The Effect of Bacillus subtilis on Bioethanol Production from Ambon Banana ( Musa paradisiaca var. sapientum Linn ) Peels by Using Fermentation Process

- The breakthrough to optimize Indonesia's energy is by utilizing abundant renewable energy sources. Its geogaphic location has the potential to become a country with the largest renewable energy source in the world. The potential energy that comes from vegetables is called bioethanol. Bioethanol is an organic fuel produced by bioethanol fermentation. It can be an alternative material for making sanitary products amid the increasing spread of COVID-19 in Indonesia. So the authors try to innovate how to use Ambon Banana (Musa paradisiaca var. sapientum Linn) peels to become bioethanol. This research aimed to investigate the effect of Bacillus subtilis in the fermentation of bioethanol production from Ambon banana peels. The methods used were pretreatment, hydrolysis, fermentation, and distillation. Bacillus subtilis and Saccharomyces cerevisiae ratio (10: 5 and 5: 5) use for obtaining high ethanol yields, as well as variations in pH 2 and 6 in the fermentation starter. The variable were designed by using Factorial Design. The result shows the yeast ratio of B. subtilis and S. cerevisiae (10: 5) resulted the highest concentration of bioethanol (6%) in 6 days. In acidic conditions (pH 2), the bacteria don’t gow optimally. The higher concentration of bioethanol (6%) was reached in pH 6. The research supports the Government Progam Reducing the dependency of fossil fuels and innovation to produce sanitary product from bioethanol.


Introduction
The world's energy demand has recently increased significantly due to the gowth in population. According to projections of the World Energy Agency by 2030 will increase world energy demand by 45% or an average of 1.6% per year [1]. Fossil fuels supply 80% of the world's energy right now. There are numerous of innovations that develop the renewable energy, such as biodiesel, bioethanol, and other products derived from renewable materials to overcome the problem.
At the conclusion of 2019, the coronavirus strain was at first found in Wuhan City, China. In Indonesia the number of covid-19 cases increases rapidly. Consequently, Indonesia take a decision Large-scale Social Limitation (PSBB -Pembatasan Sosial Berkala Besar). [2]. This policy has an impact on the economic sector and makes ethanol prices increase Bioethanol is ethanol or ethyl alcohol (C2H5OH) that produced from the fermentation of glucose (C6H12O6) from vegetable raw materials. Bioethanol is one of the biofuels is required to be used for alternative energy (PP No. 5 of 2006) [3]. The reaction formation of ethanol [4] : Biofuels are normally produced by fermentation of agicultural wastes, fruit wastes, and industrial wastes using Saccharomyces cerevisiae as nutrition for the microorganisms. The complexity of the manufacturing technique relies upon on the feedstock [5].
Journal of Vocational Studies on Applied Research, Vol.3(2), October 2021 The major challenge of production ethanol is to convert the biomass into ethanol using fast, cheap and efficient methodologies. In industrial system of ethanol production operate under non-steril condition that slow down the fermentation process [6]. The fermentation process takes starch and cellulose from plants to make Bioethanol. Bioethanol also can be made from waste materials such as potato peels, sugar canes, rice, and banana peels. The high human population causes high waste generated [7]. The major problem is the waste in the city accumulates causing an environmental problem [8]. Banana peels are organic wastes that have economic value.
The main ingedient used to make bioethanol in this study is Ambon Banana Peels. The high level of banana consumption shown from 90 million tons of bananas are produced mainly in tropical areas such as in Africa (13%), Central and South America (28%), and Southeast Asia (47%). Of the banana production, 40% is in the form of banana peel waste. This waste is decomposed easily because of the high water content. The high water content allows microorganisms to gow and accelerate decomposition. Banana peels also have various ingedients such as vitamins and minerals, besides having a lot of water content [8]. The existing research shows that banana peels contain high antioxidant activity (94,25% at concentration of 125 mg/ml) [9].
Banana peels contain monosaccharides, especially glucose (8.16%) [10]. Chemical Composition of Musa sapientum (Banana) peels shows that 10 gams of banana peel samples contain 59% carbohydrate [11]. Banana peels contain specific nutrition which depends on the stage of maturity and the cultivar. Banana peels contain an average of 6-9% protein and 20-30% fibre. [12] The fermentation process uses Saccharomyces cerevisiae and Bacillus subtilis bacteria to support the fermentation process and upgade the quality of ethanol. The the common probiotic concept states probiotics are one or more microorganisms that have beneficial effects on the host, able to persist in the digestive tract due to their tolerance to different conditions (acid & bile salts) [13].
Wild-Bacillus subtilis genus ferments 20 g/liter glucose for 48 h, producing lactate and butanediol, but not ethanol or acetate. To create the ethanologenic formula B. subtilis, reconstitution was used to disrupt the natural lactate dehydrogenase (LDH) (ldh) gene by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and dehydrogenase II gene (adhrom) liqueur under adhrom. natives of ldh. B subtilis 35 produced ethanol and buta nediol. Cell gowth and glucose utilization rates have been reduced by 70% and 65%. [14].

