Utilization of Date palm ( Phoenix dactylifera L.) wastes for bioethanol production using Pichia kudriavzevii strains

Tons of Date palm ( Phoenix dactylifera L.) fruit wastes are discarded daily by the date palm processing industries thus leading to serious environmental problems. This study aimed to investigate the potential of date palm fruit wastes as sugary feedstock for bioethanol production using yeasts. Proximate analysis of the date fruit wastes revealed a moisture content of 8.98 %; crude protein (4.39 %), ash (2.35 %), fat (0.2 %), fiber (0.8 %) and carbohydrate (84.28 %). Sulphuric acid was used for pre-treatment of the date palm fruit substrate. Fermentation was carried out under shaking and static conditions using Pichia kudriavzevii yeast strains isolated from date palm fruit wastes. Greater bioethanol yield was observed when the substrates were fermented under shaking condition. Optimization of the physical conditions improved the fermentation process faster, and significantly enhanced the production of bioethanol. An appropriate temperature of 30 o C and pH 5 produced high yield of ethanol (5 %) by Pichia kudriavzevii SGD21, whereas pH 6 for Pichia kudriavzevii SGD30 recorded a higher ethanol yield of 6 %. Under the optimal physical conditions, the fermentation process resulted in the production of 4 % ethanol after an incubation period of 96 h. Moreover, the Pichia kudriavzevii strains could be recommended for bioethanol production at 30 % inoculum size, on using sucrose as a carbon source and yeast extract as a nitrogen source. On using the Fourier-Transform Infrared (FTI) spectroscopy, the detected functional group of the produced bioethanol was O-H group. Finally, utilization of date palm fruit wastes and the yeasts strains of Pichia kudriavzevii SGD21 and Pichia kudriavzevii SGD30 can be exploited for bioethanol production and this could be an effective way for management and utilization of date palm fruit wastes.


Introduction
The amounts of greenhouse gases present in the atmosphere have increased significantly, due to the high level of pollution that emanated from the use of fossil fuels (Ballesteros et al., 2006). Several fossil energy sources (i.e. coal, oil and natural gas) are used for the production of fuel and electricity (Uihlein and Schbek, 2009). Due to the instability in the oil markets and the increase in air pollution, it is imperative to point out other options for renewable and sustainable energy sources. Bioethanol produced from bioenergy feed stocks is one of the sustainable, economic and ecologic solutions for these concerns. It can be produced from reduced sugars, stiff biomass and also from the lignocellulosic biomasses (Sims et al., 2008).
Bioethanol derived from biological origins has gained much attention in light of the growing energy crisis in recent years. Bioethanol is now being produced from different sources around the world; the most common are sugar-based plants (Sivakumar et al., 2010). Most of the conventional ethanol fermentation feedstock depends on the agricultural crops, such as sugar-based crops; starch-based crops, fruits and vegetables (Balat and Balat, 2009). Several microorganisms are used for the conversion of the different biomasses into ethanol, such as Saccharomyces cerevisiae yeast that can be used effectively to produce bioethanol.
Changes in the land-use patterns for bioethanol production that deviate from the food crops could be a threat to the global food security. For this reason, the use of a second generation of bioethanol produced from agricultural wastes such lignocellulosic materials including grasses, crop residues, leaves, sawdust, sludge woodchips, municipal solid wastes and livestock manure is increasing (Sun and Cheng, 2002;Wen et al., 2004;Staniszewski et al., 2007;Hossain et al., 2009).
Agricultural wastes are used because of their high sugar contents, as they provide abundant natural resources for the second generation of bioethanol production. Moreover, the use of these wastes does not compete with the rising food demand from the food crops (Rastogi and Shrivastava, 2017;Robak and Balcerek, 2018;Carrillo-Nieves et al., 2019). Fruit wastes including damaged fruits; peels and seeds are potential cheap alternatives used as feedstock for biofuel production (Cheng et al., 2009). The previous study conducted by Dalibard, (2018) reported that palms are one of the most useful sugary feedstock plants, because they can be used as sugar generating plants throughout the tropical world since the earliest times. According to Sriroth and Piyachomkwan, (2013), palmyra palm (Borassus flabellifer); coconut palm (Cocos nucifera), sugar palm (Arenga pinnata), wild date palm (Phoenix sylvestris), nipa palm (Nypa fructicans) and commercial date palm (Phoenix dactylifera), are all acceptable plants for sugary feedstock of palms sap.
The wild date palm (Phoenix sylvestris Roxb.) is a perennial tall tree plant, which is a monocotyledonous, dioecious and belongs to the Arecaceae family (Krueger, 2011). Date palm juicy fruit is edible, although most of the time it recognized as wastes under the tree (Islam et al., 2014). Bioethanol production from date palm fruit and sap could give a lasting solution to vast difficulties of stable energy diversification. Therefore, the objectives of the present study were to investigate the effectiveness of date palm fruit wastes as sugary feedstock for bioethanol production using yeast strains, and to explore the optimum growth conditions for these strains to potentially yield high levels of bioethanol.

