OPTIMIZATION OF BACTERIAL DOSES AND INCUBATION TIME ON ETHANOL FERMENTATION OF NIPAH FOR BIOFUEL ENERGY

7/30/2019 OPTIMIZATION OF BACTERIAL DOSES AND INCUBATION TIME ON ETHANOL FERMENTATION OF NIPAH FOR BIOFUEL E

1/16

403

OPTIMIZATION OF BACTERIAL DOSES ANDINCUBATION TIME ON ETHANOL FERMENTATION

OF NIPAH FOR BIOFUEL ENERGY

Wiludjeng Trisasiwi, Ari Asnaniand Retna Setyawati

Jenderal Soedirman University, Purwokerto, Indonesia

Abstract. Nipah (Nypa fruticans) is a species of palm trees that grows in mangrovesenvironment near the offshore. Napa is potential to produce biofuel energy. Thepurposes of this research were 1) to determine the optimum bacterial concentrationfor fermentation to produce high ethanol, and 2) to determine the optimum

incubation time for fermentation to produce high ethanol. The research had beenconducted from June until November 2009 used microbe Saccharomycescerevisiae. This research was an experimental method, factors tested includingmicrobial concentration and incubation time. The variables observed in thisresearch were reduced sugar content, total microbial (cfu/ml), and ethanol yield.

The average results showed that the highest ethanol content was produced at astarter concentration of 7.5% which produced 9.55% of ethanol. The highestethanol content was obtained from the 6 days of incubation time which produced8.98% of ethanol.

Keywords: Biofuel; Concentration; Incubation Time; Nypa fruticans;Saccharomyces cerevisiae

1

IntroductionThe decreasing of fossil energy resources in the world including Indonesia, forcingthe energy experts to look for other renewable energy as an alternative to fossilfuels. One type of biofuel that has been developed to replace the gasoline fuel isethanol (ethyl alcohol), which is made from biomass (plants) through biologicalprocesses, namely enzymatic and fermentation. Results from research during thelast 20 years has found 60 species of plants that can be used as an alternative tofuel energy, one of which is Nypa fruticansas raw material for bioethanol.

Nipah (Nypa fruticans) is a kind of palm that grows on mangrove forestenvironment or tidal area near the waterfront. This plant is intended to protect

land or sea shore from abrasion. Like the coconut tree, whole nipah plant can beused for various purposes. Napa can also be tapped to get sweet liquid from young

fruit bunches. So far utilization ofnipahsap is not optimal. Communities in thecoastal areas used nipah sap for processing sugar, such in the Village Nusadadi,District Sumpiuh, Banyumas Regency [20]. But the sugar obtained has a sense ofslightly salty and less preferred by consumers, so that processing of nipah sap intosugar was not optimal. This encourages the utilization of nipah sap to anotherproduct of the processing of bio-ethanol.

Proceedings of the Third International Conference on Mathematics and Natural Sciences

(ICMNS 2010)


2/16

Wiludjeng Trisasiwi, Ari Asnani and Retna Setyawati

404

The advantage of nipah compared with other ethanol-producing plants is that

plants can produce nipah sap 20 tons/hectare or 14,300 liters of ethanol perhectare or two times greater than sugar cane [17]. Napa potentially be used as rawmaterial for biofuels, but has not been studied intensively, so many problems thatneed to be studied. Until now, biofuels are much studied is from maize [21], fromcassava (Trusmiyadi, 2007), from rice s traw [23], and from canna starch [13].

Fermentation is one of the processes that play an important role in the processingof bio-ethanol. Microbes are commonly used in fermentation industry mainly is a

class of low levels of bacteria and fungi such as molds and yeasts [3]. Factors thatinfluence the production of ethanol are the amount of inoculants and duration offermentation. Therefore, the aims of this research are: 1). determine the optimalconcentration of microbes in the fermentation that produces high ethanol, and 2).determine the optimal incubation time on the fermentation that produces highethanol.

2 Review of Literature2.1Nipah (Nypa fruticans)Nipah (Nypa fruticans) is a plant species of palm including the family Arecaceae(palmae) that grows in the mangrove forest. This plant is the only palm speciesfrom mangrove areas. Nipah able to survive on land that is somewhat dry or dry atlow tide.

