Bioethanol Production from Olive Solid Residues by Using Rhodotorula Minuta

Bioethanol is an attractive fuel with higher potential for energy security and environmental safety. Olive solid residues were used as a raw material for the production of bioethanol through the use of different preliminary treatments . Separate treatments with cellulose, hydrochloric acid (HCl 5%), sulfuric acid (H2SO4 2%), and liquid ammonia NH4OH (20%) were used to convert cellulose and hemicellulose into monosaccharaides. The production of ethanol was observed during the fermentation process using R. minuta under anaerobic conditions. After 3 days of fermentation, lowest concentrations of ethanol of 0.233, 0.249, 0.261, and 0.275 g/ l were produced from olive waste powder sample as a result of separate pretreatment with cellulase, hydrochloric acid (HCl 5%), sulfuric acid (H2SO4 2%), and liquid ammonia NH4OH (20%), respectively, whereas the untreated sample showed ethanol yield of 0.264 g/ l.. The highest ethanol concentrations for the same samples were 0.510, 0.564, 0.737, and 0.696 g/ l, respectively, whereas that for the untreated samples was 0.445 g/ l. The highest concentration of ethanol produced (0.737 g/ l) was achieved after 3 days of fermentation of olive solid waste pretreated with H2SO4 2% at 30 o C and pH 5. The average yield of ethanol resulted from these saccharification and fermentation processes following the pretreatment of olive solid waste was 0.59 g/10 g dry olive solid residues.


Introduction
The era of renewable energy has begun and motivated countries and societies to search for sources of energy other than those dependent on fossil fuels, including solar and wind energy. In addition, the topic of biofuel production from organic materials, especially organic waste that may pollute the environment, has gained a remarkable amount of research interest worldwide [1]. Bioethanol fuel is becoming one of the best alternatives for countries that import crude oil in large amounts. Ethanol is flammable liquid; its vapor concentrations have flammability in the range of 3.3-19.0% (v/v) in air. The flammability is not a serious problem in the industrial environment if there is sufficient ventilation, which also prevents ethanol from causing severe problems as an industrial poison [2]. Bioethanol, in many ways, is superior to petroleum fuel for spark-igniting engines. It has a higher octane number than that of the conventional petroleum. Also, it is safe for storage and has a good thermal efficiency [3]. Many natural resources, including olive solid waste, date syrup wastes, and banana peels are used as raw materials for the production of bioethanol [4][5][6]. Since ancient times, the olive oil industry was established in Iraq, in particular olive mills. As for the olive solid waste, it is currently used in the manufacture of soap, charcoal, or even as fuel. Studying the use of olive solid residues for the production of ethanol is an important step in the field of waste recovery [7]. Olive waste is currently supplying global production of ethanol with 2,616,000 barrels per day, according to 2018 statistics. The advantage of producing ethanol from olive waste is that it contributes to the protection of the global environment from the increased levels of pollution [8]. The present study aims at the achievement of three main goals; (a) investigating the ability of olive oil waste to produce bioethanol; (b) evaluating the ability of Rhodotorula minuta yeast isolated from dairy products to ferment sugar to ethanol. For this purpose, different pretreatments were applied to find the most effective one in hydrolyzing cellulose, hemicellulose, and lignin into monomeric sugars, which can be then converted to ethanol; (c) using olive solid waste as an alternative biofuel to reduce the phenomenon of environmental pollution in a way that does not affect food security.

Raw materials
Before starting to grind and squeeze the olives, they were washed with water to remove impurities and dust that may be stuck to them, in addition to isolating the olive leaves from the specimens. Olive solid waste was produced through the grinding of olive (Olea europaea). Gauze cloth was used to separate olive oil from olive waste by the manual squeezing method. As a result, the mixture was composed of a liquid part, which contained crude olive oil and other liquids (Figure 1a), and olive solid residues (Figure 1b). The ligninocellulosic waste was dried in the oven, cut to small pieces, and then crushed by a grinding miller to powder form in order to increase its susceptibility to chemical pretreatment. The resulting powder was sieved with a fine 50 mesh sieve and stored in a clean container at room temperature for further use [9].

Pretreatment of olive solid waste
Raw material powder was treated with four separated pretreatments; first, pretreatment with H 2 SO 4 (2%) at a temperature of 130°C and at 500 rpm for one hour [10]; second, pretreatment with HCl (5%) at a temperature of 130°C and at 500 rpm for two hours [11],; third, pretreatment with NH 4 OH (20%) at a temperature of 70°C and at 600 rpm for 12 hours [12]; fourth, pretreatment with cellulase enzyme in a shaker incubator at 50°C at 150 rpm for three days [13]. In addition, a fifth sample was left without treatment.

