Biomass and Bioenergy 130 (2019) 105383

Contents lists available at ScienceDirect

Biomass and Bioenergy

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

Research paper

Investigation of the effect of hydraulic retention time on anaerobic digestion T

of potato leachate in two-stage Mixed-UASB system
Behzad Kamyaba,∗, Hamid Ziloueia, Bashir Rahmanianb
a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan, Iran
b
Department of Chemical Engineering, Ferdowsi University of Mashhad, Mashhad, Iran

ARTICLE INFO ABSTRACT

Keywords: In the present paper, potato leachate is introduced into the first reactor, converted to glucose through the
Biogas hydrolysis process and then into the acetic acid during the acidogenic process and as a feed entered the second
Anaerobic digestion reactor, converting to methane and carbon dioxide based on the methanogenic process. For modeling, a kinetic
UASB reactor model has been used to justify the two-stage system behavior using MATLAB software. In writing the equations
Potato leachate
for modeling in the first reactor, variations are taken with time, while in the second reactor, the equations are
Two-stage
written in solid and liquid phase and in steady state. The relationship used in most cases to represent the
Mesophilic
kinetics of bacterial growth is the equation called Monod, which is used in modeling the system. The systems
have been investigated at mesophilic temperature and the effect of the reaction's resistance on the boundary
layer has been neglected in the concentrations around the granules. According to the modeling, by increasing
the residence time and the concentration of input waste, the percentage of removal of organic compounds will
increase and fluctuate between the oxygen content of 67–81% and the methane production efficiency will
reduce (until less than 0.1 L CH4 g−1 CODremoved−1) that is agreement with laboratory model in 19th day. The
results and studies have shown that the modeling performed for a two-stage system can be used in the me-
sophilic temperature conditions, and the internal mass transfer resistance in the granules is a controlling
process.

1. Introduction
In recent years, due to the problems caused by extensive oil depen-
dence and limited energy resources, the use of biogas has been more fo-
cused [1]. The use of anaerobic digestion for energy production, urban
wastewater treatment, and agricultural and industrial waste has long been
considered in the world. Conservation of the environment, excessive in-
crease in urban and industrial wastewater, the correct use of available
energy sources, the limitation of fossil fuels, the problem of fossil fuels, and
finally the vital human need to use renewable energy is one of the most
important factors in the process of developing the anaerobic fermentation
process in the world. Anaerobic fermentation ultimately leads to the
production of methane and carbon dioxide as biogas [2–4]. Also Ni-
trification is of great importance for nitrogen removal from municipal
wastewater in the biological nutrient removal process (BNR) employed in
waste water treatment plants (WWTPs). In nitrification, ammonium is
firstly oxidized to nitrite via ammonia-oxidizing bacteria (AOB), and then
to nitrate by nitrite-oxidizing bacteria (NOB). However, due to low bio-
mass yield and sensitivity to environmental factors, nitrifying bacteria only
accounts for a small fraction of total biomass. Although nitrifying bacteria
populations are generally within the range of 4–6% of biomass for ade-
quate nitrification in nutrient removal facilities [5], a wide variation of the
fraction of nitrifying bacteria in microbial communities has been reported.
It varies from 0.39% in activated sludge [6], to 9% in a nitrifying activated
sludge SBR reactor [7], and even to over 18% in a combined activated
sludge and rotating biological contactor [8]. The difference of the per-
centage of nitrifying bacteria may be affected by operational conditions
and influent qualities. Due to the sequential oxidation property, the
growth balance between AOB and NOB plays a key role in optimization of
a nitrifying community. Nitrite is toxic to aquatic ecosystems and poses
potential threats to human health security. Furthermore, nitrite will be
converted under anoxic condition by Nitrosomonas to nitrous oxide (N2O)
[9], a lethal greenhouse gas (GHG) causing ozone depletion. Therefore,
fully understanding the population and interaction of AOB and NOB in the
nitrifying community is very important to optimize nitrification in biolo-
gical nutrient removal plants [10].
About 25% of future bio-energy is expected to be supplied from
biogas from anaerobic digestion of industrial waste, fertilizer, and

∗
Corresponding author.
E-mail address: behzadkamiab@gmail.com (B. Kamyab).

