Waste Management 62 (2017) 76–83

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

An alternative parameter to characterize biogas materials: Available

carbon-nitrogen ratio
Ming Wang a,c,⇑, Wenzhe Li b,⇑, Pengfei Li b, Shuiping Yan a,c, Yanlin Zhang a,c
a
Department of Agriculture Biological Environment and Energy Engineering, College of Engineering, Huazhong Agriculture University, No. 1 Shizishan Street, Hongshan
District, Wuhan 430070, PR China
b
Department of Agriculture Biological Environment and Energy Engineering, School of Engineering, Northeast Agriculture University, No. 59 Mucai Street, Xiangfang District,
Harbin 150030, PR China
c
Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Available carbon-nitrogen ratio (AC/N) was proposed as an alternative parameter for evaluating the
Received 14 November 2016 potential of biogas materials in this paper. In the calculation of AC/N ratio, only the carbon that could
Revised 20 February 2017 be effectively utilized in anaerobic digestion (AD) process is included. Compared with total C/N, AC/N
Accepted 20 February 2017
is particularly more suitable for the characterization of biogas materials rich in recalcitrant components.
Available online 1 March 2017
Nine common biogas materials were selected and a series of semi-continuous tests for up to 110 days
were carried out to investigate the source of available carbon and the relationship between AC/N and
Keywords:
the stability of AD process. The results showed that only the carbon existing in proteins, sugars, fat
Biogas production
Available carbon-nitrogen ratio
and hemicelluose should be considered as available carbon for anaerobic microbes. Besides, the optimal
Semi-continuous anaerobic digestion AC/N for semi-continuous AD process was preliminarily determined to be 11–15. Taken together, our
Feedstock characteristic results demonstrate that AC/N is more effective than total C/N in the evaluation of the potential perfor-
mance of AD process.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction important and advantageous methods in both waste treatment

Organic wastes should be treated timely and properly; other-
wise they will cause serious threats to the environment, such as
release of odor, reproduction of various pests, spreading of animal
pathogens and pollution of surface and ground water. Compared
with other treatment methods of organic wastes, anaerobic diges-
tion (AD) can not only effectively decrease and remove large
amounts of organic matters from the wastes, but also produce bio-
gas mainly consisting of methane (
60%) and carbon dioxide
(
40%), which can be utilized directly or converted to heat and
electricity for local energy needs (Westerman and Bicudo, 2005).
Most of organic wastes, such as crop straws, livestock manure,
municipal solid wastes and the by-products of food industry, can
be used as biogas materials (Khalid et al., 2011; Drosg et al.,
2013). Therefore, AD has been considered as one of the most
⇑ Corresponding authors at: Department of Agriculture Biological Environment
and Energy Engineering, School of Engineering, Northeast Agriculture University,
No. 59 Mucai Street, Xiangfang District, Harbin 150030, PR China (W. Li);
Department of Agriculture Biological Environment and Energy Engineering, College
of Engineering, Huazhong Agriculture University, No. 1 Shizishan Street, Hongshan
District, Wuhan 430070, PR China (M. Wang).
E-mail addresses: mwang211@163.com (M. Wang), liwenzhe9@163.com (W. Li).
and energy production.
In the past two decades, many studies have been focused on the
influence of substrate characteristics on the performance of AD
process (Drosg et al., 2013; Kafle and Sang, 2013a; Kougias et al.,
2013). Generally, total organic carbon, total Kjeldahl nitrogen and
the ratio of them (C/N) are used as basic parameters to characterize
biogas materials. In particular, the C/N of the substrate is regarded
as an effective and important parameter to evaluate AD process. It
is generally accepted that the most suitable C/N ratio for AD pro-
cess is 20–30, while too high or too low C/N ratio probably causes
acidification or ammonia inhibition in AD process, which would
result in the failure of biogas production (Molinuevo-Salces et al.,
2012; Wang et al., 2012; Sievers and Brune, 1978; Chandra et al.,
2012; Li et al., 2012). Moreover, C/N can also be used to design
the anaerobic co-digestion of two or more than two kinds of biogas
materials with a higher or lower C/N ratio than the recommended
range of 20–30 (Mata-Alvarez et al., 2014; Kafle and Sang, 2013b).
Wang et al. (2012) investigated the influence of C/N on the biogas
production in the co-digestion of dairy manure, swine manure and
wheat straw. They showed that the most stable C/N ratio for the
co-digestion is from 25 to 30, and when the C/N is 27, the biogas
yield is the maximum. In another study, the highest biogas produc-

http://dx.doi.org/10.1016/j.wasman.2017.02.025
0956-053X/Ó 2017 Elsevier Ltd. All rights reserved.
 M. Wang et al. / Waste Management 62 (2017) 76–83 77

