Production of Bioethanol From Kappaphycus alvarezii Algae by Using Pichia kudriavzevii

47 Abstract Kappaphycus alvarezii is a red algae that can be used as alternative raw material for bioethanol production. This is because K. alvarezii contains a high carbohydrate that reaches 60%. This study aims to determine the effect of fermentation nutrition and fermentation duration of hydrolysis results of K. alvarezii algae on pH changes, sugar levels, cell biomass and ethanol content. There are two methods was used in this research, namely hydrolysis method and fermentation method. The hydrolysis method was used α-amylases enzyme which is 150 KNU/L with 0.5%, 1.0%, 1.5% and 2.0% concentrations. The next method, K. alvarezii algae was fermented by Pichia kudriavzevii and used Gandasil-D® as an anorganic suplementation and yeast extract which is 0 g/L, 1 g/L, 2 g/L and 3 g/L. The results showed that K. alvarezii can be fermented into bioethanol after enzymatic hydrolysis process. The optimal ethanol content was produced at 48 hours of incubation duration. ISSN : 2580-2410 eISSN : 2580-2119

Bioethanol is an organic compound with the chemical formula C2H5OH that resulting from the fermentation process of starches, sugars and cellulose with the help of certain microorganisms (Kumar et al., 2013;Sankh et al., 2013). Bioethanol production was developed as fuel replacement fuel with fuel grade ethanol ≥ 99.5% to compensate for the scarcity of petroleum resources. It can be an alternative energy because the oxygen content was high, environmentally friendly, and its energy source was can reneweble (Kumar et al., 2013;Madigan et al., 2012).
Bioethanol produced from biomass is usually produced by biochemical processes such as fermentation process. Biomass which is used as raw material of bioethanol is corn and sugar cane where the raw material is a food material and it takes a large area to produce it. The existence of microalgae is very potential in the production of bioethanol to replace raw materials that still have high food value. Microalgae contains carbohydrates and proteins that can be used as a carbon source in the fermentation process of bioethanol formation (Nicolau et al., 2010).
Another method that can be used is fermentation. One of the potential microbes in the fermentation process is Pichia kudriavzevii. P. kudriavzevii is one of the yeast that is round, elliptical or elongated (Pandey et al., 2000). Previous studies have shown that P. kudriavzevii contains 29.3% palmitate, 8.89% stearate and oleic acid 41.9%. This yeast can also be used as a mixed starter culture on combinations between ethanol-tolerant (Nicolau et al., 2010;Ra et al., 2013). Therefore, this study aims to determine the effect of fermentation nutrition and fermentation duration of K. alvarezii algae hydrolysis results on pH change, sugar levels, cell biomass and ethanol content.

Kapppaphycus alvarezii
This study used the marien algae of K. alvarezii obtained from coastal waters of Punaga village, Mangarabombang District, Takalar regency. Algae that have been obtained are then identified that belong to K. alvarezii type.

Pretreatment Kappaphycus alvarezii Algae
Algae biomass washed and soaked in clean water for 2-3 hours. Then dried under the sun. Algae K. alvarezii that has been dried mashed using a hummer mill and sifted using a 40 mesh sieve. The resulting algae flour was weighed according to a combination of concentrations are 0.5%; 1%; 1.5% and 2% , then dissolved with 50 mL distilled water.

Hydrolysis Kappaphycus alvarezii Algae
K. alvarezii algae solution with variation concentration of 0.5%, 1.0%, 1.5%, and 2.0%. Then, the algae solution was heated using a hot plate at 100 ° C for 90 minutes. Thereafter, an enzyme α-amylase of 80 KNU/L was added. The added solution of the incubated apparatus for 24 hours, 48 hours and 72 hours. Once hydrolyzed, the algae solution is filtered to separate the natan and its supernatant. The supernatant was then centrifuged at 9,000 rpm for 10 min. Supernatant of centrifugation was taken and measured its sugar content.

Fermentation Process
K. alvarezii solution was prepared according to the optimal concentration obtained at the hydrolysis process, which is 0.5% with the addition of 150 KNU/L enzyme α-amylase and incubated for 24 hours. Then, fermentation method was added nutrients in form of Gandasil-D® as an inorganic nutrient and Yeast Extract as an organic nutrition (0.5 g/L and 1.0 g/L). After that, a starter P. kudriavzevi was activated with 5% concentration into the fermentor bottle. The fermentation process are used various fermentation durations (0 hours, 24 hours, 48 hours and 72 hours). After the incubation period of 0 hours, 24 hours, 48 hours and 72 hours, measurements of sugar levels, ethanol content and cell biomass of P. kudriavzevii.

Measurement of Cell Biomass
The cellular biomass of P. kudriavzevii was measured using the method of dry cell weight (DCW = dry cell weight). The dry weight of the cell was carried out by first centrifuging the fermentation medium sample by 50 mL at 9,000 rpm for 10 min. After centrifugation, supernatant and pellet cells are obtained. The supernatant is removed by pipette. After that, the resulting pellets are then washed by adding aquadest on the cell pellets and centrifuged at 9,000 rpm for 5 min. The pellet is then suspended with aquadest and vacuumed with a pipette and then transferred into a filter paper with a pore size of 0.47 μm which has previously been diovened to a constant weight (W1). The filter paper containing pellets is then dried in the oven at 80°C for 24 hours and weighed (W2). The DCW result is the difference between the weight of the final filter paper and the weight of the initial filter paper (W2-W1) expressed by the dry weight of the cell with gram/liter (g/L) unit.