Methods
The operating variables during the research consisted of control variables and independent variables.

• Independent Variables
The independent variables including the ratio of sample and water, pH, and ratio B. subtilis and S.cerevisiae shown in

• Experimental Process
Material Pretreatmnet -Prepare banana peels (2 kg), wash banana peels with water -Smooth the banana peel with a ginder or blender.
-Prepare eight beaker glasses, add 200/300 g of smooth banana peel according to the design and add water with a ratio of 1: 3 or 1: 2 to each beaker glass -The sample is ready for hydrolysis -Analysis of glucose Level Fermentation -Put in 100 ml Erlenmeyer filtrate, then add 12 gams of Urea as nutrition and stir until homogeneous -Variation of pH (2 and 6) by adding H2SO4 -Add Fish Probiotic containing B.subtilis 5 or 10 ml according to the variables in each sample -Make a gowth medium for fermentation.
-Fermentation in six days.

Destilation
-Separate water and alcohol -Destination the Liquid containing alcohol to obtain a higher purity -Arrange the destilation tools -Heat to a temperature of 78 o C (keep the temperature constant) for 90 minutes)

Analysis
Sample (Ambon Banana Peels) will be analyzed the percentage of glucose (%) and the product will be analyzed the physical characteristic including product volume, product yield, product density, and product Specific Gavity. Therefrence used to analyzed the level of glucose on sample is SNI 01-2892-1992 by Luff Schrool method. The percentage of Glucose is obtained by calculating based on the following equation V2 : Glucose (from Luff Shcrool Glucose Determination Table based on Na2S2O3 used (mg) ) FP : Dilution Factor W : Sample mass (mg).   Table 3 shows the volume results from the distillation. In the ratio sample: water (1: 2) resulted the highest volume was 91 ml from 4 experiments with an average of 65.75 ml. Whereas in the ratio sample: water (1: 3) resulted the highest volume was 69 ml from 4 experiments with an average of 64.25 ml. From the results obtained that the more the banana sample ratio, resulting the more volume of product. The more samples used will enrich the glucose for fermentation. Ambon banana peel as containing 68.9 g of water per 100 g. [16] Another ingedient that produces the higher volume is the water content of the sample, the more samples used will make the water involved in the fermentation process and also the possibility to pass the distillation process.  Table 4 shows the density analysis result of two types of variables (pH 2 & pH 6). The density values obtained in four experiments with a pH of 6 (0.9852 g/ml) are lower than the density values in the pH 2 (the lowest is 0.9872 g/ml.). The alcohol fermentation process can run optimally and produce a higher alcohol conversion ranges from pH 4 to 6 [17]. The density data obtained can be converted into alcohol content, where the lower the density resulted in a higher level of alcohol. The density standard of bioethanol is 0.790 g / ml [18]. From the research, the density value obtained is still far from the bioethanol density standard due to the distillation process. The study still uses simple distillation and manual temperature indicator, so that the possibility of the temperature exceeding the boiling point of alcohol increases and makes water enter the product.