tion was observed when the C/N of the mixture liquor was 20 in
the co-digestion of food waste and rice husk (Haider et al., 2015).
Therefore, C/N has been considered as an important parameter to
evaluate AD substrates in biogas production and practical
engineering.
However, in some reports, the obtained best C/N ratios for bio-
gas production were not within the recommended optimal C/N
range (20–30). Marañón et al. (2012) reported that the optimal
C/N is 16–18 in the semi-continuous co-digestion of cattle manure,
food waste and sludge. Romano and Zhang (2008) suggested that
the C/N ratio should be maintained at 15 for the co-digestion of
juice and digested sludge; Li et al. (2012) found that the corn straw
inoculated with digested sludge could be well digested for biogas
production with a C/N of 15–18, but the digestion failed when
the C/N exceeded 21. It can be seen from these studies that the
optimal C/N varies for different types of substrates to be digested,
which may be due to the different nutrient compositions of the
substrates. Because most of biogas materials are organic wastes,
they are usually rich in recalcitrant components, such as lingo-
cellulose (semi-cellulose, cellulose and lignin), which have been
proven to be hard to be utilized by anaerobic organisms in a short
retention time without any special pretreatments (Kabir et al.,
2016). Hence, a large amount of ‘‘invalid carbon” existing in these
materials is also included in the calculation of C/N. Thus, to be
more exact, this C/N ratio should be called as total C/N, which
can only present the general characteristics of the biogas material,
but cannot accurately indicate the properties of the substrate that
can be actually utilized by anaerobic organisms. Therefore, the
optimal C/N ratios obtained from different AD tests using various
biogas materials will probably be very different.
Based on the above analyses, it can be summarized that the
optimal C/N ratio for AD process may be determined by the types
of biogas materials and is not possibly unified at a specific range.
As a result, the indicative significance of C/N is rather limited. In
order to overcome this disadvantage, an alternative parameter,
namely available carbon/nitrogen ratio (AC/N), was proposed in
this study for the characterization of AD feedstock. The calculation
of AC/N only includes the carbon existing in the easily degradable
part of the feedstock, and excludes the carbon which could not be
effectively utilized by AD microbes. Based on this principle, nine
common biogas materials were selected and a series of semi-
continuous experiments lasting for 110 days were carried out, aim-
ing to investigate the correlation between their nutrient composi-
tions and methane yields, and thus to infer which carbon should be
considered as available carbon and the potential relationship
between AC/N and the stability of the AD process. The pH, volatile
fat acids, total alkalinity and ammonia nitrogen were used as the
main parameters to evaluate the stability of these digesters. The
results of this study provide a more effective parameter to charac-
terize biogas materials, which might be significant for the better
design and control of AD process in practical production.
2. Materials and methods
2.1. Substrate and materials
Nine biogas materials were used in this study, including three
livestock manures (diary manure, swine manure and chicken man-
ure), two agricultural wastes (corn straw and soybean straw), and
four other materials (food waste, cassava residue, Chinese herb-
extraction residue and corn flour). The anaerobic sludge used as
inoculum was collected from an anaerobic digester (working vol-
ume: 500 L) for cattle manure at 35 °C (pH: 7.52 ± 0.07; hydraulic
retention time: 20 days; total solid: 62.5 ± 1.3 g L1). Based on total
solid (TS), the volatile solid (VS), total Kjeldahl Nitrogen (TOC),
total organic carbon (TOC) and C/N ratio of the inocula were 81%,
2.08%, 34.83% and 16.7, respectively. The biogas materials with a
moisture content of more than 15% and the inocula were stored
at 4 °C before use, and others were stored at a ventilated and dry
place. The preparation and characterization of all the samples were
shown in Table 1 and Table 2, respectively. In order to prevent the
potential inhibition on methanogens by the ammonia and sulfur
compounds from the degradation of substrate, all the materials
were diluted to a low solid concentration before being used as
feedstock. Specifically, the chicken manure was diluted to below
3% due to its high ammonia production capacity. The total solid
content and pH value of each feedstock are also presented in
Table 2. All samples were collected in triplicate, and averaged data
of the measurements were presented.
2.2. Experimental design and procedure
2.2.1. Semi-continuous anaerobic digestion
As shown in Table 2, there were nine treatments (T1–T9) corre-
sponding to the nine biogas materials investigated in the present
work. Each treatment was performed in two replicates. Thus, eigh-
teen Erlenmeyer flasks were used as the AD digesters in the semi-
continuous experiments. The total volume and working volume
were 2 L and 1.5 L, respectively. These bottles were closed with
suitable rubber plugs, and some holes were dispersedly drilled in
the center of the plug for feeding influent, withdrawing effluent
and collecting biogas. A peristaltic pump was employed to pass
the liquid in and out of the digester. The generated biogas was col-
lected daily by aluminium gas pack (Dalian Hede Technologies Ltd.
China) and the volume was measured based on the downward dis-
placement of water. All the digesters were evenly placed in two
constant-temperature shakers (35 ± 1 °C, 120 rpm), and a time
switch within the shaker was able to control the shaking at a spec-
ified time (running 10 min in an hour).
All digesters were initially loaded with 1.5 L of inocula, and the
start-up of each digester was operated at a lower organic loading to
ensure that the AD process started successfully and the inocula
could better fit the new substrate. Then, the organic loading was

