Table 7. Concentration of soluble sugar during PKM fermentation Time of
sampling hour D-glucose
gL D-fructose
gL D-mannose
gL 0.793
±0.18 0.955
±0.08 1.739
±0.01 36
0.063 ±0.06
0.135 ±0.01
3.297 ±0.13
48 0.034
±0.00 0.080
±0.02 0.065
±0.03 72
0.018 ±0.01
0.014 ±0.00
0.146 ±0.00
96 0.007
±0.00 0.044
±0.01 0.123
±0.02 120
0.012 ±0.02
0.014 ±0.00
0.142 ±0.00
Average value ±SD
Volatile organic acid concentrations along the process of fermentation could be seen in Table 8. During fermentation, acetic acid dominates the
composition of volatile organic acids. After 72 hours the concentration of acetic acid raised, up to 87.89 mMol, compared to the acid’s concentration on the 36
hour. Table 8. Concentration of volatile organic acid mMol during PKM fermentation
Type of volatile organic acid
mMol Time of sampling hour
36 72
168 Acetic acid
67.17 31.89
87.89 55.41
Propionic acid 2.97
10.51 2.95
2.37 Iso butyric acid
0.38 0.25
0.17 0.23
n-Butyric acid 1.68
0.62 0.37
0.42 Iso valeric acid
0.33 0.22
0.20 0.18
n-Valeric acid 0.00
0.00 0.00
0.00
4.2. Discussion
Fermentation is a degradation process of complex organic substance into simpler ones, with the aid of microorganisms. This process could occur in aerobic
or anaerobic conditions Gandjar et al. 2006. During the fermentation process of PKM, a fluctuation of total microorganism occurred. In this research, lactic acid
bacteria were not detected until 24 hour of fermentation, then rise at 36 hours of fermentation. This rise of lactic acid bacteria causes the accumulation of acid
concentration in the fermentation system. The increase of acid concentration was followed by the decrease of pH fermentation. Total lactic acid bacteria drops
when the fermentation pH reaches 3 after 84 hours fermentation. The same phenomenon could be seen in the fermentation of cassava. The increase of total
lactic acid bacteria lead to the increase of titratable acidity and decreased the pH of fermentation. However, the total lactic acid bacteria decreased when the pH of
fermentation below 4 Kakou et al. 2010. This was caused by the fact that pH level is a limiting factor to microorganism growth Madigan et al. 2009.
The highest hemicellulose content in PKM is mannan. Clostridium is known to have the ability to degrade mannan; therefore a specific media, RCA,
was used to isolate and observe the total Clostridium colony change along the fermentation process of PKM. Bacteria from the RCA media were not discovered
until 12 hour of fermentation and the number continue to increase when the pH of fermentation around 4
The cell wall of PKM consists of cellulose, lignin, and hemicelluloses mannan that hard, crystalline, and water insoluble. In degradation of cell walls,
enzymes produced by microorganism must be able to penetrate into the cell. The enzymes produced by bacteria generally smaller than the enzymes produced by
fungi, making it easier to penetrate into the cell. Therefore, in the fermentation of PKM, fungi were found at 96 hours of fermentation.
. This indicates that Clostridium is capable of living in an acidic environment with a pH level below 5. Similar thing happens in the
fermentation of cassava by Brauman et al. 1996, Clostridium spp. was found when the pH below 5 which characterized by the presence of butyric acid.
In the fermentation process of PKM, polysaccharides degraded into monosaccharides. The concentration of mannose increased after 36 hours of
fermentation. The results of NDF analysis hemicelluloses, cellulose, and lignin also showed a decrease. However, if specified, the lignin content decreased
drastically at 36 hours and seen that the value of hemicellulose and cellulose increased. This is because the measuring unit used is percentage , so when one
of decline, the others will look like an increase, to fulfill the composition of 100. Therefore, to better illustrate the changes that occur during this process, the
calculation of the polysaccharide content should be in weightvolume wv. Degradation of cellulose into glucose needs the synergy of three major
enzymes, endoglucanase to breakdown cellulose molecule, as an initial act; exoglucanase acts from the non-reducing end to remove the cellobiose units; and
at the end, -glucosidase will complete the hydrolysis of celloboise to glucose
Ahmed et al. 2001. Mannan as the biggest content of hemicellulose in PKM will degrade into mannose and manno-oligosaccharides by some microorganism’s
enzyme. The degradation of mannan was done by some enzymes i.e. endo- - mannanase which cleavage -1,4-linked internal linkages of the mannan backbone
randomly; exo- - mannosidase which cleavege -1,4-linked mannosides and
produce mannose from the nonreducing end of mannans and manno- oligosaccharides; and exo- -glucosidase which will hydrolyze 1,4- -D-
glucopyranose at the nonreducing end of the oligosaccharides released from glucomannan and galacto-
glucomannan by -mannanase Moreira Filho 2008. Degradation process of mannan into mannose and mano-oligosaccharides
can be performed by aerobic and anaerobic bacteria Moreira Filho 2008. Concentration of mannose in fermentation of PKM has increased twice, at 36 and
at 72 hours fermentation. The increased of mannose concentration at 36 hours of fermentations could be the result of degradation mannan by aerobic bacteria such
as Bacillus circulans, Bacillus subtilis, Pseudomonas flourescens, Vibrio sp. Meanwhile, at 72 hours degradation of mannan into mannose was done by
anaerobic bacteria such as Clostridium thermocellum dan Clostridium cellulolytic Moreira Filho 2008; Sundu Dingle 2003.
