Kinetic and Energetic Parameters of Carob Wastes Fermentation by Saccharomyces cerevisiae: Crabtree Effect, Ethanol Toxicity, and Invertase Repression.

Carob waste is a useful raw material for the second-generation ethanol because 50% of its dry weight is sucrose, glucose, and fructose. To optimize the process, we have studied the influence of the initial concentration of sugars on the fermentation performance of Saccharomyces cerevisiae. With initial sugar concentrations (S0) of 20 g/l, the yeasts were derepressed and the ethanol produced during the exponential phase was consumed in a diauxic phase. The rate of ethanol consumption decreased with increasing S0 and disappeared at 250 g/l when the Crabtree effect was complete and almost all the sugar consumed was transformed into ethanol with a yield factor of 0.42 g/g. Sucrose hydrolysis was delayed at high S0 because of glucose repression of invertase synthesis, which was triggered at concentrations above 40 g/l. At S0 higher than 250 g/l, even when glucose had been exhausted, sucrose was hydrolyzed very slowly, probably due to an inhibition at this low water activity. Although with lower metabolic rates and longer times of fermentation, 250 g/l is considered the optimal initial concentration because it avoids the diauxic consumption of ethanol and maintains enough invertase activity to consume all the sucrose, and also avoids the inhibitions due to lower water activities at higher S0.


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One of the challenges of this century is the progressive shift from fossil energy to 52 renewable fuels. Biofuels are one of the solutions to the continuous rising of oil prices, 53 exhaustion of fossil sources, greenhouse gas emissions reduction and dependence of the 54 Middle East volatile politics. The requirements of the Kyoto Protocol and Bali Action Plan 55 encouraged the search for renewable feedstock, as sources for biofuels. Fermentation 56 processes stand out in bioethanol production since they transform simple raw materials into 57 products with aggregated value [4]. The answer to these problems could be found in second 58 generation bioethanol produced by agro-industrial residues, since its use does not compete 59 with food resources; it also allows the exploitation of raw materials with low commercial 60 value and arranges an alternative to their disposal. A wide variety of these raw materials are 61 used as carbon sources for bioethanol production, namely, sugarcane molasses, beet molasses, 62 pineapple, orange and sweet lime residues or carob industrial wastes [4,12,8,6].  The carob pod pulp exhibits a sugar content higher than sugar cane and the analysis of 67 some Turkish carob varieties showed that the most abundant sugar is sucrose with 29.9-38.4 % 68 (w/w), followed by fructose with 10.2-11.5 % (w/w) and the less abundant is glucose with  The high-level of sugar content combined with low prices makes the carob-based 71 nutrient medium an advantageous alternative to carbon sources for ethanol production. 72 Many research groups developed intensive studies to obtain efficient fermentative 73 organisms, low-cost substrates and optimal conditions for fermentation [6,17]. The persistent 74 search for different low-cost carbon sources brings as a consequence, a large variability of 75 complex polysaccharides and increases the need of understanding the hydrolysis processes 76 and how the resultant sugars are metabolized and converted in ethanol. 77 To accomplish a high ethanol yield and increased productivity the optimal 78 fermentation conditions have been subjected to substantial improvements like the integration 79 of very high-gravity (VHG) technology, by using heavily concentrated substrate. However, 80 several problems are associated to VHG technology. One of these is the incomplete induced and will result in the preferential consumption of glucose over the other carbon 92 sources [11].

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Molecular transport is a determining factor of cellular metabolism, mainly when the 94 carbon source is not the preferential one, as in the fructose and sucrose case in Saccharomyces 95 cerevisiae. Glucose and fructose use the same facilitated diffusion system but glucose has a 96 prevailing affinity, inhibiting competitively fructose transport. Invertase hydrolysis should 97 balance the monosaccharides' supply of the medium and their yeast consumption, in a way 98 that the medium osmolality remains at a minimum value during the fermentation [16]. It was 99 also shown, in the same work that regulation of the invertase activity could result in a more 100 efficient alcoholic fermentation. The glucose in carob residue substrate, at a concentration 101 above at threshold value, represses invertase synthesis and sucrose hydrolysis does not occur 102 until the glucose concentration reaches values below the threshold [6].

