b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1

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Research Paper

Anaerobic co-digestion of cattle manure and

meadow grass: Effect of serial configurations
of continuous stirred tank reactors (CSTRs)

Lu Feng a,*, Radziah Wahid a,b, Alastair J. Ward a, Henrik B. Møller a

a
Aarhus University, Department of Engineering, Blichers All
e 20, DK 8830 Tjele, Denmark
b
Norwegian University of Life Sciences, Faculty of Chemistry, Biotechnology and Food Science, P.O. Box 5003, 1432
 Norway
As,

article info
In this study, anaerobic co-digestion of cattle manure (CM) and meadow grass (MG) with
Article history: serial configurations of continuous stirred tank reactors (CSTRs) was investigated. Four
Received 20 March 2017 laboratory-scale CSTRs were operated at thermophilic condition (55
C), of which two
Received in revised form CSTRs were connected serially with equal working volumes while the remaining two
8 May 2017 CSTRs were operated as single CSTRs as controls. Improvements on bio-methane yield,
Accepted 21 May 2017 methane contents and solids reduction were observed with serial CSTR configurations.
Published online 3 June 2017 The results showed that co-digestion with 5% (w/w) MG with serial configurations of
CSTRs produced 24% more bio-methane compared with single reactor, with the total hy-
Keywords: draulic retention time (HRT) being the same. The volatile fatty acids (VFA) concentration
Bio-methane was found higher in the 1st reactor, but was reduced by 40e50% in the 2nd reactor. The
Serial CSTRs improved bio-methane yield with serial CSTR configuration with 100% CM was 8%, indi-
Meadow grass cating that the serial CSTRs process is superior in a co-digestion set up with addition of
Thermophilic MG.
Cattle manure © 2017 IAgrE. Published by Elsevier Ltd. All rights reserved.

Angelidaki, 2015). Co-digestion of animal manure with other

1. Introduction
Anaerobic digestion (AD) is a technology widely used for sta-
bilisation of animal manure while recovering bioenergy as
biogas (of which 60e70% is methane) at the same time (Møller,
Sommer, & Ahring, 2004; Nielsen, Mladenovska, Westermann,
& Ahring, 2004). The yield of biogas from animal manure is
often lower because of the poor quality of the available
organic content (Søndergaard, Fotidis, Kovalovszki, &
substrates can increase the biogas yield and offers advantages
for the management of manure and organic wastes (Li et al.,
2013; Xavier, Moset, Wahid, & Møller, 2015). During the last
15e20 years, there has been an increasing interest in Europe
and North America for the use of grass as co-substrate
(Murphy & Power, 2009). The advantages of exploiting grass
are that there is already a large font of knowledge on grass
production and harvesting (Curtis, 2006) and in extensively
cultivated agricultural areas is provides little competition as

* Corresponding author.
E-mail address: lufeng@eng.au.dk (L. Feng).
http://dx.doi.org/10.1016/j.biosystemseng.2017.05.002
1537-5110/© 2017 IAgrE. Published by Elsevier Ltd. All rights reserved.
2 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1