Nipah plant is similar to the young sago plants, but not prickly and trunked.Leaves and flowers grow from a horizontal rhizome that sank in the mud. Actuallynipah plants have stems that creep on the ground, forming roots immersed inmud, only the rosette leaves that emerges above ground. From the rhizome appearscompound pinnate leaves typical of palm, upright or nearly upright, towering up to9 m above the ground and the stem length between 1 - 1.5 m. Nipah flowersappear in auxiliary panicles, the female flowers gathered at the tip to form a ballwhile male flowers are arranged in panicles similar strands, each strand consistingof 4-5 grains of male flowers with a length of about 5 cm. Bunches of fruit can betapped approximately four to five months after the flowers grow [22].

Nipah plant has benefits in terms of economic and non economic. In terms ofeconomic, nipah plants can be used as a source of food and non food as mentionedabove. In terms of non-economic, nipah plants have intangible benefits. Rachmanand Sudarto (1992) says that the intangible benefits of palm plants include: 1). Asa buffer crop ecosystems like mangrove plants, 2). Holding soil erosion on thebanks of river discharge and resist abrasion caused by wind and tides, and 3).Some types of fish and shrimp often raise their children in the area around thenipah forests, so that the nipah forests can serve as a nursery ground or feedingground, it can even also as a place to spawn for several species of fish such asmullet, white snapper, milkfish, crabs and so on.

Nipah sap is a liquid that tastes sweet and can be processed into sugar, both sugar

molds, sugar crystals or sugar syrup [14]. It shows that nipahsap is also potential


3/16

Optimization Of Bacterial Doses And Incubation Time On Ethanol Fermentation OfNipah For Biofuel Energy

405

as a source of sweetener. Therefore, nipah sap can also be processed into

bioethanol as an alternative energy source, because it contains lots ofcarbohydrates that can be converted into ethanol by the aid of microbes. Thechemical composition of nipah sap can be seen in Table 1.

Intangible benefits of nipah plants is that the business relating to the nipah areenvironmentally friendly, because nipah plants also play an important role as abuffer ecosystems and preventing erosion. Besides low cost, because these plantsdo not need intensive care, even it can grow wild, and easily tapped because the

trees are not high.

2.2Bio-ethanol as an Alternative EnergyIn the year 2007-2010, the Indonesian government target to replace 1.48 billion

liters of gasoline with bio-ethanol in accordance with Government RegulationNo.5/2006. It is estimated bio-ethanol requirements will increase 10% in 2011 to2015, and 15% in 2016-2025. In the first period 2007-2010 for 3 years, thegovernment requires an average of 30.833 million liters of bio-ethanol per month.Currently a new bio-ethanol can be supplied as much as 137,000 liters per month(0.4%). This means that every month the government of Indonesia lack 30.696million liters of bio-ethanol as a fuel [1].

Vegetable-based fuel or bio-ethanol can reduce environment pollution; due to its

CO2 emissions are very low, making it more environmentally friendly. Bio-ethanolcan be used as a substitute for fossil fuel. Bio-ethanol with levels of 95-99% can beused as material substitution for gasoline, while level of 40% is used as asubstitute for kerosene. Bio-ethanol is a multi-purpose because it is mixed withgasoline in any composition has a positive impact.

Mixing 10% absolute ethanol with gasoline (90%), called Gasohol E10. Absoluteethanol has octane number (ON) 117, while the premium is only 87-88. GasoholE10 has ON 92 or equivalent ON Pertamax. At this composition is known as bio-ethanol octane enhancer (additive), the most environmentally friendly and indeveloped countries have shifted the use of Tetra Ethyl Lead (TEL), or Methyl

Tertiary Butyl Ether (MTBE) [1].

Advantages of bio-ethanol compared to gasoline are: (1) safe to use bio-ethanol as afuel, flash point of ethanol three times higher than gasoline, (2) fewer hydrocarbonemissions, (3) fuel consumption decreased with increasing ethanol content. Whileshortcomings: (1) cold engine is harder to do the starter, (2) ethanol reacts withmetals such as magnesium and aluminum, and (3) Nitrogen oxide emissionshigher [1].


4/16


406

2.3 Bio-ethanol ProductionIn general, bi-ethanol production technologies include 4 (four) series of processes,namely: preparation of raw materials, fermentation, distillation and purification [1].

