Environmental Technology & Innovation 19 (2020) 100867

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Biogas recirculation technology: Effect on biogas purification,

slurry characteristics, microbial activity and energy
consumption
∗
Mahmood Mahmoodi-Eshkaftaki , Ehsan Houshyar
Department of Mechanical Engineering of Biosystems, Jahrom University, P.O. Box 74135-111, Jahrom, Iran

article info a b s t r a c t

Article history: In the present study, effect of biogas recirculation on slurry characterization, microbial
Received 8 February 2020 activity and biogas purification were studied. Two digester units, one for control treat-
Received in revised form 13 April 2020 ment and another equipped with a gas compression system for recirculation treatment,
Accepted 30 April 2020
were designed and constructed. Experimental results showed that the recirculation
Available online 4 May 2020
system could improve the mixing process with a little energy consumption. The slurry
Keywords: analysis during the digestion indicated that (i) in the recirculation treatment, TC, TN,
Biogas purification COD and BOD had a step up after the first injection and then decreased while they
Anaerobic digestion efficiency continuously decreased for the control treatment, (ii) amounts of TC/TN appeared that
Methane enrichment the carbonated compounds increased more than nitrogenous compounds for recircu-
Microbial community lation treatment, and (iii) the biogas recirculation decreased pH. The biogas analysis
Stirred digester showed that the biogas recirculation could create a suitable environment for growing
the anaerobic microbes (up to 21%) in the digester that not only decreased some of the
harmful compounds like H2 S (about 0.21 %VOL) but also increased the CH4 concentration
(up to 11 %VOL). Significant correlations between the anaerobic microbial communities
and CH4 (r = 0.9), CO2 (r = −0.91), H2 S (r = −0.89), CO (r = −0.8) and O2 (r =
−0.71) for recirculation treatment documented the biogas recirculation can improve
the environmental conditions for anaerobic microbial life and is a very convenient way
for biogas purification.
© 2020 Published by Elsevier B.V.

1. Introduction

Anaerobic digestion in a suitable condition converts the organic material to biogas. The raw biogas produced from a
biogas plant is a mixture of CO2 (35–50%VOL), H2 S (500–2000 ppm), CH4 (50–65%VOL), and a little amount of CO, O2 , NH3
and H2 saturated with water vapor (Chandran et al., 2017). Presence H2 S in the biogas yields from the biodegradation
of sulfur in feed stock during anaerobic digestion (Maia et al., 2017). It needs to be removed from the biogas to avoid
facility corrosion, unnecessary production of byproducts and possible exposure to public and complaints (Maia et al.,
2017). Further, sufficient purification will necessary if biogas requires to be used in the facilities running with usual
natural gas (Vikromvarasiri et al., 2017).
The development of biogas plants to enrich CH4 production, purify the biogas production and increase their energy
performance is very important (Hosseini and Abdul Wahid, 2014). Many upgrading techniques have been developed for

∗ Corresponding author.
E-mail address: m.mahmoodi5@jahromu.ac.ir (M. Mahmoodi-Eshkaftaki).

https://doi.org/10.1016/j.eti.2020.100867
2352-1864/© 2020 Published by Elsevier B.V.
2 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867

Nomenclature
BOD: Biological oxygen demand (mg/L)
COD: Chemical oxygen demand (mg/L)
Dmix : Impeller diameter (m)
EC: Electrical conductivity (S/m)
L: Blade length (m)
NP : Power number
Ns : Impeller speed (rpm)
P: Power consumption (hp & W)
SO42− : Sulfate (kg/m3 )
T: Torque (N m)
TC: Total carbon (%)
TK: Total potassium (kg/m3 & %)
TN: Total nitrogen (%)
TP: Total phosphorus (kg/m3 & %)
TS: Total solids (kg/m3 & %)
T.VS: Total volatile solids (kg/m3 & %)
ρ: Density of slurry (kg/m3 )
Subscript
C: Control treatment
R: Recirculation treatment

