Development of a bioprocess for the production of an aquaculture biological agent

[摘要] ENGLISH ABSTRACT: Biological agents offer several opportunities to address the many challenges faced in intensivereticulated aquaculture. We therefore isolated and selected Bacillus spp. as potential biologicalagents, because this group has demonstrated an array of biological activities of possible benefit toaquaculture. They also display advantages in terms of robustness during bioprocessing and endproduct application.Natural isolates obtained from Cyprinus carpio, selected as a model high-value ornamental fishspecies, and associated culture environments, were purified and assessed for in vitro efficacybased on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease inconcentrations of ammonium, nitrite, nitrate and phosphate ions, typically found as wasteproducts in aquaculture systems. Based on suitability for aquaculture application, isolates B001,B002 and B003 were selected and further evaluated in vitro and in an in vivo trial with C. carpio.Inhibition of Aer. hydrophila growth and a decrease in concentrations of waste ions weredemonstrated in these studies. Based on 16S RNA sequence homology, the isolates wereidentified as Bacillus subtilis, B. cereus and B. licheniformis, respectively. High sequencehomology between B. subtilis and B. anthracis necessitated further safety studies on the bestisolate, B. cereus NRRL100132 (B002). The isolate was shown not to contain the anthraxvirulence genes pOX1, pOX2 or the B. cereus enterotoxin.Elucidation of the potential modes of action of a biological agent facilitates an understanding offunctionality and encourages technology uptake by end users. Competitive exclusion throughgrowth rate and competitive uptake of glucose and iron, the latter facilitated by siderophoreproduction, were shown to be key mechanisms at play in inhibition of Aer. hydrophila by the B.cereus isolate.As production cost is an important consideration in development of commercially relevantbiological products, we examined the optimization of nutrient supplementation, which has animpact on high-density production of spores by fermentation. Corn steep liquor (CSL) wasidentified as a lower cost and more effective nutrient source in comparison to conventionalnutrient substrates, in particular yeast extract and nutrient broth. The improved sporulationperformance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on organism growth andsporulation efficiency. The impact of nutrient concentration on spore yield and productivity wasmodelled to develop a tool for selection of optimal conditions. Excellent correlation with actuallaboratory fermentation data was demonstrated. A cost analysis revealed that production usingliquid phytase treated and ultra-filtered CSL was less expensive than spray dried CSL andsupported cultivation of B. cereus spores at densities higher than 1×1010 CFU ml 1.Adoption of biological agents in commercial applications is lacking, due to limitations in processand product development that address key end user product requirements such as cost, efficacy,shelf life and convenience. The development of suitable spore recovery, drying, formulation andtablet production process steps was thus performed. Key criteria used for downstream processunit evaluation included spore viability, recovery, spore balance closure, spore re-germination,product intermediate stability, end product stability and efficacy. A process flow sheetcomprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yieldedan attractive product. The formulation included corn steep liquor and glucose to enhancesubsequent spore re-germination. Viable spore recovery and spore balance closure across each ofthe process units was high (>70% and >99% respectively), with improvement in recoverypossible by adoption of continuous processing at large scale. Spore re-germination 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 affordable variable cost ofproduction.Functionality of the product, incorporating the B. cereus isolate, was investigated across a rangeof physiological conditions, including salinity, pH and temperature, based on rearing of C.carpio. Temperature had a significant influence on germination, specific growth rate and increasein cell number of B. cereus, whilst salinity and pH did not have any measurable effect on growth.Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicatedthe existence of high and low growth temperature domains. The rates of pathogenic Aer.hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate andphosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate atdifferent temperatures. This study has resulted in development of an upstream and downstream process for production ofa new B. cereus isolate (NRRL 100132) which was shown to be safe, stable, functional, robustand cost effective for application in aquaculture.

[发布机构] Stellenbosch University