Effect of durian and jackfruit seed starch combination variation on bioethanol content using Aspergillus niger and Saccharomyces cerevisiae

Durian and jackfruit seeds are around 5-15% of whole fruit, and only few is utilized as animal feed and generally become waste. Based on the Indonesian Central Bureau of Statistics, durian production in Indonesia reached 1.35 million tons in 2021 and increased by 19.40% compared to 2020. The government issued a policy to encourage the development of renewable energy sources as an alternative to fuel oil because the oil supply will run out in about 12 years. The government targets the use of renewable energy in 2025 to reach 23% by preparing a program to implement the use of bioethanol in motor vehicles and preparing the bioethanol industry in Indonesia. The purpose of this study is to get the best seeds combination to produce highest bioethanol content. Research used 50 gram 80 mesh durian and jackfruit seed starch combination and was started with multistage fermentation with addition of Aspergillus niger for 72 hours to produce glucose then continued with Saccharomyces cerevisiae for 120 hours to produce ethanol. The next stage was to distill for 6 hours to separate ethanol and water using the boiling point difference principle. The ethanol will be analyzed using refractometer. The highest ethanol content obtained was 20% from a combination of 25% jackfruit and 75% durian seed starch. This ethanol content was higher than the previous research with 100% durian seed starch which was up to 1.61% and for 100% jackfruit seed starch was up to 2.435%.


Introduction
Durian (Durio zibethinus Murr.) and jackfruit (Artocarpus heterophyllus Lamk.) are tropical plants that are widely planted in tropical regions such as Indonesia.Based on the Indonesian Central Bureau of Statistics, durian production in Indonesia reached 1.35 million tons in 2021.This number increased by 19.40% compared to the previous year which amounted to 1.13 million tons and jackfruit production in Indonesia reached 906,514 tons in 2021 which also increased compared to the previous year which amounted to 824,068 tons.Durian and jackfruit seeds are around 5-15% of whole fruit, and only few is utilized as animal feed and generally become waste [1].
Indonesia's current energy needs are still largely based on fossil fuels.Oil reserves will run out in about 12 years, gas will only last 30 years and coal can still be utilized for the next 70 years.This dependence on fossil fuels is a big problem and needs urgent solutions.One step is to utilize bioethanol as an alternative replacement [2].Bioethanol (C 2 H 5 OH) is a biochemical liquid from the fermentation process of sugar from carbohydrate sources using the help of microorganisms that can be used as an alternative to fossil fuels [3].
The utilization of bioethanol in Indonesia is currently still in the trial stage.The use of bioethanol fuel is still limitedly realized in 2 areas only, namely Jakarta and Surabaya where with a mixture of 5% bioethanol in gasoline or as E5 (Ethanol 5% and 95% gasoline).The benefits of using bioethanol can reduce greenhouse gas emissions, create potential employment in the agricultural sector and bioethanol production, and reduce imports of gasoline fuel oil.The Indonesian government targets to increase the renewable energy mix to reach 23% by 2025.To encourage it, the government has developed a program to implement the use of bioethanol in motor vehicles and prepare the bioethanol industry in Indonesia.
Durian seeds are biomass waste that is underutilized by the community, even though after research durian seeds contain carbohydrates 43.6 g -46.2 g per 100 g of durian seeds that can be converted into glucose [4].While in jackfruit, each 100 g of seeds contains carbohydrates that are also quite high, which is 36.7 g [5].Aspergillus niger fungus is more likely to break down starch into glucose because it has alpha-amylase and glucosidase enzymes, therefore starch will be broken down into glucose more optimally by Aspergilllus niger fungus [6].The process of converting glucose into ethanol by Saccharomyces cerevisiae is due to the activity of the enzymes invertase and zymase produced by the fungus.Starch degraded into glucose by the fungus Aspergillus niger as a biocatalyst will be able to accelerate the process of breaking down starch into glucose which is then followed by converting glucose into ethanol by Saccharomyces cerevisiae where these two fungi are expected to synergize in the process of ethanol formation [7][8][9][10][11][12][13]. So, the purpose of this study is to get the best seeds combination to produce highest bioethanol content.

Materials and Equipment
This research was carried out from March to August at the Biosystems Laboratory, Department of Agricultural Engineering and Biosystems, Faculty of Agriculture, University of North Sumatra.The materials used in this research are durian fruit seeds, jackfruit seeds, Aspergilus niger and Saccharomyces cerevisiae, HCl PA which will be diluted to 0.3 M, NaOH 30%, HCl 3%, H 2 SO 4 25%, CaCO 3 , sodium thiosulfate, Luff-Schoorl solution, potassium iodide, urea, ammonium sulfate, PDB and distilled water.
The tools used in this research are blender as a material crushing tool, 80 mesh sieve to filter the material, analytical scales to weigh the material, measuring cup to measure the solution to be reacted, erlenmeyer as a place of fermentation, aluminum foil as erlenmeyer cover during the fermentation process, universal pH to measure pH, distillation device to separate alcohol with fermentation results, paper to write the resulting data and mobile phone as a documentation tool during the research.