Table 1
Collection and processing of the nine biogas materials.

Name (ab.) Site of collection Processing mode Storage

DM Experimental farm of NEAU Sieving out particles (U > 0.5 cm) <20 °C
SM Cheng Gaozi Town, Harbin
CM Experimental farm of NEAU
FW Canteen of NEAU Picking out the wood sticks, lunch boxes, bones, etc., <20 °C
and then smashing to homogenity
CR Longan County Yinxin Starch Co., Ltd. – <20 °C
CHER First Affiliated Hospital, HUCM Drying and smashing to 50–80 mesh Ambient temperature and dry place
CS Hulan District, Harbin
SS Hulan District, Harbin
CF Xiangfang District, Harbin

NEAU: Northeast Agricultural University; HUCM: Heilongjiang University of Chinese Medicine; DM: diary manure; SM: swine manure; CM: chicken manure; FW: food waste;
CR: cassava residue; CHER: Chinese herb-extraction residue; CS: corn straw; SS: soybean straw; CF: corn flour.
78 M. Wang et al. / Waste Management 62 (2017) 76–83

Table 2
Characteristics of the nine biogas materials and feedstock.

Parameters DM SM CM FW CR CHER CS SS CF
No. T1 T2 T3 T4 T5 T6 T7 T8 T9
pH 7.38 7.57 7.85 6.98 7.13 7.26 – – –
TS (%) 22.20 31.40 11.50 21.80 88.10 45.50 87.40 88.30 87.20
VS (%)a 83.32 79.85 75.40 94.62 97.54 85.92 94.77 93.55 98.00
TOC (%)a 39.58 38.97 36.04 47.31 42.43 39.70 44.45 40.41 44.00
TKN (%)a 1.58 2.88 3.21 2.05 0.68 2.06 1.04 0.93 1.44
C/N 25.05 13.53 11.23 23.08 62.40 19.27 42.74 43.46 30.56
AC/N 15.49 10.51 8.53 21.69 48.42 14.56 31.13 17.53 30.31
C/N reduction (%)b 38.16 22.32 24.04 6.02 22.40 24.44 27.16 59.66 0.82
Feedstock TS (%) 6.56 6.38 2.39 4.48 5.36 6.07 6.07 6.07 6.07
pH 7.32 7.45 7.53 7.02 7.10 7.42 7.21 7.18 7.23
OLR
(HRT: 50 d) 1.01 1.02 0.36 0.85 1.05 1.05 1.15 1.14 1.19
OLR
(HRT: 35 d) 1.56 1.46 0.51 1.21 1.49 1.49 1.64 1.62 1.70
OLR
(HRT: 25 d) 2.19 2.04 0.72 1.70 2.09 2.09 2.30 2.27 2.38

TS: total solid content; VS: volatile solid content; DM: diary manure; SM: swine manure; CM: chicken manure; FW: food waste; CR: cassava residue; CHER: Chinese herb-
extraction residue; CS: corn straw; SS: soybean straw; CF: corn flour; OLR : organic loading rate, and the unit is ‘‘g VS L1 d1”; HRT: hydraulic retention time.
a
Data based on the dry matter.
b
The value is calculated by the formula of (C/N  AC/N)/(C/N) * 100%.