Data Analysis
This study used RAL (Complete Random Design) with three replications. The parameters measured were sugar content and cell biomass. Data were analyzed statistically using Analysis of Variance (Anova) with 95% confidence interval (α = 0.05). The analysis was conducted to compare the effect of acid hydrolysis process on sugar content and fermentation process effect in yielding sugar content and cell biomass from algae K. alvarezii. If there is influence then continued with Tukey test at 95% confidence level (α = 0.05) to know pair of group the same and different data in each treatment.

Sugar Levels in Hydrolysis Process
The results of the measurement of sugar content showed that algae concentration of 0.5% resulted in average sugar content of 0.56 g/g during 24 and 48 hours incubation. While, 72 hour incubation showed the average of different sugar content with other incubation duration, that is 0,47 g/g. Then concentration of 1% yields, mean sugar content of 0.56 g/g at 24 hours and 48 hours incubation. While, the duration of incubation 72 hours resulted in the average of sugar content of 0.46 g/g the concentration of 1.5% on 24 hours, 48 hours and 72 hours incubation time resulted in average sugar content of 0.46 g/g. Whereas at 2% concentration yield average of sugar content equal to 0,49 g/g during incubation period 24 hours, 48 hours and 72 hours.
Based on ANOVA test at 95% confidence interval it can be seen that the concentration of K. alvarezii algae used has significant effect on sugar content, but the duration of incubation does not affect the sugar content, then continued with Tukey test. Then, from the Tukey test it was concluded that the most optimum sugar content produced was at a concentration of 0.5% and 1% marine algae with an incubation period of 24 hours, 48 hours and 72 hours. Therefore, researchers used 0.5% concentration with 24 hours incubation duration. Research used enzymatic hydrolysis method yields sugar of 0.3 g/g of Gracillaria sp. Marine algae (Wu et al., 2014). While Gelidium amansii only produced a sugar content of 0.13 g/g (Ra et al., 2013) and a similar study was performed using Entheromorpha intestinalis marine algae producing a sugar content of 0.21 g/g (Wu et al., 2014). More study by used Saccarina latissima just able to produce 0.12 g/g sugar and Laminaria digitata produce sugar levels of 0.09 g/g (Vanegas et al., 2015). While, in other studies produced sugar content of 0.2 g/g by using the enzyme α-amylase and algae Saccarina japonica as raw materials (Jang et al., 2012).
Based on the comparison of some references, it is known that the lowest sugar content is obtained from research using Laminaria digitata algae that is 0,09 g/g and reference with highest sugar content from Gracillaria sp. of 0.31 g/g of sugar content (Vanegas et al., 2015 andWu et al., 2014). So from the comparison, it can be said that research using K. alvarezii algae, proved higher in producing sugar content of 0.56 g/g. K. alvarezii has a greater chance of producing bioethanol from the resulting sugar content.

Sugar and Ethanol Levels in Fermentation Process
This fermentation process is done during 24 hour interval, 48 hours and 72 hours. The results showed that the sugar content will decrease with the length of fermentation time and ethanol content will increasingly with the lenght the fermentation time. This means that polysaccharides contained in the algae of K. alvarezii algae produce high sugar levels at the beginning of fermentation but because of the use of P. kudriavzevii yeast causes reduced sugar content of yeast to produce ethanol. Based on previous research who said that light will be consumed by the microbes at the beginning of the fermentation period (Wu et al., 2014). The resulting ethanol content also reaches the optimum level.

Figure 1. Average Graph of Fermented Sugar Level with P. Kudriavzevii
Based on ANOVA test at 95% confidence interval it can be estimated that nutritional distance of nutrients and fermentation concentration did not significantly influence to ethanol content. So it can be concluded that the most optimal sugar content produced at hours 0 hours with sugar content of 0.56 ± 0.06 g/g while ethanol content is most generated at 48 hours incubation and ethanol content of 0.28 ± 0.50 g/g. This proves that yeast P. kudriavzevii by using the algae of K. alvarezii algae which has greater potential in producing bioethanol.

Biomass Levels in Fermentation Procces
The success of fermentation can be seen based on the microbial growth response on the fermentation medium. In this study, the measurement of yeast cell biomass P. kudriavzevii was done by using dry weight method (dry cell weight). Cell biomass measurements were performed during fermentation of 0 hours, 24 hours, 48 hours and 72 hours.
The results showed that the longer of fermentation duration so the more cell biomass produced. This means that yeast P. kudriavzevii has used carbohydrates from the algae medium to divide. Based on the research of Widodo et al. (2013) explains that the fermentation time is very influential on the activity of yeast because the longer the fermentation so the more active yeast breeding.
Based on the ANOVA test at a 95% confidence interval it can provide different values of fermented nutrients or unconverted fermented concentration to the biomass of fermentation time cells affecting the resulting cell biomass. The biomass that produces fermented nutrients does not differ significantly with inorganic supplementation and the use of producing similar biomass. It was concluded that most biomass was produced at 48 hours incubation and yielded cell biomass of 0.27 ± 0.003 g/g.

Conclusions
The enzymatic hydrolysis process yields the most optimum sugar content whic is 0.56 g/g at 0.5% algae concentration. Meanwhile, the use of yeast P. kudriavzevii in the fermentation process yields a sugar content which is 0.56 ± 0.06 g/g and ethanol content of 0.28±0.50 g/g at 48 hours incubation duration.