Bioresource Technology 156 (2014) 307313

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Semi-continuous anaerobic co-digestion of dairy manure with three crop

residues for biogas production
Jiang Li, Luoyu Wei, Qiwu Duan, Guoquan Hu, Guozhi Zhang
Biogas Scientic Research Institute of the Ministry of Agriculture, Chengdu 610041, China

h i g h l i g h t s
 Semi-continuous co-digestion of wastes under ve mass ratios can be operated stably.
 High biogas yields are achievable in mass ratio 5:5.
 Four periods were formed for the digestion.
 The N, P, S, Fe, Co and Ni improved gas production and kept the stability of AD.

a r t i c l e

i n f o

Article history:
Received 19 November 2013
Received in revised form 13 January 2014
Accepted 15 January 2014
Available online 25 January 2014
Keywords:
Semi-continuous anaerobic co-digestion
Crop straw residue
Dairy manure
Mass mixing ratio
Biogas

a b s t r a c t
The characteristics of anaerobic semi-continuous co-digestion of dairy manure (DM) with three crop
straw residues (SRs), rice straw, corn stalks and wheat straw under ve mass mixing ratios (SRs/DM)
were investigated. During the anaerobic digestion (AD) process, four periods were identied: startup, rst
stage of stabilization, second stage of stabilization, and suppression. Following the four periods, the
biogas production rate varied between 101 and 576 mL L 1 d 1. A high CH4 content and volatile solid
reduction was maintained at the SRs/DM mass mixing ratio 1:9. The highest cumulative biogas production of more than 19 L was obtained at ratio 5:5. However, ratio 9:1 performed worst in the whole process. Systematic analysis of the elements revealed nitrogen, phosphorus, and trace elements contents
were important for the AD. Overall, the semi-continuous AD is efcient within a wide range of SRs/DM
mass mixing ratios.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Anaerobic digestion (AD) is a biological process that produces
biogas from bio-degradable wastes by microorganism under poor
or no oxygen conditions. AD is gaining more attention, not only
as a solution to environmental concerns, but also as a potential
energy resource for todays energy-demanding life style. China is
one of the largest agricultural countries, which produces over
600 million ton of crop straw residues (SRs) every year, ranking
rst in the world (MOA, 2011). Rice straw (RS), corn stalks (CS)
and wheat straw (WS) are the top three crop straw wastes in China
and account for 32.3%, 25.0% and 18.3% of the total crop straw output, respectively (MOA, 2011). Thus, making use of these wastes
for biogas generation can be quite signicant. However, crop
wastes cannot be effectively degraded due to an imbalance in
Corresponding author. Address: Biogas Scientic Research Institute of the
Ministry of Agriculture, No. 13, 4th Section, South Renmin Rd., Chengdu, Sichuan,
China. Tel.: +86 28 85230701.
E-mail address: biomagongcheng2013@hotmail.com (G. Zhang).
http://dx.doi.org/10.1016/j.biortech.2014.01.064
0960-8524/ 2014 Elsevier Ltd. All rights reserved.

nutrients for microorganism and a lack of buffering capacity for

the chemical reaction (Babaee et al., 2013). This can often be overcome by co-digestion with livestock manure. Annual yield of livestock manure in China is over 2.1 billion ton, including dairy
manure (DM), swine manure, sheep manure and chicken manure
(Zhang, 2010). The use of these wastes is a major component for
producing renewable energy, and it is suitable for narrowing the
gap between the energy requirement of the industrialized world
and inability to replenish such needs from the limited sources of
energy like fossil fuels.
Studies have been done to improve biogas production from
co-digestion of livestock manure and other wastes by AD (Astals
et al., 2013; Chen et al., 2013; Saidu et al., 2013). Compared with
the digestion of single feedstock, co-digestion increases the biogas
production rate because of the better nutrient balance and
improvement of AD efciency. Generally, livestock manure contains a high total nitrogen (TN), which decreases the carbon-tonitrogen (C/N) ratios of single SRs substrates and is benecial to
co-digestion with SRs. Livestock manure is also helpful to achieve
a suitable pH during anaerobic fermentation with the production

