Effects of the carbon source on the physiology and invertase activity of the yeast Saccharomyces cerevisiae FT858

Abstract

Saccharomyces cerevisiae FT858 is an industrial yeast strain with high fermentative efficiency, but marginally studied so far. The aim of this work was to evaluate the biotechnological potential of S. cerevisiae FT858 through kinetic growth parameters, and the influence of the concentration of the substrate on the synthesis of the invertase enzyme. Invertases have a high biotechnological potential and their production through yeast is strongly influenced by the sugars in the medium. S. cerevisiae FT858 has an excellent biotechnological potential compared to the industrial yeast reference S. cerevisiae CAT-1, as it presented a low glycerol yield on the substrate (Y_GLY/S) and a 10% increase in ethanol yield on sucrose in cultures with sucrose at 37 °C. The substrate concentration directly interfered in invertase production and the enzymatic expression underwent strong regulation through glucose concentration in the culture medium and S. cerevisiae CAT-1 presented constitutive behavior for the invertase enzyme.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02335-w) contains supplementary material, which is available to authorized users.

Introduction

Microorganisms, in general, can undergo evolutionary mutations induced by adverse conditions in the environment in which they live, seeking to guarantee their survival. Thereby, some strains of the yeast Saccharomyces cerevisiae were isolated from industrial ethanol plants, showing high fermentative efficiency, as they evolved to withstand the stress conditions offered during this process. Thus, some of these strains became commercially available, being responsible for much of the Brazilian’s ethanol production, as is the case of S. cerevisiae CAT-1 and FT858 (Basso et al. 2011; Della-Bianca et al. 2013; Borges et al. 2015).

Yeasts exhibit different behaviors depending on the bioprocess to which they are subjected. Cell growth and fermentation are related not only to the presence of the sugar but also to the availability of nutrients in the medium (Santos et al. 2018). Through the monitoring of microorganisms during the industrial process, it is possible to select wild strains capable of surviving and dominating fermentation. These strains can present good fermentation characteristics, such as high ethanol productivity, low glycerol and organic acids formation, low foaming exigence, high viability, among others (Basso et al. 2011; Della-Bianca et al. 2013). Currently, only three S. cerevisiae strains selected from industrial processes are responsible for 70% of all ethanol produced in Brazil: PE-2, CAT-1 and FT858 (Borges et al. 2015).

Although the FT858 strain, as well as CAT-1, were isolated from an industrial environment, there are few published studies on its fermentative efficiency. Santos et al. (2018) evaluated the strain in different cultivation conditions and found that it has high fermentative capacity. In another study, the efficiency of the FT858 strain was compared with two other industrial strains, with this strain presenting higher ethanol production in short fermentation periods and higher sugar consumption (Morales et al. 2014).

Invertases have high biotechnological potential because they can act as hydrolyzing agents in different processes. Invertase is widely used in food and chemical industries, mainly as an additive for the production of inverted sugar. In addition, it is also important in the production of fuel ethanol, lactic acid and glycerol, medicines, paper, and as enzymatic electrodes for the detection of sucrose (Barbosa et al. 2018).

The production of invertases through yeasts is strongly influenced by the sugars in the medium. However, the nature and concentration of the carbon source are not the only important variables to be controlled in the process. To obtain the highest possible yield, conditions must be optimized in terms of nitrogen sources, pH, temperature, size and age of the inoculum (Qureshi et al. 2017; Barbosa et al. 2018).

The same yeast can present different behaviors depending on the type and variety of substrate used during a process (Batistote et al. 2010; Santos et al. 2013; Fonseca et al. 2013; Nascimento and Fonseca 2019). In addition, the transcriptional profile of the yeast S. cerevisiae is strongly affected by glucose, where the expression levels of about 40% of the genes are regulated, up or down, when this sugar becomes available to cells growing in non-fermentable carbon sources (Zaman et al. 2008; Gancedo et al. 2015).

Thus, the aim of this work was to evaluate the biotechnological potential of S. cerevisiae FT858 through kinetic growth parameters, and the influence of the concentration of the substrate on the synthesis of the invertase enzyme.

Materials and methods

Microorganisms and maintenance

The yeasts Sacharomyces cerevisiae FT858 and CAT-1 were utilized in this work. The first strain was kindly provided by Prof. Dra. Margareth Batistote from the State University of Mato Grosso do Sul (UEMS), while the second one by the São Fernando Sugar and Alcohol Distillery, Dourados, MS, Brazil. The yeasts were maintained on YPD agar (agar, 15 g L⁻¹, yeast extract, 10 g L⁻¹, peptone, 20 g L⁻¹, glucose, 20 g L⁻¹) (Camargo et al. 2018).