purification the biogas (Ryckebosch et al., 2011) such as bio-trickling filtration (Vikromvarasiri et al., 2017), biofilter
(Ryckebosch et al., 2011), diaphragm technologies (Deublein and Steinhauser, 2011), chemical absorption (Maia et al.,
2017) absorption in water (Deublein and Steinhauser, 2011). The digestion condition is one of the most effective factors
to produce a purified output biogas. It can be improved using a suitable mixing. Mixing can be accomplished by mechanical
mixers, biogas recirculation (pneumatic mixing) and slurry recirculation (hydraulic mixing). For the pneumatic mixing,
after production of sufficient biogas, 20%–25% of biogas is being recirculated into the digester for mixing purposes (Trad
et al., 2016). Mechanical mixing is the most common mixing system being used in Europe today and reported as having
the highest power efficiency per volume unit mixed while pneumatic mixing having the lowest (El-Bakhshwan et al.,
2015).
Coupling the mechanical and pneumatic mixing can affect the mixing process and its energy consumption (Mahmoodi-
Eshkaftaki and Ebrahimi, 2019a). The mixing energy consumption for liquid batches can be calculated from the energy
balance equation and empirical correlations (Mahmoodi-Eshkaftaki et al., 2016a). According to the literature for the
recirculation systems, the biogas is usually injected at the bottom of digesters (Chandran et al., 2017; Trad et al., 2016;
Luo et al., 2014). In these systems, the biogas starts to move upward towards the outlet region of the digester with
one-dimensional linear motion (just vertical motion) while biogas injection as two-dimensional, both vertical and radial
motions, will improve the mixing performance.
Effect of biogas recirculation in the digesters on removing some of the harmful compounds such as H2 S (Trad et al.,
2016; Mahmoodi-Eshkaftaki and Ebrahimi, 2019b), CO2 (Bassani, 2017) and toxic elements of the slurry (Luo et al., 2014)
has been studied. It remains to consider the effect of biogas recirculation on slurry characteristics, biogas compounds
and biological parameters. Some studies have been conducted on the microbiology of the anaerobic co-digestion system
to correlate biogas production with microbial diversity (Dearman et al., 2006; Zhang et al., 2011; Yu et al., 2014; Chen
et al., 2016). However, there is little scientific literature to clarify how development of a biogas recirculation system
can enhance the anaerobic microbial activity, biogas purification and CH4 enrichment. Therefore, the aims of the present
study are to develop a new system for two-dimensional biogas injection and study the effect of biogas recirculation on
slurry characteristics, biogas compounds and slurry microbial activity during the digestion days by focusing on the energy
consumption and biogas purification.

2. Materials and methods

2.1. Biogas plant

The anaerobic digestion tests were carried out in a laboratory batch pilot plant (Fig. 1). Dimensions of the digesters
and their mixers were determined according to the dimensions obtained by Mahmoodi-Eshkaftaki et al. (2016b). They
 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867 3

Fig. 1. Sketch of the anaerobic digestion pilot plant.

developed a stirred digester for a single-family home based on optimal power consumption. Two digester units were
designed for the experiments, one unit without gas compression system for control treatment and one unit equipped with
a gas compression system to analyze the effect of biogas recirculation on the parameters expressed. Diameter, height and
wall thickness of each digester were 0.57, 0.9 and 0.01 m, respectively, with a total volume of 0.22 m3 (Fig. 1).
The mixers were four blade marine impellers which their diameter, blade thickness, blade length and blade width
were 0.32, 0.004, 0.1 and 0.04 m, respectively. For two-dimensional biogas recirculation in the recirculation treatment,
two symmetric sparger nozzles were welded on one of the impellers, totally fixed on a hollow shaft. During the impeller
rotation, the injected biogas was moved upward with both vertical and radial motions. As shown in Fig. 1, for biogas
recirculation system, a biogas compression system equipped by a 150 W compressor with delivery pressure up to 40 bar
and reservoir capacity of 5 K, and an electric pressure switch to control its pressure was used. A water absorber filter
was used to absorb water in high-pressure pipe because the steam in biogas converted to water after compression. The
compressed biogas was injected into the bottom of digester through a hollow mixer shaft and sparger nozzles. The process
of compressing biogas and injection was done after production of sufficient biogas (after 10 days of digestion started).

2.2. Energy consumption

The power consumption by an electrical mixing system (electrical energy consumption) as the main part of the energy
consumer in a biogas plant can be calculated with Eq. (1) (Streeter and Wylie, 1975).
P = fs NP Ns 3 Dmix 5 ρ (1)
where P, Dmix and Ns are power consumed by the impeller (W), impeller diameter (0.4 m) and mixer speed (60 or 100 rpm),
respectively. According to the equation, the Ns and Dmix have the major impact on the required P to operate the mixer.
4 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867

The safety factor (fs ) was set 3 according to the reported values in the literature (Mahmoodi-Eshkaftaki and Ebrahimi,
2019b). The density of the slurry (ρ ) was 1018 kg/m3 measured by averaging the density of three diluted substrates. NP
is the power number of the impeller fully reported in the literature (Mahmoodi-Eshkaftaki et al., 2016a,b). It was equaled
to 0.35 for marine impeller with four blades. A higher mixing cost relatively need a higher torque (T ). Required torque
was calculated using Eq. (2) for the mixer used in the control treatment (El-Bakhshwan et al., 2015), and was calculated
using Eq. (3) for the mixer used in the recirculation treatment.
P
TC = (2)
2π Ns
( )
L
TR = TC + ṁV + 0.05 sin (θ)
2
PR = TR 2π Ns (3)

where TC and TR are the torques of the control and recirculation systems, respectively, PR is the power consumption
of the recirculation system, L is the blade length (0.1 m), θ is the angle of nozzles with vertical axis (30◦ ), and ṁV
is biogas injection force. Using the biogas density (1.225 kg/m3 ), inlet gas velocity (V = 287.52 m/s) and nozzle cross
section (0.000038465m2 ), the injection mass rate (ṁ) of pressurized biogas to the digester for the recirculation treatment,
obtained by the Momentum and Continuity laws, was 0.018 kg/s. Consequently, the biogas injection force was 5.17 N.
To determine the inlet gas velocity, it was assumed that there was not any change in velocity gradients during the biogas
recirculation (Chandran et al., 2017). It was calculated according to Bernoulli’s equation.
A mixing process, pausing time of 5 h and mixing time of 5 min, was selected for mixing the slurries because of
producing the most efficiency according to Mahmoodi-Eshkaftaki and Ebrahimi (2019b) researches. The P and T of the
mixers were calculated for two Ns (60 and 100 rpm), and their values were compared between the treatments.