gradually increased in the precondition of ensuring the real-time
stability of each digester. Therefore, the whole test period could
be divided into several phases according to the changes in organic
loading, and the digester was observed for at least 30 days in each
phase to ensure enough time for the digester to buffer its contents.
The TS content of each feedstock was constant through the whole
test period. Thus, the increase of organic loading could be achieved
by decreasing the hydraulic retention time. The digester number,
feedstock TS, pH and organic loading rate of each digester are
shown in Table 2. The biogas volume, methane content, pH, TS
and VS of the effluent liquid were monitored every two days to test
the stability of performance.
2.2.2. Material exchange rate
Because the initially loaded inocula of each digester were
obtained from the AD liquor of dairy manure, the components of
the inocula might have an influence on the AD performance of
the new feedstock. Therefore, it would be more accurate to evalu-
ate the AD performance if all the components in the digester were
from the studied feedstock. When the solid content of the feed-
stock was constant, the material exchange rate (MER) was mainly
determined by the hydraulic retention time and operating time.
Thus, the relationship among them could be expressed as Eq. (1):
i.d. carbon molecular sieve TDX-01: 1.5–2.0 nm) and a thermal
conductivity detector using argon as the carrier gas. Volatile fat
acids (acetic acid, propionic acid, butyric and i-butyric, valeric)
were determined by the same GC-6890N equipped with a flame
ionization detector and a capillary column (30 m  0.25 mm, Agi-
lent 1909/N-133) using nitrogen as carrier gas. The total solid
(TS), volatile solid (VS), pH (Sartorius basic pH meter PB-10, Ger-
many), total organic carbon (TOC), and total Kjeldahl nitrogen
(TKN) were determined according to standard methods (APHA,
2004). The total alkalinity (TA) was measured by titration to pH
4.5 with 0.05 M H2SO4 (Callaghan et al., 2002). Total sugar was
tested with a Fehling reagent (Lane and Eynon, 1923), total fat
was measured as the weight of the dried ethyl ether extract
obtained by prolonged extraction at 45 °C for 12 h using a Soxhlet
apparatus (Luque-Garcı´A and Castro, 2004), and total protein was
calculated as TKN  6.25 (Giuseppe et al., 2013). The contents of
hemicellulose, cellulose and lignin were determined with an auto-
matic fibre analyzer (ANKOM A2000i, US) according to the method
previously reported (Soest et al., 1991). All reagents used were of
analytical grade. All the measurements were conducted in tripli-
cate, and the averaged data are presented.

MER ð%Þ ¼ ½1  ð1  1=HRTÞt   100% ð1Þ 3. Results and discussion

where HRT represents the real-time hydraulic retention time of
semi-continuous AD, and t is the operating time (day).
2.2.3. Calculation of AC/N
The nutrient compositions of the nine samples were first ana-
lyzed as the contents of total protein, total fat, total sugar, hemicel-
lulose, cellulose and lignin. Then, the correlation between nutrient
compositions and the methane yields obtained from the semi-
continuous experiments was determined using the method of
Pearson correlation. Finally, through combining the results of cor-
relation analysis and previous related reports, it could be decided
which carbon should be included in the calculation of available
carbon.
2.3. Experimental parameters and analytical methods
The methane and carbon dioxide concentrations in the biogas
were determined with a gas chromatograph (GC-6890N, Agilent
Inc., USA) equipped with a stainless steel column (1.5 m  3 mm
3.1. Material exchange rate
The semi-continuous AD experiments were performed for up to
110 days. In the precondition of ensuring the stability of real-time
AD process, the hydraulic retention time of each digester was
decreased step by step from 50 d to 35 d, and then to 25 d, with
practical duration time periods of 30 days, 30 days and 50 days,
respectively. Therefore, according to Eq. (1), the theoretical mate-
rial exchange rate (MER) of each digester on the 100th day can
be calculated as follows:
MER ð100th day;%Þ ¼ ½1  ð1  1=50Þ30  ð1  1=35Þ30
 ð1  1=25Þ40   100%
¼ 95:14 > 95%
Thus, it is more reasonable to use the experimental data col-
lected from the 100th to 110th day for characterizing the digestion
liquor from each biogas material.
 M. Wang et al. / Waste Management 62 (2017) 76–83 79