Cultivation medium

The mineral medium contained per liter of distilled water: (NH₄)₂SO₄, 5.0 g; KH₂PO₄, 3.0 g; MgSO₄·7H₂O, 0.5 g; trace elements (EDTA, 15 mg; ZnSO₄·7H₂O, 4.5 mg; MnCl₂·2H₂O, 0.84 mg; CoCl₂·6H₂O, 0.3 mg; CuSO₄·5H₂O, 0.3 mg; Na₂·MoO₄·2H₂O, 0.4 mg; CaCl₂·2H₂O, 4.5 mg; FeSO₄·7H₂O, 3.0 mg; H₃BO₃, 1.0 mg; KI, 0.1 mg). The pH was adjusted to 6.0 with KOH before autoclaving (121 °C, 20 min). Then 1 mL of sterile-filtered vitamin solution prepared in demineralized water was added, to a final concentration per liter of D-biotin, 0.05 mg; calcium pantothenate, 1.0 mg; nicotinic acid, 1.0 mg; myo-inositol, 25 mg; thiamine HCl, 1.0 mg; pyridoxin HCl, 1.0 mg; and para-aminobenzoic acid, 0.20 mg (Verduyn et al. 1992). The sole carbon sources used were glucose, fructose, galactose, and sucrose. They were sterilized separately and added aseptically to the medium to a final concentration of 10 or 100 g L⁻¹ (Nascimento and Fonseca 2019).

Culture conditions for physiology studies

The inoculum was prepared by transferring a loopful of cells from a YPD plate to a 250 mL Erlenmeyer flask containing 125 mL of mineral medium added of each sole carbon source (glucose, fructose, galactose, or sucrose) at 10 g L⁻¹. After 12 h growth on an orbital shaker (200 rpm) at 30 or 37 °C, cultivations started by adding a certain volume of the pre-culture to 500 mL Erlenmeyer flasks containing 250 mL of mineral medium, so that the initial cell concentration in the flask was 0.1 optical density unit at 600 nm (OD_600nm) (Biospectro sp-220) (Silva et al. 2019). All cultivations were carried out aerobic in flasks stoppered with cotton, in triplicate.

Culture conditions for the evaluation of the invertase activity

The inoculum was prepared as described in the previous section, except in Erlenmeyer flasks (50 mL flasks with 25 mL of mineral medium) and the only carbon source (glucose, or sucrose) at 10 or 100 g L⁻¹. After 12 h growth on an orbital shaker (200 rpm) at 30 or 37 °C, cultivations started by adding a certain volume of the pre-culture to 125 mL Erlenmeyer flasks containing 50 mL of mineral medium, so that the initial cell concentration in the flask was 0.1 optical density unit at 600 nm (OD_600nm) (Biospectro sp-220). All cultivations were carried out in triplicate.

Sampling and sample preparation

Samples were taken every 30 min. (maximum of 4 mL per sample) and placed in an ice bath. Out of these, 2 mL were used for optical density measurement (OD_600nm) (Biospectro sp-220), followed by adequate dilutions (when necessary) and the remaining 2 mL were centrifuged (17,609×g, 5 min., 5 °C). The supernatant was frozen at − 80 °C for further determination of the concentration of sugars and extracellular metabolites. The sedimented fraction was utilized to determine the biomass concentration (Fonseca et al. 2013).

Biomass concentration and pH

The biomass pellet obtained after sample centrifugation was dried in an oven (105 °C) until constant weight. The dried cell mass (g L⁻¹) was obtained by the quotient of the difference of weighing by the volume of the centrifuged medium. Biomass concentration (X) was also indirectly determined via OD measurements performed with a spectrophotometer (Biospectro sp-220) at 600 nm. For this purpose, the measured absorbance values were converted into mass values using a linear relationship (OD units per gram dry cell mass) determined for each experiment. The pH was obtained by potentiometric measurements (Hanna) (Nascimento and Fonseca 2019; Silva et al. 2019).

