Study the Effect of pH on the Fermentation Anaerobic-Aerobic Siwalan (Borassus flabellifer L.) Sap to produce Acetic Acid

Siwalan (Borassus flabellifer L.) is a palm family that is widely planted in the Tuban area of East Java. Siwalan sap has a relatively high sugar content of about 10-15 g/100 ml. The sap is obtained by tapping the inflorescences. In general, siwalan sap is used for fresh drinks or alcoholic beverages with maximum storage in 3 days. Based on the sugar content in the sap of siwalan, acetic acid products can be made through fermentation of glucose to ethanol, then the ethanol is fermented into acetic acid. Acetic acid is widely used as a preservative of food and health drinks. The purpose of this research is to study the effect of ethanol fermentation aerobic pH on acetic acid product. Anaerobic fermentation uses saccharomyces cereviceae to produce ethanol, and aerobic fermentation uses acetobacter aceti for acetic acid production. In aerobic ethanol fermentation using pH 3; 3.5; 4 and 5. The concentration of ethanol was analyzed using GC ULTRA Scientific Gas Chromatography, DSQ II detector, and MS 220 column. Acetic acid produced from the aerobic fermentation process was analyzed using an alkalimetric method. Anaerobic fermentation uses Saccharomyces cereviceae with 1-day log phase, while aerobic fermentation uses acetobacter aceti with a 5 day log phase. Fermentation using saccaromyces cerevisiae within 24 hours in order that reduction sugar could stably decrease, optimum ethanol could be got at optimum ph 6 which could decrease 55 percent of reduction sugar concentration to produce 8,20583 %v/v ethanol. Fermentation acetic acid content using acetobacter aceti observed in 3 days at pH 6 and 30 0C will produce 6,659 g/l also shows that ph 4-6 at 30 0C will produce 6,605 g/l acetic acid. Aerobical fermentation of acetic acid in 3 days shows that ph 4-6 is highly affected by temperature at 30 0C. Statical analysis shows, in ethanol production pH and fermentation time give significant effect, but interaction from two factors has no significant effect. . This is an open acces article under the CC-BY-SA license. Article history : Received April 13, 2020 Revised April 20, 2020 Accepted April 22, 2020 Published April 30, 2020 Article Info


Material And Methods
Siwalan sap was purchased from Tasikmadu Tuban, East Java -Indonesia. Siwalan sap analyzed by HPLC Agilent 1100 series with refractive index detector in ULFPP Airlangga University Surabaya to analyzed the glucose, fructose, and sucrose content. Acetic acid produced from the aerobic fermentation process was analyzed using an alkalimetric method based on neutralization reaction and NaOH (1N) as standard solution carried out in the Department of Industrial Chemical Engineering, Vocational Faculty, ITS Surabaya.

Preculture of Cell for Ethanol Production
S.cerevisiae was obtained from Indus-trial Chemical Engineering Department, Faculty of Vocational, ITS Surabaya, Indo nesia. 180 ml of sterile Siwalan sap; 1 (g/l) of KH 2 PO 4 ; 0.5 (g/l) of MgSO 4 .7H 2 O; 1 (g/l) of (NH 4 ) 2 SO 4 ; and 10 (g/l) of yeast extract. The cultures were incubated at 30ᵒC and 125 rpm and drown every single hour for the measurement of cell growth using Hemacytometer. By plotting the number of cell against a time, the log phase for culture growth could be determined. The cell number as the variables were designed in the range of log phase, thus in time of the cell number was achieved, 1 of ml culture were inoculated to 100 ml of sterile Sugar palm juice. The culture growth was monitored during the fermentation process by counting the number of cell in the sample for every 8 hours for 48 hours. The reducing sugar formed was analyzed in aliquots by the 3.5-dinitrosalicylic acid reagent using a visible spectrophotometer detector (Cecil CE 1011) with a wavelength of 540 nm [5].

Ethanol Fermentation process
The sterile sugar palm juice was mixed with the log phase starter in incubator shaker at 32 ⁰C and 125 rpm for 24 hours (Saccharomyces cerevisiae). Inoculum concentration (cell/ml per sugar concentration) has used as variable for ethanol fermentation with inoculum concentration 5000000; 10000000; 15000000 cell/ml. The ethanol concentration from fermen ted broth was analyzed using Gas Chromatography Scientific GC ULTRA, detector DSQ II, and coloumn MS 220.

Acetic Acid Fermentation process
Broth of ethanol fermentation was added with acetobacter aceti. The addition of acetobacter aceti is 10% of the total volume of broth. Fermentation of acetic acid was carried out by aerobic process with passing air into the fermented broth. Before fermentation acetic acid by acetobacter aceti to determine time of fermentation we used preculture of cell acetobacter aceti. 180 ml of sterile broth from ethanol fermentation; 1 (g/l) of KH 2 PO 4 ; 0.5 (g/l) of MgSO 4 .7H 2 O; 1 (g/l) of (NH 4 ) 2 SO 4 ; and 10 (g/l) of yeast extract. The cultures were incubated at 30ᵒC and 125 rpm and drown every single hour for the measurement of cell growth using Hemacytometer. By plotting the number of cell against a time, the log phase for culture growth could be determined. The cell number as the variables were designed in the range of log phase, thus in time of the cell number was achieved, The culture growth was monitored during the fermentation process by counting the number of cell in the sample for every 8 hours for 120 hours. The reducing sugar formed was analyzed in aliquots by the 3,5-dinitrosalicylic acid reagent using a visible spectrophotometer detector (Cecil CE 1011) with a wavelength of 540 nm [5].