https://doi.org/10.1016/j.biombioe.2019.105383
Received 9 December 2018; Received in revised form 18 August 2019; Accepted 18 September 2019
0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
B. Kamyab, et al.
Nomenclature
COD Chemical Oxygen demand (mg COD cm−3)
HRT Hydraulic Retention Time (DAY)
VFA Volatile Fatty Acids (mg COD cm−3)
OLR Organic loading rate (mg COD cm−3 day)
other organic compounds [11]. A lot of research is being done on the
production of biogas from various substrates from industrial waste-
water to lignocellulose compounds such as fertilizers that are used as
substrates in the anaerobic process to produce biogas [12]. Biogas
fuel can be used as fuel in turbine power plants, steam engines, or
internal combustion engines to generate electrical energy [13,14].
Anaerobic digestion is the process of converting organic compounds
in the absence of dissolved oxygen to methane and carbon dioxide
[15]. The energy from transformation is small, and in fact, energy
remains stored in methane. For this reason, the growth of bacteria in
these systems has been slow, increasing the residence time in the
reactor and the volume of the desired reactors. Conversely, because
of the very low levels of sludge and cell mass, the cost of surplus
sludge disposal decreases [16,17]. The transformation of large
complex molecules present in the feed into biogas requires the pre-
sence of several groups of microorganisms [18,19]. All processes of
digestion have four stages of transformation [20,21]. Considering the
various processes that occur during anaerobic digestion, two im-
portant points should be emphasized:
The removal of organic matter during acid fermentation is limited to
the release of hydrogen. Only 30% of organic food is converted into
methane through the pathway of hydrogen-containing bacteria [22].
Acid fermentation tends to reduce alkalinity, because volatile fatty
Fig. 1. Two-stage system used in the laboratory for digestion of anaerobic potato leachate.
2
Biomass and Bioenergy 130 (2019) 105383
Q Input flow rate (cm3 day−1)
C Concentration of input waste (mg COD cm−3)
V Bio-reactor volume (cm3)
PA Partial Alkalinity (g CaCO3 l−1)
TA Total Alkalinity (g CaCO3 l−1)
acids and other intermediate products are decomposed and produce
proton. If, for some reason, the rate of acid removal is reduced by the
production of methane from the rate of acid production, instability may
occur, since methanogens develop only in good neutral alkalinity. Pure
acid production tends to reduce alkalinity, thus reducing the activity of
methane producers [20,22]. This risk can be solved by controlling the
alkalinity in biogas production systems, it is possible to control even the
composition of the percentage of the middle products of the digestive
process, such as the production of acids and advance the production of
an ideal product [23].
In this project, Potato leachate wastewater that contains high or-
ganic compounds (about 20,000 mg COD per liter) has been used as a
special substrate to produce biogas in a two-stage system. Our goal is
to design and construct an appropriate model based on the available
experimental model using the MATLAB software and compare the
results between the experimental model and the analytical model. The
proposed model consists of a two-stage system including an upflow
anaerobic sludge blanket and a mixed reactor. One of the most im-
portant aspects of this research is the environmental aspect and the
solution to the problems of various industrial wastewater, as well as
the problem of energy supply in the future and access to a renewable
energy source. These results can be helpful to achieve the practical
application of this technique.
B. Kamyab, et al. Biomass and Bioenergy 130 (2019) 105383

