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Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in approximately 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of approximately 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.

Biological products offer advantages over chemotherapeutics in aquaculture. Adoption in commercial application is lacking due to limitations in process and product development that address key end user product requirements such as cost, efficacy, shelf life and convenience. In previous studies, we have reported on the efficacy, physiological robustness and low-cost spore production of a Bacillus cereus isolate (NRRL 100132). This study examines the development of suitable spore recovery, drying, formulation and tablet production from the fermentation product. Key criteria used for such downstream process unit evaluation included spore viability, recovery, spore balance, spore re-germination, product intermediate stability, end product stability and efficacy. A process flow sheet comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded a suitable product. The formulation included corn steep liquor and glucose to enhance subsequent spore regermination. Viable spore recovery and spore balance closure across each of the process units was high (>70% and >99% respectively), with improvement in recovery possible by adoption of continuous processing at large scale. Spore regermination was 97%, whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves. The process resulted in a commercially attractive product and suitable variable cost of production.

The potential of a Bacillus cereus isolate (NRRL 100132) as a biological agent for aquaculture has been demonstrated in vitro and in vivo. The functionality of this isolate across a range of physiological conditions, including salinity, pH and temperature, based on rearing of high-value ornamental Cyprinus carpio, was investigated. Temperature had a significant influence on germination, specific growth rate and increase in cell number of B. cereus in shake-flask cultures, whilst salinity and pH did not have a measurable effect on growth. Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated the existence of high and low growth temperature domains. The rates of pathogenic Aeromonas hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at different temperatures. The present study confirmed the robustness of the B. cereus isolate (NRRL 100132) as a putative biological agent for aquaculture and further demonstrated a novel method for the assessment of in vitro biological efficacy as a function of temperature.

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