The fermentation stage
The liquid produced from the previous processes was autoclaved at a temperature of 121°C for a period of 20 minutes for sterilization from any bacteria or other microorganisms. Equipment was designed to complete and facilitate this method of fermentation, separation and disposal of carbon dioxide, as shown in Figure 2 [6].

Isolation of Rhodotorula minuta yeast from dairy products
Rhodotorula minuta is a genus of unicellular pigmented yeasts, which is part of the Basidiomycota division. It can be easily recognized by the distinct orange/ red colonies which have smooth and glossy shape when grown on SDA (Sabouraud's Dextrose Agar). This distinctive color is the result of the dyes that the yeast creates to block out specific wavelengths of light (620-750 nm) that would otherwise damage the cells. This genus can be isolated from natural environments, soil, water, milk, fruit juice and air samples [14]. R. minuta is part of the division Basidiomycota, family Sporidiobolaceae.
Yeasts were isolated from four types of dairy products purchased from Baghdad local markets. A swab was taken from each layer of the dairy product (surface, middle, and bottom) and cultured on SDA medium, then incubated for 5 days at 28°C. During this period, three colonies with morphological appearance were selected for further purification. The initial diagnosis of yeast was based on the distinct orange/ red coloration and smooth and glossy shape of the colonies, as stated before [14]. Culturing of Rhodotorula minuta yeast on agar media SDA medium was used for cultivating and maintaining the yeast R. minuta at 28 °C for 72 h. After that, the yeast was grown on the agar medium and stored at 4°C. Figure 3a shows R. minuta culture after 72 h incubation, while Figure 3b shows this genus under microscope. The diagnosis was confirmed by using vitek 2 compact system (Table 1).  Preparation of yeast culture in broth SD broth was used for preparing the culture of R. minuta. Two slants of the yeast culture were inoculated into a 100 ml borosilicate glass reagent bottle with screw cap , containing 80 ml of SD broth medium. Then, the culture was incubated at 28ºC for 72 h; these active cultures were used as inoculums for ethanol production [15].

Fermentation of raw materials
Activated R. minuta with inoculum size of 5.55×10 5 cells / ml was added to olive solid residue for each experiment (with pretreatment and without treatment) under aseptic conditions in order to prevent any contamination [16]. Distilled water (900 ml) was added to each mixture and then incubated at 30°C for a period of three days. During that, ethanol concentration was determined daily by using ethanol sensor (Vernier / USA) [17].

Influence of different pretreatment methods
The effects of different pretreatments on olive solid residues were studied. Large amounts of monosaccharaides were obtained at the end of the process, as shown in Table 2. Pretreatment of the olive solid residues sample with NH 4 OH (20%) resulted in the highest concentration of sugar (8.25 mg/ml), obtained on the second day, while pretreatment with HCl (5%) produced the lowest sugar concentration of 2.2 mg/ml after 24 h. The results show the effect of acid concentration, temperature and residence time on the biomass structure for the recovery of monosaccharides [18]. Also, the results suggest a strong alkaline effect and selective removal of lignin, without losing reducing sugar and carbohydrates, enhancing porosity and surface area of biomass and, therefore, improving enzymatic hydrolysis [19]. The results also suggest that thee pretreatments enhanced the activity of cellulase in catalyzing the process of cellulolysis, which involves the decomposition of cellulose and some related polysaccharides. Cellulases breaks down cellulose molecules into monosaccharides, such as beta-glucose, or short polysaccharides and oligosaccharides [20]. The process of hydrolyzing jeft (the word is used in some Arab country, which is olive waste dried and compact to billet shape) into its monomers was conductted. A combination of two enzymes, namely cellulase and β-glucosidase, were used in the hydrolysis process to convert glucose to ethanol [21]. The highest yield of 85.02% glucose was obtained using cellulase concentration of 15 mg/g jeft, while β-glucosidase concentration of 20 mg/ml jeft produced 512 mg/dl glucose after 8 h. Saccharification of cellulosic waste with different treatment methods was studied earlier [9]. The pretreatment of cellulosic waste with 1% of HCl and H 2 SO 4 produced 21 and 15.8 g, respectively, of reducing sugar/ 100 g of cellulosic waste. In comparison, hydrolysis with Streptomyces sp. B 167 enzymes resulted in a significantly higher amount of reducing sugar yield (25 g/ 100 g cellulotic waste).

