As an illustration of the combined effect of alcohol and initial sugar level on fermentation kin ... So is between 50 and 150 g.litre-1, and the effect of added.
J. In.il. Brew., November-December, 1984, Vol. 90, pp. 359-362
359
STUDIES ON CONJUGATE EFFECTS OF SUBSTRATE (GLUCOSE) AND PRODUCT (ETHANOL) ON CELL GROWTH KINETICS DURING FERMENTATION OF DIFFERENT YEAST STRAINS By Manuel Mota
(University ofPorto. Porto, Portugal) Pierre Streiiaiano
(EcoleNationaleSuperieured'Agronomie, E.N.S.A.T. 145. AvenuedeMum, 31000 Toulouse) Gerard Com a
(Departement de Genie Biochimique et Alimentaire ERA-CXRSH79,I.N.S.A.. Avenue de Rangueil. 31077 Toulouse Cedex) Received8 August IM3
The conjugate effect of substrate (glucose) and product (ethanol) has been studied on yeast growth during the course of fermentation. The study was made on five yeast strains Kloeckem apiculata. Saccharomycesuvarum. Saccharomyces bayanus, Saccharomyces cerevisiae UG5. and Saccharomyces cerevisiae sake. The results attest a
different action of added alcohol as compared with produced alcohol for all strains. It is also verified that kinetic behaviour, namely ethanol inhibition, varies with initial substrate concentration, and so a new parameter, the added alcohol concentration at which biomass productivity declines to one-half its value with no added ethanol, is proposed to quantify ethanol inhibition.
Key words: Yeast, growth, glucose, ethanol. Introduction
Many workers have studied the inhibitory effect of initial sugar concentration on yeast growth.21"3 Classically, inhibition due to sugar gives place in the course of growth to inhibition by cthanol. Improved alcohol fermentations may be expected when these inhibition phenomena are fully understood. It has been shown previously27 IS that produced alcohol does not act in the same way as added alcohol on growth or fermentative ability: the KIP value for added alcohol was cal culated as 105 g.litre-' against only 3-5 g.litre-> for produced alcohol in the hyperbolic relationship
Culture apparatus: eight batch vessels of 2 litres working capacity were operated at 30°C, under anaerobic conditions and agitation by circulation pump (6 litre.mn"'). A statistical analysis of the fermentation kinetics confirmed no significant deviations between the vessels.
Inoculum: yeast were grown in conical flasks with shaking during 15 h on the 20 g.litre-1, no-cthanol medium. The final cell concentration was approx. 108cellsml-', and the inoculum was always 10% of the total culture volume (i.e. 200 ml to 1800 ml medium). Biomass was evaluated by the dry matter method (filt ration on a 0-45 um millipore membrane and vacuum drying at 60°C for 24 h). Ethanoi was determined by gas-chromatography using isopropanol as internal standard. Glucose was evaluated by the enzymatic method using a Y.S.I. 27 apparatus.
where P+KIP
u (h-')is the specific growth rate, and Up, its maximal value. P (g.litrc-1) is the product concentration, and K)P is the product inhibition constant on the specific growth rate. Nevertheless the strong inhibitory effect of ethanol is known, and used e.g. in enology to obtain a muted must. However, changing the initial substrate level gives rise to different ethanol sensitivity. So, in this work a study was made of the combined effect of the substrate and product, i.e. glucose and ethanol, on yeast growth. This was done for five different strains of yeast, from the sensitive Kloeckera apiculata to the resistant Saccharomyces cerevisiae sake strain. For valid comparison of these fermentations with different initial concentrations (substrate and alcohol) biomass productivity was measured. Materials and methods
Yeast strains used were Kl. apiculata (Institut Pasteur) S. uvanim (TEPRAL 0006), S. cerevisiae strain UG5 (STREHAIANO12), S. cerevisiae sake strain (l.A.M. TOKYO) and S. bayanus (I.N.R.A.). The medium of NOVAK et al.1 was supplemented with glucose (20, 50, 100, 150,250 g.litre-') and sterile (filtration on a 0-22 um membrane) ethanol solutions were added to each vessel before inoculation to give 0, 15, 30, 50, 60, 80, 100 or 110 g.litre-1 (alcohol).
Rksults and discussion
Biomass productivity (g.Iitre-'.h '), i.e. an integral par ameter was chosen for the comparison of inhibitory effects
rather than the specific growth rate, which is a differential
parameter and so more subject to errors of determination. The productivity was measured after 10 h of fermentation, so that when the fermentation time was long, e.g. 200 h for an initial sugar level of 250 g.litre-1, the results describe only the first stage of growth. This parameter is noted
pio = xio-.xo/1° (g.litre-'.h-1) where X10 is the biomass
concentration after 10 h of fermentation time, Xo being the initial value of biomass. As an illustration of the combined effect of alcohol and initial sugar level on fermentation kin etics, figures 1-4 show the growth pattern of S. bayanus. In table I, the calculated values of Pl0 arc presented. It was noted that the best productivities are obtained when So is between 50 and 150 g.litre-1, and the effect of added cthanol is perceptible only above 50g.litre-' for these weak
values of initial substrate (SO