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of ammonia (Ashekuzzaman and Poulsen, 2011). Hence, livestock

manure is excellent raw material for anaerobic co-digestion with
SRs. As one of the main livestock manure in China, DM has buffering capacity and excess nitrogen nutrients that can support
additional carbon conversion of straws to methane gas. In fact,
co-digestion, which utilizes various raw materials, such as agricultural waste, animal manure, sewage sludge and food waste, has
been extensively applied as an effective waste management and
energy production treatment (Ashekuzzaman and Poulsen, 2011;
Curcio et al., 2010; De Vrieze et al., 2013; Larsen et al., 2013). However, these studies have mostly been carried out in batch fermentation, and the suitable mixing ratios of multi-component
substrates such as SRs and DM are largely unknown. Most previous
studies focused only on the effect of C/N ratio in the AD, omitting
the analyses of the roles of other important elements, such as
phosphorus (P), potassium (K), sulfur (S), iron (Fe), cobalt (Co),
and nickel (Ni) in the degradation of substrates (De Vrieze et al.,
2013; Podmirseg et al., 2013; Rajagopal et al., 2013; Wang et al.,
2013). For example, methanogens need Fe, Co, and Ni to make
the methane production feasible (Gustavsson et al., 2011; Uemura,
2010; Zhang and Jahng, 2012; Zitomer et al., 2008).
This study investigated how the different SRs and DM mass
mixing ratios affected the biogas-producing efciency of anaerobic
co-digestion in a semi-continuous style. The main strategy was to
determine the optimal ratio of SRs and DM, characterize the semicontinuous co-digestion of them, and evaluate the effect of the
eight elements (C, N, P, K, S, Fe, Co and Ni) of the substrate on
the biogas production.

treatment was performed in triple replicate to investigate the

effect of different mixing ratios on biogas production.
2.3. Analysis and statistics
The amount of biogas produced from each digester was recorded
every day by using the water displacement method during the
digestion period. Biogas composition (CH4 and CO2 contents) was
measured using biogas 5000 (Geotech Inc., China). pH was measured using an acidimeter (PHS-3C, DAPU, China). Chemical analysis tests were performed on the substrate of each reactor after the
experiment was completed (the same batch of DM, SRs and sludge
was used throughout testing). The TS, volatile solid (VS) were analyzed by Hach Method 8271 and 8276. Total carbon (TC), TN, total
phosphorus (TP), and total potassium (TK) were determined in
accordance with the standard methods for the examination of organic fertilizer of the Chinese agriculture industry standard (MOA,
2012). Total iron (Tfe) was determined according to the microwave
digestion atomic absorption spectrometry method (Xiao, 2006).
Total sulfur (Tsu), total cobalt (Tco), total nickel (Tni) content of
the samples were determined according to the microwave digestion
ICP-MS method (Wang et al., 2008). In the next step, principal
component analysis (PCA) was utilized for the determination of
the effect of the eight elements. PCA was performed using the
CANOCO for windows 4.5 software. Each semi-continuous experiment was deemed complete when a clearly stable trend in daily biogas volume produced was observed for at least 7 days. ANOVA was
performed to determine the signicant differences among each
treatment by using SPSS version 17.0 (SPSS China Inc.).

