Submitted in partial fulfillment of the academic requirements for the degree of Master of Technology: Biotechnology, 2009. / Production of numerous biotechnologically-important products such as
threonine is based on cultivation of bacterial cultures. Infection of these
bacterial cultures by bacteriophages has a detrimental effect in the production
of these bioproducts. Despite this, most people controlling these bioprocesses
do not recognize the early signs of bacteriophage infection. SA Bioproducts
(Ply) Ltd was no exception and has suffered tremendous loss of production
time after bacteriophages infected threonine producing E. coli strain B. This
study was aimed at developing assays to control and prevent bacteriophage
infection at this company. These included determining the source of phages
by monitoring the process plant environment, optimising the detection and
enumeration methods so as to monitor the levels of bacteriophages in the
environment, identification of bacteriophages in order to determine the
number of bacteriophages capable of infection threonine producing E. coli
strain B, treatment and of phages, and possible prevention of phage infection.
Adam's DAL method was very efficient at detecting phages in the samples
collected at various areas (sumps, odour scrubber, process water, and soil)
around the plant for 16 weeks. High levels of phages were found in the sumps
and this was identified as the source of infection. Samples collected were
grouped together according to their source. The samples were enriched and
purified in order to characterise them. The prevalent phage in all samples was
identified as a T1-like phage. Bacterial strains that grew on the plate in the
presence of phages were assumed to be resistant to phages or contained
lysogenic phages which would explain the new lytic cycles that were observed
whenever these resistant strains were used for production. UV light, green
v
indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens.
Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic
phages. This was shown by new plaque forming units that were visible on the
DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were
investigated as strategies for prevention and treatment of bacteriophage
infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At
these temperatures pfu counts in the samples were reduced significantly. At
120°C there was a complete inactivation of bacteriophages within 30 minutes.
Chemicals investigated such as sodium hydroxide and Albrom 100T were
capable of complete deactivation of bacteriophages at a very low
concentration (0.1%). Therefore, these chemicals can be used to clean the
plant area and sumps. Vitamins C, K and E solutions were investigated to
determine their inhibitory effect on bacteriophages. Vitamin C, K and E
reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and
E solutions were mixed and to determine if mixing them would enhance their
inactivation capabilities. This resulted in a reduction greater than 9 logs of
phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also
exposed to this mixture to determine effect of the vitamin mixture on its
growth. It was found that there was no effect exerted by this mixture on the
host bacteria. This proved to be an ideal mixture for combating phages during
fermentation. However, vitamin E is not cost effective for co-feeding in 200 m'
fermenters, and therefore vitamin C solution was a cost-effective alternative. It
was concluded that bacteriophage contaminated bioprocessing plant should
be properly cleaned using a combination of heat and chemicals.
Bacteriophage infection should be prevented by employing inhibitors.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:dut/oai:localhost:10321/451 |
| Date | January 2009 |
| Creators | Cele, Nolwazi |
| Contributors | Permaul, Kugen, Snyman, Francisca |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 146 p |
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