2.3. Slurry preparation

A mixture of cow manure, slurry of municipal waste and fruit waste (including grapefruit and sweet lemon) diluted
with tap water was prepared to be in the biogas plant experiments. For this purpose, 20 kg slurry of municipal waste,
obtained by a squeezing machine, was prepared for each treatment which its concentration was 1018 kg/m3 . Cow
manure (20 kg for each treatment) diluted with tap water until its concentration reached near the slurry concentration
of municipal waste. Further, 20 kg fruit waste for each treatment was cut into particulates by cutter and grinded into
small fine particles (< 4 mm) by a home type coffee grinder and diluted with tap water until its concentration reached
near the slurry concentration of municipal waste (Mahmoodi-Eshkaftaki and Ebrahimi, 2019a). These fruits were selected
due mainly to their higher buffer capacities which can be appropriate for an anaerobic digestion. As their peel contains
comparatively small amounts of organic acids and their salts, the pH values of the peel are high and these systems have
slight buffer capacities. On the other hand, their pulp contains larger amounts of organic acids and their salts having lower
pH values and higher buffer capacities. The literature showed that these fruits lead to an alkalizing effect in the human
stomach after digestion (Bailey et al., 2013; Franck et al., 2016).
The prepared substrates, substrates diluted with tap water, were combined with the same amount in the digesters for
both control and recirculation treatments to produce a suitable slurry. The digesters were filled with N2 99% at the start
of digestion for making anaerobic environment. The experiments were repeated three times for each treatment. Then the
slurry characteristics, biogas compounds and slurry microbes of the digesters were studied during the digestion time.

2.4. Physical-chemical characterization

Total solids (TS), total volatile solids (T.VS), pH and EC were analyzed in accordance with Standard Methods (Method
1684 and CEN/TS 15148). Total carbon (TC ), total phosphorus (TP) and chemical oxygen demand (COD) were measured
using titration methods. Sulfate (SO24− ) was measured with Spectrophotometer, total nitrogen (TN) was measured with
Kjeldahl apparatus, total potassium (TK ) was measured by flame photometer, and biological oxygen demand (BOD) was
measured by BD600 apparatus. The biogas compounds including CH4 , H2 S, CO and O2 were determined using a multi-
function gas detector brand GMI Ltd model GT43 (Mahmoodi-Eshkaftaki and Ebrahimi, 2019a,b) and CO2 was measured
using CO2 meter model TESTO 535.
Total anaerobic bacteria of the slurry were counted using the pour-plate method which has been fully explained in
Dohrmann et al. (2011) researches. The samples were counted by incubating for 48 h at 37 ◦ C in an anaerobic jar under 85%
N2 , 10% H2 and 5% CO2 . The media for total anaerobic bacteria were anaerobe basal agar (CM972; Oxoid, Ltd, Basingstoke,
Hampshire, UK) with 10% sterile defibrinated horse blood (Dohrmann et al., 2011; Yu et al., 2014).
 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867 5

Table 1
Energy evaluation of the treatments for two rotational speeds.
Parameters Control treatment Recirculation treatment
60 rpm 100 rpm 60 rpm 100 rpm
Mixer power consumption (W) 800.59 3706.42 896.78 3874.76
Mixer torque (N m) 2.12 5.91 2.38 6.17
Power consumption (Mixer + compressor) (W) 800.59 3706.42 1046.78 4024.76
Rule of thumb (hp/1000 gal) 32.47 150.44 36.40 157.27
Energy consumption (Mixer + compressor) (kWh/day) 320.24 1482.57 418.71 1609.90
Biogas production (m3 /day) (average) 14.35 19.44 15.35 21.25

3. Results and discussion

3.1. Energy consumption for each treatment

By substitution the values of required parameters in Eqs. (1)–(3), some parameters of energy evaluation of the
treatments were calculated for Ns of 60 and 100 rpm (Table 1). The results indicated that P and T of the control treatment
for Ns = 100 rpm were 4.63 and 2.78 times higher than that of Ns = 60 rpm. Consequently, the mixer energy consumption
of the control treatment for Ns = 100 rpm was 4.63 times higher than Ns = 60 rpm agreed with those obtained by El-
Bakhshwan et al. (2015) and Mahmoodi-Eshkaftaki and Ebrahimi (2019b). They reported that the slow-speed mixers are
less energy demanding than fast-speed mixers. The TR and PR calculated by Eq. (3) for Ns = 60 rpm were 2.38 N m and
896.78 W, respectively, and corresponding values of Ns = 100 rpm were 6.17 N m and 3874.76 W. Moreover, the power
consumption with biogas compression system was 150 W. By adding the compressor power to the calculations, total
power consumptions (Mixer + compression system) in the recirculation treatment were 1046.78 and 4024.76 W for Ns
of 60 and 100 rpm, respectively. Further, its energy consumption for Ns = 100 rpm were 3.84 times higher than Ns = 60
rpm. For two Ns in similar power consumption, the digester with recirculation system produced more torque resulting
more mixing cost than control system.
In the ranges of our rotational speeds, by increasing the Ns , both T and P increased, while it would be great to increase
T and decrease P for a mixing system. This confirmed that selecting the mixer type based only on the T and P values
Pow er(hp)
was at high-risk. Therefore, a rule of thumb Volume(1000 gal) was used as a rough guide for quickly estimating mixer power
requirements completely described in Mahmoodi-Eshkaftaki et al. (2016b) research. As calculated for this rule in Table 1,
its difference between the treatments were low and between the rotational speeds were high. The amounts of energy
consumption and rule of thumb of the mixers for both treatments confirmed that the recirculation system produces
a high missing cost with a little energy consumption. On the other hand, the biogas recirculation helps provide some
mixing to keep sediment from accumulating at the bottom of the digester (Deublein and Steinhauser, 2011). Therefore,
it can be concluded that the recirculation system improves the mixing process very well with low energy consumption.

