Synthesis of Bioethanol from Cocoa Pod Husk Using Zymomonas Mobilis

Nowadays, energy issue is one of the interesting discussions among the researchers and energy stakeholders. This is due to the reducing of the main source of world energy which consists mostly of non-renewable energy derived from fossils of living things millions of years ago. Alternatively, there are some alternative energy that promising in the future such as bioethanol from plant or fruits. For that reasons, the aim of this research is to make bioethanol from the peel of cocoa fruit. Cocoa pod husk can be used as source of bioethanol through a fermentation process with the help of bacteria. This study will examine the ethanol content in fermented cocoa pod husks. The fermentation time was 0 days, 2 days, 4 days, 6 days, 8 days. The Zymomonas mobilis bacteria is used in the fermentation process and their percentage is 8%v/v, 10%v/v, 12%v/v, 14%v/v, 16%v/v.In the process of fermentation of cocoa pod husk with Zymomonas mobilis inoculum on the 3 day to 9showed an increase. On the eleventh day in all conditions the addition of a starter was decreased. The best condition when fermentation takes place on the 8 day with a starter dose of 14% and obtained an alcohol content of 10.62%.The results of this study indicate that the addition of Zymomonas mobilis starter and fermentation time affect the levels of bioethanol produced. The addition of a starter as much as 14% and the fermentation time for 8 days are the best condition in this study.


Introduction
The depletion of fossil fuel reserves and the increasing human population is very contractive with the energy needs for human survival, economic and social activities. In the last five years, Indonesia has experienced a decline in national oil production as a result of a decline in natural oil reserves in production wells, despite the increase in population, the need for transportation facilities and industrial activities [1]. This results in an increase in demand and consumption of fuel oil which is a non-renewable natural resource. The Indonesian government still imports part of the fuel to meet domestic needs [2]. Up to now there have been quite a lot of biofuels developed from cassava, sugar cane, sugar palm, sorghum and other materials [3]. However, this plant has another use value as food. If the plant is used as raw material in producing ethanol commercially, it will result in the change of food functionality [4].
One of the most potential plants in Indonesia due to its abundant availability is cocoa. According to plantation statistics, the total area of cocoa in Indonesia in 2015 was recorded at 1,622,600 Ha with a production of 641,997 tons of cocoa and a productivity level reaching 800 kg/ha. The weight of the cocoa fruit harvested in Indonesia per Ha around 7751 kg of fruit peels and 2733 kg of wet seeds [2]. If the waste of the husk is not handled properly, it can cause environmental problems. On the other hand, the process of making bioethanol requires raw materials with less economic and do not compete with the food material [5]. Furthermore, the cocoa pod husk has a lot of cellulose allowing it to be used as raw material for bioethanol synthesis [6].
Several studies have been conducted relating to the utilization of organic waste to be used as raw material for making fuel, namely bioethanol. In bioethanol research, most people use Jathropa plant. The yields of the saccharification and fermentation processes could reach a level more than 39% [7]. Besides that, the same enzymatic method can be applied to synthesize biofuel from papaya seed. The vegetable oil from papaya seed that can be converted to biofuel is around 30% [8].
There are various types of microorganisms that are used to produce bioethanol, one of the most widely used microorganisms in the bioethanol company in the world is Zymomonas mobilis. This bacterium can surpass Saccharomyces cerevisiae within this process. The advantages of bacterium are including the growth of facultative anaerobes and having a high temperature tolerance, the ability to achieve higher conversion, resistant to high ethanol levels and low pH [9]. In fact, there are not many studies that reveal the ability of Zymomonas mobilis bacteria to convert cocoa pod husk into bioethanol. For that reason, this investigation will use the waste of cocoa pod husk as a raw material to produce bioethanol. The research aims are to gain the best composition of the volume of Zymomonasmobilis starter and the optimum day of bioethanol production.

Materials and method
The cocoa pod husk that has a yellowish-brown color and medium texture with water content around 60 -80% was used as raw material in this study. This cocoa pod husk was obtained from Kampung Cokelat, Kademangan, Blitar, Indonesia. Bacteria used Zymomonas mobilis included the nutrient broth. And, the supporting materials for analysis are 97% concentrated H 2 SO 4 , NaOH, and glucose. The equipment used are a set of hydrolysis, one set of fermentation unit, and a set of distillation apparatus. Moreover, other supporting equipment such as asthermometers, ovens, analytical balance, autoclaves, incubators and other laboratory tools are utilized in this study.

Preparation
There are two processes that carried out to prepare the cocoa pod husk before fermentation. These two processes are delignification and hydrolysis process. First, 100 grams of the cocoa pod husk were boiled at 70 0 C for1 hour and then were dried at the same temperature for 1 hour. After that, delignification of the cocoa pod husk was done by immersing and mixing this dried pod husk into 500 ml of 10% NaOH solution for 1 hour. Then, this cocoa pod husk was neutralized with distilled water to pH 8.
The cocoa pod husk as much as 100 grams from delignification process before were mixed with H2SO4 10% (v/v) of the total solution. Then, distilled water was added to the volume of 500 ml. Then the hydrolysis process was conducted with a temperature of 100 0 C for 3 hours at 300 rpm. Finally, the solution of hydrolysis was filtered and the filtrate was taken for analyzing the glucose level.