3.2. Correlation analysis between nutrient composition and methane
yield
Nutrient composition refers to the species and contents of
organic compounds in the biogas material, mainly including total
protein, total fat, total sugar, hemicellulose, cellulose and lignin.
The correlation of nutrient composition with methane yield can
indicate which carbon can be effectively converted by anaerobic
microbes. The nutrient compositions of the nine samples were
measured in triplicate and the average data were presented in
Table 3.
As shown in Table 3, diary manure, Chinese herb-extraction
residue, corn straw and corn straw are rich in lignocellulosic com-
ponents; and food waste, cassava residue and corn flour are rich in
readily degradable carbohydrate compounds as indicated by their
total sugar contents; the protein compounds are significantly
higher in swine manure and chicken manure than in other materi-
als; and food waste is particularly richer of fat than other materials,
which is mainly attributed to Chinese dietary habits. The correla-
tion analyses of nutrient compositions with methane yields were
conducted using the Pearson correlation coefficient (Table 4).
As shown in Table 4, only total fat has a significant correlation
with methane yield (P < 0.05), which is mainly attributed to the
high methane conversion rate of fat and the large variation of total
fat content (1.17–33.51%) among the nine biogas materials
(Table 3). Table 4 also shows that all the three lignocellulosic com-
ponents have a significant negative correlation with methane yield
(P < 0.05), suggesting that lignocellulosic components have very
little contribution to methane production in semi-continuous AD
process. However, the results obtained in this study are not in com-
plete accordance with the previous reports. A model to predict the
potential methane yield of lignocellulosic feedstock was developed
by Monlau et al. (2012). According to the model, sugar and protein
contents are positively correlated with methane yield, and lignin
and crystalline cellulose contents are negatively correlated with
it, all of which are similar to our present results; but hemicellulose
and amorphous cellulose have a positive influence on methane
yield, which is different from our findings in this paper. Another
previous study investigated the correlation between the nutrient
compositions of maize at different mature stages (milk, wax and
full) and their biochemical methane potential. The results also
indicated that proteins, fats, hemicellulose and cellulose are posi-
tively correlated with methane yield; however, the contribution
of hemicellulose and cellulose to methane yield is very little and
far less than that of proteins and fats (Amon et al., 2007). The dif-
ferent results obtained in these similar studies can be attributed to
the differences in samples, AD mode (batch or continuous),
hydraulic retention time and the homogeneous degree of the
feedstock.
According to the results obtained in this study and the previous
literatures, it could be concluded that the proteins, sugars (soluble
sugars and starch) and fats in the biogas materials can be effec-
tively converted by AD microbes, and hemicelluose and a part of
amorphous cellulose can be utilized, while most of cellulose and
lignin are not degradable in AD process with a shorter hydraulic
retention time (Zheng et al., 2014).
3.3. Available carbon-nitrogen ratio
According to the above discussion, the controversy only lies in
whether hemicellulose should be considered as the available sub-
strate of AD. Hemicellulose has been demonstrated to comprise
more amorphous, random and branched heterogenic polysaccha-
rides of various pentoses (xylose and arabinose), hexoses (glucose,
galactose, mannose, and/or rhamnose), and acids (glucuronic acid,
methyl glucuronic acid, and galacturonic acid). The amorphous and
branched properties make hemicellulose more degradable by bio-
logical, thermal and chemical methods than cellulose and lignin
(Ademark et al., 1998; Zhao et al., 2012). Thus, in order to expand
the practical scope of AC/N, although the content of hemicellulose
showed a negative correlation with methane yield in this assay, the
carbon in hemicellulose was still calculated as available carbon
together with that in proteins, sugars and fats. On the other hand,
it could be recognized that the carbon in cellulose and lignin can
not be utilized by anaerobic organisms in AD process with a
shorter hydraulic retention time. Thus, the AC/N can be calculated
as Eq. (2).
AC=N ¼ ðTOC  ADF  TOCadf Þ=TKN ð2Þ

Table 3
Nutrient compositions and methane yields of all the samples.

Substrate Total proteina Total fata Total sugara HCELa CELa LIGa Methane yieldb (mL g1 VS) CH4 (%)b
DM 9.00 5.61 20.06 23.93 20.98 10.80 91.7 55.45
SM 16.50 8.06 23.33 22.80 13.83 4.01 248.9 63.26
CM 17.06 8.41 22.14 21.88 10.45 7.67 269.9 49.35
FW 11.31 33.51 42.27 2.04 3.22 2.48 531.8 63.17
CR 3.06 2.92 64.23 10.40 17.17 4.68 252.4 54.55
CHER 11.81 9.16 25.99 28.75 12.03 8.97 155.1 51.12
CS 5.88 3.57 40.15 25.74 23.40 2.33 189.7 52.09
SS 5.13 1.17 22.46 18.25 39.96 15.85 73.4 49.95
CF 8.13 2.67 88.00 1.20 0.78 0.02 365.5 58.23

HCEL: hemicellulose; CEL: cellulose; LIG: Lignin; DM: diary manure; SM: swine manure; CM: chicken manure; FW: food waste; CR: cassava residue; CHER: Chinese herb-
extraction residue; CS: corn straw; SS: corn straw; CF: corn flour.
a
All the data were based on the dry matter (%).
b
Data were the mean values obtained from the 100th to 110th day.

Table 4
Correlation analysis of nutrient composition and methane yield.

