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.

PELINSKI R; CERUTTI, P; IANNONE, L; PONSONE M.L; CHULZE , S.N; GALVAGNO, M

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiologia General; 2009

Sociedad Argentina de Microbiologia General

The chlorinated isocoumarin compound, ochratoxin A (OTA), a potent nephrotoxic and carcinogenic compound is produced by several species of  Aspergillus Section Nigri  associated with grapes and it  has been reported as natural contaminant in wine and grape juice (1), (2). To study  the  biocontrol of  ochratoxigenic fungi by  K.thermotolerans,  strains isolated from grapes , in vitro tests were carried out in our laboratories and inhibition of fungal growth as well as of OTA production were achieved (3) . Thus, optimization of biomass production of these yeasts  becomes a fundamental first step in order to investigate their applicability as bio-controllers of ochratoxigenic fungi in the vineyard. K.thermotolerans  (strainLB4) ,maintained in Sabouraud ?agar medium., was grown  in semisynthetic medium in submerged cultures in shaken flasks (vol flask:vol medium, 5:1)to evaluate vegetative growth . Growth curves were done in Yeast Nitrogen Base (YNB,Difco)medium containing glucose as carbon source at 28 ºC at 200 rpm. Calculated µmax and td were 0.36 (0.29)h-1 and 1.9 ( 2.4)hrs 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, though productivity was (X fold) higher in glucose. When sugar cane molasses (58 %, w/w fermentescible sugars, FS) were employed to replace glucose in the medium, 2-3 higher biomass concentration was reached. Several categorical fermentation factors studied in aerobic cultures containing molasses :incubation temperature and time, inoculum size, vitamin (Yeast Extract) addition   and carbon source, were assayed. According to the results obtained, we decide to optimize  the fermentation conditions by means of statistically designed experiments. Various continous factors were screened using Plackett-Burmen 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 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 employing RSM, 1.1 .109 cells.ml-1 (27.98 OD640nm) 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/ARF) was calculated as x g dw.gARF. After model building and optimization, the predicted biomass optimum was verified. Validation assays in laboratory scale bioreactors are currently carried on in our laboratory.