2.3.1 Raw material for bio-ethanolThe raw material of bio-ethanol can be classified into three categories: 1). palm

sugar (sucrose), such as sugar cane, nipah sap, sweet sorghum juice, coconut sap,palm sap, cashew-fruit juice; 2). starch materials such as grain sorghum, sago,cassava, sweet potato, canna, arrowroot, dahlia tubers; and 3). Cellulose materials(lignocelluloses) include wood, straw, banana stems, etc. [15].

2.3.2

Fermentation processThe fermentation process is often defined as the cracking process carbohydratesand amino acids in anaerobic, i.e. without the need for oxygen. Compounds thatcan be broken down in the fermentation process are primarily carbohydrate,

whereas the amino acids can be fermented by only a few specific types of bacteria.Carbohydrate is the main substrate that is broken in the process of fermentation.If carbohydrate in the form of polysaccharide compounds, it will first be brokendown into simple sugars before fermentation, namely hydrolysis of polysaccharidesinto glucose. Furthermore, glucose will be split into other compounds dependingon the type of fermentation. One example is the fermentation of glucose intoalcohol through the Embden-Meyerhof Parnas (EMP) conducted by the yeast, suchas S. cerevisiae[3].

During batch fermentation system, several parameters can cause a decrease inspecific growth rate of the microbes that caused both by the concentration ofsubstrate and product of ethanol. Therefore Rakin et al. (2009), producing ethanol

using immobilized yeast cells [16]. According to Sakurai et al. (2006) and Baptistaet al. (2006) in Rakin et al. (2009), immobilized cells in fermentation processeshave been developed to reduce the inhibition caused by high concentrations ofsubstrates and products, thereby increasing productivity and yield of ethanol [16].

2.3.3 DistillationAccording Nurdyastuti (2008), alcohol produced from the fermentation process isusually still contain gases such as CO2 (resulting from changes in glucose into

ethanol) and aldehyde that need to be cleaned [10]. CO2 gas in the fermentation ofsuch typically reaches 35% by volume; so as to obtain good-quality ethanol must

be cleaned by filter-bound ethanol by CO2.

In general, the fermentation can produce bio-ethanol or alcohol with a purity ofabout 80-10% and can not be categorized as ethanol-based fuel. In order toachieve the purity above 95% so it can be used as fuel, the fermented alcohol mustgo through the process of distillation to separate the alcohol with water on thebasis of differences in boiling points of the two materials which is then condensedback [10].


5/16


407

According Musanif (2008), distillation is a separation process based on thecomponents of its boiling point, boiling point of pure ethanol at 78oC, while thewater is 100oC, by heating the solution at a temperature range of 78 - 100oC willresult in most of the ethanol evaporated, and the condensing units will beproduced ethanol with 95% volume concentration [9].

2.3.4 PurificationDistillation process carried out to obtain bio-ethanol with a high concentration.Results distillation process is not fully pure ethanol, but still contains water evenin small quantities. Bio-ethanol is used as a mixture of fuel for vehicles must becompletely dry or anhydrous so as not to cause corrosive to the machine.

Therefore, purification is necessary to eliminate water contained in ethanol [10].

Bio-ethanol can be purified by two methods, namely chemistry and physics [11].Chemical method using crushed limestone to absorb water. This method isproperly used for household-scale producers because it is simple and relativelyinexpensive cost. Its use is 7 liters of bio-ethanol needed 2-3 kg of limestone. The

mixture allowed standing for 24 hours while occasionally stirred, and then themixture is evaporated and condensed into a liquid again as ethanol 99% or more.

This bio-ethanol can be mixed with gasoline or used pure. The weakness of the useof l imestone that is the amount of ethanol that is lost is very high.

In the purification method used physics synthetic zeolite. This purification processuses the principle of surface absorption. Bio-ethanol should be used forpurification of synthetic zeolite 3A (size 3 Angstroms), which can bind more water.Advantages using synthetic zeolites: (1) the time required is shorter and (2) lostonly 10% ethanol. But it is more expensive than limestone. Therefore, the use of

synthetic zeolite is more suitable for large scale business.