2. Experimental
2.1. Digesting process of the potato waste
The system used in this project consists of a two-stage system con-
sisting of an acidogenic reactor and a methanogenic reactor as shown in
Fig. 1. Three different temperature ranges are considered in this system,
which include:
1. The first stage is mesophilic (37 °C) and the second stage is meso-
philic (37 °C)
2. First stage mesophilic (37 °C) and thermophilic second stage (55 °C)
3. First phase thermophilic (55 °C) and thermophilic second stage
(55 °C)
The first stage reactor is a glass cylindrical reservoir like a full mix
reactor with a working volume of 2 L in which the hydrolysis and
acidogenic reactions are performed and the second stage reactor of an
upstream flow of sludge bed reactor with a working volume of 0.84 L in
which the methanogenic reaction occurs. The reactor doors are covered
with plastic cork. The acidogenic reactor is equipped with a return
pump that returns fluid flow into the reactor. A 2 mm wire mesh, held
by a steel frame, is placed at a distance of 5 cm from the end of the
reactor to separate large particles from the liquid and accumulate
particles in the exterior of the reactor. Mesophilic anaerobic sludge bed
reactor upward flow with granular sludge from an up-flow anaerobic
sludge bed reactor with full scale Processed with Papermill waste,
Granulation (in the Netherlands) and Thermophilic anaerobic sludge
bed reactor Granular sludge is grafted from thermophilic anaerobic
digestion in Denmark. The reactor contents are returned at a constant
flow rate of 5 ml min−1 from the top to the bottom of the reactor to
provide good contact between the biomass and the wastewater.
2.2. Preparation of potato leachate used as substrates
from the acidogenic reactor to the methanogenic reactor. The experi-
ments showed that for a period of more than 40 days, the production
time of biogas in the methanogenic reactor is negligible. The potato
leachate is completely decomposed into acidic reactors. The pH value in
anaerobic digestion is a function of time. At the beginning of the pro-
cess, due to acid production by the acidogenic bacteria, the pH value is
reduced to 4.4, is a factor that inhibits biogas production and disrupts
digestion. Because methane-producing bacteria are highly sensitive to
PH, and in PH below 6.5 they are not capable of growth and pro-
liferation and will be destroyed. The pH value can also be increased by
increasing the concentration of NH4+ due to protein digestion. When the
amount of biogas produced reaches its steady state, the pH value will
remain in the range of 6.8–7.4. The PH variations has been shown in
Table 2.
Samples were centrifuged (3000×g) for 3 min and the supernatant
was used for alkalinity and VFA measurements. The PA values varied in
the two different reactors depending on OLRs between 0 and 3.4 g
CaCO3 l−1 in mixed reactor and 0–3.8 g CaCO3 l−1 in UASB-reactor,
respectively. Also, as we can see in Table 3, TA values varied between
1.4 and 4.3 g CaCO3 l−1 in mixed reactor and 1.1–4.8 g CaCO3 l−1 in
UASB-reactor. With increasing OLR in the UASB reactor and mixed
reactor, the PA and TA decreased in both reactors throughout the
period of the study; the pH for the UASB dropped to below 5.5 at
13.17 g COD L−1 day indicating an overload of the system; the PA
decreased to zero at this OLR, also indicating process overload; the TA
increased when process failure occurred.
2.4. Reactions
The reactions are as follows:
In the first case, the effluent in the first reactor, which is the same as
potato, turns into glucose in the presence of water. This reaction, which
is the same as the hydrolysis process, is given below.

(C6 H10 O5) + nH2 O nC6 H12 O6 (1)

The potatoes are converted into smaller particles of 0.3–5.5 cm. The
waste is collected from 100 g of potato in 150 ml of distilled water. The After this step, the acidogenic anaerobic process of glucose converts
waste is collected by mixing the sieve to remove larger solid particles. to fatty acids, which in the present project is the only fatty acid used in
The waste is diluted with a tap to a concentration of approximately 20 g acetic acid.
of oxygen per liter and stored in a feed storage tank at 4 °C. The char-
acteristics of potato waste are indicated in Table 1. C6 H12 O6 + 2H2 O 2CH3 COOH + 2CO2 + 4H2 (2)

2.3. System operation Then in the last step, called the methanogenic step, acetic acid is
converted into biogas (a mixture of methane and carbon dioxide). The
The waste is rotated in a hydrolysis reactor with a flow rate of reactions are as follows.
10 ml min−1 and sprinkled on a constant bed of potatoes. The metha-
CH3 COOH + H2 O CH 4 + CO2 + H2 O (3)
nogenic reactor feeds out of the waste begins after 24 h of rotation in
the hydrolysis reactor. After the first reactor's output enters the second
reactor, it passes through a sludge bed that is made up of a high body CO2 + 4H2 CH 4 + 2H2 O (4)
biomass. This sludge bed can be either granular or clotted. It then
In fact, this process follows the general process below.
passes through a lighter sandy bed. In other words, the sludge con-
centration decreases from the bottom to the top of the reactor. At the starch glocuse VFAs end products
top of the reactor, before the output stream is discharged, the three-
phase separator separates liquid, solid, and gas from each other. Heavy
solid particles return to the sludge bed, and liquids and gases are re- Table 1
Specifications of potato waste (with variability) as substrates
leased from the reactor. The organic load used in the second reactor
for digestion.
changes in range from 2. 2–13.2 g of oxygen per liter per day in system
1, from 4.5 to 21.3 g of oxygen per liter per day in the system 2, and PH 6.1 ± 0.6
from 1.3 to 45.6 g of oxygen per liter per day in system 3. After startup,
TS (%) 1.7 ± 0.4
changes in the amount of organic matter of the methanogenic reactor VS (% of TS) 89.4 ± 5.0
are observed with a change in the flow rate, until about 4 days after the COD total (g l−1) 20.3 ± 0.5
operation, organic loads are observed and the system becomes stable. Total carbon (% of TS) 52.6 ± 0.2
The upstream flow from the methanogenic reactor is returned to the Total nitrogen (% of TS) 1.3 ± 0.3
C N−1 ratio 40.5 ± 0.3
acidogenic reactor to be refilled and provide intermediate capacity to
Total phosphates (g l−1) 0.2 ± 0.1
prevent excessive acidification. The highest amount of organic loading Orthophosphates (g l−1) 0.03 ± 0.1
in the methanogenic reactor is determined by increasing the flow rate