Samples collection, preparation and composition of date palm fruit wastes
Date palm fruits wastes were obtained from the local markets of Agbowo, Bodija, Sango and Beere in Novel Research in Microbiology Journal, 2022 Ibadan, Oyo State, Nigeria. The fruits used were sorting wastes of dates including; fruit with texture defects, very wet fruits, fruits spoiled by microorganisms and insects. They were brought in sterile polythene bags and then transported to the Microbiology laboratory for further use.
The methods described by A.O.A.C. (2005) were used for proximate analysis of the composition of date palm fruit wastes, including moisture content; crude protein, fat, ash and total carbohydrates. The assays were carried out in duplicates.

Isolation of the yeast isolates
The yeast isolates were obtained from the date palm fruit wastes collected from Agbowo, Bere, Bodija and Sango. The collected date palm fruits were rinsed to remove sand and debris, the seeds were removed and then the flesh was grinded using a sterile mortar and pestle. About 1 g of the grinded fruit was introduced aseptically into 9 ml sterile water and then serially diluted till the dilution of 10 -6 . An aliquot of 1 ml of each appropriate dilution was dispensed into yeast extract agar (YEA) Petri plates, and then the plates were incubated at 28 ± 25 o C for 72 h. After incubation, the growing yeasts were selected, purified and then stored onto YEA slants at 4°C till further use (Kreger-van Rij, 1984).

Biochemical and microscopic identification
The yeast isolates showing remarkable characteristics were selected, and then identified according to their cultural, microscopic and biochemical characteristics; using several biochemical assays and through detecting the fermentation of 10 different sugars, in reference to Kurtzman and Fell, (1998).

Molecular identification of the yeast isolates
The DNA of the selected yeast isolates was extracted according to the procedure described by Zymo Research kit, USA. Pure culture of each yeast isolate was inoculated into yeast extract glucose broth and then incubated at 28°C for 72 h. After incubation, the broth containing the growing cells was then centrifuged to obtain the cell pellet. The cell pallet of the yeast was lysed using 50 µl of the lysis buffer, vortexed for 10 sec and then left to stand at room temperature for 5 min. The mixture was transferred to a Zymo-spin column in a collection tube, centrifuged for 1 min. at 10,000 rpm, and then the supernatant was discarded. The Zymo-spin column was transferred to a new collection tube and 200 µl of the DNA pre-wash buffer was added to the spin column, and then centrifuged for 1 min. at 10,000 rpm. About 500 µl of the DNA wash buffer was added to the spin column, centrifuged for 1 min., and the spin column was then transferred to a clean microcentrifuge tube. 50 µl of the DNA elution buffer was added, incubated for 5 min. at room temperature, and then centrifuged for 30 sec to elute the DNA (Angelov et al., 2015).

Polymerase Chain Reaction (PCR) amplification
The Internal transcribed spacer (ITS) universal primers sets that flanked the ITS1 and ITS2 regions were used to characterize the yeast isolates, including; ITS 1: 5'TCC GTA GGT GAA CCT GCG G 3' and ITS 2: 5'TCC TCC GCT TAT TGA TAT GC 3'. The PCR assay was carried out in a Gene Amp 9700 PCR System Thermal cycler (Applied Biosystem Inc., USA) with a PCR profile consisting of an initial denaturation at 94 0 C for 30 sec; annealing of primer at 55 0 C for 30 sec, at 72 0 C for 1 min., and a final termination at 72 0 C for 10 min., after which the primer was finally maintained at 4 0 C. The amplified fragments were sequenced using a Genetic Analyzer 3130×1 sequencer (Applied Biosystems, USA), according to the manufacturers' manual. The used sequencing kit was that of Big Dye terminator v3.1 cycle sequencing kit, while Bio-Edit software and Mega6 were used for all the genetic analysis, in reference to Josepa et al., (2002). The obtained sequences were compared to the National Centre for Biotechnology information (NCBI) Database by using the Basic local Alignment search tool (BLASTIN 2.2.29+); with the default settings used to find the Novel Research in Microbiology Journal, 2022 most closely similar sequences that were sorted by the E score. A representative sequence of 10 of the most similar neighbors was aligned for multiple alignments with the default settings (CLUSTAL W2) (Centre for Evolution Genomics, Arizona).