scale (Nizami, Korres, & Murphy, 2009) since the operation and
Nomenclature control of two-phase systems is complex and, as a result, in-
vestment and operational costs are higher (Boe & Angelidaki,
Symbols 2009). Furthermore, the efficiency of two-phase AD systems
B Cumulative methane yield (ml [CH4] g1 [VS]) varies depending on the substrate itself. Lindner, Zielonka,
B0 Maximum or ultimate methane yield (ml [CH4] Oechsner, and Lemmer (2016) found that the degradation
g1 [VS]) of lignocellulosic-rich substrates in a two-phase AD process
t Incubation time (d) was much lower than that of substrates with high sugar
mm Maximal methane production rate (ml [CH4] g1 content. Because of these problems with two-phase AD pro-
[VS] d1) cesses, an alternative approach operating several methano-
l Lag phase time (d) genic reactors connected in series, instead of phase
w/w Weight per weight separation, was investigated (Kaparaju, Ellegaard, &
CAi Concentration of component A at the inlet Angelidaki, 2009). Boe and Angelidaki (2009) demonstrated
CA1 Concentration of component A at the outlet that serial digestion with percent volume distributions of 90/
t Hydraulic retention time 10 or 80/20 between the two methanogenic reactors improved
Nl Normalised litre, gas volume corrected to a biogas production by 11% compared to a traditional single-
standard temperature and pressure step CSTR process. Athanasoulia, Melidis, and Aivasidis
(2012) improved biogas production by 9.5e40.1% with two
Abbreviations
methanogenic CSTRs connected in series. Thus, the serial
AD Anaerobic digestion
digestion system has be considered as a method to improve
ADF Acid detergent fibre
conversion efficiency of AD (Andriani, Wresta, Atmaja, &
ADL Acid detergent lignin
Saepudin, 2014). To date, there have been very few
BMP Biochemical methane potential
studies examining the configurations of serial CSTRs in co-
CM Cattle manure
digestion.
CSTR Continuously stirred tank reactor
In this work, four lab-scale CSTRs were operated and
DMY Daily methane yield
monitored separately. The co-substrate consisted of a mixture
HRT Hydraulic retention time
of CM and MG. Two CSTRs were connected serially with
IA Intermediate alkalinity
equally working volumes at a total HRT of 20 d while the
MG Meadow grass
remaining two CSTRs were operated as single CSTR, as con-
NDF Neutral detergent fibre
trol. The objective of this research was to investigate the effect
OLR Organic loading rate
of serial CSTRs configuration on co-digestion of CM and MG.
PA Partial alkalinity
RTD Residence time distribution
SMY Specific methane yield
2. Materials and methods
TA Total alkalinity
TAN Total ammonia nitrogen
2.1. Substrates
TN Total Kjeldahl nitrogen
TOC Total organic carbon
CM was collected from the animal facilities at Research Centre
TS Total solids
Foulum (Aarhus University, Denmark) and kept at 18
C after
VFA Volatile fatty acids
sampling. The MG was grown in meadow area (Obbekærvej 59,
VS Volatile solids
6760 Ribe, Denmark) and harvested in July 2015. After har-
vesting, the MG was left in the field and dried naturally for 3 d
before collected. The predominant species in the MG were:
reed canary grass (Phalaris arundinacea, 80%), tufted grass
animal feed due to its low forage quality (Tsapekos, Kougias, & (Holcus lanatus, 10%) and floating sweet-grass (Glyceria fluitans,
Angelidaki, 2015). However, the higher bio-fibre contents of 5%). The grass sample was cut to 10e20 mm lengths and
manure and grass often leads to lower biodegradability with a stored in airtight plastic bags at 18
C to prevent sample
typical hydraulic retention time (HRT) of 15e30 d (Hartmann, degradation.
Angelidaki, & Ahring, 2000). Thermophilic inocula were obtained from a thermophilic
Researchers are developing two-phase AD process where reactor located at Research Centre Foulum (Aarhus University,
first phase, with a shorter HRT is followed by a second phase Denmark) which had been running for more than one year
with longer HRT which improves the overall AD performance under thermophilic conditions.
(Nielsen et al., 2004; Zhang, Tao, & Dugba, 2000). Two-phase
AD systems have advantages over conventional single-phase 2.2. Biochemical methane potential (BMP)
processes, such as increased stability of the process, higher
organic loading rate (OLR) with shorter HRT and higher The BMP was determined from CM, CM þ 5% MG (w/w),
biodegradability of recalcitrant substrates (Demirer & Chen, CM þ 10% MG (w/w) and pure MG. Prior to starting the batch
