Growing environmental concerns and increasing petroleum products prices have recently stimulated interest in optimazing fermentation processes for large-scale production of alternative fuels such as bioethanol (Wyman, 2001). The yeast “Saccharomyces cerevisiae” is the major industrial ethanol producer, because it is generally acknowledged as a safe microorganism that can produce ethanol by fermentation up to 20% (v/v) from carbon sources (Cot et al., 2007). Typically, ethanol fermentation is carried out under Normal Gravity (NG) technology, which is defined as the preparation and fermentation of mashes containing 20-24% of dissolved solids (Thomas et al., 1996).  

Ethanol can be produced from many different raw materials such as corn grain (in USA), sugarcane (in Brazil), tapioca and molasses (in Thailand) (Khongsay et al., 2010). In Togo, ethanol is commonly produced in artisanal way by using palm wine, corn, sorghum or millet as raw materials for local beer brewing. Ethanol is also produced in industrial scale with sugarcane molasses and cassava chips at Anié (Togo) by a Chinese company called “Sinto’’. The Actual challenge is to use less agricultural value products/residues or wastes which are not competing with common food production.

Therefore more interest are devoted to ethanol production by developing and implementing advanced process technologies (Mielenz, 2001). Thus, wild fruits from different plants with potentially higher level of sugars can be used as an alternative low-cost feedstock (Mazmanci, 2011).

In this study, bioethanol production from two types of local fruits (Opilia amentacea and Curcubita pepo) is compared to sucrose fermentation using four strains of S. cerevisiae. The cells growth and effects on main fermentation kinetic parameters were investigated.

 

The fruits juices biochemical composition

The characteristics of raw juices extracted from 1000 g of each fruit are shown on Table 1. To extract juices of identical initial concentration of 20.0 ± 0.1 Bx, the volume of water used varied from one fruit to another. O. amentacea juice appeared on Table 1, as the richest substratum in total sugars, with a concentration of 122.7 ± 2.3 g/L corresponding to a quantity of total sugars of 147.2 ± 2.8 g/kg of fruit while the juice of C. pepo could be distinguished like the weakest in total sugar, with its concentration of 101.1 ± 2.4 g/L in total sugars, equivalent to a quantity of 111.2 ± 2.6 g/kg of fruit. The quantities of total sugars in the fruit juices were low by comparison to sucrose syrup (230 g/L) with the same initial concentration of 20.0 ± 0.1°Bx. 

 

 

With a concentration of 114.1 ± 2.7 g/L, or a quantity of 136.9 ± 3.2 g/kg of fruit, O. amentacea was also the fruit which had the most elevated content in reducing sugars. The one of C. pepo was only of 56.9 ± 3.0 g/L, or a quantity of 62.6 ± 3.3 g/kg of fruit. But, about the sucrose content, C. pepo was the best, with a concentration of 42.0 ± 0.6 g/L, or a quantity of 46.2 ± 0.7 g/kg of fruit, against a concentration of 8.2 ± 0.4 g/L and quantity of 8.2 ± 0.4 g/kg of fruit, for O. amentacea. Probably, with these contents in sugars presented in this study, O. amentacea would be the most favorable substratum to the alcohol production. Therefore, the same total soluble solids does not mean the same reducing sugar contents of which different juices should be stated.

The pHs of the fruit juices were 5.35 ± 0.08 and 3.25 ± 0.06, respectively for C. pepo and O. amentacea. These values could contribute to a better conservation of juices, then for good alcohol production.  Indeed, it had been proven that, in fermentation broth, an acidic pH allowed to avoiding contaminations or the bad reactions, due to the presence of other micro-organisms, such as the bacteria (Ban et al., 1988).

The two phytochemical tests revealed that O. amentacea juice contained more phenolic compounds (Polyphenols and flavonoïds) in relation to the one of C. pepo. However, phenolic compounds were considered as poisons to yeasts growth, so they could have negative impacts for alcohol production (Almeida et al., 2007).