Ethanol Concentration Analysis
This research using the conversion table for the density -ethanol concentration of the Badan Standardisasi Nasional to obtain the ethanol concentration. The results are shown in Figure 4.3 that shows the highest ethanol concentration obtained in the two variables was 6%. However, the average ethanol concentration obtained for the variable Sc: Bs (10: 5) ratio was higher (4.75%) compared to the Sc: Bs (5: 5) variable (4.5%). The more B. subtilis bacteria made the product have a higher average ethanol concentration. In the anaerobic fermentation process B. subtilis contains pads and adhB genes, which can help ethanol production, but the presence of the gene reduces the gowth rate of B. subtilis, and increases glucose consumption. It requires more glucose-containing materials to produce ethanol [14].

Specific Gavity Analysis
Specific Gavity is a ratio between fluid density and water, standard density at the same temperature. In this research, the result of Specific Gavity obtained an average value of 0.992. The result indicates that the yield of product distillation has not been maximum, because the Specific Gavity of the fuel from organic waste ranges from 0.734 -0.829 [19]. From the results obtained in the research, the average value of Specific Gavity was 0.992. These results indicate that the quality of bioethanol products is still not maximum. For this reason, it is necessary to do several distillations to get maximum result, because the fuel that has a high Specific Gavity value will reduce the quality of the fuels.  Figure 1 shows that the highest of the volume of bioethanol products does not always produce more ethanol content. The results show that the ethanol content fluctuates with the increasing volume of bioethanol products. Based on the result of linear equation (y = 0.25x + 3.5), it shows that a positive gadient value can make it possible to produce higher ethanol content as volume increases. However, the fermentation process also depends on the type of yeast or bacteria used, fermentation temperature, pH, sugar concentration, nutrient concentration, and other factors that can affect bacterial activity.  Figure 2 shows that the higher the density value, the ethanol content will decrease. This is shown in the linear equation obtained (y = -0.4643 + 6.7143). The linear equation obtained has a negative gadient value, so there is a possibility that the higher the density, the lower the ethanol concentration. High density indicates a high moisture content in the product because the density of the research results is closer to the density of water than the bioethanol density standard used (0.790 g / ml) [18]. A higher density value will make the consumption level of the fuel increase and a higher density will result in a smaller calorific value, thus reducing the quality of the fuel according to [20]. The alcohol concentration produced in the study shows a decrease when there is an increased density and also shows that there is a decrease in the quality of the bioethanol product and the smaller the purity value.  Figure 3 shows the relationship between product density and product volume. The figure shows that as the volume of the product produced from the distillation process increases, the density value tends to decrease. The tendency of the density value to decrease is represented by Density ( g/mL) a linear line with the equation y = -0.0003x + 0.9988. The data obtained shows a fluctuating value, but a gadient value of -0.0003 shows a tendency for the density value to decrease. High-density values (above 0.98 g / ml) indicate that the results of the fermentation and distillation processes are not optimal. To get a high density and purity value requires several times the distillation process.

Relationship Between Density and Ethanol Concentration
According to [20] shows that the distillation process takes 14 times to produce high purity and low density. So that further research is needed multilevel distillation or several distillations to get high ethanol concentrations and high volumes.

Conclusion
The results can add references to the research and development of renewable biofuels to keep energy safe. In this context, to make bioethanol use the material from Ambon banana peels with variable types of yeast (B. subtilis from fish probiotics) and the pH variable fermentation. B. subtilis from probiotics affect the results of the alcohol concentration obtained. The ratio of B. subtilis and S. cerevisiae (10: 5) resulted in the highest concentration (6%) ethanol concentration and pH affects the yield of bioethanol products. In acidic conditions (pH 2), bacteria cannot gow and develop optimally, so they cannot produce fermentation products maximally, at alkaline conditions (pH 6) where the environment supports fermentation, obtained with higher concentration ethanol of 6%.

Acknowledgment
The author would like to acknowledge the Industrial Chemical Engineering Department of Vocational School in Diponegoro University to facilitate the laboratory.