APPLICATION OF RESPONSE SURFACE METHODOLOGY TO OPTIMIZE BIOMASS PRODUCTION OF THE YEAST Kluyveromyces thermotolerans, INTENDING TO BE USED AS BIOCONTROL AGENT FOR OCHRATOXIGENIC Aspergillus SPECIES.

ROMÁN PELINSKI; PATRICIA CERRUTTI; LEOPOLDO J. IANNONE; LORENA PONSONE; SOFIA N. CHULZE; MIGUEL A. GALVAGNO

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiologia General

The chlorined isocumarin compound ochratoxin A (OTA), a potent neprhotoxic and carcinogenic compound is produced by several species of Aspergillus section Nigri associated with grapes and it has been reported in wine and grape juice. To study the biocontrol of ochratoxigenic fungy by Kluyveromyces thermotolerans, strains isolated from grapes, in vitro tests were carried out in our laboratories and inhibition of fungal growth as well as the OTA production was achieved. Thus, optimization of biomass production of these yeasts becomes a fundamental first step in order to investigate their applicability as bio-controllers of ocrhatoxigenic fungi in the vineyard K. thermotolerans (strain LB4) was maintained at 4°C in Sabouroad-agar medium. To evaluate vegetative growth, the strain was grown in semisynthetic media contained in shaken flasks (vol flask: vol media, 5:1). Growth curves were done in Yeast Nitrogen Base (YNB Difco) medium containing glucose as carbon source at 28°C at 200 rpm. Calculated µmax and td were 0.36-0.29h-1   and 1.9-2.4 h respectively. Glycerol and ethanol at the same molar C concentration as glucose were tested in YNB but only glycerol supported yeast growth aerobically reaching similar biomass production figures, through productivity was (1.5-2.0 fold) higher in glucose. When sugar cane molasses (58% w/w fermentable sugars, FS) was employed to replace glucose in the medium, 2 to 3-fold higher biomass concentration was reached. Several categorical factors studied in aerobic  cultures containing molasses  incubation temperature and time, inoculum size, vitamin (Yeast Extract)  addition and nitrogen source, were assayed. According to the results obtained, we decide to optimize fermentation conditions by means of statistically designed experiments. Various continuous factors were screened using Plackett-Burman and Fractional Factorial Designs. The results obtained showed that the remaining factors significantly affecting biomass concentration were molasses and nitrogen/phosphorous ((NH4)2HPO4, DAP) concentration and  inoculum size at the levels chosen and were picked up for the optimization step using Response Surface Modeling (RSM) and a Box-Wilson Central Composite Design was run (28°C; pHi=4.9; 200 rpm for 24 hrs). The results showed that the optimized biomass concentration achieved (1.1x109 cells ml-1; OD640nm=27.98) employing RSM, was obtained with cane molasses, 21.6 (12.5 FS)% (w/v); DAP, 0.3% (w/v) and inoculum size  (OD640nm=10-12), 2.0% (v/v). Yield (Yx/FS) values obtained were over 0.26 g g-1. After model building and optimization, the predicted biomass optimum was verified. Validation assays in laboratory scale bioreactors are currently carried out.