Sugars and extracellular metabolites

Glucose, fructose, galactose, glycerol, ethanol, and organic acids were separated using an UPLC Agilent 1290 with a Rezex ROA—Organic Acid H⁺ ion-exclusion column (8%) (Phenomenex). The column was eluted at 55 °C using water acidified with trifluoroacetic acid (TFA) at 0.005 M as mobile phase, at a flow rate of 0.6 mL min⁻¹. The volume injected was 20 μL, and the run was performed isocratically. These compounds were detected by a UV-absorbance detector at 254 nm connected in series with an Agilent 1260 Differential Refractometer (RID) coupled to a data acquisition module (Nascimento and Fonseca 2019). Sucrose was measured by the same procedure described previously, with changes in temperature and flow (25 °C; 0.3 mL min⁻¹) (Barbosa et al. 2018).

Kinetic parameters

The kinetic parameters were determined as described elsewhere (Fonseca et al. 2013; Nascimento and Fonseca 2019; Silva et al. 2019). The exponential growth phase (EGP) was identified as the linear region on an ln (X) vs. time plot for batch cultivation data. The maximum specific growth rate (µ_max) was determined as the slope of this linear region and the doubling time (DT) by the ln (2) quotient by µ_max. The biomass yield on the substrate (Y_X/S) was determined as the slope of the line on an X vs. S plot, exclusively including points belonging to the EGP. The specific rate of substrate consumption (r_S) was calculated by the quotient of μ_max by Y_X/S. The maximum biomass concentration (X_max) was indicated by the maximum dried cell mass concentration or OD_{600 nm} observed in each experiment. The product (ethanol, glycerol or acetic acid) yield on the substrate (Y_Eth/S; Y_Gly/S; Y_Ace/S) was determined as the slope of the line on a P vs. S plot. The maximum cell (P_Cel) and ethanol (P_Eth) productivities were obtained according to Eqs. (1) and (2), respectively:

$$P_{\text{Cel}}=\frac{\left(X_{\text{f}}-X_0\right)}{\left(t-t_0\right)}$$ 1

$$P_{\text{Eth}}=\frac{\left(P_{\text{f}}-P_0\right)}{\left(t-t_0\right)}$$ 2

where X_f is the final biomass concentration (g L⁻¹) and X₀ is the initial biomass concentration (g L⁻¹), while P_f is the final ethanol concentration (g L⁻¹) and X₀ is the initial ethanol concentration (g L⁻¹).

Enzyme extracts

Every 2 h, an Erlenmeyer flask was removed from the shaker for sampling and 20 mL of the culture medium was centrifuged at 1500×g for 5 min. The cell mass was resuspended in 5 mL of acetate buffer and centrifuged again to eliminate impurities. The procedure was repeated and after the last centrifugation, the cell mass was resuspended with 10 mL of buffer. The extracts were used for the invertase determination assays (Barbosa et al. 2018).

Invertase activity

The reaction mixture was composed of 0.9 mL of 0.1 M sodium acetate buffer with pH 5.0, containing 10 g L⁻¹ sucrose, and 0.1 mL of enzymatic solution. The mixture was incubated for 10 min at 50 °C. The enzymatic reaction was intermitted by adding 1 mL of DNS (3,5-dinitrosalicylic acid) and 8 mL of distilled water. The product (reducing sugar) was quantified by spectrophotometry (540 ηm), using the DNS method (Miller 1959). An invertase unit was defined as the amount of enzyme required to release 1 μmol of the product (glucose or fructose) per minute of reaction.

Statistical analysis

The Microsoft Excel 2010 program was utilized to calculate the analysis of variance (ANOVA). The Tukey test was used to determine differences between the variables temperature and substrate, in the 95% confidence interval.

Results and discussion

Physiological evaluation of S. cerevisiae FT858 on different substrates and temperatures

The kinetic parameters related to cell growth during cultivations with S. cerevisiae FT858 are shown in Table 1. The experiments were carried out on glucose, fructose, sucrose, and galactose, as the only carbon source (10 g L⁻¹) at 30 and 37 °C. Fig. S1 shows the growth kinetics, metabolite formation and sugar consumption of these cultivations.

Table 1.