2. Methods
2.1. Collection and preparation of substrates
SRs and DM were obtained from a local farm in Shuangliu
County, Chengdu, Sichuan, China. DM had a total solid (TS) of
18.4%, and was stored at 4 C. RS, CS and WS were prepared by cutting the residues into sections of 23 cm by using a grinder, with a
TS of 90.2%, 89.6% and 90.0%, respectively. Inoculum was the
anaerobic sludge, was obtained from an anaerobic digester of a
sewage plant in Chengdu City, and had a TS of 11.2%, and was also
stored at 4 C. For preservation of more than one week, DM and
sludge were stored at 17 C.
2.2. Experimental design and set-up
The experiment was conducted by using lab-scale anaerobic
digesters fabricated from 1 L polycarbonate cups with whorl cover
for discharging and feeding exibly. The cup had nozzle on the cover sealed with rubber stopper, where glass tube were inserted for
gas owing to the collection bottle full of water, forcing the water
to be pressed out. Gas volume was recorded by measuring the volume of the drain. In this work, semi-continuous fermentation was
used to determine the co-digestion of DM mixed with the three
types of SRs. The working volume of each digester was 800 mL,
including 91.69 g inoculum and an appropriate mass ratio of SRs
and DM. To obtain the best mixing ratio of the co-digestion of
DM and the three SRs, ve different mass mixing ratios at 1:9,
3:7, 5:5, 7:3 and 9:1 were tested under mesophilic conditions
(35 C) for 47 days. Tap water was added to digesters to maintain
a TS content of 8.0%. After 7 days startup, anaerobic co-digestion
was then initiated in a semi-continuous style. All reactors were
gently mixed manually for approximately 1 min prior to discharging and feeding, with a conservative organic loading rate (OLR) of
3.2 g L 1 every two days, as difculties were experienced in achieving steady state performance at an OLR of 1.6 g L 1 every day. Each

3. Results and discussion

3.1. Biogas yields and production rates at different DM/SRs ratios
The daily biogas production by the co-digestion of SRs and DM
during 47 days was recorded under ve mass mixing ratios (Fig. 1).
From Fig. 1, it can be seen that, for all the ve mass mixing ratios,
the semi-continuous co-digestion of SRs and DM could be divided
into four periods: startup, rst stage of stabilization, second stage
of stabilization and suppression. During the startup period, the biogas production increased rapidly, reaching about 400 mL d 1 on
day 7, except for ratios of 7:3 and 9:1,which was only about
100 mL d 1. It was suggested that recalcitrant polymers within
straws limited their degradation, and the lower amounts of soluble
carbohydrates in straws resulted in slow hydrolysis and fermentation. Consequently, ratio 7:3 and 9:1 showed a low daily biogas
production during the startup period.
After the feeding at OLR of 3.2 g L 1 every two days, the semicontinuous co-digestion of the ve ratios went into the rst stage
of stabilization period, and the biogas production of ratio 1:9, 3:7,
5:5, and 7:3 remained stable around 600 mL d 1 for 7 days, which
suggested the destruction of substrates. However, after day 22, the
gas production of the four ratios decreased to about 400 mL d 1 in
the second stage of stabilization. On day 32, further decrease was
observed, with a gas production of about 200 mL d 1 for the four
ratios, which indicated the suppression period came, and the
experiment was stopped on day 47. In fact, for all the SRs/DM,
the mixing ratio 9:1 performed worst, especially in the startup period, with a daily gas production of less than 100 mL d 1. In the rst
stage of stabilization period, daily gas production of SRs/DM 9:1 increased gradually, but still no more than 200 mL d 1. Interestingly,
in the second stage of stabilization period, all the SRs/DM at ratio
9:1 produced gas as much as other ratios, sometimes even more,
and it remained in this state until suppression period. This could
be explained that the substrate which was not used in the

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309

Fig. 1. Daily biogas production from the semi-continuous co-digestion of SRs and DM under ve mass mixing ratios. Each data point is the average of three independent
replications. Vertical bars represent standard deviations.