2.4 Microbes Producing EthanolBio-ethanol (C2H5OH) is the liquid from the fermentation of sugar from sources

that contain carbohydrates using microorganisms. Bio-ethanol in the fermentationprocess is formed through several metabolic pathways, depending on the type ofmicrobe involved. For the yeast S. cerevisiae and a number of others, ethanol isformed via the Embden-Meyerhof Parnas (EMP). Some of the yeast Candidatropicalis, Pichia wickerhamii can perform on xylose metabolism throughheterolactic (hexoses Mono Phosphate/HMF). Similarly, the fungus Fusariumsp.,Rhizopussp., and the bacterial genus Clostridiumwas also able to produce ethanolfrom xylose. The third line is important in fermentation of alcohol is the pathEntner-Doudoroff (ED) that are found in bacteria Zymomonas mobilis [15].

Further according Rahayu and Rahayu (1988) [15], in order to obtain the ethanol

production process that is really effective, the microbe used should havecharacteristics, including: 1). has the capability of rapid fermentation ofcarbohydrates that are relevant, 2). have the ability to perform flocculation andsedimentation, 3). genetically stable, 4). osmotoleran, especially against a solutionof high concentration of carbohydrates, 5). tolerant to ethanol, and has the ability


6/16


408

to produce high ethanol concentration, 6). cell has the capability of living is high,

so it can be used repeatedly, and 7). tolerant to temperature.

S. cerevisiaeis the yeast in the class Ascomycetes, a sub-class Hemiascomycetidae,Endomycetales orders, families Saccharomycetaceae, sub familySaccharoycoideae,and genus Saccharomyces [4]. S. cerevisiae is a unicellular organism that ismicroscopic creatures, and it uses sugar as a carbon source for metabolism(Alexopoulus and Mims, 1979), including sucrose, glucose, fructose, galactose,

mannosa, maltose and maltotriosa [7].

S. cerevisiaeis a microorganism of the most widely used in fermentation of alcohol,because it can produce high yield, resistant to high alcohol content, resistance tohigh sugar levels and remain actively engaged in activities at 4 - 32oC [6]. S.cereviceaewill metabolize glucose and fructose to form pyruvic acid through thereaction stages on the path to Emden-Meyerhof-Parnas. Then the decarboxylation

reaction occurs that converts pyruvic acid into acetaldehyde, and continueddehydrogenation reaction that converts pyruvic acid into ethanol [3].

3 Materials and Methods3.1 Materials and EquipmentThe research was conducted in June to November 2009. Materials used in thisstudy include nipah sap from Nusadadi Village, District Sumpiuh, Banyumas

Regency, S. cerevisiaeFNCC 3012 obtained from the Food and Nutrition of Inter-University Center Gadjah Mada University, medium MEA (Malt Extract Order),MgSO47H2O, KH2PO4, alcohol 70%, peptone water, dichromat potassium sulfate,potassium carbonate saturation, pH 4 buffer solution, distilled water, and othermaterials for chemical analysis of ethanol.

Tools used include tube fermentation (erlenmeyer 500 ml), incubators,spectrophotometers, centrifuges, autoclave, petri dishes, needles ose, bunsen, testtubes, beakers, pH meter, a set of distilator, and other glass equipment forchemical analysis.

3.3 Experimental DesignThis study including experimental methods, experimental design used is a

randomized block design (RBD) [5].Factors tested include microbial concentrationand incubation time.

Factors tested include the concentration of microbes (percent v/v), which is 5percent, 7.5 percent, and 10 percent, and incubation time, which is 2 days, 4 daysand 6 days. The combination treatment obtained 9 combination treatments. Eachtreatment is repeated 2 times, so the total number of trials is 18 units of theexperiment.


7/16


409

3.4 Variables and MeasurementThe variables observed in this study included the determination of ethanol,reducing sugar content, and total microbial (cfu/ml). The best treatment is basedon the highest ethanol content. Chemical analysis conducted on nipah sapmaterials that include total sugar content, salt content, pH, protein content, andtotal dissolved solids.

Observed variable measurement performed directly on each unit of the experimentas follows.

3.4.1 Ethanol content [6]Ethanol was diluted using distilled water with concentrations of 0.025, 0.05, 0.075and 0.1 percent. Prepared cup Conway, a total of 1 ml of each ethanol solution

inserted in the edge of the cup, and the other edge of the cup that was given 1 mlof saturated solution of potassium carbonate. At the center of the cup was given asolution of potassium bichromate sulfuric acid. Next, cup is sealed, and shakengently so that the two solutions at the edge of the cup mixed well, then allowed for1 hour. Solution at the center of the cup was taken using a pipette and placed in10 ml of cooked pumpkin, rinse solution remaining in a Petri dish with distilledwater, put in a flask and add distilled water up to the mark. Solution in the flask

was measured absorbance at a wavelength of 480 nm using a spectrophotometer.Created charts the relationship between alcohol concentration by absorbance, inorder to obtain regression equations to calculate the ethanol content in the sample.