Total protein Total fat Total sugar HCEL CEL LIG

r 0.243 0.743 0.503 0.766 0.792 0.740
P-value 0.528 0.021* 0.167 0.016* 0.011* 0.023*
*
Significant correlation at 0.05 level; r: Pearson correlation coefficient; HCEL: hemicellulose; CEL: cellulose; LIG: Lignin.
80 M. Wang et al. / Waste Management 62 (2017) 76–83

where TOC is the total organic carbon content in the material while Table 5 clearly demonstrates that a higher AC/N represents a
TOCadf (%) only represents the carbon content in the acid detergent higher acidification risk, which is consistent with the previous con-
fibre; ADF (%) represents the acid detergent fibre content, namely clusions about the effect of total C/N on AD process (Romano and
the sum of cellulose and lignin in the material; and TKN is the Zhang, 2008; Li et al., 2012).
abbreviation of total Kjeldahl nitrogen. All the parameters of Eq.
(2) were measured based on the dry matter of the samples. Accord- 3.5. Volatile fat acids and total alkalinity
ing to Eq. (2), the AC/N ratios of the nine materials were calculated
and shown in Table 2. The AC/N ratios of all the materials were Generally, variation of pH will occur only when the balance of
within the range of 8–50, and were found to be lower than their cor- AD process is broken, which mainly refers to the accumulation of
responding C/N ratios to some extent. For dairy manure, cassava volatile fatty acids (VFA) and the decline of total alkalinity (TA).
residue and soybean straw, the AC/N ratio was greatly reduced by However, the changes of them are not independent of each other.
38.16%, 27.16% and 59.66%, respectively, as compared with the C/ TA mainly depends on the VFA concentration for the same feed-
N ratio, which is mainly due to the abundant lignocellulosic compo- stock. Thus, the two parameters are usually monitored simultane-
nents in the three materials (Table 3). Slight differences between ously in order to accurately determine the digester stability
AC/N and C/N were only found in food waste and corn flour (Angelikadi, 1997). Specially, the ratio of VFA and TA (VFA/TA)
(Table 2), which is due to their higher contents of total sugar and can be used to measure AD process stability. When this ratio is
total fat (Table 3). lower than 0.3–0.4, the AD process is usually considered as stable
without acidification risk (Callaghan et al., 2002; Fannin et al.,
1987). Therefore, monitoring the real-time VFA/TA ratio can help
3.4. pH changes of digesters to determine whether the organic loading should be increased or
decreased.
The pH range for a AD process has been reported to be 6.5–8.2, Similar to the division according to pH in Table 5, the nine
and the optimal range is 7.0–7.5. The metabolism of methanogenic digesters were also divided into three groups according to their
bacteria would be greatly inhibited if the pH is too high or too low mean VFA/TA ratios from the 100th to 110th day (Table 6).
(Lee et al., 2009; Peng et al., 2009). The pH of a continuous AD pro- As shown in Table 6, the VFA/TA ratios of security group were
cess is mainly influenced by the organic loading and nutrient com- below 0.3, suggesting that organic loading could be further
position of the substrate. The initial pH values of all digesters were increased in digesters T1, T2 and T3. The low-risk group only con-
7.52 since the inocula added into each digester were completely sists of T4 and T8 with a VFA/TA range of 0.3–0.4, suggesting that
identical. The pH variation trends of all the digesters are illustrated organic loading for this group should be maintained or increased
in Fig. 1. step-by-step and modestly. The digesters with VFA/TA ratios
Three different trends can be observed in Fig. 1. The pH curve of
T1 was the most stable and slightly fluctuated around 7.50. This
Table 5
phenomenon could be attributed to the fact that the substrate of Ranks of potential acidification risk based on the pH at the end of experiments.
T1 was dairy manure, which was also the source of the added inoc-
Potential risk Security Low-risk High-risk
ula. The pH curves of T2 and T3 showed a gradually rising trend
until the 90th day, and then tended to stay stable at 7.7 and 7.9, pH 7.8–7.5 7.5–7.0 6.5–7.0
Treatments T1, T2, T3 T4, T6, T7, T8, T9 T5
respectively. The pH of all other six digesters showed a gradual
Operation Further increasing Maintaining the Decreasing
decrease, but it was still above 7.0 until the end of the experiment adjustment the OLR status quo the OLR
except for T5. According to the pH value of each digester at the end AC/N 8–14 15–32 40–50
of the experiment, the nine digesters were divided into three ranks
OLR: organic loading rate.
in terms of the potential acidification risk (Table 5).

0 10 20 30 40 50 60 70 80 90 100 110
8.00 8.00

HRT=50 days HRT=35 days HRT=25 days

7.75 7.75

7.52
7.50 7.50
pH

pH

7.25 7.25
T1(DM)
T2(SM)
T3(CM)
T4(FW)
7.00 T5(CR) 7.00
T6(CHER)
T7(CS)
T8(SS)
T9(CF)
6.75 6.75
0 10 20 30 40 50 60 70 80 90 100 110

Operation day (d)

Fig. 1. The change curves of pH during the whole experimental period.

 M. Wang et al. / Waste Management 62 (2017) 76–83 81

Table 6
Ranks of potential acidification risk based on the VFA/TA ratios.

