Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

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Environmental Nanotechnology, Monitoring & Management

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Influence of inoculum/substrate ratio on biogas yield and kinetics from the

anaerobic co-digestion of food waste and maize husk
H.I. Owamah a, *, S.C. Ikpeseni b, M.I. Alfa c, S.O. Oyebisi d, S. Gopikumar e,
Olusegun David Samuel f, h, S.C. Ilabor g
a
Department of Civil and Environmental Engineering, Delta State University, Oleh Campus, Abraka, Delta State, Nigeria
b
Department of Mechanical Engineering, Delta State University, Oleh Campus, Abraka, Delta State, Nigeria
c
Department of Civil Engineering, University of Jos, Plateau State, Nigeria
d
Department of Civil Engineering, Covenant University, Ota, Nigeria
e
Department of Civil Engineering, SCAD College of Engineering and Technology, Tirunelveli, India
f
Department of Mechanical Engineering, Federal University of Petroleum Resources, Effurun, P.M.B 1221, Delta State, Nigeria
g
Department of Chemistry, Federal College of Education (Technical), Asaba, Delta State, Nigeria
h
Department of Mechanical Engineering, University of South Africa, Science Campus, Private Bag X6, Florida 1709, SOUTH AFRICA

A R T I C L E I N F O A B S T R A C T

Keywords: The study reports for the first time, the influence of inoculum/substrate (I/S) ratios on biogas yield and kinetics
Co-digestion of food waste (FW) and maize husk (MH) anaerobic co-digestion. The study was investigated in six anaerobic
Biogas digesters, D-0, D-0.25, D-0.5, D-1, D-2, and D-4 at 36 ± 2 ◦ C. D-1 with I/S ratio of 1 had the largest cumulative
Inoculum
biogas yield of 9.99 L/gVS. Results from the modified Gompertz model revealed a decrease in lag phase (λ) by
Yield enhancement
Modelling
20.4% at I/S ratio 1, hence, shows that the amount of time needed for active methanogenesis to commence was
hugely decreased. Maximum specific biogas production (Rm) and maximum biogas production potential (A)
increased with increase in I/S ratio. Also, D-1 yielded the highest values of 0.37 L/gVS/day and 9.99 L/gVS for
Rm and A, respectively. A decrease in biogas yield at D-0.25 and D-0.5 was however obtained with respect to the
control experiment. The study recommends confirmation of optimum I/S ratio prior to establishment of com­
mercial biogas plants.

however, used anaerobic co-digestion of two or more organic substances

in a bioreactor to optimize the biogas yield of various substrates (El-
1. Introduction Mashad and Zhang, 2010).
Maize husk is a plant biomass produced as a bye product of intensive
Nations of the world are faced with the challenge of meeting their and widespread maize cultivation in Nigeria and many other African
fast-growing energy demand without damaging the environment countries (Owamah and Izinyon, 2015a). According to the official sta­
(Samuel and Gulum, 2019a; Samuel and Okwu, 2019b). This has tistics of the Food and Agricultural Organization of the United Nations
necessitated the exploration and exploitation of renewable energy al­ (FAOUN, 2017), maize production in the world, Africa, and Nigeria was
ternatives (Owamah, 2020; Owamah et al., 2020; Onochie et al., 2021). estimated as 1130, 84, and 11 million metric tonnes (MMT), respec­
Zhang et al. (2016) and Okwu et al. (2020) opined that the production of tively. The Federal Ministry of Agriculture, Water Resources, and Rural
biogas from waste organic substances is a viable renewable energy Development (FMAWRRD, 1988) reported that out of the 1.37 MMT of
alternative as resulting digestates are also utilized as compost for annual maize production in Nigeria, about 4.11 MMT residual wastes of
farming. Anaerobic digestion improves agricultural productivity, gen­ husks, cobs, straws, skin and trimmings are generated. Not only that
erates bio-fuel, and reduces environmental pollution (Almomani et al., these huge maize residues are discarded as wastes, the current disposal
2019; Almomani and Bhosale, 2020). Notwithstanding these great po­ technique of uncontrolled burning cause serious environmental health
tentials, development of industrial anaerobic plants have been con­ hazard and global warming. Anaerobic digestion for biogas production
fronted with many sustainability challenges such as cost-effectiveness could be an alternative for managing this agricultural waste while
and optimization of process factors and substrates. Researchers have

* Corresponding author.
E-mail addresses: [email protected], [email protected] (H.I. Owamah).

https://doi.org/10.1016/j.enmm.2021.100558
Received 12 October 2020; Received in revised form 24 July 2021; Accepted 11 August 2021
Available online 17 August 2021
2215-1532/© 2021 Elsevier B.V. All rights reserved.
H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

production from co-digestion of FW and MH is scarce or non-existent.

Nomenclature This research was therefore targeted at establishing the optimum I/S
ratio for enhanced biogas yield, using FW and MH as co-substrates. It
FW Food waste also applied the modified Gompertz model to determine the influence of
MH Maize husk these I/S ratios on the biogas yield kinetics.
λ Lag phase
Rm Maximum specific biogas production 2. Materials and methods
A Maximum biogas production potential
I-S Inoculum-substrate 2.1. Collection and preparation of substrates
C/N Carbon/Nitrogen
VS Volatile solid The FW and MH used as substrates in this study were collected from
the restaurant of Landmark University, Nigeria. They were crushed and
coalesced following the procedures outlined by El-Mashad and Zhang

Table 1
Properties of mixed substrates fed into respective digesters.
1 2 2 2 2 2
Digester FW: MH (%) (w/w, based on total weight Total weight of sample wt of FW (g) wt of MH (g) Influent VS(g/ C/N pH I/S
(g) (g) L) ratio ratio