One ml samples containing alcohol inserted into the edge of the cup, the other edgeof the cup that was given 1 ml of saturated solution of potassium carbonate. Next,stage together with the determination of standard curves of ethanol solution.

Ethanol content in the samples was calculated based on the regression equationstandard ethanol solution.

3.4.2 Reduced sugar content of Nelson-Somogyi method [19]Standard glucose solution was prepared with a concentration of 10 mg glucoseanhydrate. From the solution is dilution series, in order to obtain a solution with aconcentration of 2, 4, 6, 8 and 10 mg/100 ml. Each serial dilution of 1 ml weretaken and included in the test tube, and a tube containing 1 ml of distilled wateras blank. Into each tube is added 1 ml of Nelson reagent (mix of 25 part of Nelsonreagent A and 1 part of Nelson B), then heated in a boiling water bath for 20minutes. After warming immediately cooled in a glass containing of cold water untilthe temperature reaches 25oC. Cool all tubes plus 1 ml reagent Arsenomolybdat,stirrer until precipitate formed dissolves. All tubes mined by as much as 7 ml

distilled water, stirrer again until homogeneous. Then performed calibrationabsorbance at a wavelength of 540 nm using a spectrophotometer. From the dataobtained made regression equation showing the relationship between glucoseconcentrations by absorbance.

Samples were dissolved in distilled water to form a homogeneous mixture, andstirring with a magnetic stirrer performed for 30 minutes. If the solution turbid,


8/16


410

then performed purification by using lead acetate. Sample solution which has

clearly taken as much as 1 ml and put into a test tube, add 1 ml Nelson reagentand heated for 20 minutes in boiling water. Subsequently treated as in thepreparation of standard curve. If the calibration absorbance is too dense (a valueabove 0.8), then made dilutions of the sample solution. Sugar reduced in thesample can be calculated using the equation standard solution.

3.4.3 Total microbial pour plate method [3]A total of 1 ml sample is inserted into a test tube containing 9 ml 0.85% NaClsolution, then shake for homogeneity. Prepare a decimal dilution series to obtainthe appropriate dilution stage. Then 1 ml samples of the two final dilutions pourplate grown in a Petri dish containing the appropriate media MEAs. Furthermore,Petri dishes incubated at room temperature for 48 hours. Colonies that grew in aPetri dish calculated the amount, and the number of microbes in the samples

(cfu/ml) was calculated by multiplying the number of colonies per plate with adilution 1/factor.

3.5 Stages in the research carried out as follows:1) Sterilization of tools and media preparation. Sterilization equipment usinga glass oven at 180oC for 2 hours. Preparation of microbial growth media MEA,

sterilization performed using an autoclave at a temperature of 121oC for 15minutes.

2) Maintenance and manufacture of starter culture. Maintenance culture ofSaccharomyces cerevisiae using MEA media. Medium to manufacture the samestarter for the fermentation medium, namely: nipah sap, yeast extract (10g/l),MgSO47H2O (0.5 g/l), KH2PO4 (1.0 g/l) as much as 2 ose microbes that have been

rejuvenated incorporated into the 100 ml starter medium, then incubated for 24hours.

3) Fermentation of nipah sap.

This study used the media production unit of 150 ml each experiment and placedin a volume of 500 ml Erlenmeyer equipped with a cover of rubber and plastichoses were given to remove the CO2 gas that is formed. To maintain anaerobiccondition the hose is inserted into the water.

The composition of the substrate for fermentation is a nipah sap, Yeast extract (10g/l), MgSO47H2O (0.5 g/l), KH2PO4 (1.0 g/l). Further the media sterilized andcooled, after cold inoculated with a starter with a concentration of 5, 7.5, and 10%.

Incubation was carried out at room temperature for 2, 4 and 6 days. At the end offermentation carried out observations of the residual reducing sugar in the

medium, ethanol content, total microbial and yield of alcohol produced.