2005; Nielsen et al., 2004). Most of the studies related to two- test, the thermophilic inoculum was pre-incubated at 55
C for
phase AD have only been conducted in laboratory and 15 d to deplete the residual biodegradable organic material
pilot scale digesters. They are not yet available at commercial (degasification) (Angelidaki & Sanders, 2004).
 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1
The batch test was carried out following the procedures
suggested by Moset, Poulsen, Wahid, Højberg, and Møller
(2015). About 200 ml of inoculum was added to each 500 ml
infusion bottle, followed by the addition of substrate with a
ratio of approximately 1:1, based on volatile solid (VS) content.
A control sample with only inoculum was also included. All
bottles were tightly sealed with rubber stoppers and screw
caps and then purged with nitrogen gas for two minutes. The
bottles were incubated at 55
C for 90 d in triplicate. Biogas
production measurements were done by inserting a needle
attached to a tube with inlet to a column filled with acidified
water (pH < 2) through the butyl rubber and were calculated by
the water displaced until the relevant two pressures (column
and headspace in bottles) were equal.
Gas sample was collected using 20 ml flat bottom head-
space vial (Agilent technologies, CA 95051, USA) sealed by cap
and septa which connected to the incubation bottle and tubes
with inlet to a column filled with acidified water. Biogas from
incubator bottles travelled through the vial to the column.
Biogas produced from each sample was corrected by sub-
tracting the volume of methane produced from the inoculum
control. The biogas yield was adjusted to standard conditions
(0
C and 101.3 kPa).
2.3. Laboratory-scale reactor
The laboratory-scale experiment was carried out with a biogas
test plant system (BTP2-control, Umwelt-und Ingen-
ieurtetechnik GmbH Dresden, Germany). The biogas test plant
system consisted of four reactor units and a central control
unit. Each reactor unit was equipped with a 15L CSTR, drum-
type gas meter (TG 0.5, Ritter, Germany), Teldar® 10 l gas
sampling bag with single polypropylene septum (SKC Inc., PA
15330, USA), heating control unit (T-Controller, Umwelt-und
Ingenieurtetechnik GmbH Dresden, Germany) and an
agitator system (R100CT, Ingenieurbüro CAT, Germany). Each
CSTR reactor was equipped with a silicon heating mat which
is fitted outside the glass tube and controlled by the heating
Fig. 1 e Schematic diagram of serial CSTRs experiment.
3
control unit. Biogas composition (CH4, CO2, H2S, O2) was auto-
analysed by a gas analyser (SSM 6000 LT, PRONOVA,
Germany).
The reactors were manually fed and emptied daily by an
amount based on the different HRT of 10 or 20 d. All reactors
were set to operate at thermophilic conditions (55
C) and
filled with 10 l of thermophilic inoculum before start-up. Grass
was removed with a sieve (1 mm mesh) before adding the
inoculum to reactors. After filled by de-grassed inoculum, no
feeding and discharging occurred in all CSTRs at the start two-
weeks.
As shown in Fig. 1, reactors R1 and 1e2 (second reactor)
were fed with 95% CM þ 5% MG (w/w) from day 1e49. The
reactors were set up as serial CSTRs with the HRT set to 10 d
for each reactor. Reactors 2 and 3 (R2, R3) were run as single
CSTR and fed 95% CM þ 5% MG (w/w) (R2) or with CM only
(R3). The HRT for R2 and R3 was set to 20 d. Feeding was
stopped for around 10 d during the winter holiday. From
day 60 to day 85, only reactors R1 and R1-2 were kept as
serial configuration and changed as mono-digestion of CM.
The OLR for R1, R2 and R3 were then calculated as 9.36, 4.68
and 3.77 kg [VS] m3 d1 based on the HRT and feeding load.
R1, R1-2 and R2 were fed CM for two weeks before co-
digestion, to provide a constant environment before grass
was added.
During the experiment, pH, temperature and biogas flow
rate were measured and saved in a database at one-minute
intervals. Digestate was collected from each reactor 1e2
times a week and analysed for total solids (TS) content, VS
content, VFA, total ammonia nitrogen (TAN), total alkalinity
(TA), partial alkalinity (PA) and intermediate alkalinity (IA).
2.4. Analytical methods
TS and VS were measured according to the standard methods
(APHA, 2005). Biogas composition was analysed once a week
using gas chromatography (Agilent technologies 7890A, CA
95051, USA) equipped with a thermal conductivity detector
4 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1

