNC-alginate, alginate only, and CNF only) explored for trial batches. The fermentation profile of single strains provides a better understanding of when the cultures are grown together. Subsequently, the varying concentration of cellulose-alginate hydrogel was cast in moulds to identify the optimal casting ratio for immobilization. Among all the studied strains, T. glycolicus had the highest specific growth rate of 0.343 h-1, while R. bromii had the highest biomass accumulation of 3.89 g/L. Hence, these two strains were selected for the trial studies of multi-strain cultivation. The SEM micrographs of entrapped probiotics on cellulose hydrogels reveal the morphology of each strain immobilized during fermentation. The SEM observations also provide information about the porosity of cellulose-alginate hydrogels that functions as a matrix for immobilization. The single strain fermentation studies and SEM observations of each probiotic cell are later used as a standard reference point when cultures are grown together for multi-strain fermentation studies. In totality, the experiments demonstrate the successful cultivation of four probiotic strains with different growth profiles in a single reactor. The study aimed to reduce the manufacturing cost of multi-strain probiotics through immobilization and fermentation strategies. The work seeks to bridge the knowledge gap between probiotic production's efficiency and the applicability of nano-materials.
Various biopolymers are applicable in different fields based on their functions and properties. One example of biopolymers is the biodegradable polyester (bioplastics) known as polyhydroxyalkanoates (PHAs). The PHA bio-composites are versatile and display incredible potential as a sustainable and reliable alternative to conventional polymers (plastics). Conventionally, plastic products are derived from fossil fuels, leading to severe environmental concerns. To address the above environmental and industrial concerns, the upcycling strategy for the valorisation of food waste to produce microbial-based biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was explored in the second project of the thesis.
This study focused on the material recovery approach to address the issues of the global food waste burden, accumulation of excessive petrochemical-based PPE waste, and the severe PPE shortage. Food waste was collected and hydrolysed into glucose-rich hydrolysate to cultivate C. necator and H. mediterranei. Essentially, C. necator is reported in the literature for efficient substrate conversion, substantial biomass production and highest PHA accumulation. Similarly, H. mediterranei is a gram-negative, pleomorphic halophile that grows in high salt concentrations. Eventually, this rules out the issue of contamination and the need for sterilisation at the industrial scale. In addition to this, cells of H. mediterranei do not have any specific nutritional requirement and can be exploited for the cultivation on hydrolysate from waste streams for PHA production. Subsequently, the effect of various volatile fatty acids (VFAs), such as levulinate, butyrate, valerate, and propionate, on the fermentative production of PHBV was investigated. At the same time, the influence of different feeding strategies on biomass accumulation and corresponding intracellular PHBV production was explored. Subsequently, the biomass and PHBV yield from food waste hydrolysate was compared to the synthetic medium. The PHBV produced in this study had higher 3HV content than the synthetic medium. The fed-batch fermentation strategy in this study produced 15.65 g/L of biomass with 5.32 g/L of PHBV with 50% molar 3HV content. This is a key finding, as the molar concentration of 3HV can be modulated to suit the specification of biopolymer (film or fabric). The strategy addresses the global food waste burden and generates biopolymer PHBV, turning waste into wealth.
Similar to C. necator, the halophiles serve as commercially promising microorganisms for increasing the yield of biopolymer PHBV at lower production costs. Among the halophilic archaeal species, Haloferax has received much interest as a potential PHBV producer because of its ability to use biowastes and inexpensive renewable carbon sources as feedstock, less stringent sterilisation requirements, and simple yet convenient extraction technique. The effect of increasing the concentration of levulinic acid on the composition and production of PHBV from Haloferax mediterranei was later evaluated. Shake-flask experiments were performed to optimise the cultivation conditions, such as salt, glucose, and levulinic acid concentration. The highest growth of the halophile was observed at a salt concentration of 15% and glucose concentration of 10 g/L. Under optimised growth conditions, H. mediterranei was cultivated for PHBV production in a fed-batch bioreactor with pulse-fed levulinic acid. The maximum biomass of 3.19±0.66 g/L was achieved after 140 h of cultivation with 3 g/L of levulinic acid. A decrease in H. mediterranei growth was noticed with the increase in levulinic acid concentration in the range of 3-10 g/L. The overall yield of PHBV at 3, 5, 7, and 10 g/L of levulinic acid were 18.23%, 56.70%, 31.54%, and 21.29%, respectively. At the optimum concentration of 5 g/L, levulinic acid produced the maximum yield of 56.70% PHBV with 18.55 mol% 3HV content. A correlation between levulinic acid concentrations and PHBV production established in this study can be an essential reference for future large-scale production.
In general, the second project addresses the issues of reducing the global food waste burden and its subsequent utilisation as a renewable resource for low-cost biopolymer production. Several studies have explored the utilisation of organic wastes such as kitchen waste, vegetable waste, date palms, and synthetic food waste as the sole carbon source for the production of PHAs. To our knowledge, this is the first study to produce PHBV biopolymer through the valorisation of food waste.
In the future, research needs to focus on addressing the challenges in further development of value-added products like probiotics and biopolymers. Especially integration of techno-economic studies and the harvesting strategies that can efficiently harvest multi-strain probiotics in varying ratios need to be explored. From the aspects of biopolymer production that are relevant from a bio-medical and economic standpoint, the integration of renewable carbon sources and efficient bacterial strains appears to be a reasonable approach to reducing the production cost of biopolymers like PHA composites. Electrospinning and electro-spraying techniques seem promising to address the shortage of bioplastics for medical and industrial applications by utilizing the PHBV biopolymer produced in this study. In a nutshell, the work presented in this thesis demonstrates the relevance of CNF biopolymers for the cultivation of multi-strain probiotics in a single bioreactor. The latter part of the thesis focuses on the biorefinery approach of utilizing food waste to produce biopolymer PHBV for its respective medical and industrial applications
| Date of Award | 20 Sept 2022 |
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| Original language | English |
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| Supervisor | Sze Ki Carol LIN (Supervisor) |