3.2. Characterization of the substrates

The characteristics of the grapefruit and sweet lemon were respectively as: Energy = 1380 and 1210 kJ/kg, TC = 8.4
and 9.3%, TN = 2.3 and 2.1%, sugars = 7.31 and 15.5%, dietary fiber = 1.1 and 3.2%, fat = 0.1 and 0.3%, protein = 0.8
and 1.3%, calcium = 1 and 3%, iron = 0.1 and 5.2%, magnesium = 3 and 2%, TP = 1 and 2%, TK = 3 and 3%. From the
diluted substrates before making the slurry, three samples were selected, their physical and chemical properties were
measured, and their means have been illustrated in Table 2. As reported in the table, amounts of TS, T.VS, TN, TC and
pH were in suitable ranges for biogas production agreed with those reported by other researchers (Mahmoodi-Eshkaftaki
and Ebrahimi, 2019a,b). Amounts of TC/TN of the samples were among 15–52, which were in suitable range for biogas
production (Mahmoodi-Eshkaftaki et al., 2016a,b). For the substrates with low TC/TN ratio, practices of mixing with the
municipal solid waste or food waste can increased the TC/TN ratio favorable to anaerobic digestion (Kim and Shin, 2008).
As shown in Table 2, pH of the prepared substrates (7.5–8.2) was neutral and slightly above neutral before feeding to the
biogas plant similar to other studies (Bischofsberger et al., 2005). The buffer capacity depends mainly on CO2 concentration
in the gas phase, ammonia concentration in the liquid phase and water content in general. If the pH in the feedstock is too
high or too low so that the buffer capacity is exceeded and the pH in the digester is changed, significantly (Bischofsberger
et al., 2005).
TK, TP and SO24− in the prepared substrates were little and not toxic for anaerobic microorganisms (Fang et al., 2011).
High amount of the TK in the substrates is useful when the output wastewater of the digesters is used as liquid fertilizer
for agriculture (Wellinger et al., 2013). The high amount of SO24− can be problematic, because H2 S develops from it in a
stage before CH4 formation. It can also inhibit methane formation, because SO24− degrading microorganisms dominate as
they need less energy and do not need a symbiosis partner (Deublein and Steinhauser, 2011). The low amounts of these
parameters decreased the potential of acidification of digester during the digestion, especially for recirculation treatment
that is most likely to be at risk from acidification.
6 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867

Table 2
Physical and chemical properties of prepared substrates before slurry production.
Substrates TC (%) TN (%) TK (kg/m3 ) TP (kg/m3 ) T.VS (kg/m3 ) TS (kg/m3 ) SO24− (kg/m3 ) pH EC (S/m)
Slurry of municipal waste 45.91 2.30 0.167 0.028 3.346 7.154 0.0031 7.62 0.25
Cow manure 35.90 0.77 0.135 0.021 3.593 4.839 0.0024 7.50 0.176
Fruit waste 30.26 1.98 0.140 0.012 0.857 3.554 0.0024 8.20 0.24

The changes in TC, TN, COD, BOD and pH of the slurry were also measured during the digestion time (Fig. 2). As
illustrated for the control treatment, the amounts of the properties continuously decreased with digesting time while
for the recirculation treatment after the first injection, the TC, TN, COD and BOD had a step up. It may be due to existence
of CH4 , CO2 , NOx , O2 , . . . in the recirculated biogas (Deublein and Steinhauser, 2011; Sarker et al., 2018; David et al.,
2019), which can increase contaminations in the digester. However, these carbonated and nitrogenous compounds are
caused to an acidity environment in the digester with recirculation which is completely shown in Fig. 2 that the pH
of recirculation treatment decreased more than control treatment. During the digestion days, the amounts of TC/TN of
recirculation treatment samples increased from 30.37 to 43.9 while for control treatment samples decreased from 50.88
to 31.98. Increasing TC/TN of the recirculation treatment shows that the amounts of carbonated compounds of biogas
recirculated to the digester were more than nitrogenous compounds. Furthermore, during the digestion days, increase
amounts of BOD/COD for the recirculation treatment (differed from 0.4 to 0.6) was lower than the control treatment
(differed from 0.42 to 0.7). These results indicate that the biogas recirculation can increase COD more than BOD in the
digester which was also shown in Fig. 2.