Dig. D-0.25 75:25 30 22.55 7.5 3.5 19.6 6.8 0.25

Dig. D-0.5 75:25 30 22.55 7.5 3.3 19.3 6.6 0.5
Dig. D-1 75:25 30 22.55 7.5 4.2 20.7 7.1 1
Dig. D-2 75:25 30 22.55 7.5 3.7 23.4 7.2 2
Dig. D-4 75:25 30 22.55 7.5 3.9 22.6 7.3 4
Dig. D-0 75:25 30 22.55 7.5 3.6 20.2 6.9 0
1
based on fresh weight;
2
based on volatile solid

harnessing the energy potentials. On the other hand, in Nigeria, there is (2010). The FW and MH were combined in the ratio 3:1 based on pre­
an alarming increase in food waste (FW) generation from both resi­ vious findings (Owamah and Izinyon, 2015a).
dential and non-residential outlets owing to rapid urbanization and
population explosion (Owamah and Izinyon, 2015a). At the global level, 2.2. Physicochemical analysis of substrates
literature shows that about 30% processed food for human consumption
eventually turns out as waste, hence resulting in about 1300 billion ki­ Physicochemical characterization of the substrates was carried out
lograms (Nathan and Pragasen, 2012). Presently, one of the perturbing three times following the guidelines stipulated in APHA (2012). Labo­
environmental challenges to governments of developing nations is the ratory oven, model DHG-9053A, was used to determine the total solids
effective management of municipal solid waste, mainly food waste (TS) and the volatile solids (VS) of the substrates. Similarly, direct-
(Owamah et al., 2017). reading photometer model 7100 was used to determine the ammo­
An earlier study by Owamah and Izinyon (2015a) showed that co- nium nitrogen (NH4+-N), total kjeldahl nitrogen (TKN), chlorine (Cl),
digestion of FW and MH performed optimally at ratio 3:1 (w/w), potassium (K), sulphur (S), sodium (Na), calcium (Ca) and magnesium
respectively. Boulanger et al. (2012) reported that biogas yield from (Mg). A pH meter, model PHS-3C was used to measure the initial pH
substrates could be further improved by the addition of inoculum to give values. The physicochemical properties of the prepared substrates are
appropriate microbial profile. However, the influence of inoculum on presented in Table S1, and measured based on the wet weight samples.
biogas yield is dependent on the nature of biomass as well the inoculum/
substrate ratio among other factors (Raposo et al., 2009). Proper addi­ 2.3. Collection of inoculum and experiment for biogas production
tion of inoculum helps overcome inhibition of digesters and reduce the
time for the beginning of active methanogenesis (Raposo et al. (2009). Effluent from an anaerobic reactor digesting similar substrates was
Moreno-Andrade and Buitrón (2003) reported an increase in methane collected and utilized as the inoculum. The impact of inoculum-
content of biogas with inoculum-substrate ratio ranging from 1 to 4. The substrate ratios on the kinetics and yield of biogas production through
S/I ratio is the initial ratio of VS in the feedstock (FW and MH) to the VS six experimental designs was investigated using 10-liter computer
in the inoculum during the start of a batch digestion experiment (Feng controlled anaerobic digesters (Fig. S1) at a temperature of 36 ± 2 ◦ C.
et al., 2013). It involves the addition of some amount of inoculum to the The reactors were labeled as D-0.25, D-0.5, D-1, D-2, and D-4 and D-0 for
substrate for provision of the needed microorganisms to start the reac­ 0.25, 0.5, 1, 2, 4 and 0 inoculum/substrate ratios, respectively. The
tion. The S/I ratio was reported as a very important parameter in batch percentage mix ratios of FW to MH and other determined physico­
anaerobic digestion (Li et al., 2018). Every substrate has its appropriate chemical properties of the respective digesters’ contents are shown in
S/I ratio because of the quantity of VFA and the capacity to buffer the Table 1. A batch flow anaerobic digestion process was utilized following
accumulated VFA during the digestion process. It was reported that similar procedures by Owamah and Izinyon (2015a).
while higher S/I ratios could be toxic, lower S/I ratios could prevent The digestion ran for 44 days until biogas yield became insignificant.
induction of the enzyme essential for anaerobic digestion (Feng et al., The total biogas production for each day was measured daily from the
2013). S/I ratio could also affect the lag phase of anaerobic digestion digester’s calibrated receptacle using water displacement approach. The
(Lavagnolo et al., 2018). batch anaerobic digestions were carried out twice and the mean values
The application of inoculum for starting anaerobic digestion has of daily biogas production recorded. The analysis of percentage methane
been arbitrary. There is a need therefore to determine the optimum was done with a gas chromatograph (BUCK GC122, China).
quantity of inoculum that will be required for optimum biogas yield of
various substrates. Furthermore, literature shows that scientific infor­
mation on the best inoculum/substrate ratio for optimum biogas

2
H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

0.9
0.8

Daily biogas production(L/gVS)

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1 -5 0 5 10 15 20 25 30 35 40 45 50
Retention time (Days)

Dig. D-1 dig. D-4 dig. D-2 dig. D-0.5 dig. D-0.25 dig. D-0

0.9
0.8
Daily biogas production(L/gVS)

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1 -5 0 5 10 15 20 25 30 35 40 45 50
Retention time (Days)

Dig. D-1 dig. D-4 dig. D-2 dig. D-0.5 dig. D-0.25 dig. D-0

Fig. 1. Daily biogas yield at varied inoculum- substrate ratio.

Fig. 2. Box Plot of daily biogas yield at varied inoculum- substrate ratio.

2.4. Determination of biogas yield kinetics using the modified Gompertz to determine respective biogas production kinetics: the maximum biogas
model production potential, A (L/gVS); maximum specific biogas yield; Rm (L/
gVS/day); and lag phase, λ (days) (Owamah et al., 2020)
The daily cumulative biogas yields for the respective experimental
designs were fit into the modified Gompertz model expressed by Eq. (1)

3
H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

12

Cummulative biogas production (L/gVS)

10

0
-5 0 5 10 15 20 25 30 35 40 45 50
-2

Retention time (Days)

Dig. D-1 dig. D-2 dig. D-4 dig. D-0.5 dig. D-0.25 dig. D-0

12
Cummulative biogas production (L/gVS)

10

0
-5 0 5 10 15 20 25 30 35 40 45 50
-2

Retention time (Days)

Dig. D-1 dig. D-2 dig. D-4 dig. D-0.5 dig. D-0.25 dig. D-0

Fig. 3. Cumulative biogas yield at varied inoculum-substrate ratio.