Fermentation process
Zymomonas mobilis bacteria starter was prepared by this following procedure. There are several materials used for bacterial growth media including 6 grams of nutrient broth, 1 gram of glucose, and 500 ml of distilled water. Zymomonas mobilis was inoculated on the media and incubated at 30 0 C for 1 day. Then, 250 ml of filtrate resulting from the hydrolysis process were put in a bottle to be fermented, covered tightly with cotton and aluminum foil. The filtrate was sterilized from the hydrolysis process into the auto clave at 120 0 C for 15 minutes and cooled to 30 0 C. After wards, Zymomonas mobilis bacteria starter was added with the variables carried out of 8, 10, 12, 14, and 16 (%v/v). Then it was fermented with the run time variable of 0, 2, 4, 6 and 8 days. The fermented solution was filtered and distilled in order to purify the ethanol product.

Analysis
There are two compounds analyzed in this study that is glucose as a fermentation reactant and bioethanol product as well. Distillate product that contains ethanol compound was measured its density using pycnometer. Then, its specific gravity was calculated and the ethanol content was obtained from AOAC (Association of Official Agricultural Chemists) table based on the specific gravity value of product.
Otherwise, glucose that was a product from hydrolysis process was measured using Nelson-Somogyi method. 1 ml of sample was taken from hydrolysis filtrate and mixed with 1 ml of Nelson solution. This mixture, then was heated at 100 0 C for 2 hours. After that, the mixture was cooled into room temperature and was added by 1 ml of arsenomolybdate reagent and 7 ml of distilled water. The final mixture was measured its absorbance by Spectrophotometry UV-Vis with 510 nm of wave length. This absorbance value can be converted into glucose concentration.

Results and discussion
Cocoa pod husk is one of biomass waste having good prospect to be utilized as a feedstock for bioenergy production due to its abundant availability. Cocoa pod husk contains high carbohydrate and cellulose components as shown in table 1, so that it must be converted first into glucose via hydrolysis process [6]. This hydrolysis process using H2SO4 solution with 10% (v/v) of concentration yields the product with 9.89% (v/v) of glucose content. This result is quite smaller than Nazir et al. study which cocoa pod husk was treated by the same acid solution yet with different concentration (1.5%w/v) resulting 15.59% of glucose content as reducing sugar [10]. In the fermentation process, there is not only enoughglucoselevels that influences the reaction kinetics,butalsoother factors such as fermentation temperature,optimal pH, microbes, nutrients needed bymicrobesand reaction time [11]. Temperatureaffects the fermentation process as product formation depends on temperature. The temperature used in this study rangesfrom 31-33 0 C or room temperature. WhilethepHused for the growth of Zymomonas mobilis was around 5 [9]. Fermentation was carried out for 192 hours,and with measurements of ethanol levels once every 48 hours. The starting time of reaction at 0 hour were observed at different concentrations of inoculum as a control. The levels of ethanol had been raised along with increasing the fermentation time. For example, in the concentration of inoculum 8%, 10%, 12% and 14%, until the 192hour fermentation shows a graph that continues to increase. It is suspected that until the 192 nd hour, the bacteria are still experiencing a log phase (exponential) to stationary or there are still nutrients added to the fermentation medium such as yeast extract which is a source of nitrogen for the growth of Zymomonas mobilis [9]. So that bacterial cells will grow and divide exponentially to the maximum amount. Conversely at an inoculum concentration of 16%, fer- In the starter 14 hour to 192 hours had an ethanol content of 10.62%. Bioethanol levels that are formed in the fermentation process of cocoa pod husks are quite small on average, this is influenced by several factors, one of them is sugar content. The level of sugar produced in the hydrolysis process of cocoa peel using H2SO4 10% at 100 °C for 3 hours was 9.89%. However, several factors affected the bioethanol synthesis including the concentration of bacteria are not the same and the starter extraction when analyzed must be concurrent. Moreover, the rate of microorganism growth and the rate of production should be in line. And, after delignification, the lignin levels should be analyzed so that, we can find out what percentage of residual lignin levels that can interfere during fermentation. A few bioethanol contents in the product gives a low value of process yield as shown in figure 3. The maximum yield was achieved at 5.45% when 14% (v/v) of bacteria concentration was applied for 192 hours of fermentation.

Conclusions
Bioethanol levels that are formed in the fermentation process are influenced by several factors one of which is reducing sugar levels. Reducing sugar levels produced in the hydrolysis process of cocoa peel using H 2 SO 4 30% at 100 °C for 3 hours around 9.88%. Optimal fermentation conditions were obtained at 14% Zymomonas mobilis bacteria with 192 hours fermentation time at 30 ° C which produced the highest ethanol content of 10.62% from 25 ml of samples obtained after the distillation process.