3
B. Kamyab, et al. Biomass and Bioenergy 130 (2019) 105383

Table 2 3.2.1. Acidogenic reactor equations

PH variations for Mixed reactor, UASB reactor and biogas product. Hydrolysis rate equation for potato:
Parameter Recommended value Mixed- UASB- Biogas
Xa
Reactor Reactor product rH,p = kH,pSp
xa (5)
PH 6.8–7.8 4.4–7 5.5–7.9 6.8–7.4
In which SP indicates the concentration of potato waste for the
hydrolysis reaction, KH is the first-rate constant for hydrolysis and its
Table 3 unit is day−1.
Alkalinity variations in Mixed reactor and UASB reactor. Unstable mass conservation equations for potato:
Parameter Mixed Reactor UASB Reactor ds p
VR1 = Q(S0p S p) rH,P (VR1)
−1 dt (6)
PA (g CaCO3 l ) 0–3.4 0–3.8
TA (g CaCO3 l−1) 1.4–4.3 1.1–4.8
Sp = S0p @ t= 0

Unstable mass conservation equation for glucose:

3. Modeling
dsg
VR1 = Q(S0g Sg) + rH,g (VR1) rA,g (VR1)
The relationship used in most cases to represent the bacterial dt (7)
growth kinetics is the equation known as Monod, developed by the Sg = 0 @ t= 0
famous French microbiologist of the same name in the 1940s.
1. The removal of organic matter during acid fermentation is limited In this equation, rA, g (glucose consumption rate) and rH , g (glucose
to the release of hydrogen. Only 30% of organic food is converted into production rate) are as follows.
methane through the pathway of hydrogen-containing bacteria. Sg
rA,g = Xa,f qmax,g
K g + Sg (8)
3.1. Assumptions
Xa
1. Granules are spherical. rH,g = KH,g Sg
Xa (9)
2. The mass of the microbial cells in the penetration layer (the pene-
tration resistance from the fluid bulk to the surface of the granular- Unstable mass conservation equation for acetate:
liquid interface) is ignored and Fick's law is in place. (We do not
ds
react to the boundary layer, which is why we can confirm the mass VR1 a = Q(S0a Sa ) + rA,a (VR1)
dt (10)
transfer flux).
3. The density of the hydrolyzing bacteria in the acidogenic reactor Sa = 0 @ t= 0
varies with time, so the acidogenic reactor here is acting unsteady.
In this equation, rA, a (production rate of acetate) is as follows.
4. The whole system is in a steady state, meaning that the volumetric
flow rate of the inlet and outlet of both reactors is equal. Sa Sg
rA,a = Xa qmax,a or rA,a = 3Xa,f qmax.g
5. Establishing the following relationship K a + Sa K g + Sg (11)
starch glocuse VFAs end products Unstable mass conservation equation for hydrolysis of bacteria:
dx a,f Sa
6. The rate of consumption of glucose and volatile fatty acids follows
0
VR1 = Q(X a,f Xa,f ) + VR1Yf Xa,f qmax,a bdec X a,f VR1
dt K a + Sa (12)
the Monod type kinetics and the rate of consumption of potato
leachate is of the order of magnitude. X a = Xa 0 @ t= 0
7. In the Upflow Sludge Blanket (USB) reactor, the density of me-
thanogens is constant over time, otherwise, a transient equation for
the biomass density is also required, and equations must be entered 3.2.2. Methanogenic reactor equations
in each step. Mass conservation equations acetate granules:
8. Both reactors assume complete mixing. There is no spatial dis- This equation, which is considered in the solid phase in spherical
tribution in both reactors. coordinates, is given below. The equations written in this phase are
9. The hydrolysis and acidogenic reaction is carried out in the first used to calculate the coefficient of influence. It should be noted that
reactor, and the final concentration of acetate as an input enters the acetate is the same substrate as the input to the reactor.
second reactor. Sa
rM = qmax,M,a x a,u
10. In the second reactor, only the methanogenic reaction occurs. KM,a + Sa (13)
11. The alkalinity is neglected and reactors operate in mesophyllic
conditions. dsa
Na = Deff , In Out Cons = 0
dr (14)
3.2. Equations Effect coefficient equation:
This equation is used to consider the role of internal mass transfer
As already stated, the process is carried out in the first two reactors. within the granules.
So, we will deal with two sets of equations. The equations for the hy-
actual rate
drolysis and acidogenic process performed in the first reactor and the =
(15)
ideal rate
equations for the methanogenic process performed in the second re-
actor. Since the hydrolysis stage is limited in the first reactor and is Xa, u Sa, b 4 3
rateideal : rM = qM , m, a r
carried out at a low speed, we consider the reactions inside the reactor KM , a + Sa . b 3 (16)
as unsteady. It should be noted that the equations in the second reactor
are considered in two solid and liquid phases. rateactual = flux @ r = R (17)