Kinetic parameters of cultures performed with S. cerevisiae FT858 under different culture conditions

So (10 g L−1)	T (oC)	µmax (h−1)	Xmax (g L−1)	DT (h)	RS (h−1)	YX/S (gMCS g−1S)	YETH/S (gETH g−1S)	YGLY/S (gGLY g−1S)	YACE/S (gACE g−1S)	PCel (gDCM L−1 h−1)	PEth (gETH L−1 h−1)
GLC	30	0.40 ± 0.001bcA	1.70 ± 0.017cA	1.75 ± 0.006cdA	2.68 ± 0.008bcdB	0.15 ± 0.001cA	0.39 ± 0.004cB	0.03 ± 0.002bcB	0.01 ± 0.005abA	0.14 ± 0.001abA	0.40 ± 0.001cdA
	37	0.39 ± 0.016cA	1.13 ± 0.000fB	1.78 ± 0.074cA	3.84 ± 0.193aA	0.10 ± 0.002eB	0.46 ± 0.013bA	0.07 ± 0.003aA	0.00 ± 0.000abA	0.12 ± 0.000cB	0.43 ± 0.011bcA
FRU	30	0.37 ± 0.001cA	1.75 ± 0.008cA	1.85 ± 0.004cA	2.54 ± 0.143cdB	0.15 ± 0.008cA	0.39 ± 0.004cA	0.04 ± 0.001bB	0.01 ± 0.001abA	0.13 ± 0.001bA	0.35 ± 0.009dA
	37	0.37 ± 0.012cA	1.34 ± 0.009 dB	1.86 ± 0.058cA	3.96 ± 0.023aA	0.09 ± 0.002eB	0.39 ± 0.007cA	0.07 ± 0.004aA	0.00 ± 0.000abA	0.13 ± 0.006bA	0.47 ± 0.040abA
SUC	30	0.43 ± 0.001abA	1.71 ± 0.013cA	1.62 ± 0.003deA	3.13 ± 0.066abcA	0.14 ± 0.003cA	0.50 ± 0.002abA	0.03 ± 0.004bcdA	0.01 ± 0.000bA	0.14 ± 0.001aA	0.45 ± 0.008bcB
	37	0.46 ± 0.017aA	1.22 ± 0.030eB	1.50 ± 0.054eA	3.49 ± 0.574abA	0.12 ± 0.004 dB	0.52 ± 0.017aA	0.05 ± 0.011bA	0.00 ± 0.001bA	0.13 ± 0.003bcB	0.53 ± 0.020aA
GAL	30	0.28 ± 0.002 dB	1.94 ± 0.027aA	2.46 ± 0.017aA	1.43 ± 0.016eB	0.20 ± 0.004aA	0.36 ± 0.002cA	0.02 ± 0.001dA	0.01 ± 0.001aA	0.13 ± 0.001bcA	0.26 ± 0.002eB
	37	0.31 ± 0.001dA	1.86 ± 0.002bA	2.21 ± 0.007bB	1.87 ± 0.023deA	0.17 ± 0.003bB	0.39 ± 0.032cA	0.02 ± 0.001cdA	0.01 ± 0.001abB	0.13 ± 0.003bA	0.33 ± 0.001dA

GLC glucose, FRU fructose, SUC sucrose, GAL galactose, S substrate, T temperature, µ_max maximum specific growth rate, X_max maximum cell concentration, DT doubling time, r_S specific rate of substrate consumption, Y_X/S biomass yield on the substrate, Y_ETH/S ethanol yield on the substrate, Y_GLY/S glycerol yield on the substrate, Y_ACE/S acetate yield on the substrate, P_Cel maximum cell productivity, P_Eth maximum ethanol productivity, DCM dry cell mass. Equal small letters in the same column do not present a significant difference (p > 0.05). Equal capital letters for the same substrate do not show a significant difference (p > 0.05)

Comparing the maximum specific growth rates (µ_max), it is possible to observe that these values were not influenced by the increase in temperature in substrates glucose, fructose and sucrose. Moreover, the difference was not significant (p > 0.05) between cultivations with glucose or fructose. The highest µ_max was obtained with sucrose at 37 °C (0.46 ± 0.017 h⁻¹) and the lowest with galactose at 30 °C (0.28 ± 0.002 h⁻¹) (Table 1). Other studies carried out with industrial yeast strains revealed an increase in growth rates for cultivations at 37 °C (Della-Bianca and Gombert 2013; Nascimento and Fonseca 2019), which was not observed here with S. cerevisiae FT858.