{ [ ]}
Rm × e
At = Aexp − exp (λ − t) + 1 (1)
A

where t is time of biogas yield (days), At is cumulative biogas yield (L/

gVS). The values of A, Rm and λ were determined using the optimization
tools of Microsoft Excel 2010 version.

2.5. Statistical analysis

One-way Analysis of Variance and the Fisher’s LSD post hoc tests
were used to compare the daily yield, cumulative yield and the
maximum biogas potential from the respective inoculum-substrate ra­
tios. The tests were conducted using STATA/SE statistical software
version 13.1 at 95% confidence level. P. value < 0.05 was considered
statistically significant
Fig. 4. Box Plot of Cumulative biogas yield at varied inoculum- substrate ratio.

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H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

Table 2a
ANOVA Test Results for comparison of daily biogas yield at varied inoculum/ substrate ratio.
Source of Variation SS df MS F P-value F crit Remark

Treatment 0.517741 5 0.1035482 3.2873491 0.007981 2.2861844 Significant

Error 3.968874 126 0.031499
Total 4.486615 131

Table 2b
Fisher’s LSD Post Hoc Test Results Matrix for comparison of daily biogas yield at varied inoculum/substrate ratio.
Dig. D-0 Dig. D-0.25 Dig. D-0.5 Dig. D-1 Dig. D-2 Dig. D-4

Dig. D-0
Dig. D-0.25 Not Significant
Dig. D-0.5 Not Significant Not Significant
Dig. D-1 Significant Not Significant Not Significant
Dig. D-2 Significant Not Significant Not Significant Not Significant
Dig. D-4 Significant Significant Significant Significant Not Significant

Table 3a
ANOVA Test Results for comparison of cumulative biogas yield at varied inoculum/ substrate ratio.
Source of Variation SS df MS F P-value F crit Remark

Treatment 94.412656 5 18.882531 2.8508056 0.017902 2.2861844 Difference is Significant

Error 834.57075 126 6.6235773
Total 928.9834 131

Table 3b
Fisher’s LSD Post Hoc Test Results Matrix for comparison of cumulative biogas yield at varied inoculum/substrate ratio.
Dig. D-0 Dig. D-0.25 Dig. D-0.5 Dig. D-1 Dig. D-2 Dig. D-4

Dig. D-0
Dig. D-0.25 Not Significant
Dig. D-0.5 Not Significant Not Significant
Dig. D-1 Significant Not Significant Not Significant
Dig. D-2 Significant Not Significant Not Significant Not Significant
Dig. D-4 Significant Significant Significant Not Significant Significant

Table 4
Performance of the digesters.
Digester Inoculum-substrate Average methane content Peak biogas yield (L/ Peak methane yield (L/ Mean biogas yield (L/ Standard deviation
ratio (%) gVS) gVS) gVS) (SD)

D- 0 58.8 0.59 0.35 0.33 0.14

0 (Control)
D-0.25 0.25 46.4 0.48 0.22 0.22 0.15
D-0.5 0.5 47.2 0.57 0.27 0.31 0.14
D-1 1 66.8 0.81 0.52 0.42 0.22
D-2 2 63.8 0.73 0.45 0.36 0.24
D-4 4 62.9 0.67 0.41 0.37 0.16

3. Results and discussion Digs. D-1, D-2 and D-4 with higher inoculum-substrate ratios
exhibited better startup performances than Digs. D-0.25 and D-0.5 with
3.1. Performance of the digesters at different inoculum-substrate ratio lower inoculum-substrate ratios which is attributable to a consequent
increase in active methanogen population occasioned by increased
The combination of both FW and MH at ratio 3:1 helped adjust the C: organic load. This increased organic load could have reduced the
N ratios as presented in Tables S1 and 1, which became fairly within the duration for the multiplication of the methanogens to the required
recommended range for optimum production of biogas (Owamah and population for biogas yield (Li et al., 2014, Zhu et al., 2014; Owamah
Izinyon, 2015b). Co-digestion of the two substrates was also found to et al., 2020). Raposo et al. (2009) reported that since the addition of
improve pH (Tables S1 & 1). Haider et al. (2015) observed an inoculum to substrate enhances biogas production, ascertaining the
improvement in C/N ratio and pH through the co-digestion of food optimum inoculum to substrate ratio is imperative for large scale
waste and rice husk. The results of the daily biogas yield at the different anaerobic digestion operations. Furthermore, the results of the Analysis
inoculum-substrate ratios (Fig. 1) reveal that the addition of appropriate of Variance and the Fisher’s Post Hoc test on both daily and cumulative
inoculum dosage helped to activate microbial activities early in the di­ biogas yield showed that the difference in yield from the respective
gesters. There was a clear difference in the initial volume of biogas inoculum ratios were statistically significant (p < 0.05) (Tables 2a and b
recorded on the first 6 days of digestion in digesters D-1, D-2 and D-4 & Tables 3a and b).
when compared with digester D-0 with no inoculum (Figs. 1–4). From Figs. 1 and 2 and Table 4, the peak biogas yield of reactor D-1

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H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

10

Cummulative biogas production (L/gVS)

8

-1
-5 0 5 10 15 20 25 30 35 40 45 50
Retention time (Days)

Dig. D-1 Gompertz D-1 Dig. D-2 Gompertz D-2

Dig. D-4 Gompertz D-4 Dig. D-0.5 Gompertz D-0.5
Dig. D-0.25 Gompertz D-0.25 Dig. D-0 Gompertz D-0

10

9
Cummulative biogas production (L/gVS)

-1
-5 0 5 10 15 20 25 30 35 40 45 50
Retention time (Days)

Dig. D-1 Gompertz D-1 Dig. D-2 Gompertz D-2

Dig. D-4 Gompertz D-4 Dig. D-0.5 Gompertz D-0.5
Dig. D-0.25 Gompertz D-0.25 Dig. D-0 Gompertz D-0

Fig. 5. Modified Gompertz model plots for the varied inoculum-substrate ratios.