4
B. Kamyab, et al. Biomass and Bioenergy 130 (2019) 105383

dsa
flux @ r= R : Deff |r=R 4 R2
dr (18)
3 KM,a + Sa,b ds
= Deff a |r=R
R qmax,M,a X a,u Sa,b dr (19)
Acetate mass conservation equations of fluid in the liquid balk:
The methanogenic reactor with complete mixing (steady-state)
substrate consumption rate of the liquid bulk substrate is equal to the
rate of application of the substrate observed in the granules. Since this
equation is written in the methanogenic reactor, the spatial distribution
of the equation is ignored.
Q(sain Sa,b)
= rM
(1 ) (20)
4. Result and discussion
In this chapter, we first discuss the effect of changing the input para-
meters on the output of the system, based on the modeling performed on a
two-stage system including an acidogenic reactor and a methanogenic
reactor. The parameter whose effect on the system's output is being in-
vestigated include changes in the hydraulic retention time (HTR). Output
includes the methane production efficiency, the percentage of Chemical
oxygen demand, the concentration of acetate from the acidogenic reactor
and changes in volatile fatty acids (in the present work is acetate only). In
the following, we compare the results obtained from modeling the system
with the results of a laboratory model.
4.1. Effect of changing the hydraulic retention time on the amount of
chemical oxygen demand
Changes in the amount of Chemical oxygen demand with a hy-
draulic retention time are shown in Fig. 2. As shown in Fig. 2, the
percentage of removal of chemical oxygen demand increases with in-
creasing hydraulic residence time. The hydraulic retention time re-
presents the residence time inside the reactor. The hydraulic retention
time is obtained from Eq. (21):
V
HRT =
Q (21)
In this equation, Q is the inlet to the reactor in cubic centimeter per
day and v is the volume of the reactor used in cubic centimeter.
Hydraulic retention time unit is the day. The percentage of chemical
oxygen demand removal is obtained from Eq. (22):
CODin CODout
COD(%) = × 100
CODin (22)
With regard to the above relations, the lower the flow rate, the higher
the residence time in the reactor, thus, a greater amount of material
inside the reactor is converted and less material is released. Therefore,
the difference between the input and output concentration will be more
and more, and the percentage of COD will increase. As shown in Fig. 2,
the percentage of COD changes in the range of 50–500 cubic centimeter
per day fluctuate between the oxygen content of 67–81%. Because at
very low flow rates, the material inside the reactor is completely con-
verted and the difference between the concentration of the stream and
the output is almost constant, so the percentage of the COD will not
change much. It is worth noting that the chemical oxygen demand of the
wastewater is subject to process conditions such as temperature and al-
kalinity. As with the increase in alkalinity, the amount of chemical
oxygen demand in the wastewater, which is a measure of the amount of
organic compounds in it, is reduced. Also, with increasing temperature
during the process, the amount of COD will be reduced further. This
indicates that in the process of producing biogas, the reactor-soluble
compounds, including wastewater, are consumed [26].
In Fig. 3, changes in the percentage of removal of chemical oxygen
demand in terms of time are presented in the laboratory curve. As seen
in the figure, the chemical oxygen demand decreases by up to 31% in
the mesophylic methanogenic reactor on the nineteenth day, in this
case, the system clogs up to 13 g of COD content per liter per day. The
methanogenic reactor feeding is stopped for 2–3 days and the reactors
are cleaned. In changes in the curve after the nineteenth day, many
biological agents, such as temperature, alkalinity, reactor restart con-
ditions, wastewater treatment conditions used after cleaning the re-
actor, etc., are involved. The removal efficiency of pollutants is almost
constant after about 30 days, which indicates stabilization and the re-
covery of the refining process. The results indicate that at the beginning
of the reactor commissioning, large quantities of biomass used in a
methanogenic reactor that has not been well-balanced, was washed out
of the reactor and removed by the liquid flow from the reactor, and
simultaneously with The light sludge was washed out, and the density

Fig. 2. Chemical oxygen demand changes curve with hydraulic retention time.

