FLOCCULATION AND FERMENTATION CAPACITY OF STRAINS OF Saccharomyces STORED AT MYCOTHECA-URM

Cepas de Saccharomyces mantenidas en laMicotecaURM. fueron estudiadas en relacion a su capacidad de fermentar y flocular en una solución acuosa de melaza a 18° Brix, De estas, 11 pertenecían a S. cerevisiae y .1 a S. kluyveri. El mayor contenido de etanol (6,02% v/v) fue obtenido con la cepa 1460 y el menor (1,39% v/v) con la 2624, Los menores porcentaje de células que permanecen en suspensión(R%), 65,82%, 65,31 %, 41,66%y 55,55%, fueron obtenidos respectivamente con las cepas 2659, 2624, 2716 y 1337, indicando una mayor intensidad defloculación. Las cepas 2659 y 2716 produjeron una mayor concentración de etanol. Los resultados indican que una cepa de Saccharomyces puede o no expresar conjuntamente una buena capacidad de fermentación y floculación.


INTRODUCTION
The Micoteca-URM of the Mycology Department of the Center of Biological Sciencs (CCB) at the Federal University of Pernambuco (UFPE), registered at the Comnonwealth Mycological Institute (CMI) under the abbreviature URM (University of Recife Mycology), has presently 313 strains of yeasts, of which 12 belong to the genus Saccharomyces Meyen emend.Reess; all ofthem are preserved in mineral oil and sorne by lyophilization according lo the methods of Sherf (39) and Raper & Alexander (33), respectively.
Due to the increasing utilization of microorganisms in industrial processes and the advances in the area ofBiotechnology, the importance of culture collection has been acknowledged at the internationallevel ( 5,16 ).
The genus Saccharomyces has been widely used by

SUMMARY
Strains of Saccharomyces stored at the Micotheca-URM were studied as regards the capacity to ferment and flocculate in aqueous /IIolasses so/ution at 18: Brix.Of these, 11 belong to S. cerevisae and 1 to S. kluyveri.The highest ethano/ content (6,02 % v/v) was obtainedwith strain 1460 and the /owest (1.39% v/v) with 2624.The /owest percentage of cel/s which remained in suspension (R%) ,65.82%, 65.31%, 41.66% and 55.55% were obtained respective/y with strains 2659, 2624, 2716 and 1337; such resultsshow a greater intensity of floccu/ation.The strains 2659 and 2716, produced higher concentration of ethanol.The results show that a strain of Saccharomyces may express 01' not a/so a good capacity offermentation andfloccu/ation.the bread-making industries, animal feed, alcoholic liquors, ethyl alcohol, milk-derived products and other (21,37).
The different species and even the different samples of a same species may present or not the same morphological and lor physiological properties.Among them: capacity of alcoholic fermentation and flocculation; tolerance to ethanol and carbon dioxide; osmosensitivity and others (3,11,17,23).
Alcoholic fermentation was studied by Pasteur in 1858, but only in 1876 he did discovered the anaerobic facultative character ofthe beer yeasts.In 1897, the Buchners were pioneers in enzyrnology obtaining an extract of yeast which fermented sugars and produced ethanol and carbon dioxide (C0 2 ) , discovering the role of enzimes (endocellular substances) in the alcoholic fermentation which ineludes a sequence of enzima tic reactions triggered when a yeast with a fermentative capacity makes contact with the substratum ( 6,7,37,49).
Various kinds of raw material may be used in the alcoholic fermentation process.Sugarcane molas ses are commonly used by fermentation industries.Usually contents are as follows : 20% water, 62% sugar, 8% ashes, 3% of nitrogenous materials and 7% of other components.As an average, sugars are made up of 32% saccharose, 16% levulose and 14% dextrose.As a rule it may be safely said that molasses are a rich residue which contains 50% offermentable sugars.Thus the microorganisms which are agent of the fernlentative processes make a satisfactory use of it as an energetic source of nutrition (21 , 27,36).
The flocculation ofyeasts is defined as the capacity of spontaneous aggregation of the cells with the formation of flocs which separate from the substratum by sedimentation (7, 35, 43 , 47).This fenomenon is being used in the biological production of ethanol ,beer and yeast biomass( 8, 9, 24, 25,
In industrial processes of fermentation use is made of flocculating or non floculating species of the genus Saccharomyces.Along that line and with the intention to set al the Micoteca-URM a Bank of Yeast of Biotechnological 'nlerest, this paper has as its objectives to characterize the slrains o f Saccharomyces sto red , as regards to their capacity loferment and flocculate in an aqueus solution of molasses al 18° Brix.
Strains of Saccharomyces were seeded in Sabouraud agar + 0.5 % ofYE and left at R.T./48 h; next the cells were suspended in 10 mi of sterilized distilled water; the concentration was adjusted for 10 8 cells/m1.From each suspension 6 mI were inoculated in 54 mi of aqueous solution of molasses at 18° Brix, held in a 250 mi Erlenmeyer, and left at room temperature for 24 hours.Next the 60 mI solution was agitated by hand and 10 mI were centrifugated in a cooled centrifuge at 3.000 rpm/15 min./4°C.The superna te was analyzcd for concentration of ethanol for liquid-gaseous chromatograph and of the total reductor sugars (ART) by the DNSA (dinitrosalicylic acid) method (Miller (26)), as well as ofthe pH values as potentiometer.The sediment was used for the determination oftotal cells (C T ) .The other fraction (50ml) of the agitated solution was used for the determination of free cells (CL).From the know values of C T and CL' those of R % were calculated according to the method ofPereira lr.& Bu' Lock (30) (Scheme 1).