(0.81 L/gVS) was obtained on the 22nd day of digestion. Figs. 1 and 2 highest cumulative biogas yield of 9.12 L/gVS. While Digs. D-0.25, D-
also show that the peak biogas yields of D-0.25, D-0.5, D-2, and D-4 are 0.5, D-2, and D-4 had respective cumulative biogas yields of 4.73, 6.80,
0.334, 0.378, 0.478 and 0.432 L/gVS, respectively. These were respec­ 7.89 and 8.10 L/gVS, that of the control (Dig. D-0) was 7.17 L/gVS. The
tively obtained on the 10th, 10th, 11th and 18th day of anaerobic performance of Dig. D-0 was no surprise as food waste contains sub­
digestion. Dig. D-0 with no inoculum (control experiment) recorded its stantial amount of easily biodegradable matter; however, it is prone to
maximum biogas yield (0.59 L/gVS) on the 20th day of digestion. early acidification. Furthermore, Figs. 3 and 4 show that the addition of
As observed, while Digs. D-1, D-2 and D-4 with higher inoculum/ appropriate amount of inoculum helped to increase biogas yield as
substrate ratios had better biogas yield than Dig. D-0 (control), such was revealed in Digs. D-1, D-2 and D-4.
not the case for Digs. D-0.5 and D-0.25 with lower inoculum/substrate Xu and Li (2012) reported through investigation into the influence of
ratios of 0.5 and 0.25, respectively. This implies that while the addition (I/S) ratio on methane content of biogas produced from the anaerobic
of inoculum boosts biogas production, attention must be paid to co-digestion of expired dog food and corn stover, that there was a pos­
knowing and using the appropriate inoculum/substrate ratio for any itive relationship between inoculum-substrate ratios and the maximum
particular substrate. Figs. 3 and 4 show that Digs. D-1 yielded the biogas production and methane percentage. Similarly, Raposo et al.

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H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

The higher, the inoculum content above an optimum, the lower the
substrate content and the lower the methane yield. This notwith­
standing, better yields were obtained as inoculum/substrate ratio
increased beyond 0.5 with best performance obtained at inoculum/
substrate ratio 1. The availability of sufficient active methanogens could
also be a possible explanation for the improved biogas yield at D-1, D-2
and D-4 (Boulanger et al., 2012). However, slight decrease in biogas
yield was observed as inoculum/substrate ratio increased beyond 1,
though the yields were better than that of D-0. A possible explanation for
this may be characteristic changes of the substrate surface which during
hydrolysis affects the bioavailability of digestible materials.
Parawira et al. (2004) had previously reported that the addition of
inoculum increased the yield of biogas from solid potato waste. A similar
study on the influence of inoculum/substrate ratio on biogas yield from
co-digestion of maize and straw demonstrated that there was a signifi­
Fig. 6. Box Plot of the obtained maximum biogas potential at varied inoculum/ cant decrease in the biogas yield at inoculum-substrate ratio < 0.25 gVM
substrate ratios. per gVM of the substrate (Raposo et al., 2009). The reason for an
improvement in biogas yield at inoculum/substrate ratio >0.2 was re­
ported by Neves et al. (2004), who investigated granular inoculum on
Table 5
kitchen waste. It was concluded that the acidification of the digestion
Modified Gompertz model optimization result.
process was overcome at inoculum/substrate ratios between 0.2 and 4,
Parameter Dig. D-0; D-0.25; I/ D-0.5; I/ D-1; I/ D-2; I/ D-4; I/ which eventually, led to improved biogas yield. Also, Boulanger et al.
I/S = 0 S = 0.25 S = 0.5 S=1 S=2 S=4
(2012) reported the best production of biogas from municipal solid
A 7.95 5.06 7.46 9.99 8.32 8.82 waste occurred at inoculum/substrate ratio 2. In this study, biogas yield
Rm 0.25 0.21 0.25 0.37 0.36 0.29
increased with increase inoculum/substrate ratio and decreased slightly
Λ 5.79 5.52 5.19 4.84 5.09 5.03
after inoculum-substrate ratio 1.
Moreover, Digs. D-0.25 and D-0.5 exhibited the least content of
(2009) reported a positive outcome on the influence of inoculum/sub­ methane and recorded 46.4 and 47.2 %, respectively. Digs. D-1, D-2, and
strate ratio on the yield of biogas from municipal solid waste. In the D-4 had higher percentage methane of 66.8, 63.8, and 62.9 % with
same vein, the co-digestion of hay with soybean processing waste yiel­ inoculum/substrate ratio of 1, 2, and 4, respectively (Table 4). The
ded a positive relationship with an increase in inoculum/substrate ratio reasons for an increase in the biogas yield with increasing inoculum/
(Zhu et al., 2014). substrate ratio, as earlier adduced in this paper, could also be respon­
The mean biogas yields for Digs. D-0.25, D-0.5, D-1, D-2 and D-4 sible for the higher methane content. Therefore, findings from this study
were obtained as 0.22 ± 0.15, 0.31 ± 0.14, 0.42 ± 0.22, 0.36 ± 0.24 and are in agreement with the previous study in which FW and rice husk
0.37 ± 0.16 L/gVS, respectively. A comparison of these yields with that (RH) were co-digested at S/I ratios 0.25, 0.5, 1.0, 1.5, and 2.0 and
of Dig. D-0 (0.33 ± 0.14 L/gVS) further demonstrates that beyond the yielded specific biogas of 557, 458, 267, 97, and 71 L/kg VS, respec­
ability of inoculum addition to improve biogas production, the ratio of tively (Haider et al., 2015). The study further showed that specific
the inoculum to substrate is equally important. The addition of inoculum biogas yield at S/I 0.25 (I/S = 4) was significantly higher (p < 0.05) than
demonstrated inhibitive characteristics at inoculum/substrate ratio of what was obtained in other digesters except the digester with S/I ratio
0.25 and 0.5. This was revealed by their respective lower biogas yield 0.5 (I/S = 2). Haider et al. (2015) also reported a positive influence on
compared to that of Dig. D-0 without inoculum (Dig. D-0). This inhibi­ anaerobic co-digestion of FW and RH at substantial content of inoculum.
tion could be attributed to the release of intermediate products that are In the same vein, Elbeshbishy et al. (2012) reported that the rate of
not appropriate for the conversion by methanogenic bacteria to methane biodegradation, lag time and chances of degradation of substrates
and inadequate methanogen population. Inoculum are substrates that depended on the concentration of microorganisms in a digester. The
have previously undergone anaerobic digestion thus are rich in micro­ values of specific methane production obtained in digester D-1, D-2 and
bial population but with less methane yield. The biogas production D-4 as shown in Tables 2 are in line with documented values in literature
comes primarily from the substrates. (Marañón et al., 2012).