Production of mannan-degrading enzymes by fungi and bacteria was found to be inducible. Monomer and oligosaccharides, which are the degradation
product of mannan by the action of a basal level of hydrolytic enzymes present in the cell, act as inducers and play a key role in the regulation of mannan
biosynthesis Moreira Filho 2008. In the fermentation of PKM, the existing mannose at the beginning of fermentation at 0 hours acts as an enzyme inducer
of -mannanase. As a hydrolysis product of mannan, mannose and manno-oligosaccharides
are known as a special nutrient for lactic acid bacteria such as Bifidobacterium sp. and Lactobacillus sp. It is possible that the growth of lactic acid bacteria in the
PKM fermentation influence by mannose concentration. Phothichitto et al. 2006 have been isolated Bacillus circulans from soil. This B. circulans have ability to
hydrolyze mannan and could promote growth of Lactobacillus reuteri AC5.
Lignin was the most degraded polysaccharide after 168 hours of fermentation. However, the greatest lignin degradation process occurs in 36 hours
of fermentation. Probably, after 36 hours of fermentation the amounts of oxygen in the fermentation system dwindle. This is indicated the by increase of the total
anaerobic bacteria. Banner et al. reported that conversion of lignin into a product gas slower in anaerobic than in aerobic condition see Vicuna 1988. This result
also indicates that the fermenting microorganism has the ability to degrade lignin. Martani et al. 2003 showed that Micrococcus and Bacillus are able to degraded
lignin from black liquor, by product of pulp industry, about 75 and 78 respectively within 14 days of incubation.
The degradation of polysaccharides caused the increase of protein content of PKM. After 168 hours fermentation, the protein content was increased only
4.79. The increased in crude protein value of degraded PKM was partly due to ability of enzyme to increase the bioavailability of the protein hitherto
encapsulated by cell walls. Glucose, fructose, and mannose, product of degradation polysaccharide,
converted to pyruvate via the glycolysis pathway and pyruvate is split to yield acetyl-CoenzimA. In aerobic condition, acetyl-CoA will enter the Krebs cycle. In
anaerobic condition, acetyl-CoA is reduced into acetic acid or other fermentation products using NADH derived from glycolysis reactions as electron donor
Madigan et al. 2009. The increasing acetic acid concentration at 72 hours of fermentation showed the accumulation of acetic acid as a product of anaerobic
fermentation. Butyric acid was a major end product of fermentation by Clostridium. The
fermentation product was influenced by the duration and the condition of the fermentation. In fermentation of PKM, the concentration of butyric acid is very
few. It is because during fermentation of PKM, the pH was acid around 4. The same result also showed by Zu and Yang 2004, who tried to fermente xylose by
Clostridium tyrobutyricum on pH between 5.0 and 6.3. At pH 6.3, the
fermentation gave a high butyrate production than at low pHs 5.7. At low pH, the fermentation produced more acetate and lactate as the main products, with
only a small amount of butyric acid. This situation associated with changes in the
activities of several key enzymes. The activities of phosphotransbutyrylase PTB, which is the key enzyme controlling butyrate formation, and NAD-independent
lactate dehydrogenaseiLDH, which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast,
cells at pH 5.0 had higher activities of phosphotransacetylase PTA, which is the key enzyme controlling acetate formation, and lactate dehydrogenase LDH,
which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Some species Clostridium also produce
acetone and butanol as fermentation product. The synthesis of acid will ceases when the pH of medium drop and the neutral product such as butanol and acetone
begin to accumulate Madigan et al. 2009. At the beginning of fermentation 0 hour, the concentration of acetic acid
was higher than at 36 hours. This is probably because concentration of acetic acid at the beginning of fermentation was a product from PKM fermentation during
storage in the warehouse. This probability supported by decreasing of pH of PKM from 5.25, when recently come from the factory Lampung, to 4.56 after storage
for a year at 0 hour. A decrease in pH indicates that acid accumulation in PKM is the product of fermentation.
V. CONCLUSIONS AND RECOMENDATION