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In general, the lower affinity of hexose transporters for fructose, when comparing to 104 glucose, explains the residual fructose prevalence at the end of fermentation. However, the 105 role of sugar transport systems in efficient fermentation processes remains unsolved [14]. 106 In this work, carob waste fermentations with low and high initial sugar concentrations 107 were performed and the kinetic and energetic parameters of cell growth, as well as the 108 consumption rates of glucose, fructose and sucrose hydrolysis were calculated in each of the 109 media with different initial sugar concentrations.

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The establishment of the best technological conditions to achieve the highest ethanol 111 productivities and yields for 2 nd generation biofuel production, using carob industrial wastes 112 as raw-material, was a major goal of the present work. In order to identify the factors that 113 limit the fermentation efficiency, Crabtree effect, invertase repression and ethanol toxicity 114 were studied in the present work, using kinetics and energetic approaches.  The strain was maintained on solid YEPD medium (peptone 20 g/l, yeast extract 10 g/l, 123 glucose 20 g/l, agar 15 g/l). Inocula were made in 250 ml shake flasks, containing 50 ml of 124 liquid YEP medium (yeast extract 5 g/l, peptone 10 g/l) supplemented with carob extract.

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The cultures were incubated in an orbital shaker (NeifoPentlab, Portugal), at 150 rpm and 126 30ºC, until it reach late exponential growth phase. These cultures were used as inocula to get 127 a initial cell concentrations of about 1 x 10 7 cells/ml.   (Table 1). Between 20 and 100 g/l, transitions between the exponential and the 209 stationary phases were very abrupt and correspond to the exhaustion of sugar in the culture 210 ( Fig. 1, A, B and C). At the highest tested concentrations (250 to 350 g/l), the sugars were no 211 longer the limiting factor when the stationary phase was reached, because at this point there 212 were still sugars available in the medium (Fig. 1, F, G and H). In these cases, we submit that 213 ethanol was the factor limiting growth. At concentrations higher than 250 g/l the cells were 214 not even able to consume all the sugars added and, consequently, the final biomass decreased.

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In relation to specific growth rates (µ), longer exponential growth phases were found with 216 increased sugar concentrations (Fig. 1), but with decreasing µ values (Table 1). Several 217 physiological mechanisms, underlying this decrease, were identified. The initial sugar 218 concentration affected the biomass yield factor, i.e., less biomass was produced per gram of 219 sugar consumed, due probably to the osmotic stress (Table 1). 220 The energetic efficiency of the sugar catabolism affected also the value of µ. During 221 the first 3 hours of culture, for S 0 below 150 g/l, there was sugar consumption but no ethanol 222 was produced (Fig. 1), which indicates that oxygen was available and the catabolism was 223 completely oxidative. After that time ethanol begun to be produced and the catabolism was 224 progressively fermentative. This change in the efficiency of energy metabolism determined 225 the decrease in the yield factor biomass/sugar from 0.13 to 0.02 g/g (Table 1) Table 1. In this table, a positive sign was added to q E when there was net 237 production of ethanol, and a negative one, when there was net consumption. As mentioned 238 before, ethanol was always produced during the growth phase, at any initial sugar 239 concentration. From 20 to 100 g/l of initial total sugar an increase of q E , from 0.4 to 1.0 g of 240 ethanol per g of biomass per hour was observed. That was the maximal production rate 241 reached because at higher initial sugar concentrations the rate decreased ( Table 1). The  This Crabtree effect hypothesis was supported by the yeasts behavior because, when all the 246 glucose had been consumed, cytochromes synthesis was derepressed, and ethanol was in fact 247 oxidized. At 20 g/l of initial sugar, after glucose exhaustion, the yeasts were completely 248 derepressed and were able to perform a diauxic growth, consuming all the ethanol that has 249 been produced (Fig. 1A). A similar pattern could be observed at 50 up to 200 g/l of initial 250 sugars, but with an apparent lower derepression, as measured by the specific rates of ethanol 251 consumption, that decreased from 26 mg of ethanol per gram of biomass per hour at an initial 252 glucose concentration of 6.1 g/l ( Fig. 2A) to 10 mg of ethanol per gram of biomass per hour at 253 an initial glucose concentration of 44.8 g/l. (Fig. 2C, Table1). Apparently, this low 254 consumption did not provide energy enough to synthesize new biomass and, although ethanol 255 consumption could be measured, no increase in biomass could be detected (Fig. 1 B, C, D Table 1). It may be argued that, once glucose was exhausted, derepression should take place was consumed during the stationary phase ( Fig. 1 and Table 1). On the contrary, at these high 263 concentrations the alcohol continued to be produced by the metabolically uncoupled cells, 264 unable to grow but yet able to ferment (Table 1). However, the specific ethanol production 265 rates were much lower (0.11 to 0.13 g/g.h) than those of the exponential phase (0.67 to 266 0.84g/g.h) (   Table 2). Up to 200 g/l total sugar, all the ethanol was produced during the 271 exponential phase. However, at these low sugar concentrations, after glucose exhaustion, 272 ethanol was completely consumed when S 0 was 20 g/l and in significant amounts at 50 and 273 274 not consumed but produced by the uncoupled stationary cells, in an amount similar or even 275 higher than that produced by the exponential cells (Table 1 and 2). It must be remarked that at  It can be observed that sucrose concentration decreased immediately after inoculation 291 at the lowest initial sugar concentration (20 g/l), indicating that active invertase was present.