(TCD) and helium as the carrier gas. Alltech® CTR 1 double

column (Grace, Maryland 21044, USA) was used. The temper- 3. Results
ature of oven, injector port, and detector was 120, 150, and
150
C, respectively. Dissolved VFA was determined using a 3.1. Substrate characteristics
gas chromatograph (Agilent technologies, CA 95051, USA)
equipped with a flame ionisation detector (FID) and helium as Table 1 lists the main characteristics of the CM, MG and
the carrier gas. A DB-1 Column with a length of 30 m and in- inocula used in batch and CSTRs. MG had the highest TS, VS
side diameter of 0.53 mm was used. The temperatures of (75.36 ± 0.21%/68.39 ± 5.35%) and carbon content (34.4%).
initial oven, injector port, and detector were 100, 285, and Higher cellulose (35%) and hemi-cellulose (33%) contents were
300
C, respectively. The following temperature programming observed in MG than in CM (24 and 17%). CM contained
of oven was set: 100
C hold 1 min, ramp to 120
C at approximately 16% lignin whereas MG contained only 6%.
10
C min1, hold 5 min; ramp to 220
C at 30
C min1, hold Two different mixture ratios (95% CM þ 5% MG, 90%
3 min. CM þ 10% MG) (w/w) were applied in batch tests before the
TAN was determined weekly from digestate using semi-continuous experiment in order to determine suitable
photometry (Spectroquant Kit, Merk, NJ 07033, USA). Total mixture ratio. The TS contents of the 95% CM þ 5% MG (w/w)
organic carbon (TOC) was analysed according to EN 13137 and the 90% CM þ 10% MG (w/w) mixtures were 12.52 ± 0.02
(DIN, 2001). Total Kjeldahl nitrogen (TN) was determined ac- and 15.42 ± 0.18, respectively.
cording to APHA (2005). Crude protein content was calculated
by determining total organic nitrogen and multiplying by a
3.2. BMP assay
factor of 6.25 (Hattingh, Thiel, & Siebert, 1967).
As shown in Fig. 2, final BMP values were ranked as follows:
Samples for fibre analysis were dried (48 h at 60
C) and
[MG] > [90% CM þ 10% MG] > [CM þ 5% MG] > [CM]. 5.50 and
milled to a particle size of 0.8 mm using a Cyclotec™ 1093 mill
3.48% higher BMP values were obtained with 5 and 10% grass
(FOSS, MN 55344, USA). Fibre fractions, neutral detergent fibre
addition, respectively. The specific parameters fitted with the
(NDF), acid detergent fibre (ADF) and lignin (ADL) were ana-
modified Gompertz model are shown in Table 2. The coeffi-
lysed according to the Van Soest, Robertson, & Lewis (1991)
cient of determination (R2) ranged from 0.965 to 0.973, the
method. From these fractions, hemicellulose, cellulose and
maximum methane production rate (mm) from 12.73 to
lignin contents were calculated. The hemicellulose content
15.89 ml g1 [VS] day1 and the lag phase from 0.45 to 1.13 d.
was calculated as the difference between NDF and ADF, the
Statistical analysis showed that there was no significant dif-
cellulose content as the difference between ADF and ADL, and
ference (p < 0.05) in BMP between CM, MG and a mixture
the lignin content was assumed to be equal to ADL. TA, PA and
thereof.
IA were measured at day 21, 27 and 48 by titration with HCl
Based on the ultimate bio-methane potential (B0) and in
(0.1M), which consists of two end points during titration pro-
order to start up with a TS content that will not create physical
cess: the first to pH 5.75 is due to the existence of bicarbonate
problems in the digesters, a mixture of 95% CM and 5% MG
and is known as PA; the second to pH 4.3 corresponds to TA
was used in the semi-continuous experiment.
(Jantsch & Mattiasson, 2003). The IA, which is related to the
VFA concentration, is estimated from the difference between
3.3. Serial CSTRs co-digestion
TA and PA.

Process performance during the AD period is illustrated in

2.5. Calculations and statistics
Fig. 3. All mean values are based on data covering day 30e49
and are summarised in Table 3.
The average DMY and SMY, biogas composition and VS
reduction were calculated based on the data acquired after 30
days of batch digestion. The modified Gompertz model (Eq. (1)) Table 1 e Chemical composition.
was used to fit the measured methane yield (Lo et al., 2010):
Parameters CM MG Inoculum
TS (%) 9.21 ± 1.2 75.36 ± 0.21 4.48 ± 0.20

 
m e VS (%) 7.54 ± 0.71 68.39 ± 5.35 3.63 ± 0.30
B ¼ B0 exp  exp m ðl  tÞ þ 1 (1) Ash (%) 2.33 ± 0.50 3.17 ± 0.35 1.45 ± 0.03
B0
pH 7.29 ± 0.11 ND 8.24 ± 0.01
where B represents the cumulative methane yield (ml [CH4] Acetic(mg l1) 6368.77 ± 187.74 ND 215.27 ± 7.02
g1 [VS]) at time t, B0 is the maximum or ultimate methane Propionic(mg l1) 1511.48 ± 81.15 ND 25.06 ± 0.71
yield (ml [CH4] g1 [VS]), mm is the maximal methane produc- TVFAs(mg l1) 9019.42 ± 359.39 ND 251.55 ± 9.07
TAN(g l1) 1.94 ± 0.14 ND 1.33 ± 0.03
tion rate (ml [CH4] g1 [VS] d1), l is the lag phase time (d), t is
Cellulose(% TS) 23.99 34.95 ND
the incubation time (d) and e is equal to 2.72. The significance
Hemicellulose 16.57 32.57 ND
of the bio-methane yield was obtained using one-way ANOVA (% TS)
analysis with a significance level of 0.05. Microsoft PowerPoint Lignin(% TS) 15.51 6.18 ND
2010 (Microsoft, WA 01060, USA) and Origin 8.0 (Origin Lab, Protein (% TS) 5.75 17.69 ND
WA 01060, USA) were used for graphing and modelling, and TOC (%) 3.34 34.40 ND
JMP 13.0 (SAS Institute Inc, NC 27513, USA) was used for sta- TN (%) 0.92 2.83 ND

tistical analysis. ND: Not determined.

