93-102.

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In media 1 and 2, pH increased from the beginning of cultivation until 12th day with the
highest pH were 10.3 and 10.2, respectively (fig.1). The increasing of pH was caused by
presence of sodium bicarbonate (NaHCO3) in medium. Dodium bicarbonate was dissolved into
Na+ and HCO3 (bicarbonate ion). Na+ was used as micro nutrient by S.platensis while HCO3
was converted in form CO2 and OH- with help of enzyme carbonic anhydrase (Reuter and
Muller 1993); Jaiswal, et al 2005; Badger et eal 1994). CO2 formed was utilized as carbon
source for photosynthesis. Ion OH- accumulated in medium caused alkaline in pH (Richmond
and Grobbelaar 1986; Grobbelaar 2004; De Morais and Costa, 2007)
The increasing in pH was related with photosynthetic activity, where the higher the rate
of photosynthetic activity, the higher the pH medium (Andrade and Costa, 2007). In Fig.1,
S.Platensis reached the point of death on the 14th day and 16th day respectively in medium I and
II after it reached the highest pH at 12th day.
While in medium III-VII, pH decreased at first time of cultivation and then when up.
This phenomenon might be caused by activity of bacteria in medium. The more concentration of
digested vinasse were added, the more amount of bacteria was contained in medium. Based on
principle that was proposed by Oswald et al, 1957), at beginning of cultivation oxidation bacteria
in medium converted organic compounds of wastewater into CO2 via respiration. CO2 formed
reacted with water to form carbonate, so medium to be acidic. Then, carbonate was used by
S.platensis for photosynthetic process and released OH-, so pH medium gradually increased.
Besides activity of bacterial oxidation might participate in the system, S.platensis also
utilized organic carbon as source of carbon to produce CO2 through respiration. This growth
called hetetrophic growth, whereby energy and carbon source derived from organic carbon such
as glucose. Carbon dioxide formed caused acidic in pH medium. Furthermore, S. platensis used
CO2 for photosynthesis. This growth was called photoautotrophic and heterotrophic processes
that took place simultaneously in cell were called mixotrophic growth (Marqueez et al, 1993).
Ogbonna and Tanaka (1998) stated that addition of organic carbon would reduce the
production of photosynthetic pigments, so rate of photosynthesis was slowly. Carbon dioxide
was the main of carbon source needed in photosynthesis. In this study, carbon dioxide was
obtained from NaHCO3. The more digested vinasse was added into medium, the more slowly
rate of photosynthesis went on in system (Fig.1).
 Production of biomassa in media I and II declined after 14th and 16th day, respectively.
This declined phase was called deth phase because a lot of algal cells were death. This phase was
occurred due to the age old culture and the limitation of light and energy supply. Membran cell
of S.platensis broke (lysis), so organic materials in cell were out and dissolved into medium
(Fogg and Thake, 1987). This phenomenon might cause decreasing in pH medium. This could be
shown in Fig.1, growth of S.platensis decreased and pH of medium decreased too.
The mimum growth rate (umax) of S.platensis on each medium could be seen in Table 2.
The greater the concentration of digested vinasse, the smaller the maximum growth rate. Media
II, with the addition of the smallest concentration of digested vinasse (0.8% v/v or 242 mg/L
COD), had the highest umax of the others (medium III-VII). However, medium II had less umax
than media I (without digested vinasse addition) although medium II had more time exponential
phase than medium I. That was caused by dark color and turbidity medium that reduced
penetration of light into medium. Richmond (1988) confirmed that organic carbon and light were
two important factors that influenced the growth of mixotrophic microalgae.
Bioresource technology 149 (2013)
Bacteria involved in biogas production used ammonia/ammonium as nitrogen sources.
However, in large amount of these caused inhibition or toxic for bacterial growth. Deublein and
Steinhauser (2008) reported that, ammonia concentration of 80 mg/L was start inhibition and that
of 150 mg/L was toxic for bacteria. Whereas, ammonium concentration of 1500–10,000 mg/L
was start inhibition and that of 30,000 was toxic for bacteria. Composition of ammonia and
ammonium was depended on pH condition of substrate. Ammonia and ammonium had
permanent equilibrium:
𝑁𝐻4 → 𝑁𝐻3 + 𝐻 +
𝑁𝐻4+ + 𝑂𝐻 − → 𝑁𝐻3 + 𝐻2 𝑂

(Sung and Liu, 2003). Ammonia was more toxic than ammonium.
The more acid of pH condition, the more concentration of ammonium in the substrate, so
400/7 variable generated ammonium in too large amount and it had inhibiting effect to bacterial
activity. Variable of 700/7 also generated less total biogas than variable of 600/7 and more that
than control variable. That was caused by amount of ammonium formed during fermentation. At
COD/N of 700/7, ammonium formed was not enough to be used as nitrogen source by bacteria
so that the growth of bacteria was disturbed and finally biogas formed in little amount.
Niu et al. (2013a) reported that at mesophilic temperature, ammonium concentration less
than 5000 mg/L caused COD removal efficiency over 60% and carbohydrate removal efficiency
of 80%. Increasing of ammonium concentration from 5000 mg/L to 15,000 mg/L, the VFAs
accumulation increased sharply from 2000 to 15,000 mg/L, and COD, carbohydrate, protein
removal efficiency gradually decreased. Ammonium concentration over than 4000 mg/L, biogas
production slightly decreased. Whereas ammonium concentration of 16,000 mg/L caused biogas
production ceased. Niu et al. (2013b) reported that at thermophilic temperature (55 •} 1 _C), the
inhibition concentration of ammonium started from 4000 mg/L. Increasing ammonium
concentration from 4000 to 6000 mg/L, the VFAs accumulation increased sharply from 5000 to
25,000 mg/L. This increasing of ammonium concentration was quite sensitive to biogas
composition (methane production decreased from 0.20 to 0.05 L/g VS). Moreover, synergy
inhibition of ammonium (8000 mg/L) and VFAs (25,000 mg/L) caused biogas production almost
ceased. Whereas Sung and Liu (2003) reported that inhibiting NH4 concentration of 5770 mg/L
caused 64% reduction in methane production. From results of Niu et al. (2013a, 2013b), can be
concluded that ammonium concentration of 5000 mg/L was maximum concentration allowed in
the digester at both thermophilic and mesophilic temperature.
Biogas production process was consisted of three phases that were hydrolysis,
acidogenesis, acetogenesis and methanogenesis. Niu et al. (2013a) reported that hydrolysis
inhibiton was occurred at ammonium concentration of 5500–15,300 mg/L. Whereas acidogenesis
and methanogenesis were occurred at ammonium concentration of 6500–15,000 and 4800–
13,000 mg/L, respectively. Among the three types of anaerobic bacteria, the methanogenic
bacteria was the least tolerant to ammonium inhibition (Niu et al., 2013a).
Vinasse contained organic materials such as acetic acid, lactic acid and glycerol (Yavuz,
2007). Therefore, bacteria in the digester transformed these organic materials easily to be biogas.
From Fig. 1(a), biogas began to form at 1st and reached maximum point of biogas production
daily at 3rd fermentation. After the 3rd day of fermentation, biogas production daily was drop
sharply. The inhibiting of ammonium cannot be a reason because all of variables had the same
profile, so this phenomenon might be caused by presence of phenolic compounds in the vinasse.
Vinasse generated from ethanol industry that produced ethanol from molasses, contained
phenolic compounds in high level (Martin et al., 2002; Garcia-Garcia et al., 1997). Phenolic
compounds were very difficult to be degraded through biological activity and had antimicrobial
and phytotoxic properties, so that disturbed decomposition process of organic materials in the
digesters. Phenolic compounds disturbed bacterial activity in ways: (1) reaction with membrane
cell, (2) inactivation of essential enzymes, and (3) destruction and inactivation of material
genetic functions (Brannen and Davidson, 1993).
Speece (1996) stated that ammonium concentration in the digester must be maintained in
excess of 40–70 mg/L to prevent a reduction in the activity of the biomass. That means, activity
of biomass needed 40–70 mg/L ammonium every day in the digester. In this work, digesters
were operated in 60 days, so ammonium excess needed in 60 days = 60x70 = 4200mg/L. Based
on this theory, variable control (COD/N of 1436/7) produced 3220mg/L but activity of biomass
needed 4200 mg/L. Thus, biogas formed was just in little amount because nitrogen source
(ammonium) not enough to build cell structure by bacteria.
Vinasse contained COD in large amount so that VFAs easily to be produced in the
system. Accumulation of VFAs caused decreasing in pH. pH condition of substrate could be
increased by accumulation of NH4+ that was produced by degradation of protein and urea.
Accumulation of ammonium in 700/7 variable was too little, so decreasing in pH was occurred
easily. In this work, authors maintained pH substrate in neutral condition by using NaOH 2 N
each two days. The fluctuation of pH condition in big gap might hamper the bacterial activity in
the digester. Whereas, 600/7 had accumulation of ammonium concentration in appropriate
amount, which was 5089 mg/L. According to Niu et al. (2013a, 2013b), ammonium
concentration above of 5000 mg/L was start inhibition. Biogas was produced optimally at
ammonium concentration not more of 5000 mg/L.
IJE 27 (2014)
%TS
In anaerobic digestion, organic materials were decomposed by microorganisms through
four stages, namely:hydrolysis, acidogenesis, acetogenesis and methanogenesis. The product of
the process was biogas.
This research was done to investigate the effect of water addition on total solid (TS %) in
the initial substrates. Variation of vinasse:water ratio caused change in solid concentration and
COD content of substrates (Table 3).
Putri et al. reported that water molecule was needed to support the hydrolysis reaction at
acetogenesis stage. At stage of hydrolysis, hydrolytic microbe degraded complex organic
compounds (carbohydrate, protein, fat) into simple organic compounds (glucose, LCVFA, amino
acid). At acetogenesis stage, acetogenic bacteria converted ethanol, propionate acid and butyric
acid into acetic acid [1].
Budiyono et al. reported that the ratio cattle manure:water:rumen fluid produced the
biggest total biogas which has TS between 7.4 – 9.2% [2]. Budiyono’s result confirmed the
report of Zennaki et al. that solids concentration 7 – 9% in the substrate produced biogas
optimally [17]. Below total solids level of 7%, process degradation of materials into biogas was
unstable while total solids level of 10% caused an overloading of the fermenter [2, 27]
In this research, variable R13 (TS 7.015±0.007%) produced the biggest total biogas and
had the fastest biogas production rate. The result of this study was similar with those of the other
authors [2, 17, 27]. This research, authors did not use solid waste such as cattle manure but used
liquid waste, vinasse. Authors conclude that total solid level of either solid waste or liquid waste
need to be considered.

pH Profile
In anaerobic digestion, the optimum condition of acidity for biogas production is in the
range 6.8 to 8.2. Biogas production rate will decline at pH condition higher or lower than the
range [10]. In the early stages of fermentation process, large amounts of organic acid are
produced by acid-forming bacteria, so that pH in the digester can reach under 5. Then, the
fermentation process continuous and pH value gradually becomes neutral. Decomposition of
protein produces NH4-N that makes pH neutral in the digester [28].
 Acidogenic bacteria produce acetate, hydrogen gas, carbon dioxide and few other VFA
such as propianic and butyric acid. These products make pH drop. A low pH value inhibits the
activity of microorganisms that are involved in the biogas production especially methanogenic
bacteria [10,[29].
Vinasse contained variety of organic substances such as acetic acid, lactic acid and
glycerol [30]. In this research, biogas was formed quickly in the beginning of fermentation, but
after 4-12th day (Figure 2 and Figure 3(b)), biogas was decreasing and finally ran out. Acetic
acid, lactic acid and glycerol were simple organic compounds which can be easily converted by
microorganisms into biogas. The drop in pH decreased biogas production rate (Figure 3). This
phenomenon was caused by ratio COD:N of substrate. The good COD:N range to produce
biogas optimally is 350:7 – 1000:7 [10]. In this research, vinasse obtained had COD:N ratio =
1436:7.
Carbohydrate-rich substrates were good producers of VFAs and protein-rich substrates
were good buffering capacity due to production of ammonia. So, if ratio between carbohydrate
(C) and protein (N) are appropriate, biogas will be produced optimally [31].
This was caused by activity of acidogenic and methanogenic bacteria. The more organic
compounds entered into digesters caused the faster acidogenic bacteria growth rate. Organic
compounds will be converted into fatty acid and it caused pH to decrease. Whereas,
methanogenic bacteria could not grow well at low pH. This condition caused biogas production
low although COD removal was big. This phenomenon could be concluded that the fermentation
process was dominated by acidogenesis process and activity of methanogenic bacteria did not
grow well in the system (Figure 3).
Iqbal Syaich tekkim ije kinetics
Effect of yeast
Total biogas from variables of TY5, TY6, TY7, TY8, TY9 was 220, 333, 370, 421, 374
mL respectively. By addition of yeast (TY5-TY9), total biogas was increased as much as 22.91,
81.97, 56.12, 53.09, 42.21 % respectively compared T5-T9 (without yeast addition). Yeast
Saccharomyces cerevisiae converted glucose into ethanol, acetic acid and butyric acid [23]. The
reaction can be seen in Equations (3) and (4). In Equation (3), 4 mol of glucose were converted
to 2 mol of acetic acid and 3 mol of butyric acid. Whereas in Equation (4), 1 mol of glucose was
converted to 1 mol ethanol and 1 mol of acetic acid.

Meanwhile, in biogas production processing, ethanol, acetic acid and butyric acid were resulted
from acidogenesis phase. The acetic acid formed could be converted into methane and carbon
dioxide by methanogenic bacteria directly. However, the methanogenic bacteria could not
convert ethanol and butyric acid into methane. Hence, the methanogenic bacteria needed help of
acetogenic bacteria to change ethanol and butyric acid into acetic acid and hydrogen. Then,
acetic acid was converted to be methane [24]. In TY5-TY9, the yeast would convert glucose into
ethanol, acetic and butyric acid. These compounds were intermediate products of biogas. Thus,
total biogas formed was more than that in substrates without yeast addition (T5-T9).
Figure 1 showed the results of the batch test used to investigate the effect of initial pH on
biogas production with yeast addition. When the pH was 5, the total biogas was 220 mL.
Whereas, when the pH was above 5, the quantity of biogas produced substantially increased. The
most biogas production (421 mL) was reached at initial pH of 8. In addition, when the pH was 9,
the total biogas was lower than that when pH of 8. Lin et al. [23] stated that yeast produced
ethanol, acetic and butyric acid with composition depended by initial pH. The best initial pH for
ethanol production was 5, the composition of ethanol, acetic acid and butyric acid was 65.54,
1.63, 0.02 %, respectively. Furthermore, at initial pH of 6, the composition of them was 48.80,
9.00, 17.05 %, respectively. We concluded that, the more alkaline of pH, the less of ethanol and
the more acetic and butyric acid formed. Thus, in this work, initial pH of 5 – 7 produced ethanol
in high concentration. The high amount of ethanol was in the system, methanogenic bacteria
were death. Whereas, initial pH of 8 – 9 produced ethanol in concentration which was still
tolerance for methanogenic bacteria, and high acetic and butyric acid which were used as raw
materials to produce biogas.
The substrate pH of TY5, TY6, TY7, TY8, TY9 was changing during fermentation, from
5, 6, 7, 8, 9 to 5.43, 5.46, 5.63, 5.74, 5.71, respectively. By presence of yeast, the final pH was
lower than that at no yeast addition (Figure 1 and Table 1). The ethanol, acetic and butyric acid,
which were produced by yeast activity, were accumulated in the system. Thus, the substrates of
TY5-TY9 were more acidic than substrates of T5-T9.

TS Removal
The total solid (TS) removal was analyzed to know the effect of initial pH and yeast
addition on organic material removal. The initial and final TS were shown in Table 1. In
addition, Figure 4 presented the TS removal for all variables. The more the final TS value, the
less the TS removal value. The TS removal in TY5-TY9 (12.628-66.669 %) was more than that
in T5-T9 (13.265-33.716 %). The yeast helped the anaerobic bacteria to degrade organic
materials into biogas. Both without and with yeast addition, the TS removal increased when the
initial pH was increased from pH 5 to 6. Furthermore, at pH 7, the TS removal value was the
least. When the initial pH was increased from pH 7 to 9, the TS removal increased. The biggest
of TS removal was at pH of 9.
Yeast Saccharomyces cerevisiae grew optimally at pH of 5. In that condition, the yeast
produced ethanol, acetic acid and butyric acid with composition of 65.54, 1.63, 0.02 %
respectively. When the pH was above 7, the ethanol production decreased and the acetic and
butyric acid increased. Although TS removal at initial pH 5-6 was higher than that at pH 7, the
biogas production at pH 7 was more than that at pH 5-6. That might be caused by ethanol
production in large amount. Hence, that disturbed methanogenic bacteria so that the biogas
production was just little at pH 5-6. When pH of 7, the Saccharomyces cerevisiae still grew but
its growth rate was lower. The yeast also produced high acetic and butyric acid. The
methanogenic bacteria converted these compounds into biogas. Thus, the biogas production was
higher than that at pH 5-6, although the TS removal value was low. The pH of 8 was the best
condition, because Saccharomyces cerevisiae produced compounds with high acetic and butyric
acid which was higher than that at pH 7. Anaerobic bacteria, especially methanogenic bacteria,
grew well in substrate of tofu wastewater at initial pH of 8. Therefore, the biogas formed was the
highest of all variables. The methanogenic-bacterial activity was hampered at pH above 8.
However, the hydrolysis bacteria contained in rumen fluid still can live at the condition. Hence,
total biogas at pH 9 was lower than that at pH 8, although the TS removal was higher than at pH
8. We concluded that at pH 5-6, the organic materials removal was dominant by yeast
Saccharomyces cerevisiae. At pH 8-9, the organic materials removal was dominant by hydrolysis
bacteria (Clostridium sp.). Whereas at pH 7, both of microbes, Saccharomyces cerevisiae and
Clostridium sp. could grow well but not optimally, so that the TS removal was low although the
biogas formed was high enough.
Iqbal Syaich Tekkim IJRER

Organic acid was divided into two kinds which was not dissociated acid and dissociated
acid. Composition of them in the substrates was depended on pH value. The more acid of pH
value, the more the presence of not dissociated acid in the substrate. The presence of not
dissociated acid disturbed methanogenesis phase because that organic acid was penetrated into
bacterial cell and denatured protein of bacteria [3]. Furthermore, Brannen and Davidson [23]
reported that protein, nucleid acid and fosfolipid in the bacterial body were damaged by dropping
pH (< 7.0). Therefore, ratio of VW:TW of 60:40, 80:20, 100:0 produced biogas in little amount
which was 19.96, 18.77, 10.64 mL/g COD respectively.
Substrate with VW:TW ratio of 20:80 and 40:60 produced biogas of 159.01 and 120.22
mL/g COD respectively. The pH value of that was decreasing from 7.0 until 5.1 and 4.9
respectively. That condition was bothering bacterial activity during fermentation, but anaerobic
bacteria still can adapt in this condition until 14 – 16 day of fermentation. This phenomenon was
caused by final pH in 20:80 and 40:60 that was higher than final pH in substrate of 60:40, 80:20,
100:0 (4.0, 3.9, 3.9). The best ratio value was 20:80, because its COD/N ratio was the closest to
the optimum range which was 1042/7 and generated biogas was the most of all variables.
Addition of vinasse waste as co-substrate of tofu-processing wastewater was caused not
only COD/N substrate in good range, but also supplying micronutrients in the system. Vinasse
contained micronutrients needed by anaerobic bacteria which were K+, Na+, Ca2+, Mg2+ of 6.5,
0.35, 9.0, 2.5 g/kg TS respectively. This ion increased the efficiency of organic fermentation
[24]. In this research, fermentation of substrate mixing VW and TW with composition of 20-40%
VW volume was better on biogas production rate than fermentation of VW or TW alone.
Addition of too high amount of vinasse into the tofu-processing wastewater decreased biogas
yield.
 V7-2798
At substrate of VR (Vinasse+Rumen), initial pH of 7 generated the more biogas yield
(3.74 mL/g VS) than the other variables, which were initial pH 6 (3.19 mL/g VS) and initial pH
8 (3.43 mL/g VS). Some authors reported that pH condition of neutral produced biogas
maximally. According to Metcalf (2003), the best range pH condition to produce biogas was 6.9-
7.3. Anderson and Yang (1992) stated that 6.4-7.6 was the optimum range in anaerobic
digestion. Budiyono et al. (2013) reported that pH neutral (7) was the best pH condition needed
anaerobic bacteria to do degradation process and generate biogas. Thus, pH neutral was the
optimum condition in biogas production process.
From Fig. 3a, variable with initial pH of 6, 7, 8 had the same pH profile daily, but initial
pH of 7 generated the most of total biogas. Based on that, lag period, that time needed bacteria to
adapt in substrate, was the main point which was at the first two day.