S. cerevisiae FT858 presented the lowest affinity for the substrate galactose since they had the lowest µ_max values at 30 and 37 °C (0.28 ± 0.002 h⁻¹; 0.31 ± 0.001 h⁻¹, respectively) (Table 1). Close related values were reported for S. cerevisiae CAT-1 under the same culture conditions (0.28 ± 0.001 h⁻¹; 0.35 ± 0.000 h⁻¹) (Nascimento and Fonseca 2019). It occurs because the galactose catabolism by the cell involve more metabolic reactions in relation to the other studied substrates. Other factors, such as the transport system and accumulation of intermediates in the metabolic pathway, are responsible for slowing cell growth (Ostergaard et al. 2000; Ideker et al. 2001; Bro et al. 2005; Fonseca et al. 2013; Nascimento and Fonseca 2019).

Regarding the substrate consumption rate (r_S), the influence of the temperature increase is perceived in the cultures with glucose, fructose and galactose, where at higher temperatures higher r_S were obtained. The sucrose substrate did not show any significant difference (p > 0.05) (3.13 and 3.49 h⁻¹) (Table 1). The behavior of this parameter is related not only to temperature but also to the substrate transport system used by yeast.

The transport of common monosaccharides (glucose or fructose) in S. cerevisiae is carried out by the process of facilitated diffusion (Bisson et al. 2016). The increase in temperature promotes the acceleration of metabolism and the uptake of available substrates, and as a result, the increase in r_S. In Fig. S1, it is observed that the cultures carried out at 37 °C reached the stationary phase in less time. It was more evident in the cultivations with glucose and fructose.

Since the galactose catabolism pathway is more complex than the other evaluated substrates, as much as the temperature promotes the acceleration of the yeast metabolism, it becomes limited by the increase in the concentration of metabolic intermediates that inhibit the flow through the Leloir pathway (Hong et al. 2011).

Similar behavior also occurs in the cultivations with sucrose. Since the predominant mechanism of sucrose consumption in S. cerevisiae occurs after the hydrolysis of sucrose by extracellular invertase, which produces glucose and fructose that enter the cell through facilitated diffusion transporters (Basso et al. 2011). Thus, the consumption of this substrate is also limited by the catabolic repression in the expression of invertase, generated by the concentration of glucose released in the culture medium. The dynamics of this process regulates the consumption of sucrose by yeast throughout the cultivation, as can be seen in Fig. 1 (Alipourfard et al. 2019; Nascimento and Fonseca 2019).

Fig. 1.

Kinetics of substrate consumption and cell growth during cultivation of the industrial yeast S. cerevisiae FT858 in mineral medium with sucrose 10 g L⁻¹ at 30 °C. (Cross mark) X (biomass, g/L); (filled circle) S (total substrate, g L⁻¹); (filled diamond) SUC (sucrose, g L⁻¹); (filled triangle) FRU (fructose, g L⁻¹); (filled square) GLC (glucose, g L⁻¹)

The products formed with the most significant concentrations during the cultivations were ethanol, glycerol, and acetate (Fig. S1; Table 1). The product yield on the substrate (Y_P/S) were calculated for ethanol (Y_ETH/S), glycerol (Y_GLY/S) and acetate (Y_ACE/S) (Table 1).

With the increase in temperature, only the cultivations with glucose and fructose showed a significant difference (p > 0.05) for the Y_GLY/S values. For all evaluated substrates, S. cerevisiae FT858 showed lower Y_GLY/S values when compared to those obtained with the S. cerevisiae CAT-1 (Nascimento and Fonseca 2019).

S. cerevisiae CAT-1 and other yeast strains have the characteristic of producing less glycerol because of its great resistance at some adverse conditions found in the process (Basso et al. 2008). A major concern during industrial fermentation processes is that the cells convert the sugar consumed into the production of glycerol, which is related to the stress caused to the cells during fermentation (Basso et al. 2011; Borges et al. 2015). In this context, S. cerevisiae FT858 is more efficient than S. cerevisiae CAT-1 because it presents lower Y_GLY/S values.

The highest Y_ETH/S values were achieved with sucrose at both temperatures (average of 0.51 g_ETH/g_substrate) (Table 1). Only for the glucose substrate Y_ETH/S showed a significant difference (p < 0.05) between 30 and 37 °C. The substrates fructose and galactose, at both temperatures, and glucose at 30 °C, the difference was not significant (p > 0.05) between them. The Y_ETH/S obtained in this study for all tested substrates were higher than those reported in the literature for S. cerevisiae CAT-1 (Nascimento and Fonseca 2019). It is important to underline that a 10% increase in Y_ETH/S was obtained when sucrose was used as substrate (Y_ETH/S = 0.53 g_ETH g⁻¹_substrate for S. cerevisiae FT858 against Y_ETH/S = 0.44 g_ETH g⁻¹_substrate reported for S. cerevisiae CAT-1) at 37 °C. The ethanol productivity (P_Eth) was higher for the S. cerevisiae FT858 cultivations with fructose and sucrose at 37 °C (0.47 ± 0.040 and 0.53 ± 0.020 g_ETH L⁻¹ h⁻¹, respectively).

