Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biosystems Engineering

Committee Member

Yi Zheng, Committee Chair

Committee Member

Caye Drapcho

Committee Member

Terry Walker

Committee Member

Mark Blenner

Abstract

DHA/EPA producing marine protists have recently become more attractive due to their additional high-value products, including extracellular enzymes, extracellular polymeric substances (EPS), and astaxanthin. In this study, Thraustochytrium striatum ATCC 24473 was studied and the cultivation conditions were been optimized to achieve high biomass and fatty acid yield. Results showed that seawater salinity, neutral pH and mild rotation speed favored biomass growth, while better lipid accumulation was observed under high C/N ratio, higher salinity or high pH. Comparison in different carbon sources showed starch and glycerol improved fatty acid accumulation the most, and ammonia chloride was the best nitrogen source for fatty acid accumulation. In fed-batch cultivation, 12-day incubation under nitrogen depletion condition elevated the content of total fatty acids from 28 to 38% compared to the batch. The qualitative and quantitative analysis of enzymes from T. striatum indicated that at least seven extracellular enzymes including lipase and six polysaccharases (i.e., amylase, cellulase, xylanase, chitinase, pectinase, and κ-carrageenase) were formed during cultivation. The carbon and nitrogen concentrations and salinity significantly affected the kinetics of enzyme activities. Enzyme-specific polysaccharide substrates including starch, CMC, xylan, κ-carrageenan, pectin, and chitin did not induce the production of corresponding enzymes by T. striatum. Moreover, with the investigation of components of EPS under different growth phases, enzymes appeared to correlate with the production and monosaccharide composition of EPS from T. striatum. The EPSs from T. striatum mainly contained polysaccharide (41-64%, w/w) and protein (25-40%, w/w) contents. The monosaccharide profile of the EPS polysaccharide consisted of glucose, galactose, arabinose, and trace amount of xylose. Bioactivity tests showed that EPSs had anti-tumor, antioxidant, anti-inflammatory, and antibacterial activity. The comprehensive studies of astaxanthin formation from T. striatum indicated that glucose plus yeast extract/peptone was the optimum combination for both cell mass and astaxanthin production. In the batch model low C/N ratio and temperature condition, 100% ASW salinity and neutral pH favored cell mass growth, however, high C/N ratio, acidic or strong alkaline pH, high salinity and high cultivation temperature improved astaxanthin accumulation. In addition, the presence of H2O2, blue/white light illumination and oxygen favored astaxanthin accumulation, while dry air can only improve cell growth. In the pulsed fed-batch cultivation, the highest cell mass, astaxanthin and total carotenoids concentrations were achieved after 15 days of cultivation. No growth inhibition observed when corn stover hydrolysate was utilized as a carbon source for fed-batch cultivation, but 25% of astaxanthin content reduction was found compared with glucose-based fed-batch process. Finally, a Monod-equation based biokinetic model with/without substrate inhibition was established, and it can simulate substrate consumption, cell mass growth, total fatty acid accumulation, and astaxanthin formation rate under glucose concentration from 2.5-50 g/L.

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