Potential risk Security Low-risk High-risk

1
Total VFA (mg L ) 500–1000 1000–1500 1000–1500
TA (mg CaCO3 L1) 5000–9000 3500–5000 2000–3500
VFA/TA <0.3 0.3–0.4 >0.4
Treatments T1, T2, T3 T4, T8 T5, T6, T7, T9
Operation adjustment Further increasing the OLR Maintaining the status quo Decreasing the OLR
AC/N 8–14 22, 15 15–48

OLR: organic loading rate; VFA: volatile fatty acids; TA: total ammonia.

exceeding 0.4 were classified into high-risk group, and thus organic
loading should be reduced for these digesters.
Additionally, it can be observed from Table 6 that the VFA/TA
ratio of the digestion liquor was positively correlated with the
AC/N of the feedstock. This result is mainly due to that a lower
AC/N usually represents a lower sugar and a higher protein content
in the sample, which results in less VFA and more ammonia pro-
duction after the AD process, and ammonia is obviously able to
improve the TA value of the digestion liquor (Jindřich et al., 2012).
3.6. Ammonia nitrogen concentration
Numerous studies have shown that an excessive ammonia
nitrogen (AN, NH+4-N) concentration can inhibit the methane pro-
duction of methanogens. A previous study reported that the inhibi-
tion would start when the AN concentration exceeds 1500 mg L1,
and it would lead to a toxic effect on the methanogens when the
AN concentration reaches 3000 mg L1 (Calli et al., 2005).
As shown in Fig. 2, the initial AN concentrations of all digesters
were 800 mg L1. Except for the AN curves of T2 and T3, all other
AN curves showed a slight declining tendency after the test started,
and then tended to be stable from the 70th day. The lowest AN
concentration below 100 mg L1 was observed in T5 at the end of
the test, which might be due to the fact that there was only
0.67% of TKN in cassava residue (Table 2). T2 and T3 showed an
opposite trend compared with other digesters, and the highest
AN concentration was observed in T3, although the feedstock TS
and organic loading in T3 were only half of those in other digesters,
which is due to the high TKN in chicken manure as shown in
Table 2. Using the AN content of effluent (mg L1) and the TS of
feedstock (g L1) of each digester, the potential AN production rate
(ANPR, mg g1 TS) could be calculated according to Eq. (3) and the
results are shown in Table 7.
ANPR ðmg g1 TSÞ ¼ AN ðmg L1 Þ=Feedstock TS ðg L1 Þ ð3Þ
Table 7 shows that all the AN concentrations were below the
toxic level (3000 mg L1) of methanogens, except for the three
AN concentrations calculated from chicken manure. That is to
say, all the materials investigated in this study except for chicken
manure could be applied for biogas production in wet semi-
continuous AD process (feedstock TS  10%) without ammonia
toxicity. Furthermore, slight ammonia inhibition might only occur
in swine manure and food waste when the feedstock TS was
80 g L1 and 100 g L1, respectively (AN > 1500 mg L1, Table 7).
However, the inhibition would be weakened in actual AD process
due to the acclimation of methanogens to the gradually elevated
AN concentration in the digestion liquor (Bonoli et al., 2014). All
the AN concentrations calculated from chicken manure were
higher than the toxic level for methanogens due to the higher
ANPR of chicken manure as shown in Table 7. Therefore, the feed-
stock TS of chicken manure should be diluted to less than 6% with-
out any pretreatment of ammonia removal before AD, which is
similar to the result of Lv et al. (2011).
3.7. Stability analysis
Acidification and ammonia inhibition are the two main causes
for the failure of methane production in AD process. The former
usually occurs with the accumulation of volatile fatty acids, which
is usually due to the overloading of feedstock; while the latter is
closely related to the characteristics of the substrate. However, a
slightly higher ammonia content could improve the buffer capacity

2500
T1(DM) T2(SM) T3(CM) T4(FW) T5(CR)
T6(CHER) T7(CS) T8(SS) T9(CF)

2000
AN Concentration (mg/L)

1500

1000

500

0
0 10 20 30 40 50 60 70 80 90 100 110
Operation day (d)

Fig. 2. Effective carbon-nitrogen ratio and TS ammonia production.

82 M. Wang et al. / Waste Management 62 (2017) 76–83

Table 7
AN production rate and theoretical AN concentrations of AD liquor with different TS contents of feedstock in semi-continuous AD process.