Preparation of Siwalan Sap
Siwalan sap is obtained from tapping siwalan trees in Tuban, East Java, Indonesia, filtered and sterilized at 121ᵒC and 15 psi for 15 minutes. the sap content of palm sap was analyzed using High Performance Liquid Chromatography (HPLC). The results of High Performance Liquid Chromatography (HPLC) can be seen in Table 1 2 % Sugar content in siwalan sap was quite low, because the sugar content of siwalan sap is influenced by varieties of siwalan plants, plant age, plant health and soil. The sweet taste of siwalan sap was reduced. So that the type of soil on the siwalan plantation can affect the sweetness level of the siwalan sap. Other causes of sugar content in siwalan sap is siwalan sap had not done pre-treatment process, so that the siwalan sap will be spontaneously fermented and effect the decrease of sugar content in siwalan sap.

Ethanol Fermentation Process.
Ethanol production from fermentation siwalan sap using saccharomyces cereviceae has inoculum concentration for variable. Saccharomyces cereviceae growth analysis by applying counting chamber system to detect a well-done fermentation. From Figure 1 can be seen saccharomyces cereviceae growth curve which show a cell growth of 426666666,7 cell/ml within 24 hours and at this stage balance growth or logphase with a constant growth is faced by the microorganism.

pH effect on ethanol production
Detecting pH effect upon batch fermentation by comparing pH performance in conversion of sugar into ethanol process by residual sugar analysis by DNS system applying acid reagent 3-5 dinitrisalisilate to detect reducing sugar content. Reducing sugar curve fermented by s,cereviciae for each ph can be see in figure 2. In Figure 2 sugar concentration curve which is declining during microorganism development, which shows that on batch fermentation reducing sugar within medium is used to produce carbon for saccharomyces cereviceae to synthesize energy through ethanol fermentation, highest cell growth at ph 6 is 15200000000 cells/ml sample. In this reasecarh unaerobic fermentation for 24 hours is taken to make reduction sugar decline steadily, From this research shows ph 6 can decrease 55 percent of reducting sugar concentration converted into ethanol of 8,20583 percent v/v through analysis of chroma-tography scientific gas GC ULTRA dengan detector DSQ II dan column MS 220. (Universitas Surabaya).

Acetic acid fermentation and pH effect in the fermentation
Acetic acid was form from ethanol, using acetobacter aceti to convert ethanol to acetic acid. Time of fermentation using log phase from acetobacter aceti. From this research log phase acetobacter aceti in 120 hours or 5 days (data not shown), however the other study mentioned that time of fermentation of acetobacter aceti is 72 -80 hours [7]. Analyze acetic acid using acidi alkalimetri method with pp indicator [9]. In this study pH and temperature effect was analysis. From this research acetic acid form in 120 hours at pH 4; 5; 6; 7 with two different temperature 25 o C and 30 o C.  Figure 3 shows pH and temperature effects toward acetic acid produced, highest acetic acid concentration was on pH 6 and temperature 30 o C which is 6,659 g/l, this is in accordance with [8] stating that acetic acid was optimum at pH 5,5 and 30 o C, higher than this will cause acetobacter aceti damaged cells, lower than this will cause inactive acetobacter aceti, thus fermentation will not happen.

Statistical Analysis for Ethanol and Acetic Acid Production 3.5.1 Ethanol production
Producing alcohol from sugar fermentation uses 2 factors, pH with four levels pH 4, pH 5, pH 6 and pH 7, the second factor is time of fermentation with five levels are 0, 6, 12, 18 and 24 hours. following are test on effec t of experimantal factors, first is testing interaction effect on ph factor and observation time, if an unsignificant result, next test will be on similarity effect of ph and observation time factors, hypothesis test for the existance of interaction effect of ph factor and observation time, anova chart test statistic, the test is against ho, meaning that at least there is one hour of different orservation it can be detected from the test on parametre in this case factors like ph and observation time will have a significant effect but interraction between these two factors will not have any effect on acetic acid formation. Interraction of ph 4 and 5 and 7 but in the test the interaction has no significant effect toward produced ethanol percentage, it could be seen that ph 6 will produce higher ethanol percentage for all factor levels of observation time in comparison to all other, thats why ph 6 more suggested due to higher ethanol percentage produced.

Acetic Acid Production
From the analysis we can conclude that variable temperature give P-value = 0,036 < α (α = 0,05), this is indicating that temperature has effect for % acetid acid concentration, meanwhile pH has P-value pH = 0,103 > α, this is indicating that pH has no effect in acetid acid production. From figure 4 shows temp 30 o C will produce higher acetic acid concentration percentage than of 25 o C while pH 4 and 6 will produce the same result at 30 o C except pH 7 which will decline sharply.

Conclusion
Palm juice can be unaerobic fermented produce acetic acid by adding sacca-romyces cerevisiae and aerobic fer-mented by adding acetobacter aceti, first fermentation using saccaromyces cerevisiae within 24 hours in order that reduction sugar could stably decrease, optimum ethanol could be got at optimum ph 6 which could decrease 55 percent of reduction sugar concentra-tion to produce 8,20583 %v/v ethanol using analysis of gas chromatography scientific GC ultra and detector dsq II and column ms 220. Fermentation acetic acid content using acetobacter aceti observed in 3 days at ph 6 and 30 ⁰C will produce 6,659 g/l also shows that ph 4-6 at 30 ⁰C will produce 6,605 g/l acetic acid. Aerobical fermentation of acetic acid in 3 days shows that ph 4-6 is highly affected by temperature. Statistical analysis shows, in ethanol production pH and fermentation time give a significant effect, but not significant for the interaction both of the two factors. Statistical analysis from acetic acid production is tem-perature giving a significat effect to acetic acid production (P value = 0,036 < α), while pH has no significant effect to acetid acid production (P value = 0,103 > α).