Method
The research method is non factorial completely randomized design with three treatments and three repetitions.The treatments are symbolized by X, Y and Z which X is combination of 25% durian seed starch and 75% jackfruit seed starch in mass base.Y is combination of 50% durian seed starch and 50% jackfruit seed starch in mass base.And Z is combination of 75% durian seed starch and 25% jackfruit seed starch in mass base.

Sampel Preparation.
Washing durian seeds and jackfruit seeds, slicing with a thickness of 2-3mm, removing mucus on the seeds with salt, then soaked with lime water for 1 hour.Then dried and pulverized to 80 mesh.

Preparation of fermentation materials.
Weighing each ingredient according to the treatment, then mixed and put into a 1 liter jar.Added distilled water as much as 300ml, then added Aspergillus niger and allowed to stand for 3 days to obtain glucose.

Fermentation.
Adding saccharomyces cerevisiae as much as 3% of the ingredients, mixed well then allowed to stand for 5 days.The fermentation results will be filtered and then distilled.

Distillation.
The distillation process will be carried out at a temperature of 78 -80 ° C, because the boiling point of ethanol is 78.3 ° C and the boiling point of water is 100 ° C.So with this ethanol will be faster.So with this ethanol will evaporate faster.

Ethanol content test.
In testing the ethanol content produced, it can be measured using a refractometer.The working principle of the refractometer is light refraction.The solution will be dripped on the glass and then illuminated by light.According to Snellius law, light will experience refraction whose angle of incidence is equal to the angle of refraction.

Glucose levels
Samples before fermentation will be analyzed for glucose content using the Luff-Schoorl method.This method is one method that is widely used in analyzing the glucose content of a material because in addition to being frequently used, this method also has an error rate of 10%.  1 shows that glucose average level from sample X was 19.8%, while sample Y was 26.3% and sample Z was 31.65%.The highest glucose content was found in sample Z which was up to 31.65%.Table 1 indicates that the higher seed starch composition, the higher glucose content that will be produced and vice versa.

Yield
Yield is the ratio of the material produced to the total raw material used as base material.The yield is expressed in percent (%).There are two yields that will be analyzed, namely fermentation yield and distillation yield.

Fermentation yield.
Fermentation is a process of converting starch into alcohol with the help of Sacharomyces Cerevisiae.The fermentation time is 7 days.Fermentation yield is the percentage between the volume of fermentation that has been filtered divided by the volume before filtering.

Figure 1. Fermentation yield
Based on the Figure 1, it can be seen that the highest average yield is found in sample Z fermentation with an average percentage of 34.75% while the lowest yield is in sample X fermentation with a percentage of 31.42%.Fermentation yield can be influenced by sugar concentration, additional nutrients, fermentation pH, temperature, and also the time required in the fermentation process.
Based on the three samples, the yield of fermentation results that look not so different, this is because the treatment of the three samples is the same, which is at the same volume, the same temperature, and at the same fermentation pH.

Distillation yield.
Distillation is the process of separating solutions with the principle of boiling point difference.The yield of distillation is the percentage between the volume of distillate divided by the volume of fermentation that has been filtered.

Figure 2. Yield from distillation
Based on the Figure 2, the highest average distillation yield is in samples X and Y, which is 9.16% and the lowest in sample Y is 7.5%.Based on the three samples, the distillation yield is not so different, this is because the volume used in each treatment is the same, the time for distillation for each treatment is also the same, namely 6 hours, as well as the temperature and pH given to each sample treatment is also the same.

Ethanol Content
Based on the research that has been done, the ethanol content obtained can be seen in Figure 3. Based on the Figure 3, it can be seen that the highest ethanol content is found in sample Z (75% durian seed starch and 25% jackfruit seed starch) with a percentage of 20% while the lowest ethanol content is found in sample X (25% durian seed starch and 75% jackfruit seed starch) with a percentage of 4%.From the three samples, it can be seen that the difference in ethanol content produced is directly proportional to the percentage of starch content contained in the seeds.The higher the starch content of the main raw material, the higher the glucose content and of course the ethanol content will also be higher.Sample Z with a combination of 37.5 grams of durian seed starch and 12.5 grams of jackfruit seed starch produces the highest ethanol content, this is because the glucose content produced is also higher than the other two samples.High glucose levels are influenced by the starch content in the raw material, namely the higher the starch content in a raw material, the higher the glucose content that will be produced with the help of Aspergillus niger.With a glucose level of 31.6%, after fermentation it will produce 20% ethanol.Based on the results of research that has been done, ethanol with a content of 1% -5% can be used as a flavoring agent in medicines, so that the taste of the medicine solution is more preferred.Ethanol with a content of 18% or more can be used as a preservative in medicine.While the use of ethanol with a content of 20% is used to increase the solubility of medicines with the main solvent of water in drinking medicines.