DM SM CM FW CR CHER CS SS CF
1
ANPR (mg g ) 10.06 22.61 86.87 17.54 0.97 4.48 2.56 5.15 6.89
AN (mg L1)a 604 1356 5212 1052 58 269 154 309 413
AN (mg L1)b 805 1809 6950 1403 78 358 205 412 551
AN (mg L1)c 1006 2261 8687 1754 97 448 256 515 689

AN: ammonia nitrogen (NH+4-N); ANPR: AN production rate; DM: diary manure; SM: swine manure; CM: chicken manure; FW: food waste; CR: cassava residue; CHER:
Chinese herb-extraction residue; CS: corn straw; SS: soybean straw; CF: corn flour.
a
60 g L1.
b
80 g L1.
c
100 g L1 (TS content of feedstock).

Fig. 3. Risk ranks divided with the terminal pH and VFA/TA of each digester.

of the digestion liquor to neutralize the volatile fatty acids that
could not be utilized immediately by the methanogens (Jindřich
et al., 2012).
The potential risk to methane production could be pre-
evaluated with the pH, VFA/TA and AN concentration of the efflu-
ent liquor. According to Table 7, ammonia inhibition would almost
never occur for all the samples except for chicken manure in wet
semi-continuous AD process. Therefore, only pH and VFA/TA were
used to evaluate the potential risk of acidification. According to
Table 5 and Table 6, the correlation between AC/N and risk of each
digester could be described as Fig. 3.
As shown in Fig. 3, the deeper the color is, the higher the risk of
failure in the semi-continuous AD process will be. The potential
risk within the red zone is the highest due to the higher VFA/TA
and lower pH, and only cassava residue is included in this zone,
suggesting that the organic loading rate of the digester fed with
cassava residue should be reduced gradually from the present level
of 2.09 g VS L1 d1 (Table 2) to prevent the failure of biogas pro-
duction. Five digesters are included in the low-risk zone (yellow
and pale yellow). The pH values of all of them are between 7.00
and 7.25, which approximate to the bottom line of the suitable
pH in AD process for biogas production. The VFA/TA ratios of this
zone are also relatively lower, suggesting that these digesters have
weak real-time buffer capacity to resist the change of ion balance.
Thus the organic loading rate of these yellow digesters should be
kept at the current level of 1.70–2.38 g VS L1 d1 (Table 2) and
needs strict monitoring. The digesters in the green zone in Fig. 3
are far from risk of acidification due to the higher pH (7.5–8.0)
and lower VFA/TA ratio (<0.3). Three digesters of T1, T2 and T3
are included in the green zone, indicating that the organic loading
of these digesters can be further increased. However, due to the
higher NH+4-N concentration, the organic loading rate of T3 fed with
chicken manure can not be further increased from the current level
of 0.72 g VS L1 d1 (Table 2) to prevent ammonia inhibition. Addi-
tionally, chicken manure could only be digested at a low TS con-
centration of feedstock due to its higher ANPR, which can easily
lead to ammonia inhibition when the feedstock TS is greater than
6% (Table 7), while the lower concentration of feedstock will lead
to a serious waste of dilution water in practical engineering. There-
fore, the digesters of T1 and T2 fed with dairy manure and swine
manure were assessed to have better stabilities than other diges-
ters, suggesting that T1 and T2 would be able to bear a higher
organic loading with a low risk of acidification and ammonia
inhibition.
According to the above analyses and the AC/N ratios of dairy
manure and swine manure, the optimal AC/N for semi-
continuous AD process could be preliminarily determined to be
11–15. In other words, the AD process of the substrate with the
most suitable AC/N should be able to bear a higher organic loading,
and there would be no ammonia inhibition with a higher TS con-
tent of the feedstock (6% < TS < 10%). Furthermore, a lower organic
loading is required for the AD process to prevent acidification
when the AC/N is above 15; and when the AC/N is below 11, the
TS concentration of the feedstock must be controlled at a low level
to avoid ammonia inhibition.
4. Conclusions
The AC/N ratios of all the biogas materials investigated in this
paper are lower than their corresponding total C/N ratios due to
the exclusion of the recalcitrant carbon in cellulose and lignin from
the total carbon. From the perspective of the demand of AD
microbes, AC/N would be more intelligible and accurate to charac-
terize the biogas materials rich in recalcitrant components than
total C/N. Our results show that the optimal AC/N ratio for semi-
continuous AD is 11–15. AC/N can also explain the fact that anaer-
obic digestion tests with different samples often produce different
optimal C/N ratios for AD process. Thus, AC/N could be a more
effective parameter to evaluate AD process than total C/N.
Acknowledgements
This work was mainly supported by the National Science and
Technology support projects of China for the 12th five-year-plan
 M. Wang et al. / Waste Management 62 (2017) 76–83
(No. 2011BAD15B04) and the Fundamental Research Funds for the
Central Universities (No. 2662015QD034). This study was also par-
tially support by the National Natural Science Foundation of China
(No. 51376078), foundation of Fundamental Research Funds for the
Central Universities (No. 2015PY077).
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