Evolutions of brix and yeasts population during fermentation period

The curves of Figures 1 and 2 showed the profiles of Brix and the number of the living and dead yeast cells variations, during the fruit juices fermentation. After the inoculation, there is a continuous dropping of the Brix for each strain. However, the dropping of the Brix show three phases: a lag phase at the beginning, followed by an acceleration phase, and then finishing by the deceleration phase. It is known that the Brix reduction is favorable to ethanol pro-duction under anaerobic conditions.

 

 

 

The maximum viable cell numbers during the fermentation

The yeasts proliferation varied from one medium to another (Table 2). The values of the maximal viable cells number of every strain were significantly different (P ≤ 5.0%) in the enriched media as well as in the non-enriched media. Among the three different media investigated, C. pepo juice showed the best yeasts cells growth. So, the maximal viable cells number is obtained in the non-enriched medium with S2 (14.30 ± 0.05 × 10⁷0.02 × 10⁷/mL cells). But O. amentacea juice is the medium in which yeasts cells growth was the poorest. 

 

 

 

The final P_exp values from sucrose media are two or three times higher than those of fruit juices. The maximum P_exp values with enriching approach are 50.60 ± 0.57 g/L (equivalent to 60.72 ± 0.68 g ethanol/kg of fruit) for O. amentacea fruits using S4 (Table 5) and 46.3 ± 1.46 g/L (equivalent to 50.93 ± 1.61 ethanol/kg) for C. pepo fruits using S3 (Table 4). The maximum P_exp value with sucrose medium enriched were 105.97 ± 1.99 g/L (equivalent to 460.97 ± 8.66 g ethanol/kg sucrose NG), reached with S2. From Tables 3, 4 and 5, it can be deduced that ethanol production with O. amentacea juice was slightly better than that with C. pepo. In addition, ethanol produced using S2, S3 and S4 gave better P_exp values relatively to those obtained with S1. It is known that high sugar consumption can lead to a good ethanol production (Khongsay et al., 2012 and Khongsay et al., 2010). So, the weak sugars quantity and the presence of poisonous substances such as phenolic compounds in fruit juices (Almeida et al., 2007), might likely have impacts on the outputs of ethanol production. Therefore, a preliminary detoxification process could contribute to the improvement of ethanol production from these kinds of raw materials (Mazmanci, 2011). Unfermentable carbon source’s existence in the juices might also explain why the ethanol yields were low in the juices. 

 

 

 

Ethanol productivity (Qp: g/L/h)

With sucrose, the best productivity (Qp) obtained was 2.208 ± 0.041 g/L/h with S2, and the lowest one was 1.456 ± 0.048 g/L/h with S4. Without nutrients, the best Qp reached with S2 in sucrose mashes was 0.756 ± 0.018 g/L/h, while the lowest one was 0.624 ± 0.011 g/L/h obtained with S1 (Table 3). For C. pepo enriched juice, the best and the lowest Qp obtained by S3 and S4 were 0.643 ± 0.020 g/L/h and 0.461 ± 0.008 g/L/h, respectively. But in the absence of enriching nutrients, C. pepo juice best productivity was 0.225 ± 0.009 g/L/h with S2 and the lowest is 0.187 ± 0.008 g/L/h by S4 (Table 4). In the case of O. amentacea enriched juices, it was S2 which gave the best productivity of 0.408 ± 0.009 g/L/h and the lowest one of 0.294 ± 0.011 g/L/h was given by S1. Without the enrichment of O. amentacea juices, the best productivity of 0.203 ± 0.009 g/L/h was given by S3, and the lowest one of 0.154 ± 0.006 g/L/h by S4 (Table 5). So, to get high ethanol productivity, the fermentation time must be kept in a short time (Khongsay et al., 2010).