5
B. Kamyab, et al.
Fig. 3. Laboratory curve of chemical oxygen demand changes with time.
of the bulk density began to increase. But over time, the degree of
biological mass washing as well as the speed of increasing sludge
density have been reduced. This suggests a granulation of sludge. Over
time, the sludge is applied to the characteristics of stability.
4.2. Effect of the change of hydraulic retention time on methane production
efficiency
Chart of the methane production efficiency changes with the hy-
draulic residence time is shown in Fig. 4. As seen in the figure, the
methane production efficiency decreases with increasing residence time
inside the reactor. This decrease is initially high, but after the residence
time inside the reactor increases, due to the larger size of the granules
inside the methanogenic reactor, and as a result of increasing the in-
ternal mass transfer resistance within the granules, the rate of pro-
duction of Methane gas decreases. Also during the disintegration of
solids in the mixture and hydrolysis of microbial anaerobic digestion
process and their use by methane-producing bacteria in higher re-
sidence time, it produces biogas [24,25].
In Fig. 5, experimental curve of the changes in methane production
efficiency are presented in terms of time. As it is seen in the figure, the
methane gas production rate decreases with time to less than 0.1 L of
Fig. 4. Changes in methane production efficiency with hydraulic retention time.
6
Biomass and Bioenergy 130 (2019) 105383
Fig. 5. Laboratory curve of changes in methane production efficiency with
time.
methane per gram of chemical oxygen demand, at this time, the eclipse
occurs in the reactor. So the machines will shut off and the reactors will
be clean. Then the system starts up again. As it is seen, after cleaning
the system, methane gas production efficiency has increased, but after a
while, due to eclipse, production output is reduced.
4.3. Effect of changing the hydraulic retention time on the concentration of
acetate output from the acidogenic reactor
The graph of changes in the concentration of acetate output from
the acidogenic reactor with the hydraulic retention time is shown in
Fig. 6. It is observed that in the low flow stream, the concentration of
acetate is low and almost constant in the output of the acidogenic re-
actor. By decreasing current flow and thus increasing residence time,
the acetate concentration of the output of the acidogenic reactor gra-
dually decreases until it reaches a constant value at low flow rates. In
other words, in low flow current, there is virtually no current in the
reactor, so the concentration of acetate from the reactor will be very
low [26,27].
B. Kamyab, et al. Biomass and Bioenergy 130 (2019) 105383

Fig. 6. Concentration curve of output acetate with hydraulic retention time.

4.4. Effect of hydraulic retention time on input organic load of organic inputs decreased.

Chart organic load changes with the hydraulic retention time is

given in Fig. 7. As seen in the figure, the input organic load decreases 4.5. Effect of changing the hydraulic retention time on the concentration of
with increasing residence time. The organic load is obtained from the acetate output from the methanogenic reactor
equation (23):
In Fig. 9, the acetate concentration changes from the methanogenic
Q×C C reactor to the hydraulic residence time are shown. This reduction in the
OLR = = concentration of the output from the methanogenic reactor is justified
V HRT (23)
by increasing the residence time, increasing the reproduction of the
Changes in Fig. 7, are justifiable using equation (23). microbes and thus increasing the size of the granules. Increasing the
Laboratory results from organic load changes with time in the la- size of the granules leads to an increase in the internal mass transfer
boratory are shown in Fig. 8. The amount of organic load increased up resistance, and thus the concentration of the acetone output from the
to the nineteenth day, and then, due to the clogging of the reactor and methanogenic reactor decreases. The graph of the laboratory model is
other biological agents present in the laboratory conditions, the amount shown in Fig. 10. As seen in this figure, the concentration until the

Fig. 7. Curve of organic load changes with hydraulic retention time.