beginning was degraded later. Moreover, these results indicated

that the co-digestion of SRs and DM could signicantly delay the
attainment of the highest daily gas production at the mass mixing
ratios of 9: 1.
In this work, feeding was carried every two day. It was observed
that, the daily biogas volume before feeding was always higher
than that after feeding, with a gap of more than 150 mL d 1 for
all the ve ratios, especially in the suppression period. This could
be caused by imported fresh air during periodic feeding, where
oxygen might inhibit the activity of anaerobic microbiology. Furthermore, stirring during discharging and feeding might have had
an adverse effect on microbiology in digester with the destruction
of localized pockets of high acetate concentration for the methane
production and the growth of methanogens (Sindall et al., 2013).
Among the three types of straw, WS/DM had the highest gas
production on the rst day, with more than 400 mL, which suggested it had intense microbial activity and rapid substrate decomposing reaction at the beginning. Biogas from the RS/DM mixing
ratios of 1:9, 3:7, 5:5, and 7:3 reached their peak yield values at
813, 788, 850 and 953 mL d 1 on day 18, 22, 22 and 18, respectively (Fig. 1, rst stage of stabilization period). The digestion of
RS/DM 9:1 produced substantial biogas only after day 21 i.e., later
than other RS/DM combinations, but, by the end, it had a peak of
about the same size to other ratios (802 mL d 1 on day 32, Fig. 1
second stage of stabilization period). This can also be attributed
to a high amount of indigestible RS and slow decomposition. Compared to RS/DM, CS/DM had a relatively lower and later peak yield.
WS/DM has its own daily biogas production characteristics, with a
peak yield value range from 700 to 820 mL d 1 for the ve ratios.
But WS/DM 9:1 increased rapidly only after day 26 (Fig. 1). Liew
et al. (2012) reported that WS contained more lignin, leading to a
more recalcitrant structure than RS and CS, and thus limiting the
degradation of this lignocellulose.
The cumulative biogas productions by the co-digestion of SRs
and DM at ve mixing ratios are shown in Fig. 2. For all the mixing
ratios, CS/DM 5:5 had the highest biogas yield at 19,428 mL after
47 days of digestion, but this was not signicantly different from
WS/DM 5:5 (19,127 mL (p > 0.05)). In fact, in this work, ratio 5:5
showed for the best results in the nal cumulative biogas produc-

tion among the ve ratios, no matter which SRs was co-digested

with DM. However, CS/DM 9:1 had the lowest value at
10,523 mL, nearly 46.2% lower than CS/DM 5:5. In addition, the
cumulative biogas production for ratio 9:1 also was the worst in
the ve ratios of all the SRs /DM. This was consistent with the daily
biogas production data (Fig. 1).
The biogas production rate of SRs and DM at ve mass mixing
ratios in four periods are shown in Fig. 3. For RS/DM, ratio 1:9
had the highest biogas production rate in the startup period, which
reached 236 mL L 1 d 1, and showed an increase of about 50.4%
compared with other RS/DM ratios (101123 mL L 1 d 1)
(p < 0.05). This suggested that setting RS/DM mass mixing ratio
at 1:9 could make the AD start smoothly. For CS/DM in the startup
period, ratio 7:3 had the highest biogas production rate, suggesting
this ration improved the destruction of CS at the beginning of the
AD. And for WS/DM, there was almost no difference among the ve
ratios in the startup period. In the rst stage of stabilization, for RS/
DM, ratio 1:9 kept stay on the top, but ratio 3:7, 5:5, and 7:3 also
reached a relatively high biogas production rate, which was 454,
520 and 453 mL L 1 d 1, respectively, leaving RS/DM 9:1 in the
bottom, which was drastically lower, averaging approximately
121 mL L 1 d 1. These results veried slower biogas production
rate was accompanied by more straws in the substrates. CS/DM
and WS/DM had a similar trend with RS/DM in the rst stage of
stabilization, except for WS/DM 7:3, averaging 32.3% less than
WS/DM 1:9, 3:7, and 5:5, but 44.6% more than WS/DM 9:1, as
showed in Fig. 3 (p < 0.05). In the second stage, stabilization period,
RS/DM 5:5 and 9:1 produced biogas faster than the other ratios,
with rates of about 525 mL L 1 d 1. In the suppression period, ratio
7:3 reached the highest biogas production rate in the ve ratios of
SRs/DM, while ratio 1:9 reached the lowest (220230 mL L 1 d 1),
which indicated ratio 7:3 had signicant substrate degradation,
even there was some suppression for other ratios.
In the whole process, the biogas production rate reached highest at different mass mixing ratios for different periods. But in the
rst stage of stabilization, the biogas production rates of all the ratios were always higher than that in the other periods, except for
SRs /DM 9:1. According to the results from Fig. 2 and Fig. 3, the
mass mixing ratio 9:1 was not feasible for the semi-continuous

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J. Li et al. / Bioresource Technology 156 (2014) 307313

Fig. 2. Cumulative biogas production from the semi-continuous co-digestion of SRs and DM under ve mass mixing ratios. Each data point is the average of three
independent replications.