 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1
Fig. 2 e Experimental bio-methane yield from batch test.
3.3.1. Daily bio-methane yield (DMY)
As shown in Fig. 3a, the DMY increased rapidly from 5 L to
10e12 l [CH4] d1 (R1) and 8e9 l [CH4] d1 (R2) between day
1e10 as a result of adding grass. No stagnant period was
observed at the beginning. This may be because the inoculum
used in the CSTRs was taken from a full-scale reactor fed the
same substrates. From day 11e20, the DMY from R1 and R2 fell
significantly, congruent with a decrease in pH and methane
content. The methane content from R1 was only 33% at day 15,
which indicates a lower methanogenic activity as a result of a
higher OLR. The lowest DMY from R1 was 1 l at day 18 and
from R2 it was 2.9 l at day 19. After day 20, a recovery of bio-
methane production from digesters R1 and R2 was observed.
The DMY from R1 reached 10 l [CH4] d1 while it was lower
from R2 reaching a steady 5e6 l [CH4] d.
Compared with R1, the DMY from R1-2 was more stable
during the whole experiment. The DMY gradually increased
from 2 l to 4 l [CH4] day1 during the first 20 d and remained
stable in the range of 5e6 l [CH4] d1. The average specific
methane yield (SMY) from the serial CSTRs in co-digestion
was 132.52 ± 14.80 Nl kg1 [VS] (as shown in Table 3), which
was 24% higher than single CSTR co-digestion
(106.91 ± 11.05 Nl kg1 [VS]).
3.3.2. Process stability
The total and individual VFA profiles of each reactor during
AD are shown in Fig. 4. The VFA concentration from R1 and R2
increased from day 1 to day 20, with high acetic acid content
(50%). The total VFA concentration in R1 reached a maximum
Table 2 e Kinetic parameters for mono-/co-digestion of CM and MG.
Samples BMPa (ml [CH4] g1 [VS])
B0 (ml [CH4] g
CM 231.55 ± 8.06
95%CM þ 5% MG 239.6 ± 9.68
90%CM þ 10%MG 244.28 ± 4.66
MG 254.57 ± 12.72
a
Experimental BMP.
5
value of 15 g l1 after day 20, which gradually fell to 11 g l1 at
day 47. During the same period, the total VFA from R2 reached
10 g l1 at day 21 and gradually fell to 7.5 g l1. As a result of
VFA accumulation, pH dropped from 7.2 to 6.3 in R1 and to 6.8
in R2. From day 20e50, the pH value from R1 and R2 gradually
increased to 7.2, which due to the reduction of VFA concen-
tration. When fed effluent from R1, there was approximately
50% reduction in VFA in R1-2. Hill, Cobb, and Bolte (1987)
suggested that a propionic acid to acetic acid ratio higher
than 1.4 indicated impending digester failure. From day
30e49, the acetic acid contents for R1 and R2 fell by about 50%,
while the concentration of propionic acid gradually increased.
The propionic acid to acetic acid ratio for R1, R2 and R3 was in
the range 0.2e0.6 with an average ratio (day 30e49) of
0.36 ± 0.08, 0.82 ± 0.13, 0.55 ± 0.10 and 0.53 ± 0.02 for R1, R1-2,
R2 and R3, respectively.
TA, IA/PA and IA/TA ratios at day 27, 40 and 47 are shown
in Fig. 5. The average TA values for R1, R1-2, R2 and R3 were
11.07 ± 1.37, 10.44 ± 1.57, 13.10 ± 0.39 and 10.38 ± 1.57 g l1,
respectively. The IA/PA ratio for R1 and R2 gradually fell from 3
to 1.5 and from 1.5 to 1 from day 27e47, respectively. The IA/
PA ratio measured at day 47 (R1, R1-2 and R2) were ranged in
the suggested value for a stable process (Lopes Pereira,
Montenegro Campos, & Motteran, 2013; Shofie, Qiao, Li,
Takayanagi, & Li, 2015).
The TAN concentration (shown in Fig. 6) from all digesters
rose for the first 20 days and stabilised during the remaining
period. The average TAN values from R1, R1-2, R2 and R3 at
day 30 were 2.73, 2.49, 2.52 and 2.69 g l1, respectively.
In this study, the TS and VS of the effluent (Fig. 7) showed a
significant increase until day 40, which suggests that TS and
VS accumulated inside the digesters. This is partly due to the
higher input of TS in the hemi-solid state (Abbassi-Guendouz
et al., 2012), and also due to the lignocellulosic contents of CM
and MG which degrades slowly in the AD process.
The average effluent TS and VS values for the serial CSTR
co-digestion (shown in Table 3) were 8.12 ± 1.33% and
6.56 ± 1.15%, respectively, corresponding to a 35.03% TS
