ETHANOL FERMENTATION POTENCY OF WILD YEAST ON BAMBOO RHYZOSPHERE

Bamboo plant rhizosphere known as source of simbiotic useful microorganisms, including yeast. Wild yeast explored should be tested it’s adaptability to new ecology especially nutritional source availability. The research aim to get potential yeast which can work well during fermentation process in apple juice substrate. We were isolated yeast from three different locations: Ketawanggede District, Karangploso District, and Lowokwaru District. All locations was located around Malang city. Result showed that there were found 13 isolates yeast: Protomyces sp, Agaricostilbum


INTRODUCTION
Yeast is one of microorganisms belong to unicellular fungi.Based on the type of yeast metabolism, yeast can be divided into two groups: fermentative yeast and oxidative yeast.Fermentative types can perform alcoholic fermentation, which breaks down sugar (glucose) into alcohol, lactic acid and gas.
Yeast has been used for industrial processes such as the manufacture of alcoholic beverages, fermented tape, the manufacture of animal feed, cosmetics, and antibiotics (Tanaka, et., al., 1990;Ardhana, M.M. and G.H. Fleet, 1989).Yeast in the future can be developed as renewable resources, because some types of yeast capable of producing alcohol from a variety of different types of carbohydrates.Various exploration will find yeasts especially the types of yeast that have potential in the fields of industry, particularly in the production of bioethanol (Lansane, B.K., G. Vijayalakshi, M.M. Krishnaiah, 1997).
Research on yeast is mostly done in exploration of various ecosystems in Indonesia.It is believed that the amount of yeast in nature is much higher than yeast that has been known for.Research has been done that yeast exploration conducted on the district of Jember, East Java Some yeast isolates were obtained among other Candida sp. 1, Candida sp. 2, Candida sp. 3, Debaryomyces sp. and Kloeckera sp.(Muhibuddin, A and I.R. Sastrahidayat, 2015;Ivanesthi, IR., S. Nurhatika, dan A.Muhibuddin, 2016).
The purpose of this research was to observe the potential of wild yeast from bamboo ethanol fermentation, because it's potential (Singhal, P., 2013) from February till June 2016.Soil samples obtained from bamboo plant roots in three locations: Ketawanggede District, Karangploso District, and Lowokwaru District.

Materials
The laboratory equipments were used in the research consisted of plastic Materials used in the research include YMA media (Yeast Malt Agar), YMB media (Yeast Malt Broth), SB media (Saboroud Broth), alcohol 70%, NaOCl, distilled sterile water, spirits, matches, composite soil samples, (NH4)2SO4, H2SO4, and apples.

Research Design
The research was conducted using 14 treatments and each treatment was repeated three times.
The treatments were:

Soil Samples Exploration
Samples isolated by collect composites soil samples from the bamboo plant rhizosphere.Soil sample was taken at 15 cm depth (Ashliha et al., 2014).

Yeast Isolation and Purification
10 grams soil sample put to the Erlenmeyer that contained 90 ml of distilled water.The mixture then homogenized and deposited ± 5 minutes.Supernatant was taken at 10 ml and put in a flask contained 40 ml of media YMB.Then incubated on top orbital shaker at room temperature for three days.For dilution taken 1 ml suspension from the Erlenmeyer contained isolates from YMB, then put into a test tube that contained 9 ml sterile distilled water and gained up to 10 -5 serial dilution.After that, taken 0.1 ml suspension and inoculated on YMA media with spread methods.Yeast grown on YMA media incubated at room temperature for about 3 days and purification to obtained pure colonies.Observations were made on the characteristics of the macroscopic colonies grown on media (Ashliha et al., 2014).

Yeast Identification
Yeast isolates were identified to the genus by referring to the identification guide book "The Yeast a Taxonomic Study".Observations were made macroscopically and microscopically.Macroscopic observation was based on the appearance of colony morphology upon isolation and purification include the shape, texture, color, surface, elevation, and the waterfront.Microscopic observations on yeast include cell shape, size, type of budding, presence or absence of hyphae or pseudohyphae and spore types were obtained from isolates (Widiastutik et al., 2014).Microscopic observation use preparation cultured on glass objects and characters seen in microscope (Ashliha et al., 2014).

Yeast Growth
Yeast growth in liquid media test to know the mechanism of carbohydrate utilization by the yeast.One ose colonies of YMA was added to test tubes that contained 10 ml SB media and grown for 24 hours.Yeast oxidative in SB media will form a layer or pellicle on the surface of the media, while the 123 fermentative yeast will form a layer or pellicle on the basis of media (Jumiyati et al., 2012).

Substrate Fermentation
5 kg apple was washed with tap water.Then the apples crushed using a blender with add water ratio 2:1 (w/v).and then squeezed to separate the fiber and fruit extract.Pasteurization apple juice at temperature of 65-70º C for 15-20 minutes.Then added 1% (NH4)2SO4 (v/w) and 10% sugar (v/w) to the apple juice.Then added H2SO4 or NaOH in apple juice to adjust the pH to the range 4.5-5 for optimum growth of yeast (Sigit et al., 2014).

Yeast Activators
Apple juice prepared in the Erlenmeyer flask for diluted up to 100 ml.Then added 1% (NH4)2SO4 (v/w) in apple juice.Apple juice heated up to boiling and cooled in a state phase.Then added H2SO4 or NaOH in apple juice to adjust the pH optimum conditions growth of yeast.Add 20 ml the apple juice solution to the Erlenmeyer.After that, added one ose yeast isolates that taken from propagation on YMA media in apple juice solution and incubated on an orbital shaker for 24 hours (Santi, 2008).

Fermentation Test
100 ml fermentation substrate was added to the bottle fermentation.After that, added 6% yeast activator solution.Then covered the bottle fermentation with aluminum foil to exclude the contamination by other microbes.The fermentation process was conducted for 72 hours (Sigit et al., 2014).