The fermentation stage
Different pretreatments were applied to olive solid waste to increase sugar content. Several fermentation experiments were conducted to determine the suitable number of cells for inoculating the fermentation medium that contains monosaccharides. Based on the haemocytometer method [22], four different initial yeast cell numbers (5.55×10, 5.55×10 3 , 5.55×10 5 , 5.55×10 7 cells/ml) were used in the fermentation medium. Based on the fermentation experiments, it was found that the best inoculum size was 5.55×10 5 cells/ml, which increased ethanol yield by about +0.2 g/l each 24 h, while the inoculum size of 5.55×10 7 cells/ml increased ethanol yield by about +0.1 g/l.. Therefore, the inoculum size of 5.55×10 5 cells/ml was used in all the following experiments of ethanol production ( Table 3). High ethanol production depends on high cellulose and hemicellulose concentrations. Increases in cellulose and hemicellulose content are best affected by removal of non-cellulose components by pretreatment. The presence of lignin is a key limiting factor for the saccharification of lignocellulosic feedstocks [23]. The mean concentrations of ethanol produced from olive solid waste samples, without pretreatment, were 0.264, 0.301, 0.445, and 0.373 g/l, respectively, after each day of the four days incubation period, while they were 0.261, 0.310, 0.737, and 0.100 g/l, respectively, during pretreatment with H 2 SO 4 (2%). The statistical comparison made between the days of pretreatment of samples with H 2 SO 4 showed significant differences in the yield gained in these days at p≤ 0.05, as listed in Table 3. Also, the mean concentrations of ethanol produced from olive solid waste samples pretreated with cellulase were 0.233, 0.399, 0.510 and 0.361 g/l, respectively, after each day of the four days incubation period, while they were 0.275, 0.528, 0.696 and 0.446 g/l, respectively, for samples pretreated with NH 4 OH. The statistical comparison made between the days of pretreatment of samples with cellulase and NH 4 OH showed significant differences in the yield of ethanol (p≤ 0.05), as listed in Table 3.
The mean ethanol concentrations produced from olive solid waste samples pretreated with HCl 5% were 0.249, 0.435, 0.564 and 0.412 g/l, respectively, after each day of the four days incubation period. The statistical comparison revealed significant differences between these values at p≤ 0.05 (Table 4). After 3 days of fermentation of olive waste, significant differences were observed in ethanol yield among all pretreatments (p≤ 0.05). The higher production of ethanol, after three days, was 0.737 g/l in olive waste samples pretreated with H 2 SO 4 2%, while the lowest production was 0.445 g/l in untreated samples (Figure 4). The amount of ethanol produced was decreased in the fourth day. It has been reported that the fermentative activity of non-Saccharomyces yeasts is manifested in the presence of small amounts of oxygen, which lead to an increase in cell biomass and a decrease in ethanol yield   [24]. Although ethanol is a final product of anaerobic fermentation of sugars by yeast, it is toxic to yeast cells and induces stress responses, such as the expression of heat shock proteins and the accumulation of trehalose [25]. A 6% concentration of ethanol was reported to decrease the growth rate of the cells by 50% [26].
A similar study [27] studied the use of rice cake to produce ethanol using a collection of yeasts, including Rhodotorula minuta. Rice cake was mixed with raw starch digesting enzymes of Aspergillus niger, Lactobacillus fermentum, R. minuta, Rhodotorula mucilagnosa, Candida krusei, and Kodamara ohmeri into different fermenting chambers. R. minuta showed the highest efficiency of ethanol production (52.06%) at temperature of 30°C and pH of 2.58. Similar results were recorded by another study [13] which applied separated hydrolysis and fermentation processes on hydrolysate that undergoes detoxification. Issatchenkia orientalis was the most efficient in producing of ethanol when supplemented with glucose. Using simultaneous saccharification and fermentation processes following pretreatment of olive mill solid wastes (OMSW), the average ethanol yield was 3 g/100 g dry OMSW. The results are comparable to those obtained elsewhere [28], in which one gram of extracted olive pomace was used as substrate for bioethanol production. Enzymatic hydrolysis of extracted olive pomace pretreated with dilute acid hydrolysis was investigated. The enzymatic hydrolysis and bioconversion were firstly carried out by separate hydrolysis, fermentation, and presaccharification processes, followed by simultaneous saccharification and fermentation. The latter process showed a relatively higher bioethanol fermentation yield (0.46 gg-1) when compared to the separate hydrolysis and fermentation processes.
Fermentation experiments showed that there are many factors that could affect ethanol production from olive waste. These factors include sugar content produced during pretreatment, type of yeast used in fermentation process, pH value during the simultaneous saccharification and fermentation processes, and temperature.

Conclusions and Recommendations
The different pretreatments caused different levels of efficiency in converting the lignocellulosic material in the treated olive waste into monosaccharaides. Pretreatment of raw material with H 2 SO 4 for 24 h and NH 4 OH for 48 h demonstrated the highest sugar conversion rates in relation to contact time. The average ethanol yield was 0.59 g/10 g dry olive solid waste. The yeast R. minuta has a promising future for bioethanol production.