Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

The microbial ecology of cow slurry in vermiculture beds

The suitability of cow slurry as a substrate for vermicomposting by Eisenia fetida was investigated. Particular attention was given to the effects of the earthworm on the decomposition and stabilisation of the slurry; and to the interactions between E. fetida and the microflora of the substrate. Assessment of the chemical and microbiological changes in cow slurry stored under forced aeration, and subsequently in shallow trays, showed that neither method was suitable for the treatment of slurry. A comparison of two methods of vermicomposting showed that top-feeding of slurry was more efficient in promoting earthworm growth and cocoon production than the mixing of slurry with solid materials. Management practices were found to have an important influence on the efficiency of the process. An investigation o:f the effect of E. fetida. on the decomposition of slurry indicated that the presence of this earthworm enhanced the stabilisation of the substrate and increased the plant-available nitrogen content. Specific nutritional interactions were observed between E. fetida and micro-organisms in sand/cellulose microcosms. The earthworms were found to be feeding directly upon the cells of certain micro-organisms. Other species were found to be toxic to E. fetida.. A technique was developed :for the production of axenic E. fetida., and the use of such earthworms in :feeding experiments confirmed the importance of some micro-organisms in earthworm nutrition. The seeding of vermiculture beds with one such micro-organism stimulated earthworm growth and consumption of the substrate. Vermicomposted mixtures of cow slurry and spent mushroom compost were shown to have potential application as casing materials in mushroom cultivation. The findings of this study indicate the suitability of vermicomposting as a method for the stabilisation of intensively-produced cow slurry, and give some indication of the importance of micro-organisms in the nutrition of E. fetida.

The housefly Musca domestica as a mechanical vector of Clostridium difficile

Background - Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. Aim - To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. Methods - M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. Findings - M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4 h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/− 66.9 for vegetative cell suspension and 288.2 +/− 83.2 for spore suspension). After 1 h, this had reduced (21.2 +/− 11.4 for vegetative cell suspension and 19.9 +/− 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/− 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/− 0.58. C. difficile could be recovered from fly excreta for up to 96 h. Conclusion - This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas.