Folder ISR, 1-s2.

0-S18766
PH is a very interesting indicator in the stabilization process it shows us the good
behavior of the anaerobic digestion [10]. Anaerobic processes are strongly influenced by changes
in pH. The anaerobic digestion takes place optimally near neutrality pH 7 with optimal value
between 6.5 and 7.5 [11].
As can be seen in Fig. 1, the pH evolution, for all ratios (ISRs), can be divided into three
main parts. The first part lasted 10 days and characterized by a drop in pH from 7.5 to a
minimum value of 6.5. During the anaerobic acid-phase stage of complex organic substrates,
mainly constituted by carbohydrates, proteins and lipids, these are converted basically to volatile
fatty acids (VFA) and to a lesser extent to other low molecular weight compounds [12].
In the second part, the pH values increased. This increase is due to the fatty acids
consumption by bacteria acetogenesis [13] to produce the biogas precursors. And finally, the
third part from the 22nd day, during which we noted a pH values stabilization between 6.8 and
7.6. It is explained by the stabilization process of anaerobic digestion and it reached of all the
anaerobic digestion stages. These results were noted by [14,15] in their researches on anaerobic
digestion of organic waste.
EFFECT OF INOCULUM TO SUBSTRAT RATIO

Biogas production rates of GPM were highly unstable as I/S ratio increased from 1.45 to
4.3 and showed no significant difference at higher I/S ratios. I/S ratio showed significant effect
on biogas production accumulation where biogas production accumulation increased from
0.4427 to 1.00783 Nm3kg-1VS with I/S ratio increase. Modified Gompertz regression of
experimental data showed better simulation of biogas production accumulation than first order
kinetic regression.
ENVIROMENTAL TECH VOLUME ISSUE 2016

The optimal I/S range for biogas production was 1.5-2.0 in digestion of alage and
municipal waste, [15][16] it was suggested that optimal I/S change slightly with the kind of
substrate and higher I/S was insignificant in enhancing the methane production.

The relation of I/S and biogas yield was shown in Fig. 1. The biogas yield increased from
220.5 to 433.4 mL/gVS with I/S in the range of 0.95-2.2 and decreased when I/S exceed 2.2, the
highest biogas yield (433.4 mL/gVS) was achieved at I/S of 2.19. It suggested that I/S was an
important factor in anaerobic digestion of FW.

The high methane content could be attributed to that FW were rich in lipids and proteins
and methane content of biogas generated by lipids and proteins was higher than that of generated
by carbohydrates.[20][21] For the methane content which was less than 70%, most of it came
from the bioreactors which I/S were below 1.5, probably because low I/S resulted in low
cumulative biogas production and part of CO2 did not transfer into CH4.

The concentration of ammonia and VFA decreased as I/S value raised at a certain load of
FW or inoculum, it suggested that high I/S value was contributed to reduce the accumulation of
ammonia and VFA.

The VFA composition under different I/S value was similar, it suggested I/S value had no
influence on VFA composition, maybe because the VFA composition was highly dependent on
the type of substrate.[25]

The biogas production was respectively up to 254.1, 346.8, 329.0 mL on the first day, but
no biogas was generated in next few days and biogas production was recovered after 6, 13, 8
days. The abundant protein and lipid in fish waste may cause the risk of ammonia or LCFA
inhibition in anaerobic digestion. VFA has no direct inhibitory effect on methane production,
but high concentration of VFA can provoke a drop on pH, resulting in the decline of activity of
methanogens and methane production.[29-31] Considering that ammonia concentration and lipid
content of FW (10.8%) were far away from the inhibition threshold, but the VFA concentration
was at a high value, the possibility of VFA inhibition were higher than that of LCFA and
ammonia inhibition. The inhibition phenomenon appeared in the bioreactors with low I/S. Liu et
al.[32] also observed the lag on biogas production caused by VFA inhibition at low I/S ratio and
high organic load.

It was easy to see that daily gas production gradually declined and then had two
fluctuations. The biogas production declined in days 1-9 perhaps because of the accumulation of
VFA and the drop of pH, the increase of biogas production after 10 days may be owed to the
hydrolysis of ammonia, which caused pH value going up.
OPTIMIZATION OF INOCULUM TO SUBSTRAT RATIO

For optimization of I/S ratio for co-digestion of fleshings and the mixture of tannery
sludge (primary and the secondary sludge), experiments were carried out in batch reactors by
varying the I/S ratio as reported in Table 1. The biogas generation was monitored on daily basis,
and cumulative biogas generation is presented in Table 4 and it compares the cumulative biogas
generated with different I/S ratios with a constant substrate input of 7.5 g of VS.

As the I/S ratio was increased from 0.25 to 2.30, there was an increase in biogas
generation of 1087–2933 ml was observed. Daily biogas generation for different I/S ratios is
presented in Fig. 2. It was also observed that the substrate utilization rate (SUR) increased with
increase in the amount of inoculum. Irrespective of I/S ratio, the maximum biogas generation
was observed for the residence time of 35–45 days. However, the batch reactors were operated
for residence time of 50 days wherein biogas generation was ceased.

When I/S ratio decreased, methanogenic activity slow down resulting in decrease in
generation of biogas was observed. Similar findings were observed by Hashimoto (1989) for the
batch fermentation of wheat straw when I/S ratio was below 0.25, methane yield was reduced
drastically. Chynoweth et al. (1993, 2001) reported that at an F/M ratio of 0.5 maximum
conversion rate of substrate was reached, and Owen et al. (1979) reported 1.0 as a standard for
organic fraction of solid waste.

In this study, when I/S ratio increased from 0.25 to 2.30, 145–391 ml of biogas
generation per gram of VS added was observed. The increase in biogas generation for I/S ratio of
0.25–2.30 is reported in Table 4, and it is evident that when I/S ratio increased from 0.50 to 1.00
a substantial increase in biogas generation was observed whereas for I/S ratio increased from
1.00 to 2.30, though an increase in biogas generation of 1.5–2.12% was observed; however, it is
marginal w.r.t amount of inoculum added. The use of more inoculum amount in co-digestion
process has no significant influence on biogas generation beyond certain point. However,
excessive use of inoculum leads to an increase in digester volume unnecessarily required for the
co-digestion. Hence, it was observed from the present studies that the I/S ratio of 1.0 wherein
maximum conversion rate of substrate was observed w.r.t amount of inoculum added beyond
which the increase was marginal only.

The methane content of biogas is 70% irrespective of I/S ratio. With this as the basis, the
methane yield (ml) at different I/S ratios was deducted from the volume of the biogas generation.
Then, the methane yield (ml) was converted as the methane yield per gram of VS added by
dividing the methane yield by VS added, and the data are presented in Table 4. The methane
yield varied from 101.5 to 274.7 ml/g of VS added, and the variations of specific methane
production rate for different I/S ratios are presented in Fig. 3. A methane yield of 211 ± 6 ml/g of
VS added was observed for anaerobic digestion of maize waste by Raposo et al. (2006). Methane
yield depends on lipid, protein, and carbohydrate content present in the substrate (Neves et al.
2008; Shanmugam and Horan 2009). In this study, the methane yield was up to 268.1 ml/g of VS
added was observed as fleshings one of the substrates selected for the study contains mainly
lipids. The lipids are attractive substrates for anaerobic digestion. Similar observations have been
noticed by Gunaseelan (1997) and reported that 0.11–0.42 m3 methane yield per kg of VS added
depending on the composition of carbohydrates: lipids: protein present in the waste.

Variations in pH and oxidation reduction potential (ORP)

At the end of digestion period, the pH and ORP for different I/S ratios were observed and
the same are presented in Fig. 4. The pH varied from 7.1 to 7.3 which is an indication that
souring of digesters did not occur during the co-digestion irrespective of I/S ratio. Oxidation
reduction potential is a measure of the degree of metabolic reactions that take place during
anaerobic digestion process. The ORP was ranged from -409 to -351 mV. The methanogens can
develop in an environment where pH is 6.5–7.6 and ORP is -300 mV. The biogas generation was
influenced by methanogenic activity and I/S ratio maintained for the reactors. The biogas
generation depends on the conversion of substrate into volatile fatty acids (VFAs) and the further
conversion of VFA into methane.

Conclusion

For various I/S ratios, i.e., for 0.25, 0.50, 0.67, 1.0, 1.5, 2.00, and 2.30 studied in this
study, an increase in biogas generation of 1087–2933 ml was observed.

When I/S ratio increased from 1.0 to 2.3, the increase in biogas generation was 1.5–
2.12% and the increase in I/S beyond 1.0, significant increase in biogas generation was not
observed. The use of more inoculum amount in co-digestion process has no significant influence
on biogas generation and it ultimately leading to increase in digester volume. For the I/S ratio of
1.0, maximum conversion rate of substrate was observed which resulted in generation of 383 ml
of biogas per gram of VS added.

Considering I/S ratio as 1.0 as the optimum, additional reactor volume required beyond
the optimum ratio of 1.0 resulted an additional volume of 33, 67, and 87%, respectively, required
for I/S ratio of 1.50, 2.0, and 2.30, and the net incremental benefit in terms of bio-energy
generation is 1.57, 2.09, and 2.09% only. Similarly for I/S ratios below 1.0, i.e., for 0.667, 0.5,
and 0.25, the reduction in reactor volume is 22, 33, and 50%. For these I/S ratios, the decrement
in terms of bio-energy generation is 50.7, 53.8, and 62.1%. Hence optimization of I/S ratio is not
only beneficial for arriving the reactor volume but also maximizing the benefits of bio-energy
generation also.
30-14 LAWAL
The result showed no biogas produced from the controlled biodigester over the entire
digestion period. Fig. 1 (a and b) show biogas production rates and accumulation plots of SPM
over the digestion period, respectively. It can be seen from Fig. 1a that R1, R2 and R3
biodigesters needed 30, 29 and 27 days to complete digestion, respectively. It appeared that as
I/S ratio increased, digestion period decreased and biogas production rates increased. It can also
be observed that curves of biogas production rates for R1, R2 and R3 biodigesters exhibited
similar pattern over the entire digestion period; R3 biodigester exhibited the max. biogas
production rates followed by R2 and R1 biodigesters (Fig. 1b). The peak (maximum) biogas
production rate occurred on the 5th day for all biodigesters in the order of R3 (0.30526 Nm3/kg
volatile solids (VS) days), R2 (0.15308 Nm3/kg VS days) and R1 (0.11009 Nm3/kg VS days).
ANOVA showed that inoculum:substrate (I/S) ratio had a significant effect (P < 0.0372) on
biogas production rates.

The total accumulated biogas produced was found to be in the order of 1.46784, 0.88177
and 0.57195 Nm3/kg VS in R3, R2 and R1 biodigesters, respectively (Fig. 1b). Raghunathan et
al. (2008) reported similar biogas production accumulation values (1.1 to 0.382 Nm3/kg VS) for
gelatin solid waste. Cumulative analysis of the results showed that 80% of biogas produced in
biodigesters R1, R2 and R3 accumulated on the 14th, 15th and 14th day of digestion period,
respectively.
2_foster_perez_romero

there was an increase in the production with the increase in the inoculum contents and the
decrease of total solids percentage.

The higher total acid concentrations were observed in the start up phase between the days
1 and 15 (Figure 7b). In this stage, the high molecular weight compounds were converted into
intermediate VFA, mainly propionic and butyric acids by acidogenic bacteria. Acetic acid, the
major intermediate in methanogenesis was formed through the degradation of propionic and
butyric acids and through the oxidation of hydrogen, process involving different acetogenic
populations. The methanogenesis and methane gas production increased while the carbon
dioxide and VFA concentrations decreased.

Figure 8a shows the evolution of biogas (CH4, CO2 and H2), as percentage. The initial
methane production increased when the production of hydrogen (20% of total volume of biogas)
decreased. Methane can only be formed by specific methanogenic bacteria which use acetic acid
or hydrogen. Considering only the stabilization phase (20-60 days), the biogas produced had
higher methane concentration of 49.9% during the stabilization phase (20-60 day).

Figure 8b shows the biogas and methane production (as L/day) and the evolution of
cumulative biogas and methane (as L). Higher production of biogas was observed in the start up
phase. The reactor reached the higher daily generation of biogas (4.3 L/day) in the first 20 days
and higher values of methane yield of 0.22 LCH4/gVS. After the day 20, the process was
characterized by methanogenic phase or stable phase, where the acidity continuous increases,
fundamentally due to butyric acid, and decreasing the acetic acid. The cumulative biogas and
methane yield showed a slowly increase, day after day, until reached maximum values of 85.1 L
and 13.8 L, respectively.
BioRes_08_2_2487_Feng

The F/I ratio has been found to be one of the most parameters in batch anaerobic digestion
(Chudoba et al. 1991). Each feedstock has its suitable F/I ratio because the amount of volatile
fatty acids (VFA) and the capacity to buffer the VFA cumulative in the anaerobic process
(Lesteur et al. 2010). It was reported that a higher F/I ratio may be toxic, while a lower F.I ratio
may prevent induction of the enzyme necessart for biodegradation (Prashanth et al. 2006). The
lag phase of anaerobic digestion could also affected by this factor ( Chen and Hashimoto 1996)
10.1016@0961

Rapid progress of anaerobic batch fermentation for methane depends primarily on the
maintenance of a favourable pH range. It has been reported that the production of methane is
active only in the range pH 7.5-8.5, whereas acid production can go on between pH 6 and 10.”
This study showed that addition of a high proportion of inoculum to give an I/S ratio of 67,
resulted in successful batch fermentation of Parthenium. A high volume of inoculum accelerated
the rate of gas production and fermentation was completed in a shorter time than other inocula
volumes tested. Thus, short-term batch fermentation of Parthenium at 26 _+ 2°C may be
possible with a high volume of inoculum. This might be the possible reason for short-term
fermentation of substrates like Gliricidia leaves for 28 days at 32”C,* lakeweed (Lagarosiphon
major) and luceme hay for 17 days at 37’C,‘* aquatic weeds for 35 days at 37YY and Pistia for
30 days at 35°C.‘4
10.1016@j.jenvman

The inoculum concentration should always be high compared to the concentration of the
substrate (VS basis), and the F/M should be recognized as one of the major parameters affecting
the results of anaerobic assays (Neves et al., 2004).
The H2S concentration in biogas increased with an increase in the F/M ratios (0.5e2.0)
under both mesophilic and thermophilic conditions (Fig. 2).
Gunaseelan (1995) also reported a significant increase in methane yield with an increase
in the inoculum to substrate ratio (i.e., decrease in the F/ M ratio) during anaerobic digestion of
Parathenium at room temperature (26 _C).