Estimation of the total reducing sugar
The fruit palm wastes total glucose content was determined according to the method adopted by Miller, (1959). About 3 ml of 3-5-dinitrosalicylic acid (DNS) reagent was added to 3 ml of glucose sample in a lightly capped test tube, and then the mixture was incubated in a water bath for 5-15 min. at 90 o C until the red-brown color disappeared. After cooling, 4 ml of dist. water was added to the samples and then the absorbance was measured at 540 nm using a spectrophotometer (721G Visible Spectrophotometer, China). The reducing sugar concentration was determined from the standard curve prepared using glucose, and then multiplied by the dilution factor. Different concentrations of glucose were prepared ranging from 1 mg/ ml to 10 mg/ ml. About 1 ml of DNS was added to 1 ml of each glucose concentration in the test tube, and then the mixtures were heated in a boiling water path for 10 min. The test tubes were cooled rapidly after which the optimum density of the sample was read against the blank using the spectrophotometer at 540 nm. A graph of absorbance against the concentration was then plotted (Miller, 1959).

Pre-treatment by steam explosion (Auto hydrolysis)
The date palm fruit wastes were washed; oven dried, blended into powder using a mortar and then kept in zip-lock bags for further use. About 100 g dry powder of date palm fruit wastes were pre-treated by subjecting them to a high pressure 121 o C for 30 min. using an autoclave, according to Tutt et al., (2014).

Acidic pre-treatment
With reference to Yousif and Abdulhay, (2017), approximately 100 g dry powder of date palm fruit wastes were pre-treated with 0.6 % H 2 SO 4 , and then heated at 121 o C for 15 min. The pH was adjusted to pH 5 using NaOH.

Preparation of yeasts cell suspension
The yeast inoculum was prepared in Yeast extract potato dextrose (YEPD) broth (LAB M). A 24 h old culture of each yeast isolate was added individually to 10 ml of autoclaved molasses fermentation medium, and then the tubes were shaken gently to form homogeneous suspensions (Taouda et al., 2017).

Fermentation of hydrolyzed date palm wastes
An aliquot of l g of the blended date palm fruit wastes sample were aseptically added into sterile 2 l Erlenmeyer flask, covered with an aluminum foil, and then allowed to ferment for 5 d on a rotary shaker (MON 106 MODEL, MON Scientific). Samples of 100 ml were withdrawn aseptically at 24 h interval, and estimated for determination of the residual sugar and ethanol contents in the fermentation media (Yousif and Abdulhay, 2017).

Recovery of bioethanol and determination of its content
The distillation process was used to separate the bioethanol from the fermentation broths. Each fermented broth was placed on the electric heating mantle machine (LAB Heating Mantle 51 Model) at 78 o C, and then water flow was connected. The distillation product was collected in a round bottom flask (Wakil et al., 2013).
Ethanol content of the fermented samples was determined using a gravimetric method in reference to Wakil et al., (2013). The specific gravity was determined using a density bottle. An aliquot of 100 ml of the fermentation samples was distilled individually using an electric heating mantle (LAB Heating Mantle 51 Model), which was used to collect the distillate through a glass condenser. The dry weight of the empty density bottle was determined. The distillate was poured into a 25 ml volume dry density bottle and then weighed. Equal volume of dist. water was poured into a dry density bottle and then weighed. The weight of the empty dry density bottle was subtracted from both of the weight of density bottle containing the fermented sample distillate and that of dist. water. The specific gravity was determined by dividing the weight of the distillate of the fermented sample by the weight of the dist. water, according to the following equation of Wakil et al., (2013): Where; W1=weight of the density bottle plus weight of the distillate of the fermented sample; W2=Weight of the density bottle plus weight of the dist. water; W = Weight of the empty density bottle The specific gravity was used to determine the concentration of bioethanol using the Ethyl Alcohol Conversion Table ( Wakil et al., 2013).

Determination of the effects of various physical parameters on bioethanol production
The fermentation process was carried out using the selected two yeast strains, where their activities were known to vary with respect to the inoculum concentration; pH, nitrogen and carbon sources, and the incubation temperature. It is therefore imperative to optimize the fermentation conditions for the yeast cells so that the bioethanol production efficiency increases. Various factors were investigated that affect the bioethanol production from molasses.