7
B. Kamyab, et al.
Fig. 8. Input-organic load variation curve with time in laboratory model.
reactor reaches a state of shock, increases. After the nineteenth day, the
amount of acetate concentration decreased with time due to eclipse and
the shutdown of reactors and due to cleaning. It should be noted that in
the present study, due to the simple simplification, acetic acid is con-
sidered as a volatile fatty acid [18,19].
4.6. Concentration time curve by changing the hydraulic retention time in
the acidogenic reactor
The concentration changes diagram in the acidogenic reactor is
generally presented in Fig. 11. This chart is derived from the base data
used in modeling. Then, according to the modeling, the concentration
changes with the hydraulic residence time have been investigated.
Three samples of the resulting graphs in a specific flow rate and a
concentration are shown in Fig. 12.
As you can see, In Fig. 12, with increasing hydraulic residence time,
the intensity of increasing the concentration of germs and acetate
Fig. 9. The concentration of acetate output of methanogenic reactor with hydraulic retention time.
8
Biomass and Bioenergy 130 (2019) 105383
decreases and the intensity of decreasing the concentration of glucose
and starch increase. As shown in Fig. 11, the density of germs has not
reached a constant value. Because little time has been considered and
biological processes have a long time scale and in order to see the
constant concentration phenomenon, we need to take more time.
5. Conclusions
In the present study, the results of a two-stage system, including a
methanogenic and acidogenic reactor, were compared and investigated.
It should be noted that the amount of pollution above 2 g of oxygen per
day per liter causes the lack of digestion of bacteria and acidification of
the environment and low alkalinity which results in the loss of alkali-
nity, the lack of granulation of sludge and the prolonged startup of the
primary sludge. Investigating the removal of chemical oxygen demand
of leachate from the reactor, in short detention time, it is clear that the
decomposition of organic matter occurs in the active region of the
B. Kamyab, et al. Biomass and Bioenergy 130 (2019) 105383

Fig. 10. Concentration changes of fatty acid output from methanogenic reactor with time in laboratory model.

Fig. 11. Concentration curve with time for acidogenic reactor.

reactor, where the microorganisms are most present. As the hydraulic

retention time increases, the percentage of pollutant emissions in-
creases until after a while these changes are fixed. In other words, at the
beginning of the reactor commissioning, large quantities of biomass
used in a methanogenic reactor that does not have a good density are
washed out of the reactor and is removed by the liquid flow from the
reactor and, at the same time as the sludge was washed, the density of
the bulk density has begun to increase. But over time the severity of the
biomass washing as well as the speed increases the density of the sludge
is reduced. This suggests a granulation of sludge. Over time, the sludge
is applied to the characteristics of stability. The results of methane
production efficiency with increasing residence time, compatible with
the laboratory model. As a result of system modeling, gas production
efficiency decreases with increasing residence time. In the laboratory
model, the production efficiency decreases over time until the eclipse
occurs in the reactor. It should be noted that in the laboratory model, in
reactors, eclipse occurs and the changes in the resulting graphs are due
to this, and it is not possible to provide such conditions in the modeling.
The results obtained for organic load changes, the concentration of
acetate output from the acidogenic reactor and the concentration of Fig. 12. Concentration curve with time in the acidogenic reactor by changing
acetate output from the methanogenic reactor with the hydraulic re- the hydraulic retention time.
tention time were obtained as expected.