Ethanol yield (Yp/s: g/g) and yield efficiency (Ey: %)

The experimental Yp/s values (0.428 ± 0.008 g/g to 0.527 ± 0.006 g/g) and the Ey: values (79.29 ± 1.40 % to 97.67 ± 1.10 %) (Tables 3 and 4)), in relation to the final sugars consumptions, were slightly lower than what were hypothetically the theoretical predicted. The ethanol concentrations produced were also slightly lower than the calculated theoretical ethanol concentrations. Many factors explained this decrease in the expected results. First, the production of by-products such as organic acids, other alcohols, ketones, etc. was harmful for the good productivity. Secondly, the use of parts of sugars by the yeasts to get energy, useful and compulsorily to their growth was also seen as a source of output loss (Ballerini and Alazard-Toux, 2006). The previous reported Yp/s of cane molasses (Reed, 2002), dried sweet potato chips/flour (Woolfe, 1992), and cassava chips/flour (Balagopalan et al., 1987) were 265 to 272 g ethanol/kg, 280 to 320 g ethanol /kg, and (420 to 450 g ethanol /kg), respectively.

In the present study, the maximum ethanol yields calculated were 41.296 g ethanol /kg for O. amentacea fruits and 36.134 ethanol g/kg for C. pepo fruits. Ameyapoh et al. (2006) produced bioethanol from Manguifera indica L. (Anacardiacea) mash of 6°Bx with four strains of S. cerevisiae (AYS01, AYS02 AYS001 and L2056). They got outputs (g/L ethanol) of 7.83; 6.52; 5.21 and 8.65, respectively. On the other hand, beer production achieved by Dahouenon-Ahoussi et al. (2012) from Sorghum bicolor and Musa acuminata mashes, gave the output of 4.86%Vol, equivalent to approximately 37 g ethanol /L. Our results were better than those obtained by Ameyapoh et al., (2006) and Dahouenon-Ahoussi et al. (2012). However, they were lower than those obtained with Washintonia robusta fruit juice containing 105 g/L of reducing sugars, and which succeeded to a maximal production of ethanol of 25 g/L, equivalent to 71.42 ± 1.4 g of ethanol/kg (Mazmanci, 2011).

The improvement of ethanol production yields by using other different microorganisms such as Zymomonas mobilis (Claassen et al., 1999), Saccharomyces bayanus (Castellar et al., 1998), Saccharomyces pastorianus (Fujii et al., 2001), Kluyveromyces fragilis (Szambelan et al., 2004) or genetically modified microorganisms (Ostergaard et al., 2000), will be investigated in the future.

The outputs of bioethanol production obtained in this study were 50.60 ± 0.57 g ethanol/L (equivalent to 60.72 ± 0.68 g ethanol/kg of fruit) and 46.3 ± 1.46 g/L (equivalent to 50.93 ± 1.61 g ethanol/kg of fruit), respectively for O. amentacea and C. pepo. Moreover, all the other fermen-tation kinetic parameters mesured in this study showed clearly that the media supplementation with the mixture of nutrients (urea, monosodium glutamate, potassium dihy-drogen phosphate, and magnesium sulfate heptahydrate) improved significantly the batch ethanol production performances. However, the maximal concentration of ethanol produced with sucrose (105.97 ± 1.99 g/L) was comparatively more elevated. All the results we obtained suggested that S4 is the most suitable strain for ethanol production under normal gravity(NG). The use of these conditions can be very attractive in fuel ethanol production domains.

The use of C. pepo and O. amentacea fruits as alternative carbon sources for ethanol production was proposed. Ethanol production yields for the two fruits were very low. However, as these fruits were abundant and inedible, so their eventual exploitation for bioethanol production could be a source of added value for the local population.

This would decrease the use of edible products for fuel production. But, fruit juices detoxification and supple-mentation with little quantity of sucrose or other nitrogen sources and/or other nutrients as well as other fermen-tation processes should be further investigated to improve and complete sugar consumption under normal gravity and very high gravity conditions.

The author(s) have not declared any conflict of interests.

The authors are grateful to their co-workers and to Université de Lomé (UL). We thank Mr. Senyo AMEVOR for helping in the reading of the manuscript and Mr. Yao HOUEKOU for his technical support.