reduction and 38.02% VS reduction (15.36% TS reduction and
16.29% VS reduction for R1). For the single CSTR co-digestion,
the TS reduction was 27.53% while VS fell by 33.36%.
3.3.3. Serials CSTRs mono-digestion
From day 60e85, R1 and R1-2 were fed CM and the reactors'
performance was compared to the single reactor (R3). Without
MG addition, the integrated DMY (Fig. 3a, day 60e85) from R1
and R1-2 fell slightly from 12 to 7 l [CH4] d1. The VFA, TS and
VS contents gradually decreased for R1-2, while the VFA
Modified Gompertz model
1
[VS]) mm (ml g1 [VS] d1) l (d) R2
215.03 ± 8.61 13.02 ± 2.18 0.99 ± 1.22 0.976
219.90 ± 9.40 15.61 ± 3.38 1.09 ± 1.39 0.965
227.42 ± 8.40 15.89 ± 2.94 1.13 ± 1.20 0.974
240.96 ± 10.23 13.31 ± 2.47 0.45 ± 1.46 0.970
6 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1
Fig. 3 e Process data from CSTRs: (a) DMY, (b) pH value, (c) Methane content.
concentration in R1 remained constant at 10 g l1. As shown in
Table 3, the average DMY and SMY were 7.39 ± 0.62 l d1 and
102.96 ± 8.22 Nl kg-1 [VS], respectively (day 75e85). The
average SMY was 7.6% higher than the AD of CM with single
CSTR.
The propionic acid concentration for R1-2 rose from day
60e80, commensurate with the gradual acetic acid reduction.
The average propionic acid concentration and propionic acid
to acetic acid ratio were 3150.25 ± 163.47 mg l1 and 1.88 ± 0.26,
respectively, during this period. This ratio was higher than the
maximum value suggested by Hill et al. (1987) for avoiding
digester failure. However, the performance of R1-2 was
generally more stable than R1, which had a lower propionic
acid concentration and propionic acid to acetic acid ratio.
With the serial CSTRs configuration, the improvement in bio-
methane yield was lower without MG addition.
4. Discussion
The results present in this study demonstrated that the bio-
methane yield could be improved by serial CSTRs configura-
tion both operated under co-digestion and mono-digestion.
Compared with single CSTR, serial digestion under same
retention time has 24% higher SMY when fed with mixture of
CM and 5% (w/w) MG, accompanying with lower VFA con-
centration and higher TS/VS reduction. According to Boe and
Angelidaki (2009), the improved bio-methane yield was
mainly attributed to the fact that the 2nd reactor (R1-2) was
fed only with the effluent from 1st reactor, which contained
all important microbial groups and also VFA, nutrients and
residual organics. Kaparaju et al. (2009) suggested that the
main reactor (the first reactor) in a serial digestion system
must be sufficiently large to maintain a stable process with a
lower VFA level. In this experiment, however, higher VFA
concentration was found from 1st reactor of the serial CSTRs,
which were reduced to at the 2nd reactor. The result indicates
that the 1st reactor acts as both hydrolysis/acidogenic and
methanogenic reactor while the 2nd reactor mainly worked as
methanogenic reactor. Furthermore, with serial CSTRs
configuration, intermediate process inhibitors were partly
removed to the next reactor which maintains the process
more stable compared with single reactor. The improved bio-
methane yield was lower after changed to mono-digestion of
cattle manure compared with serial co-digestion (7% higher
than mono-digestion with single CSTR). Bio-methane pro-
duction from 2nd reactor accounted to 34% of the total pro-
duction when fed with CM while higher bio-methane recovery
was observed from 2nd reactor which constituted to 40% of
the total bio-methane yield when fed with CM and 5% (w/w)
MG.
Many studies have evaluated digester performance using
the VFA concentration. The increased concentration of pro-
pionic acid (or propionic acid to acetic acid ratio) could be a
good indicator for digester imbalance (Ahring, Sandberg, &
Angelidaki, 1995; Nielsen, Uellendahl, & Ahring, 2007). In this
study, the concentration of propionic acid from reactors R1,
R1-2 and R2 was increased from 1000 mg l1 to 2500 mg l1.
According to Hajarnis and Ranade (1994), an AD system with a
propionic acid concentration as high as 2750 mg L1 has a high
 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1 7