3.3. Performance of the recirculation system

Average amounts of biogas production of the treatments were reported in Table 1. The amounts were in agreement
with those reported by Marcos et al. (2012). As shown the amounts of biogas production of the recirculation treatment
were nearly similar to the control treatment. Therefore, it becomes important to determine the suitable treatment which
produce a fuel more purified.

3.3.1. Biogas purification

To determine the effect of biogas recirculation on biogas quality, amounts of CH4 , H2 S, O2 , CO and CO2 were measured
during 50 days according to previous literature (Miah et al., 2016). The biogas compounds of the treatments versus
digestion time are illustrated in Fig. 3. The digestion time was selected somehow that the CH4 reached to its maximum
amount. As shown in Fig. 3(a), with slurry digestion over time in the control treatment, (i) CH4 reached to the maximum
amount on 45th day of digestion (68.47%VOL) and then decreased, (ii) CO2 decreased with high intensity, and (iii) H2 S, O2
and CO decreased generally with low intensity. Furthermore, for the recirculation treatment in Fig. 3(b)(i) CH4 reached
to the maximum amount on 40th day of digestion (82.65%VOL) and then decreased, (ii) CO2 , CO and O2 decreased with
high intensity, and (iii) H2 S felled zero. Total amounts of biogas compounds were close to the amounts reported in the
literature (Kumar et al., 2020).
The results showed that the CH4 production in the recirculation treatment was lower than the control treatment before
25th day, and after that it kept a steady increase rate to reach a peak on the 40th day of digestion. The O2 concentration
in the recirculation treatment was more than that of the control treatment before 15th day, and after that it felled. The
results appeared that the biogas recirculation increased CH4 production up to 11%VOL and decreased O2 up to 1%VOL in
comparison with the control treatment agreed with those obtained by Sarker et al. (2018). It is noteworthy that the O2
amounts in the samples taken from the biogas were lower than 5%VOL (Fig. 3).
The maximum amounts of CO2 production for the treatments were the highest on 5th day of digestion (about 43%VOL).
The mean amounts of CO2 production for two treatments were close together, since the researchers have reported
the biogas recirculation led to decrease CO2 (Deublein and Steinhauser, 2011; Sarker et al., 2018; David et al., 2019).
Furthermore, average amount of CO production for the recirculation treatment was 0.02%VOL higher than the amount
in the control treatment which was a weak point for the recirculation system. The results indicated that CO and CO2
could not be well solved in the slurry after biogas recirculation due mainly to high amounts of carbonated compounds in
the biogas. Therefore, these contaminants were not decreased from the biogas outlet which was disagreement with the
finding of David et al. (2019). However, the amount of CO concentration for two treatments was lower than 0.045%VOL,
and thus, its effect on biogas efficiency was very low.
Average amount of H2 S production of the recirculation treatment was 0.21%VOL lower than the amount in the control
treatment. For the recirculation treatment during the digestion days 10–30, until the slurry was saturated H2 S reduced
with high intensity, and then its removal speed decreased because H2 S is more soluble than CO2 and CO (Deublein
and Steinhauser, 2011; David et al., 2019). According to the literature, CO2 is ∼20 times, H2 S is ∼72 times, and NH3
is ∼39 000 times more soluble than CH4 at standard temperature and pressure (Sarker et al., 2018). This implies the
biogas recirculation can remove H2 S more than CO2 , which is in confirmation with results obtained in this research.
The recirculation of biogas production into digester is not recommended when high levels of H2 S are handled because
 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867 7

Fig. 2. Changes the slurry characteristic during the digestion time for the control and recirculation treatments.
8 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867

Fig. 3. Changes of biogas compounds during the digestion time, (a) control treatment, (b) recirculation treatment.

the slurry quickly becomes contaminated with elementary sulfur which causes operational problems (Zhao et al., 2010;
Yeo-Myeong et al., 2017), for example, produce an acidity environment in the digester. H2 S can decompose to sulfate
and sulfur according to the following reactions: 2H2 S + O2 → 2S + 2H2 O + 3O2 → 2H2 SO4 ; or by direct reaction:
H2 S + 2O2 → H2 SO4 . Totally, it was concluded that the biogas recirculation significantly eliminated H2 S.
The higher amount of CH4 and lower amounts of H2 S and O2 in the recirculation treatment in comparison with the
control treatment confirms the significance of biogas recirculation. Furthermore, the low amount of H2 S in the outlet
biogas simplifies the CO2 absorption for two stage biogas purification process because H2 S is more easily absorbed
than CO2 and more difficult to desorb (Deublein and Steinhauser, 2011). Therefore, for high purified biogas, the biogas
 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867 9

Fig. 4. Total anaerobic bacteria counted from the slurry sampled for the control and recirculation treatments.

recirculation can be used for pre-cleaning of the biogas to remove such impurities which is a prerequisite for chemical
absorption. According to the literature, by improving the digestion conditions such as improve substrates and biogas plant;
the microbial activities may be improved and thus purify the biogas. These issues are carefully considered in the following
sections.