Influence of the substrate concentration on the invertase production by Saccharomyces cerevisiae FT858 and CAT-1

For a better evaluation of the biotechnological potential of S. cerevisiae FT858, the activity of the invertase enzyme produced in cultures with glucose or sucrose as the only carbon source, at low (10 g L⁻¹) and high (100 g L⁻¹) concentrations was investigated. Results obtained with S. cerevisiae CAT-1 were utilized as a comparative parameter.

Growth kinetics, metabolite formation and substrate consumption were obtained for S. cerevisiae FT858 and CAT-1 during cultivations with 100 g L⁻¹ carbon source (Fig. S2). The kinetic growth parameters of these cultivations were compared with those obtained here with 10 g L⁻¹ carbon source for S. cerevisiae FT858 and the data reported in the literature (Nascimento and Fonseca 2019) for S. cerevisiae CAT-1 at the same carbon source concentration (Table 2).

Table 2.

Comparison of kinetic growth parameters of cultivations with S. cerevisiae FT858 and CAT-1 in high and low substrate concentration

So	Strain	[] (g L−1)	µmax (h−1)	Xmax (g L−1)	DT (h)	PCEL (gDCM L−1 h−1)	Sfinal residual (g L−1)	Reference
GLC	FT858	10	0.40 ± 0.001c	1.70 ± 0.017d	1.75 ± 0.006c	0.14 ± 0.001b	0c	This work
		100	0.35 ± 0.002e	2.88 ± 0.028a	1.95 ± 0.009a	0.12 ± 0.001c	39.8 ± 2.522b	This work
	CAT-1	10	0.44 ± 0.007ab	2.00 ± 0.012e	1.58 ± 0.025de	0.08 ± 0.000a	0c	Nascimento and Fonseca (2019)
		100	0.41 ± 0.001c	2.37 ± 0.032b	1.71 ± 0.004c	0.10 ± 0.001d	50.5 ± 1.784a	This work
SUC	FT858	10	0.43 ± 0.001b	1.71 ± 0.013d	1.62 ± 0.003d	0.14 ± 0.001ab	0c	This work
		100	0.37 ± 0.000de	2.29 ± 0.012c	1.87 ± 0.001b	0.09 ± 0.001d	39.3 ± 1.412b	This work
	CAT-1	10	0.42 ± 0.014a	2.00 ± 0.030d	1.64 ± 0.055e	0.08 ± 0.001b	0c	Nascimento and Fonseca (2019)
		100	0.38 ± 0.010d	2.85 ± 0.007a	1.86 ± 0.052b	0.12 ± 0.000c	54.5 ± 3.634a	This work

GLC glucose, SUC sucrose, S substrate, [] substrate concentration, µ_max maximum specific growth rate, X_max maximum cell concentration, DT doubling time, P_Cel maximum cell productivities, DCM dry cell mass, equal letters in the same column there is no significant difference (p > 0.05)

Cultivations were carried out up to 24 h. As only the concentration of the carbon source was changed, while the concentrations of the other relevant components of the mineral medium (e.g. nitrogen source) were maintained the same, the conditions were not sufficient to sustain the cell growth until the total consumption of the carbon source at the high concentration (Table 2; Fig. S2). Therefore, for these experiments, only the growth parameters were calculated.

As S. cerevisiae FT858 was isolated from an ethanol industrial plant, its fermentative capacity is strongly related to the invertase enzyme activity since the cleavage of sucrose into glucose and fructose is the fundamental step for the sucrose metabolism by the cells (Basso et al. 2008; Marques et al. 2016).