risk of lowering methane production when the pH falls below

Mono-digestiona

8932.45 ± 1000.06
6.5. This can explain the reduction in bio-methane yield from

1643.02 ± 213.90
R1 between day 10e20. However, a reduction in bio-methane

95.70 ± 8.87

50.32 ± 4.55
3.61 ± 0.33

7.23 ± 0.03

2.69 ± 0.17
yield was also observed for R2 when the propionic acid con-
centration was between 2000 and 2500 mg l1 with a pH value
around 7.0. In terms of TAN, according to Varel, Isaacson, and
e

e
0
Bryant (1977), the AD process could be inhibited by a TAN
concentration in excess of 1.7 g l1 in the thermophilic tem-
102.96 ± 8.22

perature regime with a high OLR of CM and pH value above 7.8.

7.39 ± 0.62

In another study, a severe inhibition was observed at a TAN

Sum

concentration of 4.0 g l1 (Angelidaki & Ahring, 1993).

From a mass flow perspective, using serially paired CSTRs
e

in place of a single CSTR is often advantageous, although this

Serial mono-digestionb

7122.04 ± 543.67 advantage becomes smaller with the addition of each suc-
1010.49 ± 83.18
2nd reactor

cessive CSTR and therefore a balance between performance

56.55 ± 1.12

and capital expenditure must be reached to determine the

2.48 ± 0.34

7.43 ± 0.02
9.16 ± 0.57

7.09 ± 0.41

2.63 ± 0.05

optimal number of reactors in a CSTR cascade. A single CSTR

reactor has a distinct residence time distribution (RTD) which
e

describes material flowing through the reactor. Assuming a

single feeding event per day, an operational schedule that
involves removal of a portion of the (partially) digested ma-
2343.77 ± 591.80
9792.95 ± 901.61
1st reactor

terial prior to a corresponding amount of substrate being

66.48 ± 4.27

50.09 ± 2.84
4.91 ± 0.32

7.24 ± 0.07
8.69 ± 0.60

7.04 ± 0.58

2.62 ± 0.10

added at each feeding event (allowing for losses to the gas

phase) and that the reactor is completely mixed, a portion of
7.59c

the previous days addition will be flushed out of the reactor

e

at the next feeding event, this portion being equal to 1/HRT.

Thus, a considerable amount of material is ejected from a
2423.08 ± 402.99
8443.18 ± 658.61
Co-digestiona

single CSTR in a much shorter time than the nominal HRT

106.91 ± 11.05

27.53 ± 5.48

33.36 ± 5.72
50.29 ± 1.57

and it follows that unless this material is rapidly degraded to

5.66 ± 0.58

9.06 ± 0.68

7.05 ± 0.61

2.52 ± 0.18
7.25 ± 0.1

biogas, some of the methane potential will be lost. The CSTR

11.71d

design also means that some material is still left in the

e

reactor long after the nominal HRT has been reached. How-
ever, as the organic matter is being converted to gas over
time it can be assumed that any material left in the reactor a
132.52 ± 14.8
14.02 ± 1.57

long time after addition will be non-digestible and of little

Table 3 e Parameters from one-/serial co-/mono-digestion AD process.

Sum

interest in terms of biogas production. What this means is

that the nominal HRT is simply an average and tells us
nothing of the distribution of material ejected from the
Calculated as an average value based on the digestion from day 70e85.
Calculated as an average value based on the digestion from day 30e49.
7746.35 ± 226.65
891.89 ± 105.27

reactor over time, and that loss of material soon after addi-
Serial co-digestiona

35.03 ± 10.61

38.02 ± 10.87
2nd reactor

57.38 ± 1.56

tion is not fully compensated for by material which is

5.62 ± 0.49

7.28 ± 0.02
8.12 ± 1.33

6.56 ± 1.15

2.49 ± 0.25

retained for much longer periods. By splitting the single CSTR

into two CSTRs, the RTD is altered in a way that affects the
e

distribution of material over time but without changing the

actual average HRT.
Comparison of co-digestion with mono-digestion.

The tanks-in-series model (Levenspiel, 1999, pp. 257e366)

13482.66 ± 1573.97

Comparison of seiral CSTRs with single CSTR.

can be used to examine mass flow through serial CSTRs,

2238.84 ± 500.51
1st reactor

assuming each reactor demonstrates mixed flow (i.e. the

89.82 ± 19.47

10.58 ± 0.83
15.36 ± 6.66

16.29 ± 7.11
47.19 ± 2.01
8.40 ± 1.82

6.96 ± 0,19

8.86 ± 0.75

2.73 ± 0.16

reactor contents are completely homogenized). For a single

CSTR, the addition of a material (A) can be used to calculate
28.71d
23.96c

the concentration of A at the CSTR outlet over time (Eq. (2)):