3.3.2. Effect of biogas recirculation on the microbial activity

The previous studies demonstrate that anaerobic microbial activity orients the anaerobic digestion. Majority of these
studies focused on correlating microbial communities with slurry properties, digestion conditions and biogas production
(Dearman et al., 2006; Zhang et al., 2011; Yu et al., 2014). The relationship between anaerobic bacteria activity and biogas
compounds was determined in the current study. For this purpose, anaerobic microbial communities were analyzed for
both treatments. Total colony counts of anaerobic bacteria of the slurry sampled from the treatments have been illustrated
in Fig. 4 versus the digestion days.
It was revealed that during digestion days 25–35, the anaerobic microbial communities of slurries for the recirculation
treatment strongly increased more than that of the control treatment and then remained nearly stable. The results of
both treatments showed that until the anaerobic microbial communities increased to maximum values, their trends
were similar to CH4 production trends. After that by staying the anaerobic microbial community stable at the most
community, the CH4 increased and CO2 decreased during the digestion days. The correlation coefficients between the
anaerobic microbial communities and CH4 , CO2 , H2 S, CO and O2 concentrations produced for the recirculation treatment
were 0.9, −0.91, −0.89, −0.8 and −0.85, and for the control treatment were 0.94, −0.97, −0.85, −0.71 and −0.7,
respectively, all significant at p < 0.05. In other words, by increasing the anaerobic microbial communities in this research,
the CH4 concentration increased and CO2 , H2 S, CO and O2 concentrations decreased. It showed that the main parts of the
anaerobic microbial communities were methanogenic bacteria.
To check the validity of this statement, culturable microorganisms were studied as an acceptable method. Therefore,
three samples were taken from the slurry of each treatment in the digestion day 30 where the anaerobic microbial
communities obtained their maximum values. The samples were serially diluted and cultivated with the pour-plate
method (Dearman et al., 2006; Zhang et al., 2011). A plate with the most colonies was selected for each treatment.
Five morphologically distinct colonies were picked up from the plate for single-colony transfer technique and were
named as MC1, MC2, MC3, MC4 and MC5. All five isolates did not show any growth in the aerobic medium. So, those
were confirmed as strict anaerobes, because methanogens are obligate anaerobes. The five isolates were observed for
morphological traits using Gram reaction. Gram reaction results of methanogenic isolates were studied for the samples
according to Ravichandran et al. (2016) researches. For the sample of recirculation treatment, MC1, MC2, MC4 and MC5
were confirmed to be Gram-negative isolates, and MC3 alone was confirmed as Gram-positive while for the sample of
control treatment, MC1, MC2 and MC4 were confirmed to be Gram-negative isolates, and MC3 and MC5 were confirmed
as Gram-positive. Owing to this, most of the methanogens are classified as Gram-negative due to lack of peptidoglycan
layer, it can result the recirculation treatment can produce CH4 more than another treatment. These confirmed that the
biogas recirculation can create an environment to increase the anaerobic microbial activity, especially methanogens, and
thus enrich CH4 by purifying the biogas from H2 S and O2 without meaningful changing the CO2 .

4. Conclusion

The force analysis showed that the equivalent resistance force caused from injected biogas on the mixer was 5.17
N and its required torque was 0.26 N m which was very low and negligible, therefore the energy consumption of the
10 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867

control and recirculation treatments were seen as nearly equal. To produce the biogas in two treatments, the slurries
were composed of cow manure (20 kg), fruit waste (20 kg) and slurry of municipal waste (20 kg) diluted with tap water.
The physical and chemical properties of the prepared substrates and slurries were useful for high biogas production. The
slurry analysis during the digestion days showed that (i) for the recirculation treatment after the first injection, TC, TN, COD
and BOD had a step up and then decreased, (ii) the biogas recirculation led to an acidity environment in the digester, and
(iii) amounts of TC/TN for the recirculation treatment increased and for the control treatment decreased confirming that
the amount of carbonated compounds of recirculated biogas was more than nitrogenous compounds. The biogas analysis
showed that the biogas recirculation could purify the biogas by decreasing some of its harmful compounds and enrich
CH4 concentration by creating a suitable environment for growing the anaerobic microbes without meaningful changing
the CO2 . Significant correlations between the anaerobic microbial communities and biogas compounds especially for the
recirculation treatment documenting that the biogas recirculation has no harmful effects on anaerobic microbial life in the
digester, and it can purify the biogas production with low power consumption. Although the study provides good insights
into the effect of biogas recirculation on biogas improvement, further experiments on different substrates are needed
to better understand their effects. It is suggested from this study that sweet lemon wastes may be contained essential
oils, which inhibit the mechanism of anaerobic microbial community. The pretreatment processes for the removal of the
inhibitory phenolics can be studied in future researches.

CRediT authorship contribution statement

Mahmood Mahmoodi-Eshkaftaki: Conceptualization, Methodology, Investigation, Project administration, Data cura-

tion, Formal analysis, Writing - original draft. Ehsan Houshyar: Methodology, Investigation, Visualization, Writing - review
& editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgment

The authors are sincerely thankful to the Iran National Science Foundation (ANSF) (Grant number ‘‘97007783’’) for
financial support.