Table 6a
ANOVA Test Results for comparison of maximum biogas potential at varied inoculum/substrate ratio.
Source of Variation SS df MS F P-value F crit Remark

Treatment 93.027531 5 18.605506 2.7998274 0.0196612 2.2861844 Difference is Significant

Error 837.2994 126 6.6452333
Total 930.32693 131

Table 6b
Fisher’s LSD Post Hoc Test Results Matrix for comparison of maximum biogas potential at varied inoculum/substrate ratio.
Dig. D-0 Dig. D-0.25 Dig. D-0.5 Dig. D-1 Dig. D-2 Dig. D-4

Dig. D-0
Dig. D-0.25 Not Significant
Dig. D-0.5 Not Significant Not Significant
Dig. D-1 Significant Not Significant Not Significant
Dig. D-2 Significant Not Significant Not Significant Not Significant
Dig. D-4 Significant Significant Significant Not Significant Significant

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H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

Table 7
Present study compared with previous studies.
S/ Substrate Mean biogas yield (mL/gVS) Temperature Methane Digestion Inoculum used Reference
N /duration of content mode
digestion

1 FW;80 g VS/L. day 448.9 ± 6.6 37 ◦ C/60 days 61.1 ± 0.8 Semi- Seed Sludge Zhang et al.
continuous (2020)
2 FW; 80 g VS/L. day + 484.6 ± 32.6 37 ◦ C/60 days 64.2 ± 1.5 Semi- Seed Sludge Zhang et al.
Trace Metal continuous (2020)
3 75% FW + 25% SS, 80 g 413.4 ± 29.3 37 ◦ C/60 days 64.8 ± 2.0 Semi- Seed Sludge Zhang et al.
VS/L continuous (2020)
4 75% FW + 25%SS, 80 g 413.4 ± 29.3 37 C/60 days
◦
64.8 ± 2.0 Semi- Seed Sludge Zhang et al.
VS/L continuous (2020)
5 75% YW + 25% FW, 80 165.4 ± 15.6 37 ◦ C/60 days 61.0 ± 2.0 Semi- Seed Sludge Zhang et al.
g VS/L continuous (2020)
6 56.3% YW + 18.7% FW 164.7 ± 22.7 37 ◦ C/60 days 61.3 ± 1.5 Semi- Seed Sludge Zhang et al.
+ 25% SS, 80 g VS/L continuous (2020)
7 50% YW + 50%FW, 80 296.0 ± 19.9 37 ◦ C/60 days 63.5 ± 1.3 Semi- Seed Sludge Zhang et al.
g VS/L continuous (2020)
8 37.5% YW + 37.5% FW 232.4 ± 46.7 37 C/60 days
◦
63.9 ± 2.1 Semi- Seed Sludge Zhang et al.
+ 25% SS, 80 g VS/L continuous (2020)
9 25% YW + 75%FW, 80 360.0 ± 30.2 37 ◦ C/60 days 64.1 ± 1.5 Semi- Seed Sludge Zhang et al.
g VS/L continuous (2020)
10 18.7% YW + 56.3% FW 314.9 ± 17.1 37 ◦ C/60 days 64.4 ± 1.7 Semi- Seed Sludge Zhang et al.
+ 25% SS, 80 g VS/L continuous (2020)
11 FW; Phase I (0–33 days) 387 ± 129 37 C/60 days
◦
Not stated Semi- Anaerobic sludge collected from Shi et al.
continuous an anaerobic digester in the lab (2020)
treating food waste
12 FW; Phase II) 546 ± 52 35 ± 1 ◦ C/(34–60 73.7 ± Semi- Seed Sludge Shi et al.
days 2.6% continuous (2020)
13 FW; Phase III 531 ± 28 35 ± 1 ◦ C/(61–92 69.3 ± Semi- Seed Sludge Shi et al.
days) 2.6% continuous (2020)
14 FW; Phase IV 161 ± 119 35 ± 1 ◦ C/(93–101 Not stated Seed Sludge Shi et al.
days) (2020)
15 Catering waste and 559 ml L− 1 d− 1), 30 ◦ C/60 days Not stated Semi- Cattle manure Tayyab et al.
treated parthenium continuous (2019)
biomass
16 Catering waste and 386 ml L− 1 d− 1 30 ◦ C/60 days Not stated Semi- Cattle manure Tayyab et al.
untreated parthenium continuous (2019)
biomass
17 Food Waste and 205 to 345 ml/g VSadded 35 ◦ C/30 days 50–80% Batch Anaerobic sludge Uma et al.
Switchgrass (2020)
18 Food Waste and 180 to 296 ml/g VSadded 55 ◦ C/18 days 50–80% Batch Anaerobic sludge Uma et al.
Switchgrass (2020)
19 FW 520 L/kgVS (cumulative) 35 ± 1 ◦ C /20 days 50–80% Batch Seed bacterial culture El-Mashad &
Zhang (2010)
20 FW 657 L/kgVS (cumulative) 35 ± 1 C /30 days
◦
53.7% Batch Seed bacterial culture El-Mashad &
(Average) Zhang (2010)
21 (32% of food waste + 411 35 ± 1 ◦ C /20 days 53.7% Batch Seed bacterial culture El-Mashad &
68% manure) (Average) Zhang (2010)
22 (32% of food waste + 455 35 ± 1 ◦ C /30 days 53.7% Batch Seed bacterial culture El-Mashad &
68% manure) (Average) Zhang (2010)
23 (48% of food waste + 504 35 ± 1 0C /20 days 53.7% Batch Seed bacterial culture El-Mashad &
52% manure) (Average) Zhang (2010)
24 (48% of food waste + 531 35 ± 1 C /30 days
◦
53.7% Batch Seed bacterial culture El-Mashad &
52% manure) (Average) Zhang (2010)
25 Food waste 445 ml/gVS added (cumulative) 50 ± 2 ◦ C/28 days 73% Batch Anaerobic Sludge Zhang et al.
(Average) (2007)
26 FW at F/1 = 4 784 ml/gVS 50 ± 2 ◦ C/28 days 66.1 Batch Microbial Inoculum Liu et al.
(2009)
27 FW and Phyllanthus 0.44 (L/g VS added) 25–30 C/
◦
at HRT 53.75 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
emblica 40 days (2013)
28 FW 0.34 (L/g VS added) 25–30 ◦ C/ at HRT 54.63 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
40 days (2013)
29 Phyllanthus emblica 0.31 (L/g VS added) 25–30 ◦ C/ at HRT 34.77 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
40 days (2013)
30 FW and Phyllanthus 0.39 (L/g VS added) 25–30 ◦ C/ at HRT 54.52 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
emblica 30 days (2013)
31 FW 0.31(L/g VS added) 25–30 ◦ C/ at HRT 54.98 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
30 days (2013)
32 Emblica 0.33 (L/g VS added) 25–30 ◦ C/ at HRT 40.90 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
30 days (2013)
33 FW and Phyllanthus 0.30 (L/g VS added) 25–30 ◦ C/ at HRT 53.78 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
emblica 20 days (2013)
34 FW 0.30 (L/g VS added) 25–30 ◦ C/ at HRT 54.68 Semi-batch Anaerobic mesophilic sludge Panyadee et al.
20 days (2013)
35 Phyllanthus emblica 0.29 (L/g VS added) 46.20 Semi-batch Anaerobic mesophilic sludge
(continued on next page)

