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Title: Pullulan production from agro-industrial wastes
Author: Barnett, Christian
ISNI:       0000 0001 3446 3984
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2000
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This study was divided into two parts. The first part of the investigation concentrated on assessing two agro-industrial waste products for suitability for pullulan production by fermentation with Aureobasidium pullulans. The second part concentrated on trying to elucidate the biosynthetic processes involved during pullulan biosynthesis both in vztro and in vivo. The two waste materials investigated as possible fermentation substrates were potato starch waste (PSW) and olive oil waste waters (OOWW). New methodologies were developed to characterise both the media and monitor their subsequent fermentation. OOWW are a by-product of the olive oil industry that cause environmental problems mainly related to their phytotoxicity. Fermentations using OOWW proved unsuccessful for the synthesis of pullulan, yielding only 2.8 g/L after 7 days. The development of a capillary electrophoresis method allowed the simple phenol compounds present in OOWW to be monitored throughout the fermentation. Subsequent fermentation of OOWW withv4. pullulans showed that all the detectable phenol compounds were removed within four days. A. pullulans was shown to lack the full compliment of enzymes to degrade raw PSW, therefore various hydrolysis regimes were developed to produce hydrolysates with a range of oligosaccharide composition. Analysis of the hydrolysates by capillary electrophoresis enabled the characterisation of the oligosaccharides in the fermentation media. Batch fermentations, in both shake flasks and fermenters, were undertaken to investigate the effect of supplementing PSW with sources of nitrogen, phosphate and yeast extract. These showed hydrolysed PSW to be a good substrate for both A. pullulans growth and pullulan synthesis. The highest yields were obtained from PSW containing predominately maltose, yielding up to 60 g/L exopolysaccharide of which was 90% was pullulan. The second part of the study concentrated on investigating pullulan biosynthesis in vivo and via cell-free lysates. Initially investigations were carried out by incubating A. pullulans cell lysates with uridine diphosphoglucose (UDPG), adenosine triphosphate and various possible promoters. Some in vitro biosynthesis was obtained which was shown to be time, substrate and protein dependent. The reaction was stimulated by Mn²⁺, Mg²⁺ and Ca²⁺, and inhibited by Zn²⁺. Investigations using A. pullulans wall/membrane extracts showed no pullulan biosynthetic activity, however a significant amount of glycogen synthase activity was detected. The in vivo studies employed both specific and non-specific action of a number of protein glycosylation inhibitors to assess their effect on pullulan biosynthesis. The action of the inhibitors was targeted at various hypothesised stages of pullulan biosynthesis, resulting in a possible synthetic system from UDPG to pullulan via lipid intermediates involving various organelles.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Aureobasidium; Starch; Olive oil; Biosynthesis