Once a hydrolysate with a high content of sugars and a low level of microbial inhibitors is obtained, this medium may be inoculated with microorganisms to produce ethanol, xylitol or other metabolites. Most of the industrial ethanol is nowadays produced by fermentation of carbohydrates. This process is influenced by many variables. First, the microorganism used. The most commonly microorganisms used for industrial production of ethanol are Saccharomyces yeasts as they quickly ferment hexoses into the bioproduct and they have high ethanol tolerance. However, these yeasts do not metabolize pentoses, thus limiting their application to hydrolysates rich in D-xylose. On the other hand, yeasts capable of assimilating pentoses are known, such as Pachysolen tannophilus, Candida tropicalis, Candida shehatae, Candida parapsilosis, Pichia stipitis, Candida guilliermondii, Kluyveromyces marxianus, Kluyveromyces fragilis and Debaryomyces hansenii. Nevertheless, it should be noted that some of them provide mainly ethanol, others generate xylitol while some yeasts synthesize products or mixtures thereof. P. stipitis and C. shehatae have been reported as the most suitable yeasts for the transformation of D-xylose to ethanol. Roberto et al. (1991) analyzed the performance of four microorganisms (P. stipitis, P. tannophilus, C. utilis, and C. tropicalis) on a lignocellulosic hydrolysate with 20 kg/m3 of D-xylose obtained by subjecting sugar cane bagasse to SE pretreatment (190°C-5 min) with acid impregnation . Fermentation with P. tannophilus NRRL 2460 achieved ethanol concentrations lower than 3 kg/m3, whereas with the yeast P. stipitis CBS 5773 the ethanol concentration reached 9 kg/m3. Furthermore, Toivola et al. (1984) investigated the capacity of 200 types of yeast to ferment D-xylose to ethanol, and found that C. shehatae, P. stipitis and P. tannophilus were among the six species capable of generating ethanol with yields above 5% . Moreover, while the first two yeasts (C. shehatae and P. stipitis) generated ethanol concentrations between 5.9 and 6.6 g/L (starting from 20 g/L D-xylose) the third solely produced 2.1 g/L ethanol.
Initial inoculum concentration also affects the course of fermentations. This variable should be set according to the substrate concentration and, especially, to microbial inhibitors . With respect to the substrate concentration, it is desired to be as high as possible (as far as it does not affect the microorganism) to further reduce bioproduct separation costs. Concentrations of D-xylose higher than 100 kg/m3 are not desirable for fermentation with P. stipitis , although Roberto et al. (1991) obtained 30 kg/m3 ethanol from 145 kg/m3 D- xylose .
Numerous compounds have been described that inhibit yeast fermentation. Their influence on a given microorganism depends on the specie to be inoculated, the amount of biomass introduced, and concentrations of inhibitors and other components of the culture medium. Synergetic effects among species are important in explaining inhibitions. HLW and SE pretreatment can produce toxic compounds as depicted in Figure 4.
Numerous articles highlight the inhibitor effect of acetic acid. The toxic character of the acid is more pronounced the lower the pH of the culture . It has been reported that furfural and 5-hydroxy-methyl-furfural are not critical microbial inhibitors because of their relatively low concentrations in hydrolysates. What is more, increasing the amount of inoculum is an effective way of reducing the inhibiting effect of these compounds. With respect to lignin phenolic derivatives, the most toxic are the less substituted acids since their lipophilic character favors its transport into the cell thereby causing the subsequent release of protons.
Figure 4. Products and by-products obtained during the acid hydrolysis of lignocellulosic biomass [4, 106].
The biochemical use of biomass to ethanol can be performed using sequential hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The latter could bring about higher yields and lower ethanol production costs (by reducing investment) also avoiding the enzyme inhibition by D-glucose [108, 109]. Another advantage of SSF schemes is the resistance to microbial contamination. However, thermotolerant yeasts (Kluyveromyces marxianus, Kluyveromyces fragilis, Candida acidothermophilum, etc.) are required for SSF. These yeasts must be capable to work at sufficiently high temperatures (even above 40°C), close to the optimum temperatures of enzyme functions.
Scarce literature deals with the fermentation of sugars from olive endocarps to produce ethanol or xylitol. Ballesteros et al. (2001) focused on the utilization of cellulosic fraction of this biomass . After a SE pretreatment step, the resulting solid was subjected to simultaneous saccharification and fermentation (SSF) using a thermally acclimated yeast (K. marxianus CECT 10895) capable of fermenting D-glucose. The highest yield of ethanol (58.8%) was achieved by pretreating (steam explosion, 210°C-4 min) acid-impregnated olive stones. The above conditions resulted in a culture medium with a concentration of 12.9 g/L ethanol. The presence of lignin in the pretreated solid could explain the low ethanol yield. To solve this problem, Cuevas et al. (2015) tested Organosolv pretreatments using mixtures of ethanol and water with or without the presence of acid catalyst (H2SO4) . The pretreated solid was composed of 83.3% cellulose and 17% lignin. The SSF of this solid with the thermotolerant yeast S. cerevisiae IR2-9a completely hydrolyzed the cellulose fraction, and the final ethanol concentration was greater than 45 g/L.
Regarding sequential saccharification and fermentation (SHF) schemes, Cuevas et al. (2009) applied them for ethanol and xylitol production after LHW pretreatment of olive stones . The variation in the severity of pretreatment (Log R0 between 3.23 and 4.39) had no significant effects on overall yields of ethanol, but affected those of xylitol. The highest yield was 0.25 g ethanol/g sugar.
The SHF scheme was also assayed by Saleh et al. (2014) with olive stones pretreated with dilute sulfuric acid (195°C-5 min-0.025 M), . After pretreatment, the prehydrolyzed liquid (rich in D-xylose) was separated from the solid, and this was subjected to enzymatic hydrolysis. Subsequently both hydrolysates were fermented separately with P. tannophilus ATCC 32 691. As a whole, 9.2 g xylitol and 10.3 g ethanol were produced from 100 g of olive stones.