4) DistillationAfter completion of fermentation, in fluids that contain ethanol is distilled to purifythe ethanol. Distillation carried out using a distillation flask with a temperature


9/16


411

bath (water bath) 78 - 80oC, connected with condenser at a temperature 18 - 20oC.

The result of distillation is collected and then measured the volume andconcentration of ethanol.

4 Results and Discussion4.1 Analysis of nipah sap

The analysis of the raw material from nipah trees taken from Nusadadi Village,District Sumpiuh, Banyumas Regency, result the average chemical content of thenipah sap are presented in Table 1.

Table 1 Results of chemical analysis of nipah sap

Chemical composition Total

Total solids (Brix)pH

Total sugar content (%)Soluble protein content (%)Salinity (%)

22.706.50

13.300.250.31

The results of the analysis indicate that total sugar contain of nipah sap is quitehigh which reached 13.3 percent, thereby potentially as raw material for makingethanol. In the manufacture of ethanol via fermentation either by yeast or bacteria,

the concentration of sugar contained in the materials will affect production. If toolow, then the ethanol produced little. Conversely, if too high sugar content in the

substrate is inhibited microbial growth. This is thought to cause high sugar highosmotic pressure in the fermentation medium, so that cells can undergoplasmolysis. According Fardiaz (1989), sugar is a preservative against microbialdamage [3]. In addition to reducing sugar function water activity (Aw), so thatmicrobes are blocked, also led to increased osmotic pressure outside the cell.

4.2 Results of Fermentation ProcessThe result of bio-ethanol fermentation process of nipah sap using S. cerevisiaemicrobes with different concentrations of starter and variety of incubation time arepresented in Table 2.


10/16


412

Table 2 The average value of variable fermentation ofS. cerevisiaeat different

concentrations of starter and incubation timeTreatment Reduced sugar(%)

Totalmicrobial(cfu/ml)

Bio-ethanol (%)

5% starter 0.1871 8.35 106 3.927.5% starter 0.1535 1.07 108 9.5510% starter 0.1548 8.27 106 8.93

Incubation 2 days 0.2168 1.83 106 5.68Incubation 4 days 0.1454 2.15 108 7.73Incubation 6 days 0.1332 1.88 106 8.98

4.3 Reducing sugar content.Reducing sugar content in the media at the end of fermentation, described theamount of sugar left over and not converted into ethanol. The average value ofresidual reduced sugar content fermented by S. cerevisiae at differentconcentrations of starter is presented in Fig. 1 and at various time of incubation ispresented in Fig. 2.

0

0,05

0,1

0,15

0,2

5 7,5 10

Starter concentration (%)

Reducin

gsugar(%)

Fig. 1 The average value of reducing sugar content in various concentration ofstarter.

These results indicate that the higher the concentration of starter, reducing sugarcontent remaining in the fermentation medium is progressively decreasing. Thismeans that sugar as a source of carbon converted into ethanol by microbes S.cerevisiae. More and more starter that is used means that the higher microbialpopulation in the medium led to more and more sugar that is used by microbes forgrowth and for production of ethanol, so that by the end of the fermentation ofsugar is left fewer and fewer. According to the Moat and Foster (1979), sugar is a


11/16


413

source of carbon and energy used for microbial growth and metabolite production

[8].

0

0,05

0,1

0,15

0,2

0,25

2 4 6

Incubation time (days)

sugarreducing(%)

Fig. 2 The average value of reducing sugar content in various incubation times.

The longer the incubation time then the levels of reducing sugar in thefermentation medium is progressively decreasing. This means that the longer the

fermentation, the sugar used by microbes to grow and produce metabolites,including ethanol.

4.4 Total microbialThe number of microbes during the fermentation process can be calculated. Theaverage number of microbes S. cerevisiae at different concentrations of starter ispresented in Figure 3 and at various time of incubation is presented in Figure 4.


12/16


414

6,85

6,9

6,95

7

7,05

5 7,5 10


Totalmicrobial(logcfu/ml)

Fig. 3 Average total microbial at vary of starter contentration.

The average number ofS. cerevisiae at a concentration of 5% starter, 7.5% and10% respectively 8.35 x 106; 1.07 x 108; and 8.27 x 106 or in log form is 6.9222,7.0295, and 6.9173.

