Strategies to control bacteriophage infection in a threonine bioprocess

Abstract

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.