Document Type

Dissertation - University Access Only

Award Date

2008

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology

Abstract

This dissertation comprises a complete literature review pertaining to the production of polyhydroxyalkanoates (PHA) by microbial based cultures. The literature review covers all aspects involved in PHA production from maintenance and genetics of the microbes producing PHA to isolation and characterization of the PHA. Medium chain length polyhydroxyalkanoates (mcl-PHA) were produced by Pseudomonas resinovorans from condensed com solubles and sodium octanoate. An ethanol-modified supercritical fluid extraction (SFE) procedure was developed for extraction of mcl-PHA. A temperature and pressure of 60°C and 9000 psi, respectively, was identified as suitable extraction conditions when 15% (v/v) ethanol modifier was utilized. Polymer obtained from ethanol-modified SFE was compared to that obtained from Soxhlet extraction. Similar extract characteristics (purity, cell dry weight%) were obtained from both methods. Extracts obtained by modified SFE was positively identified as PHA by analysis with FTIR, GC/MS/FID, and 13C-NMR. Pseudomonas putida KT217 is a medium-chain-length polyhydroxyalkanoate (mcl-PHA) producer. To reduce PHA production costs, we developed a less expensive medium to grow P. putida KT217 to a high cell dry weight (CDW). P. putida KT217 was grown in aerated shake flasks on a condensed com solubles (CCS) medium at concentrations ranging from 100-600 g/L. The CCS provided several organic acids and carbohydrates that were utilized for growth. The 400 g/L CCS level produced approximately 6 g CDW/L, at a growth rate of 1.03 h- 1, and maximum cell population of 4x 109 CFU/mL. Because CCS is low in nitrogen, ammonium hydroxide was added at a level of 1.73 g/L, and this significantly increased the CDW(> 20 g/L) produced in an aerated bioreactor. Therefore, a nitrogen supplemented CCS medium could provide a cheap source of nutrients for production of P. putida KT217 and PHA. Pseudomonas putida KT2 l 7 was then grown on a complex medium comprised of CCS in fed-batch mode with carbon sources that are co-products of the biodiesel industry to assess the organism's capability to produce medium-chain-length polyhydroxyalkanoate (mcl-PHA). The growth phase was carried out in a medium containing 400 g/L condensed com solubles (CCS), supplemented with ammoniu hydroxide as a nitrogen source. Following the exponential phase, co-products of the biodiesel industry (soapstock and glycerin) were fed into the reactor to trigger PHA production. When glycerin was added to the bioreactor (75 g/L total addition), the final cell dry weight (CDW) and PHA content were 30g/L and 31 %, respectively. The monomeric composition in the PHA formed was relatively uniform throughout fermentation with 3-hydroxydecanoate dominating. When a total of 153 g/L of sunflower soapstock was added to the bioreactor in a fed-batch manner, the final CDW and PHA content were 17 g/L and 17%, respectively. Following addition of soapstock the monomeric composition of the polymer changed dramatically, with the 3- hydroxyoctanoate monomer becoming dominant and greater unsaturation present in the PHA.

Library of Congress Subject Headings

Agricultural chemicals Agriculture Analytical chemistry Microbiology

Format

application/pdf

Number of Pages

250

Publisher

South Dakota State University

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