Type of microbe S. cerevisiae has a growth pattern as follows, namely the

increasing number of starter used, the number of microbes at a concentration ofstarter increased to 7.5 percent. Progressively increasing the amount of starterused, the number of microbes tends to decline. This was expected, because of theincreasing number of microbes in the fermentation medium will lead tocompetition in getting the nutrients, so many microbes that inhibited growth.

According Fardiaz (1988), the pattern of microbial growth in batch culture willaccelerate the competition in obtaining nutrients, because in the system during thefermentation process no additional nutrients [2].

0

12

3

4

5

6

7

8

9

2 4 6


Tota

lmicrobial(logcfu/ml)

Fig. 4 The average number of microbes in various incubation times.


13/16


415

The longer the fermentation the average number of microbes was increasing, butthe subsequent decline. The average number ofS. cerevisiae in fermentation for 2days, 4 days, and 6 days, respectively, are 1.83 x 106; 2.15 x 108; and 1.88 x 106or in log form 6.2613, 8.3329, and 6.2747.

The results showed that the length of fermentation will affect the growth ofmicrobes. From these data shows that the number of microbes increased untilfermentation for 4 days, further decrease the number of microbes. It is possible,

after 4 days of fermentation microbes entering the stationary growth phase , so thatno increase in the number of microbes. No increase in the number of microbes,because the nutrients in the medium decreases, and also caused inhibition ofgrowth by the presence of ethanol produced.

4.5 Bio-ethanol contentAverage levels of ethanol produced during fermentation byS. cerevisiaeat differentconcentrations of starter are presented in Fig. 5 and at various time of incubationis presented in Fig. 6.

0

2

4

6

8

10

12

5 7,5 10


Ethanolcontent(%)

Fig. 5 The average value of ethanol content in various starter concentrations.

More and more starter concentration used does not necessarily produce ethanol is

high. The results showed that the highest ethanol produced at a concentration of7.5% starter that is using S. cerevisiae, and at higher concentrations of ethanolproduced decreased. This is indicated by the number of microbes that tend to

decrease with the large amount of starter.

The higher the concentration of starter used in the fermentation process, notnecessarily produce high ethanol. The results showed that the highest ethanol,produced at a concentration of starter S. cerevisiae at 7.5%, and at a higherconcentration of starter, the fuel decreases. It is suspected that the higher theconcentration of starter has increased competition for nutrients between microbes,


14/16


416

resulting in lower productivity. This is indicated by the number of microbes that

tend to decrease with the large number of starter.

Optimal concentration of starter was 7.5% and ethanol produced by 7.55%. Rakinet al. (2009) reported that the maximal ethanol production in fermentation of cornmeal hydrolyzates of 10.05% was produced at a concentration of 5% starter [16].

0

2

4

6

8

10

2 4 6


Ethanolconcentration

(%)

Fig. 6 The average value of ethanol content in various incubation times.

The longer the incubation time, the resulting ethanol content increased. Thehighest levels of ethanol produced in the incubation for 6 days with ethanolcontent of 7.84%. Results of research from Putri and Sukandar (2008) showed thatethanol production from canna starch using S. cerevisiae, the maximum ethanolconcentration achieved during fermentation at the 24th, and began to decline at the

36th hour [13]. This is because at the hour of the 24 yeast entered the phaseexponentially, while at the 36th hours yeast cells begin to enter stationary phase.

5 Conclusion and Suggestion5.1 ConclusionFrom the research results can be summarized as follows:1. Optimal concentration of starter that produces the highest was 7.5%, the

resulting ethanol content of 9.55%.

2. Optimal time of incubation of the highest ethanol yield was 6 days, levels ofethanol produced by 8.98%.

5.2 SuggestionBio-ethanol production from nipah sap can be improved by optimizing theproduction process conditions, such as the pH, the concentration of sugar (sourceof C) and a source of protein and some minerals. Need to do research by using a


15/16


417

continuous fermentation method, meaning no addition of nutrients and no

nutrient regulation and the fermentation conditions regulation.

References

[1] Bustaman, S. (2008), Sago-based Bio-ethanol Development Strategy inMaluku, Perspektif, 7(2), 65 79.

[2] Fardiaz, S. (1988), Fermentation Physiology, Inter-University Center for Foodand Nutrition, Bogor Agricultural University in collaboration with the Instituteof Information Resources, Bogor Agricultural University.