9
B. Kamyab, et al.
Acknowledgment
The authors are indebted to Biotechnology Laboratory of Isfahan
University of Technology, Isfahan Municipal Waste Factory, Isfahan,
Iran for their financial assistance and support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biombioe.2019.105383.
References
[1] M.A. Sathianathan, Biogas;Achievement and Challenge, Association Agencies for
Rural Development, New Dehli, 1975.
[2] M.H. Gerardi, The Microbiology of Anaerobic Digesters, John Wiley & Sons, New
Jersey, 2003.
[3] K.C. Khandelwal, S.S. Mahdi, Biogas Technology, fourth ed., McGraw-Hill, New
Dehli, 1992, pp. 1–3 2.
[4] Margarida F. Temudo,Robbert Kleerebezem,Mark van Loosdrecht,Influence of the
PH on(Open) Mixed Culture Fermentation of Glucose: A Chemostat Study,
Department of Biotechnology, Delft Univercity of Technology, Published online 9
March 2007 in Wiley Interscience.
[5] P.H. Nielsen, T.R. Thomsen, J.L. Nielsen, Bacterial composition of activated slud-
ge—importance for floc and sludge properties, Water Sci. Technol. 49 (2004)
51–58.
[6] H.M. Dionisi, A.C. Layton, G. Harms, I.R. Gregory, K.G. Robinson, G.S. Sayler,
Quantification of Nitrosomonas oligotropha—like ammonia-oxidizing bacteria and
Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR,
Appl. Environ. Microbiol. 68 (2002) 245–253.
[7] B.K. Li, S. Irvin, K. Baker, The variation of nitrifying bacterial popula- tion sizes in a
sequencing batch reactor (SBR) treating low, mid, high concentrated synthetic
wastewater, J. Environ. Eng. Sci. 6 (2007) 651–663.
[8] S.J. You, C.L. Hsu, S.H. Chuang, C.F. Ouyang, Nitrification efficiency and nitri-
fying bacteria abundance in combined AS-RBC and A2 O systems, Water Res. 37
(2003) 2281–2290.
[9] B.B. Colliver, T. Stephenson, Production of nitrogen oxide and dinitrogen oxide by
autotrophic nitrifiers, Biotechnol. Adv. 18 (2000) 219–232.
[10] A. Sepehri, M.H. Sarrafzadeh, Activity enhancement of ammonia-oxidizing bacteria
and nitrite-oxidizing bacteria in activated sludge process: metabolite reduction and
CO2 mitigation intensification process, Appl. Water Sci. 9 (2019) 131.
10
Biomass and Bioenergy 130 (2019) 105383
[11] H. Nielsen, J.B. Al Seadi, T. O.Popiel, P, The future of anaerobic digestion and
biogas utilization, Bioresour. Technol. 100 (2009) 5478–5484.
[12] A. Lehtomäki, S. Huttunen, T.M. Lehtinen, J.A. Rintala, Anaerobic digestion of grass
silage in batch leach bed processes for methane production, Bioresour. Technol. 99
(2008) 3267–3278.
[13] R.P.J.M. Raven, K.H. Gregersen, Biogas plants in Denmark: successes and setbacks,
Renew. Sustain. Energy Rev. 11 (2007) 116–132.
[14] A. Keshtkar, B. Meyssami, G. Abolhamed, H. Ghaforian, M. K.Asadi, Mathematical
modeling of non-ideal mixing continuous flow reactors for anaerobic digestion of
cattle manure, Bioresour. Technol. 87 (2003) 113–124.
[15] L.T. Angenent, K. Karim, M.H. Al-Dahhan, B.A. Wrenn, R. Domíguez-Espinosa,
Production of bioenergy and biochemicals from industrial and agricultural waste-
water, Trends in Biotechnology 22 (9) (2004) 477–485.
[16] M.H. Gerardi, The Microbiology of Anaerobic Digesters, John Wiley & Sons, New
Jersey, 2003.
[17] Widodo, S., "Determination of Volatile Solids, Biogas and Methane Potentials from
Municipal Organic Waste and Mixed Materials as Basic for Designing a Biogas
Reactor", University College of Boras, School of Engineering.
[18] V.A. Vavilin, B. Fernandez, J. Plastsi, X. Flotats, Hydrolysis kinetics in anaerobic
degradation of particulate organic material: an overview, Waste Manag. 28 (2008)
pp939–951.
[19] R. Borja, A. Martin, E. Sánchez, B. Rincón, F. Raposo, Kinetic modelling of the
hydrolysis, acidogenic and methanogenic steps in the anaerobic digestion of two-
phase olive pomace (TPOP), Process Biochem. 40 (2005) 1841–1847.
[20] H. Bouallagui, Y. Touhami, R. Ben Cheikh, M. Hamdi, Bioreactor performance in
anaerobic digestion of fruit and vegetable wastes, Process Biochem. 40 (2005)
989–995.
[21] Sue Baldwin, Anthony Lau, Max Wang, “Development of a Calculator for the
Techno-Economic Assessment of Anaerobic Digestion Systems.” Chemical and
Biological Engineering, University of British Columbia Vancouver, BC, 2009.
[22] P. Chermisinoff Nicholas, Biotechnology for Waste and Wastewater Treatment,
Noyes, New Jersey, 1996.
[23] J.-I. Horiuchi, T. Shimizu, K. Tada, T. Kanno, M. Kobayashi, Selective production of
organic acids in anaerobic acid reactor by pH control, Bioresour. Technol. 82
(2002) 209–213.
[24] S.J. Hashemian, A. James, Gas – solid – liquid separator in anaerobic treatment of
wastewater, Water Res. 24 (No. 3) (1990) 381–382.
[25] A.P. Buzzini, L.J. Patrizzi, A.J. Motheo, E.C. Pires, Preliminary evaluation of the
electrochemical and chemical coagulation processes in the post-treatment of ef-
fluent from an upflow anaerobic sludge, J. Environ. Manag. (2007) doi:10.1016.
j.jenvman. 2005.10.017.
[26] L.W. Hulshoff Pol, W.J. De Zeeuw, C.T.M. Velzeboer, Lettinga, Granulation in UASB
reactors, Water Sci. Technol. 15 (8–9) (1983) 291–304.
[27] J.H.F. Pereboom, Size distribution model for methanogenic granules from full scale
UASB and IC reactors, Water Sci. Technol. 30 (12) (1994) 211–221.