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The invertase activity was so high in this condition that the concentration of glucose in the 293 culture increased, because its production by sucrose hydrolysis was higher that its 294 consumption by the cells (see Fig. 2A). This immediate sucrose hydrolysis was also present at 295 100 g/l fermentation, although at a lower rate, as shown by the rate of sucrose disappearance 296 and by the fact that the concentration of glucose did not increase. However, at fermentations 297 performed at higher S 0 (see the case of 250 g/l in Fig. 2C) sucrose was not immediately 298 hydrolyzed and only when glucose had been consumed, sucrose hydrolysis showed a high rate. 299 Anyway, at this sugar concentration the invertase activity was enough to hydrolyze all the 300 added sucrose (Fig. 2C). At even higher S 0 concentrations, 350g/l (Fig. 2D), it was observed 301 that invertase activity increased very slowly, even when glucose concentration was very low, 302 indicating that, although derepression may have taken place, either the enzyme was not being 303 synthetized, due to the action of the accumulated ethanol (about 40 g/l) and/or its activity was 304 being inhibited by the high osmolality of the medium (water activity, a w of 0.964) value [3,6].

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This dependence of invertase activity on the water activity of the culture has been previously 306 reported [17]. In any case, with S 0 equal to 350 g/l the invertase activity was so low that 307 sucrose was not completely hydrolyzed and 52 % of the initial sucrose concentration 308 remained in the culture, even after 96 hours of fermentation.

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As mentioned, invertase activity could be quantified as the specific rate of sucrose 310 hydrolysis, calculated as described in Material and Methods, and this rate was taken as a 311 indirect measure of the amount of enzyme synthesized. When these rates were related with the 312 corresponding glucose concentrations in the culture, as depicted in Fig. 3, it could be 313 observed that, whatever is the initial concentration of total sugar in medium, invertase 314 synthesis seems to be repressed at glucose concentrations higher than 40 g/l (Fig. 3).  318 Taking in consideration the data of Table 2 it can be concluded that, from all the sugar 319 concentrations assayed, 250 g/l is the best concentration to be used in industrial processes for 320 ethanol production from carob wastes sugars. At this sugar concentration the diauxic behavior 321 is not present at all and ethanol is not consumed during the stationary phase (Fig. 1F).

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Crabtree effect may be at its maximum, glucose respiration is almost completely repressed 323 and, therefore, catabolism is completely fermentative, with an ethanol/sugar yield of 0.42 g/g, 324 near the maximum (Table 2). Working at this S 0 concentration had another advantage, ethanol 325 is produced by both cells, exponential (82 %) and stationary (18 %). Although invertase is 326 initially repressed, the complete glucose consumption enables its derepression. The 327 determined water activity (a w ) of 0.964 is not low enough to inhibit strongly the hydrolysis of 328 sucrose, as happens at higher concentrations, and sucrose can be completely consumed. At 329 this optimal concentration of 250 g/l, the metabolic rates are slower than at lower S 0 values, 330 due to physiological reasons that have been analyzed above, and the ethanol productivity, 331 although not the highest, is close to the maximal obtained (Table 2). Another remarkable 332 advantage is the high final concentration of ethanol attained, close to 100 g/l in these assayed           Gro wt h p a ra me t e rs a t e xp o n e n t ia l p h a s e S0 (g/l) µ A C C E P T E D Fig. 3