[3] Fardiaz, S. (1989), Food Microbiology, Ministry of Education and CultureDirectorate General of Higher Education, Inter-University Center for Food andNutrition, Bogor Agricultural University.

[4] Frazier, W.C. and W.C. Westhoff (1978), Food Microbiology, McGraw HillPublishing Co. Ltd. New Delhi, India.

[5] Gomez, K.A. and A.A. Gomez (1995), Statistical Procedures for AgriculturalResearch, Second Edition, Translators: Sjamsuddin, Endang, and J.S.Baharsjah, University of Indonesia (UI-PRESS).

[6] Kartika, B., A.D. Guritno, D. Purwadi, and D. Ismoyowati (1992), EvaluationGuidelines for Agricultural Products from the Industry, Inter-UniversityCenter for Food and Nutrition, Gadjah Mada University, Yogyakarta.

[7] Lewis, M.J. and T.W. Young, (1990), Brewing, Chapman and Hall. New York.[8] Moat, A.B. and J.W. Foster (1988), Microbial Physiology, Second Edition, John

Wiley & Sons, New York.

[9] Musanif, J. (2008), Bio-ethanol, www.agribisnis.deptan.go.id., downloadJanuary 14, 2009.

[10] Nurdyastuti, I. (2008), Technology of Bio-ethanol Production Process, Journalof Bio-fuel Development Prospects, 75-81.

[11] Pranowo. D. K. (2007). Bio-ethanol 99.5%, Purify it with Limestone,www.trubus-online.com., download January 16, 2008.

[12] Prihandana, R. (2007), Cassava Bio-ethanol Fuel Future, Agromedia, Jakarta.[13] Putri, L.S.E. and D. Sukandar (2008), Starch Conversion Canna (Canna edulis

Ker.) into Ethanol through Acid Hydrolysis and Fermentation, Biodiversitas,9(2), 112 -116.


16/16


418

[14] Rachman, A.K. and Y. Sudarto (1992), Nipah Source New Sweetener,Kanisius, Yogyakarta.

[15] Rahayu, E.S. and K. Rahayu (1988), Alcoholic Beverage ProcessingTechnology, Inter-University Center for Food and Nutrition Gadjah MadaUniversity, Yogyakarta.

[16] Rakin, M.; L. Mojovic; S. Nikolic; M. Vukasinovic and V. Nedovic (2009), Bio-ethanol production by immobilized Sccharomyces cerevisiaevar. ellipsoideus

cells, African Journal of Biotechnology, 8(3), 464 471.

[17] Smith, D (2006), Nypa palm: ethanol super-crop?Biofuel Review, Singapore, 15June 2006, download Sunday, February 15, 2009.

[18] Steinkraus, K.H. (1983), Handbook of Indigenous Fermented Foods, MarcelDekker, Inc. New York.

[19] Sudarmadji, S., B. Haryono and Suhardi, (1997), Procedure Analysis of FoodAgriculture, Liberty, Yogyakarta.

[20]Tresnawati, H. (2009), Motivation for Women Processing of Nipah-Sugar InImproving Household Income of Nusadadi Village, District Sumpiuh,Banyumas Regency, Thesis, Faculty of Agriculture, Jenderal SoedirmanUniversity, Purwokerto.

[21]Trifosa, D. (2007), Corn Starch Conversion Being Bio-ethanol, Thesis,Department of Chemistry, Bandung Institute of Technology.

[22] Wikipedia (2009), Nipah, www.wikipedia.org., downloaded May 5, 2009.[23] Wulandari, A. (2007), Preliminary Study of Water Juice Fermentation of Rice

Straw Being Bio-ethanol with Commercial Yeast, Thesis, Department ofChemistry, Bandung Institute of Technology.

Details of authors

WILUDJENG TRISASIWIUniversitas Jenderal Soedirman,Purwokerto, Indonesia

ARI ASNANI

Universitas Jenderal Soedirman,Purwokerto, Indonesia

RETNA SETYAWATIUniversitas Jenderal Soedirman,Purwokerto, Indonesia

Documents

OPTIMIZATION OF BACTERIAL DOSES AND INCUBATION TIME ON ETHANOL FERMENTATION OF NIPAH FOR BIOFUEL ENERGY