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H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

Table 7 (continued )
S/ Substrate Mean biogas yield (mL/gVS) Temperature Methane Digestion Inoculum used Reference
N /duration of content mode
digestion

25–30 ◦ C/ at HRT Panyadee et al.

20 days (2013)
36 Food waste and rice Highest specific biogas yield of 37 ± 1 ◦ C/30 days Not stated Batch Fresh dung Haider et al.
husk 584 L/kg VS at S/I ratio 0.25 (2015)
37 75% FW and 25%Maize Peak daily 0.81 L/gVS; Average 37 ± 1 ◦ C/44 days 66.8 Batch Anaerobic mesophilic effluent This study
Husk yield 0.42L/gVS at Inoculum/
Substrate ratio1

3.2. Modified Gompertz modelling of experimental data microbial acclimatization. Lag phase (λ) was also found to decrease with
increase in inoculum/substrate ratio but for D-2 and D-4 (Tables 3). The
3.2.1. Effect of inoculum/substrate ratio on the maximum biogas increase in the population of active methanogens could be associated
production potential (A) with a reduction in the lag phase with increase inoculum/substrate ratio
Fig. 5 shows the plots of the modified Gompertz model simulation of (Boulanger et al., 2012).
experimental data from digesters D-0 to D-4 while Fig. 6 presents the box
plot of the maximum biogas production potential estimated using the 3.2.3. Influence of inoculum-substrate ratio on the maximum specific
modified Gompertz model. Table 5 shows the summary of the predicted biogas production (Rm)
kinetic parameters from the simulation curves. Tables 3 shows that inoculum/substrate ratio affected maximum
The results presented in Table 5 reveal that inoculum/substrate ratio specific biogas production (Rm). Dig. D-0 (control) had Rm of 0.25 L/
varied negatively with the maximum biogas production potential (A). In gVS/day. Dig. D-0.25 with inoculum-substrate ratio of 0.25 had Rm of
other words, an increase in inoculum/substrate ratio above the opti­ 0.21 L/gVS/day showing a decrease in maximum specific biogas yield.
mum, resulted in a corresponding decrease in the value of (A). There was Also, no noticeable change in Rm with increase in inoculum/substrate
however a slight decrease after inoculum/substrate ratio 1 to 2 and 4. ratio to 0.5 as compared with the control was obtained. However, with
Contrary to the expectations that the value of A and inoculum/substrate an increase in inoculum/substrate ratio to 1, as obtained in Dig. D-1, a
ratio should increase with increase in the amount and activity of huge rise (32.4%) in Rm i.e. from 0.25 to 0.37 L/gVs/day was obtained.
methanogens, the value of A for ratios greater than 1 was surprisingly Improvement in Rm was also obtained at inoculum/substrate ratio of 2
not as high as that of 1, underpinning the importance of determining the and 4 when compared with the control experiment. The design of large-
appropriate inoculum/substrate ratio before the establishment of large scale anaerobic plants requires the determination of Rm values of sub­
scale biogas plants. strates to be digested. Moreover, the Rm values increased with increasing
The slight decrease in (A) at inoculum/substrate ratio greater than 1 maximum biogas production potential (A), indicating an increase in the
is attributable to reduction in substrate quantity available for biodeg­ rate of attainment of substrate degradation. Furthermore, at inoculum/
radation since the quantity of inoculum was now greater than the sub­ substrate ratio of 1, maximal rate of biogas yield and substrate con­
strates. According to Boulanger et al. (2012), it could be that the amount sumption was obtained.
of active methanogens was gradually outweighing the available sub­ Digs. D-0.25 and D-0.5 with high lag phase (λ), low (Rm) and (A)
strates for biodegradation. Dig. D-0 (control) had an (A) value of 7.95 L/ values (Tables 3) could be categorized as failed or inhibited digesters.
gVS while Dig. D-0.25 and D-0.5 with lower inoculum-substrate ratios of Inoculum/substrate ratios of 0.25 or 0.5 inhibited methanogenesis
0.25 and 0.5, respectively, recorded (A) values of 5.06 L/gVS and 7.46 possibly as a result of reduced bioavailability and poor hydrolysis
L/gVS, respectively. The highest value of A (9.99 L/gVs) was however (Hashimoto, 1989). Considering the complex nature of the energy-
obtained at inoculum-substrate ratio of 1. Although a slight decline was economic considerations as well as growing global interest in the
recorded at inoculum/substrate ratio of 2 and 4 as explained earlier, the application of biogas for real life benefits, the authors hope to investi­
maximum biogas potentials of 8.32 L/gVs and 8.82 L/gVs where still gate the energy returned on energy invested in further studies. The
higher than the values of lower inoculum/substrate ratios. Compared comparison of the results of this present study with previous ones
with the control, D-1 with inoculum/substrate ratio of 1 produced the (Table 7) shows that the results of this study followed the trend of
most significant maximum biogas production enhancement of 20.4 %. previous reports. Apart from the study of Zhang et al. (2007) in which
While the Analysis of Variance results for the maximum biogas potential methane content of over 73% was obtained, the purity of biogas ob­
(Table 6a) shows that the difference for the respective ratios is statisti­ tained in the study is higher than most in the literature.
cally significant, the post hoc test (Table 6b) showed that only ratios, 1,
2 and 3 were significantly different from that of D-0. However, the 4. Conclusion
difference is more significant between the various ratios which but­
tresses our previous submissions. While D-1 was significantly higher Buttressing the earlier reports that the addition of inoculum could
compared to D-0, D-2 and D-4 were significantly lower. enhance biogas production from various substrates, this study has
demonstrated that inoculum/substrate ratio of 1 could greatly enhance
3.2.2. Effect of inoculum/substrate ratio on lag phase (λ) the yield and methane content of biogas produced from the anaerobic
From Tables 3, inoculum/substrate ratio showed a negative rela­ co-digestion of FW and MH. Biogas yield was found to vary with inoc­
tionship with lag phase (λ). As inoculum/substrate ratio increased, λ ulum/substrate ratios. While D-1, D-2 and D-4 experienced increased
decreased. The highest λ of 5.79 days was recorded in Dig. D-0 (control) biogas yield with respect to D-0; D-0.25 and D-0.5 showed inhibitive
which had no inoculum. It was closely followed by λ of 5.52 days in Dig. tendencies. The hugest increase in biogas yield was observed at inoc­
D-0.25 with inoculum-substrate ratio of 0.25. The least λ of 4.84 days ulum/substrate ratio 1. Furthermore, the inoculum/substrate ratio
was recorded at inoculum-substrate ratio 1 (Dig. D-1). Lag phase (λ) of varied inversely with the lag phase for microbial acclimatization. Lower
5.09 and 5.03 days was obtained from D-2 and D-4 with inoculum- inoculum/substrate ratios of 0.25 and 0.5 had longer lag phase of 6.52
substrate ratios of 2 and 4, respectively. A decrease in λ of 16.4% was and 4.99 days respectively, while that of 1 was 4.84 days. The inoculum/
obtained at inoculum/substrate ratio 1 in comparison with the perfor­ substrate ratio of 1 also produced the highest maximum biogas pro­
mance of Dig. D-0, signifying a 16.4% reduction in the time taken for duction potential (A) and maximum specific biogas production rate