Table 2 shows that the strains showed very different behaviors in relation to the production of biomass in cultivations with 100 g L⁻¹ substrate. With the glucose substrate, S. cerevisiae CAT-1 presented a lower X_max (2.37 g L⁻¹) and a higher µ_max (0.41 h⁻¹) compared with S. cerevisiae FT858 (X_max = 2.88 g L⁻¹; µ_max = 0.35 h⁻¹). In sucrose, the opposite was observed in relation to X_max. For S. cerevisiae CAT-1 it reached 2.85 g L⁻¹, which was superior to the 2.29 g L⁻¹ found for S. cerevisiae FT858. The µ_max were very close (0.38 h⁻¹ for S. cerevisiae CAT-1 and 0.37 h⁻¹ for S. cerevisiae FT858) (Table 2).

Production of invertase by S. cerevisiae FT858 and CAT-1 was evaluated from glucose and sucrose to investigate the effect of the concentration of these substrates on the production of this enzyme (Fig. 2).

Fig. 2.

Invertase production by the industrial yeasts S. cerevisiae CAT-1 and FT858 during cultures in mineral medium with 10 g L⁻¹ and 100 g L⁻¹ substrate (a glucose 10 g L⁻¹, b sucrose 10 g L⁻¹, c glucose 100 g L⁻¹, d sucrose 100 g L⁻¹). Blue bars correspond to S. cerevisiae CAT-1 and red bars correspond to S. cerevisiae FT858

It was observed that, at the concentration of 10 g L⁻¹ (Fig. 2a, b), the yeasts only presented considerable enzymatic activity after 8 h of cultivation. For glucose, the greatest activity was observed with 12 h for CAT-1 (20.4 ± 0.15 U mL⁻¹) and with 10 h for S. cerevisiae FT858 (12.3 ± 1.06 U mL⁻¹). For sucrose, the greatest activities were observed in 10 h of culture for both strains (S. cerevisiae CAT-1 = 39.4 ± 3.93 U mL⁻¹; S. cerevisiae FT858 = 49.6 ± 0.11 U mL⁻¹). From this time on, a considerable reduction in activity was observed but maintained stable between 20 and 30 U mL⁻¹ up to 24 h of cultivation.

In the cultures with 100 g L⁻¹ of the carbon sources (Fig. 2c,d), the two strains showed a very low invertase activity. There was no invertase activity for S. cerevisiae FT858 in glucose. On the other hand, S. cerevisiae CAT-1 presented little activity after 12 h of cultivation. In sucrose, there was a greater production of invertase. However, still at very low levels. S. cerevisiae FT858 showed higher activity compared to S. cerevisiae CAT-1 (5.62 ± 0.03 and 4.81 ± 0.20 U mL⁻¹, respectively). In a previous study with S. cerevisiae FT858, substrate concentration affected the fermentative behavior of the yeast, leading to a greater loss of viability and ethanol concentration (Santos et al. 2018).

The invertase activity was measured in whole cells. Therefore, only periplasmic and regulated invertases were considered, where the SUC2 gene is glucose repressed at a concentration equal or superior to 20 g L⁻¹ and induced at a concentration below to 2 g L⁻¹ (Gancedo et al. 2015). Thus, in both substrates at 10 g L⁻¹, the enzymatic activity was more evident when the cultivations tend to reach the stationary phase, i.e., when the carbon source is practically exhausted from the medium. For the S. cerevisiae FT858 cultivations started with 10 g L⁻¹ glucose, at 8 h of growth, the concentration of glucose was 4 g L⁻¹ while at 10 h it dropped to approximately 0.1 g L⁻¹. This indicates that only after the decrease in glucose concentration the expression of the SUC2 gene is induced.

The gene repression generated by the glucose concentration was also observed in cultures with 10 g L⁻¹ sucrose. It occurred because the levels of glucose/fructose in the medium increased over time, regulating the production of invertase by the yeast. At 6 h of cultivation with S. cerevisiae FT858, the glucose concentration was 2.6 g L⁻¹, while at 8 h it was 0.4 g L⁻¹. At 10 h, time that the highest Invertase activity was observed, all sugars had already been consumed.

It has been reported that in S. cerevisiae when glucose is not abundant or even absent in the medium, SUC2 expression occurs at a baseline level. In an environment rich in sucrose, the basal level of the invertase generates a glucose/fructose pool around the cells, causing the maximum expression of SUC2. In addition to this sugar pool, changes in concentration throughout the cultivation were also related to gene expression. When the glucose/fructose concentration accumulates above a limit, SUC2 is repressed, leading to the consumption of the hexoses already available. This invertase expression dynamics, through the glucose balance, is what optimizes the sucrose consumption by the yeast (Marques et al. 2016).