Decreasing in pH was caused by production of VFAs (Volatile Fatty Acids) during
fermentation. Vinasse obtained in this study was produced from ethanol industry that produced
ethanol from molasses. In the process of producing ethanol, molasses was hydrolyzed and
fermented with help of Saccaromyces cerevisiae. Then, ethanol formed was separated from broth
using distillation. The bottom product of distillation was called vinasse so that vinasse contained
many simple molecular chain compounds (Budiyono et al., 2013). These compounds were
degraded easily by bacteria so that VFAs was formed in large amount. Accumulation of VFAs
caused drop in pH (Fig. 3). Sumardiono et al. (2013) reported that during degradation process of
vinasse in the anaerobic digesters, pH profile of substrate had decreasing trend from beginning
until ending of process. That was caused by high carbohydrate content of vinasse. Substrates that
were rich carbohydrate generated VFAs in large amount. Yadvika et al. (2004) stated that
concentration of VFAs in digester was no more than 2000 mg/L that was good for fermentation
process.
During fermentation process, there are two kinds of organic acid in the substrate, which
are not dissociated acid and dissociated acid (Deublein and Steinhauser, 2008). The ratio
composition of them is depended on pH substrate. The more acid condition of substrate, the
more amount of not dissociated acid. In this study, pH substrate was drop (Fig. 3), so the kind of
organics acid that was dominant, was not dissociated acid. Presence of not dissociated acid
hampered the methanogenic bacterial activity. Not dissociated acid was penetrated into cell and
denatured the protein of bacteria (Deublein and Steinhauser, 2008). Whereas, According to
Brannen and Davidson (1993), the inhibitory mechanism of bacterial activity by organic acid
(acetic acid, propionic acid, butyric acid) was related with acid-base equilibrium. Acid-base
equilibrium in cell of bacteria was in neutral condition of pH. Organic acid penetrated into cell,
disturbed acid-base equilibrium so that bacterial cell was lysis. Changing in pH could spoil
protein, nucleic acid and phospholipid in cell bacteria. At pH condition less than 7, presence of
acetic acid of 1000 mg/L could hamper degradation process. Whereas, iso-butyric acid and iso-
valeratewere 50 mg/L; propionic acid was 5 mg/L (Deublein and Steinhauser, 2008).
Initial pH of 7 was the optimum initial pH condition at substrate with or without urea
addition. Addition of urea did not change the pH profile (Fig. 3b). It showed that VFAs was
generated rapidly and in large amount so that bacteria involved biogas production was death.
Elbeshbishy and Nakhla (2012) stated that accumulation of VFAs in large concentration was
toxic for methanogenic bacteria.
At initial pH of 6, at substrate of VRU biogas formed had 1.4 times greater than that at
substrate of VR. Whereas, at initial pH of 7 and 8 were 1.7 and 1.1 times greater than at VR.
Budiyono et al. (2013) stated that at urea addition, biogas formed had 52.47% greater than that at
not urea addition.
That might be caused by microorganism (Saccaromyces cerevisiae) that might be
presented in vinasse. In ethanol industry, this microorganism actively converts glucose into
ethanol and CO2 at pH of 5. Between pH of 6 and 7, S. cerevisiae might more actively at pH of 6

than pH of 7. Based on that, initial pH of 6 was faster to produce biogas firstly than initial pH of
7, because S. cerevisiae actively generated ethanol (liquid) and CO2 (gas). Biogas formed from
th th
initial pH of 6 might be dominated by carbon dioxide (CO2). However, in the 4 and 6 day of

fermentation, initial pH of 6 generated biogas that was less than initial pH of 7. S. cerevisiae
might be involved in the system just at first time fermentation.
World applied sciences journal 26 (2013)

Ammonia (NH ) formedammonium ( 3 NH4 ) depend on + pH condition. Ammonium

had less toxic than ammonia. Ammonium will be toxic just in high concentration. Ammonium
concentration of 1,500-10,000 mg/L was inhibition start and that of 30,000 mg/L was toxicity
concentration [31].
Substrate with COD/N ratio of 400/7 might contain nitrogen total that was too much, so
that ammonia/ammonium formed caused toxicity for bacterial activity. Whereas, substrate with
COD/N ratio of 700/7 contained nitrogen total that was not in appropriate amount yet.Although,
COD/N of 400/7 and 700/7 was included in optimum range that was stated by Speece [17]. The
good COD/N ratio in this study was 500/7 - 600/7.