Zt
1 t= t
CA1 ¼ e t e =t CAi dt (2)
H2S concentration (ppm)

t1
0
Methane content (%)
SMY (Nl kg1 [VS])

Improvement (%)d
Improvement (%)c

where CAi is the concentration of component A at the inlet, CA1

Effluent VS (%)
Effluent TS (%)

is the concentration of component A at the outlet and t is the

Reduction (%)

Reduction (%)

VFAs (mg l1)

DMY (Nl d1)
Parameters

TAN (g l1)

HRT.
For two serially connected CSTRs (tank 1 preceding tank 2)
the concentration of A at the second CSTR outlet (CA2) can be
pH

d
b
a

calculated (Eq. (3)):

8 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1

Fig. 4 e Total and individual VFA concentrations.

Fig. 5 e TA, IA/PA and IA/TA ratios (Day 27e48).

 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1 9

CSTR are also not accounted for in mass flow models. There-
fore, in terms of chemical environment, there is potentially a
negative effect in the first stage and potentially a positive ef-
fect in the second stage.
Estimating CSTR performance from batch test data is not
straightforward. The batch assay is a highly dynamic pro-
cess, starting with a single load much greater than that
experienced during a typical CSTR feeding event. This can
lead to inhibitory levels of VFA forming during the early
days of batch digestion and even a severe reduction in pH
(unpublished data). The gas/methane production curves
therefore take an approximate first order or sigmoidal form
(the latter when a lag phase is evident at the start of the
assay). Frequent (and sensible) feeding of a CSTR will nor-
mally allow steady state to be reached if input mass and
composition remain constant, with a much more stable
Fig. 6 e TAN concentration. chemical environment that is conducive to biogas produc-
tion. These differences in environment between batch and
continuous digestion processes mean that, for example, the
mm derived from batch tests cannot be used to estimate this
parameter in a CSTR, and due to the unavoidable loss of
material from the CSTR design as described above (espe-
cially a single CSTR), the continuous process should achieve
a SMY somewhat lower than the B0 found in a batch assay.
The problems of transferring batch assay rate parameters to
a continuous process has been demonstrated by Batstone,
Tait, and Starrenburg (2009), who attributed this to high
initial loading and dynamic conditions within batch assays
and by Moset, Al-zohairi, and Møller (2015) who stated that
the reaction rate is dependent on inoculum to substrate
ratio, inoculum activity and acclimation, all of which differ
greatly between batch and CSTR digestions. Batch assay
data is best used to compare reaction rates between sub-
strates relative to each other, and also when these are
digested in the same batch, but not to determine absolute
rate values.
Fig. 7 e Effluent TS and VS concentrations.

5. Conclusion
2 3
Zt Zt Co-digestion of CM and MG with serial CSTRs set up increased
1 t t 6 1 t t 7
CA2 ¼ e =t2 e =t2 4 e =t1 e =t1 CAi dt5dt (3) the methane yield by 24% compared with single CSTR. The
t2 t1
0 0 methane content, TS and VS solids reduction were also higher
in serial digestion. Compared with single mono-digestion
The models show that at 20 days (i.e. the nominal HRT
system, the effect of serial CSTRs configuration was more
chosen in this work) after A was added, a serial CSTR config-
pronounced for the co-digestion system. Application of serial
uration of 10 þ 10 days HRT will retain 10.18% more of A when
CSTRs has advantages since the extra costs on arrangement
compared to a single CSTR of 20 days HRT. However, this is
and operation are lower, especially for the existing biogas
assuming A is not reactive and passes through the system
plant with multiple reactors.
unchanged and unhindered. In an AD process, a portion of the
organic input material will be degraded and lost to the gas
phase; this portion becomes larger over time, as determined
by degradability coefficients and reaction rates. Also, the se- Acknowledges
rial CSTR configuration described here did not have similar
chemical environments in each of the sequential CSTRs, with The authors wish to thank the buildings manager, Mogens
high VFA concentration and low pH in R1 causing a degree of Møller Hansen and lab-technician, Britt Amby Malthesen at
inhibition (at least in the co-digestion experiment) which Biogas Plant, Foulum, Aarhus University for assistance in this
reduced the methane concentration. However, the advan- work. This work was financially supported by Innovation fund
tages outlined by Boe and Angelidaki (2009) regarding the Denmark (1377-00040A, Nomigas project) and DCA-Danish
supply of VFA and nutrients from the first CSTR to the second Centre for Food and Agriculture (1305-00018B).
10 b i o s y s t e m s e n g i n e e r i n g 1 6 0 ( 2 0 1 7 ) 1 e1 1

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