Enzyme activity values above 1 U mL⁻¹ were found considerable. Thus, for the cultivations with the carbon source at 100 g L⁻¹ (Fig. 2c, d) it can be considered that there was a subtle invertase activity for S. cerevisiae CAT-1 in glucose, and for both strains in sucrose.

The hydrolytic activity of invertases occurs predominantly in sucrose concentrations of 50 g L⁻¹ or less (Barbosa et al. 2018). Moreover, for both strains, the invertase activity became more evident after 12 h, when the cultures approached the concentration of 50 g L⁻¹ (Table 3). Due to the osmotic stress, to which the yeasts were exposed, there was a great repression of invertase expression in these cultivations due to the increase in the concentrations of glucose/fructose in the medium (Table 3).

Table 3.

Dynamics of sugar concentration of cultivations (100 g L⁻¹) after sucrose hydrolysis

Time (h)	S. cerevisiae FT858			S. cerevisiae CAT-1
	GLC (g L−1)	FRU (g L−1)	SUC (g L−1)	GLC (g L−1)	FRU (g L−1)	SUC (g L−1)
8	15.0 ± 0.106	15.6 ± 0.198	55.6 ± 0.573	9.1 ± 0.099	13.8 ± 0.276	70.9 ± 0.014
10	14.7 ± 0.042	15.0 ± 0.064	52.0 ± 0.672	13.6 ± 0.594	13.8 ± 0.368	61.1 ± 1.386
12	14.7 ± 0.170	13.7 ± 0.099	44.9 ± 0.990	19.9 ± 0.396	19.8 ± 0.410	44.0 ± 0.361
14	14.6 ± 0.127	14.4 ± 0.085	37.5 ± 0.573	18.7 ± 0.339	13.1 ± 0.297	40.4 ± 0.269
24	1.3.0 ± 0.113	20.9 ± 0.000	5.3 ± 0.028	12.8 ± 0.057	18.7 ± 0.127	22.9 ± 0.184

GLC glucose, FRU fructose, SUC sucrose

There are indications that the invertase produced by S. cerevisiae CAT-1 is of a constitutive nature. It is suggested because the invertase activity was still evident in an extremely rich medium, with glucose as the sole carbon source, which was not observed for S. cerevisiae FT858 (Fig. S2).

Another aspect observed was the different dynamic behavior of the sucrose catalysis (Table 3). In the cultivation times that showed higher invertase activity (12, 14 and 24 h), the glucose concentration for S. cerevisiae FT858 remained regulated and close to 15 g L⁻¹. For S. cerevisiae CAT-1, this concentration fluctuated considerably, reaching values very close to 20 g L⁻¹, which would suppress the expression of SUC2 (Gancedo et al. 2015). After 14 h of cultivation S. cerevisiae FT858 increased invertase activity in relation to S. cerevisiae CAT-1.

There are several signaling pathways involved in the response to nutrients in Saccharomyces spp. The Rgt1 network also participates in controlling the expression of hexose transporter genes according to the level of glucose availability, so that cells express only the transporters with the greatest affinity for available glucose (Zaman et al. 2008). Gancedo et al. (2015), in their study of SUC2 expression in S. cerevisiae observed that both Rgt1 and Mth1 play an important role in blocking invertase transcription in the absence of glucose, suggesting that the main mechanism is the binding of the Rgt1 complex -Mth1 into an Rgt1 binding site on the SUC2 promoter.

Conclusion

It was concluded that the culture conditions directly interfered in the yeast physiology. S. cerevisiae FT858 has an outstanding biotechnological potential since the kinetic growth parameters observed were very close to those obtained with S. cerevisiae CAT-1 under similar cultivation conditions. It underlines the low glycerol yield on substrate (Y_GLY/S) (even at 37 °C) and a 10% increase in the yield of sucrose into to ethanol obtained with S. cerevisiae FT858 when compared to S. cerevisiae CAT-1 at 37 °C. The concentration of the substrates directly interfered in the production of invertase for both S. cerevisiae FT858 and CAT-1. The enzymatic expression underwent strong regulation through the concentration of glucose in the culture medium. S. cerevisiae CAT-1 presented constitutive behavior for the invertase enzyme.

Electronic supplementary material

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Author contributions

VMN: conceptualization, methodology, investigation, writing—original draft. GTUA: investigation. RSRL: methodology, visualization, formal analysis. GGF: project administration, supervision, visualization, writing—original draft, review & editing.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.