Effect of initial pH
In this assay, the effects of different pH values were studied, while the other physical parameters were kept constant of (10 %, 30°C and 4 d); respectively. The different pH values used were; 4.0, 4.5, 5.0, 5.5 and 6.0. The assay was conducted according to the method adopted by Arslan et al., (2021).

Inoculum concentration effect
In reference to Mojovic et al., (2006), various inoculum concentrations such as 10 %, 20 %, 30 % and 40 % were prepared using diluted YEPD broth medium, while the other physical parameters including substrate content; temperature and the time were kept constant at 10 %, 30°C and 4 d; respectively. The fermentation process was carried out as described earlier.

Effect of co-culture of the selected yeast strains on bioethanol yield
According to Eladpum et al., (2012), an aliquot of 500 ml of the fermentation medium was used. A 48 h old culture (5× 10 8 cfu/ ml) inoculum of the 2 selected yeast isolates were inoculated simultaneously, and then incubated at 30 o C on a shaker at 100 rpm as described before. Samples were taken aseptically at 24 h interval for 5d, and estimated for determination of the bioethanol content in the fermentation media.

Impact of different carbon and nitrogen sources on bioethanol production
Different carbon sources such as glucose; sucrose and fructose were used, while the yeast concentration and the other physical parameters including substrate content; temperature and the incubation time were kept constant of at 10 %, 30°C and 4 d; respectively, in reference to Hosny et al., (2016). Similarly, different nitrogen sources including yeast extract; malt extract, urea and peptone were used individually, and the fermentation was processed as described before at substrate content, temperature and the time of 10 %, 30°C and 4 d; respectively (Hosny et al., 2016).

Effect of agitation on bioethanol yield
To study the impact of agitation on bioethanol production; the fermentation process was carried out at pH 5.0 and a temperature of 30°C in a volume of 100 ml medium, in reference to the method conducted Novel Research in Microbiology Journal, 2022 by Armanul, (2014). A 48 h old inoculum of each yeast strain was added individually to the fermentation medium, and then the flasks containing the same sugar concentration were kept both under shaking at 120 rpm and at a static condition, and thereby the bioethanol content was determined after incubation.

Determination of the functional group of bioethanol
The functional group of bioethanol was determined using the Fourier-Transform Infrared Spectroscopy (FTIR) (ALPHA). The infrared spectrum analysis was used to identify the vibration signs of the bioethanol recovered from the wastes of date palm. The infrared spectrum of the bioethanol was recorded by passing a beam of infrared light through the sample. When the frequency of the IR was the same as the vibrational frequency of a bond or collection of bonds, absorption took place. Examination of the transmitted light revealed how much energy was absorbed at each frequency (or wavelength), according to Arslan et al., (2021).

Statistical analysis
The experimental data were analysed using Analysis of Variance (ANOVA) to determine the means with SPSS version 23 and the level of significance was set at p ≤ 0.05. A bar chart was also used to compare the effects of the physico-chemical parameters on bioethanol yield.

Yeast isolates obtained from the date palm wastes
A total of 61 yeasts isolates were recovered from the date palm fruit wastes on YEA medium. All of the isolates were cream colored, with circular and irregular shapes, dull and shiny appearances and entire margins. The cell morphology of the isolates observed under the light microscope showed that the majority of cells were ovoid, apiculate and elongated with budding.

Biochemical identification of the yeast isolates
Results recorded in Table (1) showed that most of the selected yeast isolates expressed variation in their fermentation of the 10 tested sugars. Almost all isolates utilized glucose; sucrose, dextrose, maltose, fructose, mannitol, sorbitol and inulin. However, none of the isolates fermented galactose.

Molecular identification
According to results of the PCR amplification, both of the selected yeasts isolates were identified as Pichia kudrivavzevii SGD21 and Pichia kudrivavzevii SGD30. They were assigned accession numbers of OK33443 and OK334444, respectively. The phylogenetic tree demonstrated that both selected strains were not from the same ancestor, although there was a noticeable similarity between them (Fig.  1).

Proximate analysis of date palm fruit waste
Results of the proximate analysis of date palm fruit wastes showed the presence of high amounts of carbohydrate content of (84.28 %), moisture content (8.98 %), crude protein (4.39 %), crude fat (0.2 %), fiber (0.80 %) and ash (2.35 %), which made them suitable substrates for the production of bioethanol. The values were recorded in means of duplicates.