9
H.I. Owamah et al. Environmental Nanotechnology, Monitoring & Management 16 (2021) 100558

(Rm). Okwu, M.O., Samuel, O.D., Otanocha, O.B., Balogun, P.P., Tega, O.J., Ojo, E., 2020.
Design and development of a bio-digester for production of biogas from dual waste.
World J. Eng. 17, 247–260.
Declaration of Competing Interest Onochie, U.P., Ikpeseni, S.C., Igweoko, A.E., Owamah, H.I., Aluma, C.C., Augustine, C.,
2021. Optical properties of zinc sulphide thin films coated with aqueous organic dye
The authors declare that they have no known competing financial extract for solar and optoelectronic device applications. J. Electron. Mater. 50 (5),
2576–2583.
interests or personal relationships that could have appeared to influence Owamah, H.I., 2020. Biogas yield assessment from the anaerobic co-digestion of food
the work reported in this paper. waste and cymbopogon citratus. J. Mater. Cycles Waste Manage. 22 (6), 2012–2019.
Owamah, H. I., Alfa, M. I., & Onokwai, A. O., 2020. Preliminary evaluation of the effect
of chicken feather with no major pre-treatment on biogas production from horse
References dung. Environmental Nanotechnology, Monitoring & Management, 100347.
Owamah, H.I., Izinyon, O.C., 2015a. Development of simple-to-apply biogas kinetic
Almomani, F., Bhosale, R.R., Khraisheh, M.A.M., Shawaqfah, M., 2019. Enhancement of models for the co-digestion of food waste and maize husk. Bioresour. Technol. 194,
biogas production from agricultural wastes via pre-treatment with advanced 83–90.
oxidation processes. Fuel 253, 964–974. Owamah, H.I., Izinyon, O.C., 2015b. Optimal combination of food waste and maize husk
Almomani, F., Bhosale, R.R., 2020. Enhancing the production of biogas through for enhancement of biogas production: experimental and modelling study. Environ.
anaerobic co-digestion of agricultural waste and chemical pre-treatments. Technol. Innovation 4, 311–318.
Chemosphere 255, 126805. Owamah, I.H., Izinyon, O.C., Igbinewekan, P., 2017. Characterization and quantification
APHA, 2012, American Public Health Association: “Standard Methods for Examination of of solid waste generation in the Niger Delta Region of Nigeria: a case study of Ogbe-
Water and Waste-water”; 22nd Edition, Washington DC, USA. Ijoh community in Delta State. J. Mater. Cycles Waste Manage. 19 (1), 366–373.
Boulanger, A., Pinet, E., Bouix, M., Bouchez, T., Mansour, A.A., 2012. Effect of inoculum Panyadee, S., Petiraksakul, A., Phalakornkule, C., 2013. Biogas production from co-
to substrate ratio (I/S) on municipal solid waste anaerobic degradation kinetics and digestion of Phyllanthus emblica residues and food waste. Energy Sustain. Dev. 17
potential. Waste Manage. 32, 2258–2265. (5), 515–520.
Elbeshbishy, E., Hafez, H., Nakhla, G., 2012. Viability of ultrasonication of food waste for Parawira, W., Murto, M., Zvauya, R., Mattiasson, B., 2004. Anaerobic batch digestion of
hydrogen production. Int. J. Hydrogen Energy 37 (3), 2960–2964. solid potato waste alone and in combination with sugar beet leaves. Renewable
El-Mashad, H.M., Zhang, R., 2010. Biogas production from co-digestion of dairy manure Energy 29 (11), 1811–1823.
and food waste. Bioresour. Technol. 101 (11), 4021–4028. Raposo, F., Borja, R., Martin, M.A., Martin, A., de la Rubia, M.A., Rincon, B., 2009.
Federal Ministry of Agriculture, Water Resources and Rural Development, 1988. Influence of inoculum to substrate ratio on the anaerobic digestion of sunflower oil
Agricultural policy for Nigeria: Strategies for implementation. FMAWRRD, Lagos: cake in batch mode:process stability and kinetic evaluation. Chem. Eng. J. 149,
Nigeria. 70–77.
Feng, L., Li, Y., Chen, C., Liu, X., Xiao, X., Ma, X., & Liu, G., 2013. Biochemical methane Samuel, O.D., Gulum, M., 2019a. Mechanical and corrosion properties of brass exposed
potential (BMP) of vinegar residue and the influence of feed to inoculum ratios on to waste sunflower oil biodiesel-diesel fuel blends. Chem. Eng. Commun. 206,
