Date of Award

31-8-2024

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Sugumaran Karuppiah

Keywords

Microbial Polysaccharides, Dextran, Sugarcane Juice, Sugarcane Molasses, Response Surface Methodology, Kinetic Modelling, Downstream Processing, Scale-Up

Abstract

Microbial exopolysaccharides (EPS) synthesized by microorganisms have a wide range of applications from thickener agents in the field of food to formulations in the field of pharmaceuticals. Owing to their rheological, structural, and physicochemical characteristics in addition to its biodegradability, low antigenicity, antioxidant activities, etc., several EPS have gained research focus in recent years. Dextran is one such example of EPS, which is synthesized by lactic acid bacteria (LAB). However, its production is limited to utilization of sucrose medium with a yield of 0.3-0.4 g dextran/ g sucrose. Hence, this thesis work focuses on dextran production using sucrose-rich alternate feedstocks and its optimization.

Sugarcane juice (SOJ) and sugarcane molasses (SCM) are enriched sources of vitamins, minerals and carbohydrates that proves to be an effective medium for LAB growth and dextran synthesis. The presence of melanoidins, negatively charged heteropolymer, in SCM greatly affects the production and recovery of dextran from fermentation broth. Therefore, SCM pretreatment with activated charcoal (TCM) resolves this issue. After conventional optimization, Leuconostoc mesenteroides produced a maximum titer of 44.5 ± 0.5 g/L and 42.0 ± 2.2 g/L dextran on SOJ and TCM media respectively. However, only 0.29-0.4 g/g of dextran could be obtained. So, further optimization by statistical method (Central-Composite Design) improved the dextran titer to 78.1 ± 1.9 g/L (SOJ) and 60.0 ± 2.0 g/L (TCM), resulting in a 9.8-fold and 9.0-fold increase on SOJ and TCM media, respectively when compared with unoptimized media.

The produced dextran yield was also compared with that of the optimized sucrose medium (94.5 ± 2.0 g/L). In the case of sucrose medium, around 9.5-fold increase in production has been observed. Also, the yield was improved to 0.47 ± 0.01, 0.39 ± 0.01, and 0.4 ± 0.01 g/g in sucrose, SOJ and TCM media, respectively. The characterization studies such as NMR, FTIR, and GPC, of the produced polymer confirmed it to be dextran. With the help of rheology study, the dextran solutions were found to be pseudoplastic (n = 0.89 for dextran from SOJ media, and n = 0.62 for dextran from TCM media at 10 % w/v) that can affect fermentation performances such as mass transfer rate, mixing, aeration and downstream processing.

On the basis of economic yield and molecular mass, dextran production using TCM media proved to be cost-effective. Hence, further understanding about kinetic parameters for dextran production on TCM media was studied using various mathematical models. A maximum specific growth rate of 0.35 h-1 was obtained using logistic equation. From Leudeking-Piret model, dextran production was found to be predominantly growth associated with a specific productivity of 3.79 g/(g.h).

Interestingly, from the literature survey, it was observed that the functional properties of dextran is usually affected by its recovery conditions, aeration, agitation, etc. So, in this study, dextran production in a 3L fermenter was carried out under previously optimized conditions to study the influence of aeration and agitation on the dextran titer, its molecular mass and broth rheology.

It was found that the aeration did not significantly affect dextran titer but on increasing impeller speed (50 to 150 rpm), not just the titer (55 to 60 g/L), but also its molecular mass improved from 2780.6 kDa to 3930.6 kDa. Apart from fermentation conditions, recovery of dextran also plays an important role on the overall cost as well as functional properties of dextran. By optimizing the downstream processing, the yield of dextran was found to improve from 0.4 g/g to 0.47 g/g on using 1:4 v/v solvent supernatant ratio of ethanol.

In addition, DES was also able to successfully precipitate 70.0 ± 2.5 g/L of dextran at a ratio of 1:2 v/v supernatant to solvent. The obtained dextran was efficiently utilized in the bioremediation and biorefinery processes. For industrial production of dextran, large throughput is necessary which can be achieved by transferring the optimized conditions to large-scale fermenters and by proposing certain scale-up strategies. During scale-up, based on constant P/V strategy, an agitation of 54 rpm was estimated for efficient dextran production in a 2000 L working volume.

Download

Share

COinS