Production of bioethanol from date palm fruit wastes through steam explosion and acid pretreatments
Results of the production of bioethanol by Pichia kudrivavzevii SGD21 and Pichia kudrivavzevii SGD30 through steam explosion and acid pretreatment of date palm fruit wastes demonstrated different bioethanol concentrations, initial and final pH values, initial and final reducing sugars, as presented in Tables (2 and 3 Where; A: Acid production; AG: Acid and gas production; G: Gas production; -: No growth; +: Growth. All assays were carried out in duplicates Novel Research in Microbiology Journal, 2022

Impact of initial pH value on bioethanol yield
The fermentation reaction was sensitive to changes in pH value. The optimum pH recorded for Pichia kudriavzevii SGD21 was pH 5, which corresponded to alcohol production level of 5 %. On the other hand, the optimum pH for Pichia kudriavzevii SGD30 was pH 6, which recorded the highest alcohol production of 6 %, as demonstrated in Fig. (2).

Effect of inoculum concentration on bioethanol production
Results of the production of bioethanol using different inoculum concentrations are presented in Fig. (3). Ethanol production was at 0 level when no inoculum was used; however, it was significantly improved as the inoculum concentration increased from 10 % to 30 % for P. kudriavzevii SGD21 and P. kudriavzevii SGD30. On the other hand, when the used inoculum concentration rose to 40%, the level of bioethanol production significantly decreased.

Effect of co-culture of both yeasts strains on bioethanol yield
On co-fermentation, both yeasts strains were inoculated simultaneously. Results presented in Table  (4) show that different bioethanol concentrations; initial and final pH, initial and final reducing sugars were recorded on co-inoculation of both yeast strains, with the highest productivity (4 %) of bioethanol content observed when a substrate concentration of 10 % and initial pH 5.0 were employed.

Impact of different carbon and nitrogen sources on bioethanol production
As demonstrated in Fig. (4), fructose was the best carbon source for bioethanol production, with maximum yield of 12 % recorded by Pichia kudriavzevii SGD21, and a yield of 9 % exhibited by Pichia kudriavzevii SGD30. Compared to fructose, glucose and sucrose expressed lower bioethanol yields by both yeast strains. On the other hand, Fig. (5) shows that yeast extract was the best nitrogen source for bioethanol production by both yeast strains with a maximum yield of 7 %; compared to peptone, malt extract and urea.

Effect of agitation on bioethanol level
Fermentation was carried out under shaking and static conditions. Greater bioethanol yield was recorded in the fermentation medium when fermented under a shaking condition, as shown in Fig. (6).

Evaluation of the functional group of bioethanol
The Fourier Transform Infrared (FTIR) spectrum of the produced bioethanol is demonstrated in Fig.  (7a-b). The recorded results showed the presence of peaks ranging between 3810-3500 cm -1 , which were indicative of the presence of O-H stretching free alcohol groups that were not inter molecularly bonded. Additional peaks ranging between 3500-3200 cm -1 also indicated the presence of O-H groups; however, they were inter-molecularly bonded. A single peak at 2369 cm -1 indicated the presence of a strong carbon dioxide group.