References

Bailey, D.G., Dresser, G., Arnold, J.M., 2013. Grapefruit-medication interactions: Forbidden fruit or avoidable consequences? Can. Med. Assoc. J. 185,
309–316. http://dx.doi.org/10.1503/cmaj.120951.
Bassani, I., 2017. Hydrogen Assisted Biological Biogas Upgrading. Technical University of Denmark, DTU Environment, Kgs. Lyngby.
Bischofsberger, W., Dichtl, N., Rosenwinkel, K.H., Seyfried, C.F., Bohnke, B., 2005. Anaerobtechnik. Springer, Berlin, Germany.
Chandran, J., Yogaraj, D., Manikandan, K., Jeyaraman, P., 2017. Optimization of biogas recirculation velocity in biogas mixing anaerobic digester with
the feed of 8% TDS using CFD. Int. J. Mech. Eng. Technol. 8, 596–606, http://www.iaeme.com/IJMET/index.asp.
Chen, R., Roos, M.M., Zhong, Y., Marsh, T., Roman, M.B., Ascencio, W.H., Uribe, L., Lorio, L.U., Kirk, D., Reinhold, D.M., Chavarria, J.A.M., Ruiz, D.B.,
Pereira, J.F.A., Montero, W.R., Srivastava, A., Liao, W., 2016. Responses of anaerobic microorganisms to different culture conditions and
corresponding effects on biogas production and solid digestate quality. Biomass Bioenergy 85, 84–93. http://dx.doi.org/10.1016/j.biombioe.2015.
11.028.
David, B., Nicolas, D., Pierre, L., Gilles, H., 2019. A compact, economic scrubber to improve farm biogas upgrading systems. Sep. Purif. Technol.
http://dx.doi.org/10.1016/j.seppur.2019.02.054.
Dearman, B., Marschner, P., Bentham, R.H., 2006. Methane production and microbial community structure in single-stage batch and sequential batch
systems anaerobically co-digesting food waste and biosolids. Appl. Microbiol. Biotechnol. 69, 589. http://dx.doi.org/10.1007/s00253-005-0076-9.
Deublein, D., Steinhauser, A., 2011. Biogas from Waste and Renewable Resources, an Introduction. WILEY-VCH Verlag GmbH & Co. KGaA, Boschstr
12, 69469 Weinheim.
Dohrmann, A.B., Baumert, S., Klingebiel, L., Weiland, P., Tebbe, C.C., 2011. Bacterial community structure in experimental methanogenic bioreactors and
search for pathogenic clostridia as community member. Appl. Microbiol. Biotechnol. 89, 1991–2004. http://dx.doi.org/10.1007/s00253-010-2955-y.
El-Bakhshwan, M.K., Abd El-Ghafar, S.M.A., Zayed, M.F., El-Shazly, A.E., 2015. Effect of mechanical stirring on biogas production efficiency in large
scale digesters. J. Soil Sci. Agric. Eng. Mansoura Univ. 6, 47–63.
Fang, C., Boe, K., Angelidaki, I., 2011. Anaerobic co-digestion of desugared molasses with cow manure, focusing on sodium and potassium inhibition.
Bioresour. Technol. 102, 1005–1011. http://dx.doi.org/10.1016/j.biortech.2010.09.077.
Franck, C., Frédérique, O., Andres, G.-L., François, L., Luis, N., Patrick, O., 2016. Phylogenetic origin of limes and lemons revealed by cytoplasmic and
nuclear markers. Ann. Bot. 11, 565–583. http://dx.doi.org/10.1093/aob/mcw005.
Hosseini, S.E., Abdul Wahid, M., 2014. Development of biogas combustion in combined heat and power generation. Renew. Sustain. Energy Rev. 40,
868–875. http://dx.doi.org/10.1016/j.rser.2014.07.204.
Kim, S.H., Shin, H.S., 2008. Effects of base-pretreatment on continuous enriched culture for hydrogen production from food waste. Int. J. Hydrog.
Energy 33, 5266–5274. http://dx.doi.org/10.1016/j.ijhydene.2008.05.010.
Kumar, V., Kumar, P., Kumar, P., Singh, J., 2020. Anaerobic digestion of Azolla pinnata biomass grown in integrated industrial effluent for enhanced
biogas production and COD reduction: Optimization and kinetics studies. Environ. Technol. Innov. http://dx.doi.org/10.1016/j.eti.2020.100627.
Luo, J., Lu, X., Liu, J., Qian, G., Lu, Y., 2014. Biogas recirculation for simultaneous calcium removal and biogas purification within an expanded granular
sludge bed system treating leachate. Bioresour. Technol. 173, 317–323. http://dx.doi.org/10.1016/j.biortech.2014.09.128.
 M. Mahmoodi-Eshkaftaki and E. Houshyar / Environmental Technology & Innovation 19 (2020) 100867 11