biogas production. Bioresources, 8(2), 2487-2498. 682–694.
Food and Agriculture Organization of the United Nations, 2017. Food and Agriculture Samuel, O.D., Okwu, M.O., 2019b. Comparison of Response Surface Methodology (RSM)
Organization Statistical Pocketbook World Food and Agriculture. FAOUN, Rome: and Artificial Neural Network (ANN) in modelling of waste coconut oil ethyl esters
Italy. production. Energy Sources Part A 41, 1049–1061.
Haider, M.R., Zeshan, Yousaf, S., Malik, R.N., Visvanathan, C., 2015. Effect of mixing Shi, X., Zuo, J., Li, B., Yu, H., 2020. Two-stage anaerobic digestion of food waste coupled
ratio of food waste and rice husk co-digestion and substrate to inoculum ratio on with in situ ammonia recovery using gas membrane absorption: performance and
biogas production. Bioresour. Technol. 190, 451–457. microbial community. Bioresour. Technol. 297, 122458. https://doi.org/10.1016/j.
Hashimoto, A.G., 1989. Effect of inoculum/substrate ratio on methane yield and biortech.2019.122458.
production rate from straw. Biol. Wastes 28, 247–255. Tayyab, A., Ahmad, Z., Mahmood, T., Khalid, A., Qadeer, S., Mahmood, S., Andleeb, S.,
Lavagnolo, M.C., Girotto, F., Rafieenia, R., Danieli, L., Alibardi, L., 2018. Two-stage Anjum, M., 2019. Anaerobic co-digestion of catering food waste utilizing
anaerobic digestion of the organic fraction of municipal solid waste–effects of Parthenium hysterophorus as co-substrate for biogas production. Biomass Bioenergy
process conditions during batch tests. Renewable Energy 126, 14–20. 124, 74–82.
Li, L., He, Q., Zhao, X., Wu, D.i., Wang, X., Peng, X., 2018. Anaerobic digestion of food Uma, S., Thalla, A.K., Devatha, C.P., 2020. Co-digestion of food waste and switchgrass for
waste: correlation of kinetic parameters with operational conditions and process biogas potential: effects of process parameters. Waste Biomass Valorization 11 (3),
performance. Biochem. Eng. J. 130, 1–9. 827–839.
Li, Y., Zhang, R., He, Y., Zhang, C., Liu, X., Chen, C., 2014. Anaerobic co- digestion of Xu, F., Li, Y., 2012. Solid-state co-digestion of expired dog food and corn stover for
chicken manure and corn stover in batch and continuous stirred tank reactor (CSTR). methane production. Bioresour. Technol. 118, 219–226.
Bioresour. Technol. 156, 342–347. Zhang, R., Elmashad, H., Hartman, K., Wang, F., Liu, G., Choate, C., Gamble, P., 2007.
Liu, G., Zhang, R., El-Mashad, H.M., Dong, R., 2009. Effect of feed to inoculum ratios on Characterization of food waste as feedstock for anaerobic digestion. Bioresour.
biogas yields of food and green wastes. Bioresour. Technol. 100 (21), 5103–5108. Technol. 98 (4), 929–935.
Marañón, E., Castrillón, L., Quiroga, G., Fernández-Nava, Y., Gómez, L., García, M.M., Zhang, Z., Guo, L., Wang, Y.i., Zhao, Y., She, Z., Gao, M., Guo, Y., 2020. Application of
2012. Co-digestion of cattle manure with food waste and sludge to increase biogas iron oxide (Fe3O4) nanoparticles during the two-stage anaerobic digestion with
production. Waste Manage. 32 (10), 1821–1825. waste sludge: impact on the biogas production and the substrate metabolism.
Moreno-Andrede, I., Buitron, C., 2003. Influence of the initial substrate to Renewable Energy 146, 2724–2735.
microorganisms concentration ratio on the methanogenic inhibition test. Water Zhang, Q., Hu, J., Lee, D., 2016. Biogas from anaerobic digestion processes: research
Science and Technology 48, 17-22. updates. Renewable Energy 98, 108–119.
Neves, L., Oliveira, R., Alves, M.M., 2004. Influence of inoculum activity on the bio- Zhu, J., Zheng, Y.i., Xu, F., Li, Y., 2014. Solid-state anaerobic co-digestion of hay and
methanization of a kitchen waste under different waste/inoculum ratios. Process soybean processing waste for biogas production. Bioresour. Technol. 154, 240–247.
Biochem. 39 (12), 2019–2024.

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