Discussion
For effective bioethanol production, the efficiency of delignification depends mainly on the type of pretreatment. In this study, sulphuric acid (H 2 SO 4 ) was used for pre-treatment of the fruit waste substrates. This is in accordance with the previous work of Praveen Kumar et al., (2017), who used agricultural waste materials as alternative sources of cellulosic materials feedstock during bioethanol production.
It was observed that date palm fruit wastes have a high amounts of carbohydrate content of (84.28 %), moisture content (8.98 %), crude protein (4.39 %), crude fat (0.2 %), fiber (0.80 %) and ash (2.35 %), which made them suitable substrates for the production of bioethanol. It is known that date fruit contains less amount of fat, more fiber and high ash content. The higher ash content expresses the presence of high mineral contents, and thus date palm is also a good source of several nutrients. These nutrients support the microbial growth during the fermentation process for bioethanol production (Awan et al., 2018).
The pH of date palm fruit filtrates decreased as the fermentation days increased. This is in line with the study conducted by Wakil and Onilude, (2011), where a decrease in pH was observed in fermented weaning blends with the increase in the fermentation period. Moreover, the decrease in pH was also recorded by Wakil and Osamwonyi, (2012); to be one of the characteristics of the process of fermentation.
The reducing sugar of fermenting filtrates significantly decreased as the fermentation days increased, in agreement with the previous study of Teck-Yuan et al., (2011). The recorded maximum yield of alcohol observed with the increase in sugar fermentation is supported with the findings of Bhatti et al., (2019), who stated that more sugar consumption increases the bioethanol yield. Similarly, Rani et al., (2006) also reported a decrease in the level of reducing sugar as the fermentation progressed.
The effect of co-culture of P. kudriavzevii SGD21 and P. kudriavzevii SGD30 in the fermentation medium was observed, where the highest bioethanol yield was recorded after 48 h of fermentation. These low results obtained when both yeasts strains were inoculated simultaneously in the fermentation medium may be attributed to the competition for nutrients between them. Similar results had been made on coculture of Zymomonas mobilis with Pichia stipitis during the previous study conducted by Fu et al., (2009), which may be attributed to oxygen deprivation in the Z. mobilis cells. The effect of initial pH on bioethanol yield was observed. The optimum recorded pH value was 5.0, which corresponds to the highest alcohol production. Each microorganism has its optimum specific pH that enhances its specific enzymes to catalyse the required reactions. P. kudriavzevii SGD21 had the maximum recorded bioethanol concentration of 6 % at pH 6, while P. kudriavzevii SGD30 had the maximum bioethanol level of 5 % at pH 5. It is generally known that yeasts favor slightly acidic environment (Hosny et al., 2016). According to Mansi and Mita, (2016), maximum specific alcohol was produced at pH 5.0. Moreover, the same authors reported that an initial pH of 5.0 of the fermentation media had great influence on bioethanol production. However, any increase or decrease in the initial pH of the fermentation medium from 5.5 causes marked decreases in the bioethanol yield.
In this study, ethanol production significantly improved as the quantity of inoculum increased from 10 % to 30 % in the fermentation media inoculated with P. kudriavzevii SGD21 and P. kudriavzevii SGD30. On the other hand, when the inoculum amount was raised to 40 %; the bioethanol production was significantly decreased. Currently, an inoculum concentration of 30 % was recorded as an appropriate concentration. In contrast to this study, Arslan et al., (2021) predicted that production of bioethanol from date palm fruit wastes can be increased by using 25 % as an initial inoculum concentration. Addition of Novel Research in Microbiology Journal, 2022 sucrose to the fermentation medium as a carbon source had the highest bioethanol yield compared to glucose and fructose, in agreement with Bhatti et al., (2019). On the other hand, yeast extract was the best nitrogen source for bioethanol production with a yield of 7 %; followed by peptone, malt extract and urea, in accordance with the previous study conducted by Hosny et al., (2016). Greater bioethanol yield was observed when the substrate was fermented under shaking conditions, which may be attributed to the proper distribution of the nutrients available for utilization by the fermenting yeasts. This correlates with the study of Armanul et al., (2014) who recorded that all yeast isolates produced better bioethanol yield under shaking conditions; during fermentation of pineapple peels as substrate.

The
Fourier Transform Infrared Spectrophotometer (FTIR) analysis of the produced bioethanol confirmed that the ethanol group (C 2 H 5 OH) was present. According to Manzoor et al., (2020), who stated that during FTIR analysis; if the beak region is observed between 3.200-3.600 cm -1 ; this confirms the presence of an ethanol group. This result is similar to the recent work of Arslan et al., (2021), who recorded the presence of an alcoholic functional group in the bioethanol produced from fermentation of wastes of the date palm fruit. All the optimized physical conditions improved the fermentation process and significantly enhanced the production of bioethanol. An appropriate temperature of 30 o C, pH of 5 and 6 for P. kudriavzevii SGD21 and P. kudriavzevii SGD30; respectively, fermentation time of 96 h, inoculum size of 30 %, sucrose as C source, yeast extract as N source, and fermentation under agitation; all resulted in maximum bioethanol production by the tested yeast strains.

Conclusion
In this study, it was revealed that both P. kudriavzevii SGD21 and P. kudriavzevii SGD30 strains were tolerant to 20 % ethanol concentration, mesophilic (30 o C) and can survive at various pH ranges (2, 4, 6, 8 and 10). The highest bioethanol concentration was obtained when the physical factors of the fermentation process were optimized. It was observed that 30 % inoculum concentration was the best concentration for high bioethanol production. Finally, these currently tested yeast strains could be used at the industrial level to obtain an increased yield of bioethanol on fermentation. Accordingly, fermentation may be considered as an effective way of date palm fruit wastes management and utilization.