Mahmoodi-Eshkaftaki, M., Ebrahimi, R., 2019a. Assess a new strategy and develop a new mixer to improve anaerobic microbial activities and clean
biogas production. J. Cleaner Prod. 206, 797–807. http://dx.doi.org/10.1016/j.jclepro.2018.09.024.
Mahmoodi-Eshkaftaki, M., Ebrahimi, R., 2019b. The effect of blade angle of turbine impellers on anaerobic digestion efficiency in stirred digesters.
Energy 178, 772–780. http://dx.doi.org/10.1016/j.energy.2019.04.183.
Mahmoodi-Eshkaftaki, M., Ebrahimi, R., Ghasemi-Pirbaloti, A., 2016a. Design and Construction a Batch Biogas Plant and Study on the Effects of Mixer
Type, Mixer Speed and Digestion Conditions for Methane Enrichment (A dissertation for the degree of Doctor of Mechanical Engineering of
Biosystems). Shahrekord University, Iran.
Mahmoodi-Eshkaftaki, M., Ebrahimi, R., Ghasemi-Pirbaloti, A., 2016b. Design of stirred digester with optimization of energy and power consumption.
Environ. Prog. Sustain. Energy 36, 104–110. http://dx.doi.org/10.1002/ep.12451.
Maia, D.C.S., Niklevicz, R.R., Arioli, R., Frare, L.M., Arroyo, P.A., Gimenes, M.L., Pereira, N.C., 2017. Removal of H2 s and CO2 from biogas in bench scale
and the pilot scale using a regenerable Fe-EDTA solution. Renew. Energy 109, 188–194. http://dx.doi.org/10.1016/j.renene.2017.03.023.
Marcos, A., Al-Kassir, A., López, F., Cuadros, F., Brito, P., 2012. Environmental treatment of slaughterhouse wastes in a continuously stirred anaerobic
reactor: Effect of flow rate variation on biogas production. Fuel Process. Technol. 103, 178–182. http://dx.doi.org/10.1016/j.fuproc.2011.12.035.
Miah, M.R., Rahman, A.K.M.L., Akanda, M.R., Pulak, A., Rouf, M.A., 2016. Production of biogas from poultry litter mixed with the co-substrate cow
dung. J. Taibah Univ. Sci. 10, 497–504. http://dx.doi.org/10.1016/j.jtusci.2015.07.007.
Ravichandran, M., Munisamy, P., Natarajan, S.D., Varadharaju, C., 2016. Rare detection and identification of methanogenic bacteria from diverse
ecological niches in India for carbon balance and management in our environment. Int. J. Adv. Res. 4, 1174–1186. http://dx.doi.org/10.21474/
IJAR01/2508.
Ryckebosch, E., Drouillon, M., Vervaeren, H., 2011. Techniques for transformation of biogas to biomethane. Biomass Bioenergy 35, 1633–1645.
http://dx.doi.org/10.1016/j.biombioe.2011.02.033.
Sarker, S., Lamb, J.J., Hjelme, D.R., Lien, K.M., 2018. Overview of recent progress towards in-situ biogas upgradation techniques. Fuel 226, 686–697.
http://dx.doi.org/10.1016/j.fuel.2018.04.021.
Streeter, V.L., Wylie, E.B., 1975. Fluid Mechanics. McGraw-Hill, New York.
Trad, Z., Fontaine, J.P., Larroche, C., Vial, C., 2016. Multiscale mixing analysis and modeling of biohydrogen production by dark fermentation. Renew.
Energy 98, 264–282. http://dx.doi.org/10.1016/j.renene.2016.03.094.
Vikromvarasiri, N., Champreda, V., Boonyawanich, S., Pisutpaisal, N., 2017. Hydrogen sulfide removal from biogas by bio-trickling filter inoculated
with Halothiobacillus neapolitanus. Int. J. Hydrog. Energy 42, 18425–18433. http://dx.doi.org/10.1016/j.ijhydene.2017.05.020.
Wellinger, A., Murphy, J., Baxter, D., 2013. The Biogas HandBook Science, Production and Applications. In: Woodhead Publishing Series in Energy,
no. 52, Woodhead Publishing Limited, Cambridge CB22 3HJ, UK.
Yeo-Myeong, Y., Shihwu, S., Hang-Sik, S., Jong-In, H., Hyun-Woo, K., Dong-Hoon, K., 2017. Producing desulfurized biogas through removal of sulfate
in the first-stage of a two-stage anaerobic digestion. Biotechnol. Bioeng. 114, 970–979. http://dx.doi.org/10.1002/bit.26233.
Yu, D., Kurola, J.M., Lahde, K., Kymalainen, M., Sinkkonen, A., Romantschuk, M., 2014. Biogas production and methanogenic archaeal community in
mesophilic and thermophilic anaerobic co-digestion processes. J. Environ. Manage. 143, 54–60. http://dx.doi.org/10.1016/j.jenvman.2014.04.025.
Zhang, Y., Zamudio Canas, E.M., Zhu, Z.W., Linville, J.L., Chen, S., He, Q., 2011. Robustness of archaeal populations in anaerobic co-digestion of dairy
and poultry wastes. Bioresour. Technol. 102, 779. http://dx.doi.org/10.1016/j.biortech.2010.08.104.
Zhao, Q., Leonhardt, E., MacConnell, C., Frear, C., Chen, S., 2010. Purification Technologies for Biogas Generated by Anaerobic Digestion. CSANR
Research Report 001.