Variable Observations
The exploration data was analysed by Diversity Index (H '), Evenness Index (E), and the Dominance Index Observation of physical characteristics such as color observed by observe color changes during fermentation.Physical characteristics such as smell was observed during fermentation.Both above characteristics were observed every 24 hours.Observation cells numbers was observed every 24 hours.The number was calculated using a UV-VIS spectrophotometer with a wavelength of 600 nm (turbidimetry methods).pH and physical characteristics such as temperature was observed using T1 900.
The ethanol percentage was measured using picnometer and rotary evaporator.The rotary evaporator was used to gain the ethanol solution from the fermentation medium, while the picnometer was used to measure the ethanol value once evaporation was performed.The ethanol value measurement was done by obtaining the specific weight with picnometer.First, the picnometer was filled with aquades, then both were sealed and weighted, resulting in W2; the picnometer was thereafter emptied, the remaining aquades absorbed with acetone.The picnometer was dried in an oven and thereafter weighted for the second time, resulting in W1.The weight of aquadest (W) was obtained by substracting W2 to W1.

Data Analysis
Quantitative data analyzed descriptively using charts.The qualitative data analyzed descriptively.

pH
Decreasing of pH from all treatments indicates that there are fermentation process on apple juice.The highest decrease at observation between 0 hours to 24 hours was using Agaricostilbum sp1.
The highest decreasing of pH between observation at 24 hours to 48 hours was using Komagataella sp.isolates with a pH of 4.48 to 3.49.
The highest decrease in pH between observation at 48 hours to 72 hours in the fermentation treatment with added activators of Debaryomyces sp2 with pH 3.93 to 3.68 on a 24-hour observation.
Decrease in pH is generally influenced fermentation process by the yeast that produces a byproduct such as alcohol and carbondioxide that acidic chracter (Kartohardjono 2007 in Azizah et al.,2012).

Temperature
The fermentation process begins with temperature increase, and after the highest point the temperature will decrease to approximately the same temperature as the beginning of the fermentation process.Test fermented for 72 hours were performed using apple juice show the temperature increasing and decreasing, although still in the range of 26-27ºC.
Temperature fluctuations is not significant as long as possible because the fermentation bottle cap was opened every 24 hours for measurement of temperature and pH.It indirectly provides different treatment for their mixing process when the electrode was inserted.The measuring process indirectly affect the discharge of carbondioxide is a byproduct of the fermentation process.Carbondioxide was formed by the natural fermentation process can affect the temperature during the fermentation process.The loss of carbondioxide indirectly affect to temperatur.Yeast can produce alcohol in an optimal temperature range of 30-33ºC (Azizah et al., 2012).

Smell
Fermentation test was conducted for 72 hours showed a change it's smell when compared to observation 0 hours.The changes generally occurs on all substrates with each treatment and the addition of 13 yeast isolates tested.Control unchanged smell due to the absence of additional yeast isolates.The changes as well be an indication of the formation of alcohol during the fermentation process.

Color
Substrate fermentation test that using a solution of apple cider shows fawn at 0 hours of observation on the overall treatment with the addition of yeast isolates.Control treatment was a different color than the other treatments on observations 0 hours because she did not isolate the addition of yeast.Colors were observed at 0 hour show differences in observation 72 hours, so that indicates a change in nutrition during fermentation test.Azizah et al. (2012) mentions that the fermentation substrate will change

Optical Density
Observations commonly show OD values indicate that the observation 0 hours until 72 hours of observation yeast still in the range of numbers 1, except for the treatment with the addition that the isolates Agaricostilbum sp1 observation 72 hours reached a value of 2. The increase in OD up to 72 hours of observation on the all treatment added of activators showed that the alcohol level produced during fermentation test can still be tolerated by the yeast, so the yeast is still the potential to produce higher alcohol because yeast can still breed.Azizah et al. (2012) states that a certain level of alcohol can inhibit the growth of yeast.
OD values among treatment showed no significant difference.The results of cell counting using turbidimetry method is strongly influenced by the cell size and color density of a test solution.

Relations Between Media Growth and Fermentation Result
Protomyces sp has obligate fermentative characteristics and the resulting alcohol level is 4.6%.Agaricostilbum sp1 has facultative fermentative characteristics and the resulting alcohol level of 9%.Debaryomyces sp1 has facultative fermentative characteristics and the resulting alcohol level is 9.3%.The highest of alcohol produced from the fermentation test with the addition of Agaricostilbum sp3 isolates that have oxidative characteristics.This can occur because unscrew bottle fermentation process when observation of pH and temperature.Bottle fermentation which opened indirectly remove carbon dioxide contained in the bottle fermentation, and causing the entry of oxygen into the bottle fermentation.Excessive carbon dioxide in the bottle fermentation can inhibit the growth of yeast.In addition, the alcohol level produced by yeast metabolism will be more on aerobic conditions that do require oxygen.The conditions according to the oxidative properties of yeast, so that the resulting alcohol level higher than treatment with the addition of other isolates.

CONCLUSION
(C) with each Ludwig et al. (1998) in Sidiyasa et al. (2006) as follows: Exploration data based on Jukri et al.

(
total individuals of all species Pi = The proportion of an individual species in relation to all types E of individuals in a type N = Amount of individuals all types

Table 1 .
Diversity Index

Table 5 .
Microscopic Characteristic of Yeast Udenimyces sp2 have a facultative fermentative characteristics as well as the resulting alcohol level of 5%.Agaricostilbum sp3 has a facultative fermentative characteristics and the resulting alcohol level is 11.6%.Trigonopsis sp2 has obligate fermentative characteristics and the resulting alcohol level is 5.4%.