RESULTS AND DISCUSSION
Different results were achieved with the strains of Saccharonyces as regards the capacity of fernlentation and flocculation in an aqueous solution of molasses al 18° Brix.The concentration of ethanol produced by the strains of Saccharomyces after 24 hours of fermentation varied from 1,3 9 % v/v with strain 2624 to 6.02 % v/v of ethanol after a 24 hours fermentation .Lima (20), operated with this same strain in a fermentation tube with sugarcane molasses at 15° Brix , with fermentation times from 1 to 12 hours and obtained ethanol concentrations which varied from 1,18 % v/v (1 hour fermentation) to 5,53 % v/v (11 hours fermentation) .The variation of results of fermentation with different strains of Saccharomyces is stated in the literatures as evidenced by different works (4,14,31,38,51).
To determine the percentage of ART the glucose standard curve was used, the correlation rate was r = 0.9974 showing a good relation between the variables.The equation Y= 1.041 X -0.153 was used to obtain glucose concentrations of the superna te.
At time O the concentration of ART was 15,62 % w/v an and pH 5,30 ; after 24 hours of fermentation, ART concetration varied from 11 ,60 % w/v to 2,90 w/v and pH from 4.29 to 5 (Fig. 1).ART concentration and pH values "iere inversely proportional to the ethanol produced by the :;trains.
Values of R % were calculatedto determine indirectly the intensity offlocculation which is• represented by 100 -R %; thus lesser values of R %, indicate higher rate of flocculation.The Fig. 1, shows that the rates ofR % derived from strains of Saccharomyces varied from 41,6 to 92, 41 %.
Among the strains wich flocculated better, strains 2659 and 2716, produced high concentrations of ethanol (Fig. l).According to Seiko et a1.(38), yeasts with such characteristics are suitable for continuous fermentations in the production of ethanol with cell recycling.Strains 1337 and 2624, produced low concentrations of ethanol (Fig. l)and are not ~uitable for processes of alcoholic fermentation.
S.cerevisiae 2624, produced flocs made up ofpseudomycelium exclusively (Fig. 3) and low concentrations of ethanol(1.39%v/v)(Fig.l).These results confirm data in Rose & Harrison (35), where strains which form flocs by pseudomycelium are the best for industrial fermentations.
Among the strains which did not flocculate, the 1460, 2658, 2689,2690, produced higher concentrations os ethanol (Fig. l) and this recomends their use in fermentation proeesses.
The results ofthis work already tested back the information in Rose & Harrison (35), which state that the capacity to ferment and to flocculate may be expressed or not jointly for the same strain of Saccharomyces.
Fontana et al. (9), noticed that the pH range from 5 to 5,5 and the temoperature from 15 to 28°C, are conditions suitable for flocculations.Conditions of R. T. (28°C ± 1°C) and pH 5,3 of the aqueous solutions of molasses at 18° Brix, in which this work took place, are the same indicated by those authors for the flocculations .
The results ofthis work show that there is no relations between fermentation an flocculations; strains of S. cerevisiae may or may not present joining capacity to ferment and flocculate;strains of S.cerevisiae 2659 and 2716, are known as yeast with a good fermentation and flocculation capaeity; strains 1460, 2658, 2689, 2690, are yeasts with a good fermentation capacity but do not flocculate; the types of flocs differ from one S.cerevisiae to other.
The characterization of yeasts from the standpoint of their capacity to ferment and to flocculate shows how important they are for applied research in alcoholic fermentation industries.This in itself plainly justifies the need for the implementation of the yeast bank of biotechnological interest at URM-Mycotheca.