3.2. pH, biogas composition and organic solids degradation

Fig. 3. Biogas production rate in four periods (startup, rst stage of stabilization,
second stage of stabilization and suppression) from the semi-continuous codigestion of SRs and DM under ve mass mixing ratios. Each data point is the
average of three independent replications.

co-digestion of SRs and DM. Possible reasons for the low carbon to
biogas efciency in the SRs/DM 9:1 reactors include the following:
(1) Imbalance of C/N in the mass mixing ratio 9:1of SRs/DM; (2)
Lack of trace nutrients through the presence of the ratio; (3) Organic overloading, resulting in a reduction in the methanogenic activity; and (4) High levels of carbohydrates in the straws that
converted carbon to excessive acid and occurred before methanogenesis (Mata-Alvarez et al., 2000).

pH is one of the key factors in AD, and the growth of methanogens can be signicantly inuenced by the pH level (Rajagopal
et al., 2013). The pH value reected the changing processes in
the digesters with similar trends in all the mixtures during the
47 days of semi-continuous co-digestion (Fig. 4). On day 2, the
pH values decreased from 6.80 to 5.25 with SRs percentage
increasing in the ve mixing ratios, and WS/DM 9:1 had the lowest
pH value (5.25). But from day 13, the pH values increased in all the
mixtures, and then remained at approximately 7.00 until the end
of the experiment. This stability conrmed that the biogas production of each mixture reached the methanogenesis stage. It further
indicated that excess acid was not formed in the suppression period, as moderate pH was recorded. However, the pH of SRs/DM 9:1
valued from 5.50 to 5.25 before day 23, showing the buffering
capacity of DM did not work, as SRs composed 90% of the total substrate mass. According to the results from both daily and cumulative biogas production, it could be concluded that the optimal pH
values for the co-digestion of SRs and DM ranged from 6.80 to
7.20, as ratio 5:5 showed. These results are consistent with Abouelenien et al. (2010) who found that with an ammonia-stripping
unit (here similar to SRs), methane was successfully produced from
the treated chicken manure at a pH of approximately 6.70.
The methane content of the biogas from all the SRs/DM ratios
varied among the four periods, from 29.3% to 40.1% for the startup
time, 50.464.5% for the rst and second stage, and 50.130.2% for
the suppression, which showed a relatively big deviation over the
entire digestion (Fig. 4). This was consistent with the result from
daily biogas production in Fig. 1. In the rst 5 days, the methane
content had a faster increase only for ratio 1:9 of RS/DM and
WS/DM, from 40.6% to 55.3%. This increase could be observed in
all the ratios of CS/DM, indicating that CS reached the methanogenesis stage earliest among the three SRs. The methane content of
other four ratios of RS/DM and WS/DM were below 40% in the rst
5 days, some even had a value of 29.3%. Reason for this might be
that, compared to RS and WS, CS had more easily degradable organic matter, which boosted the activity of the microbiology in

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311

Fig. 4. Methane content (line and symbol) and pH values (symbol) from the semi-continuous co-digestion of SRs and DM under ve mass mixing ratios. Each data point is the
average of three independent replications.

the startup time (Liew et al., 2012). When the easily degraded matter was used up, CS/DM got a similarly methane content as RS/DM
and WS/DM, as Fig. 4 showed. However, unlike their methane content, the difference for the three SRs was not evident in the biogas
production rate or daily biogas production in the startup time.
After day 35, the methane content decreased dramatically for all
the treatments, especially for WS/DM 1:9, which was only 30.7%
on day 36. As pH was normal, this could be the result of inhibitors
generated with the digestion going on, thus affecting the growth of
methanogens during the AD process (Chen et al., 2008; Madsen
et al., 2011).
In order to determine the amount of co-substrate that had been
degraded in the co-digestion experiments, VS and TS analysis of all
the ratios were carried out at the end of the digestion. The calculated reductions for each ratio are reported in Fig. 5A and B. The
ANOVA results indicated that just as the VS reduction for different
ratios of RS/DM, the population means of TS reduction for different
ratios of RS/DM and WS/DM were not signicantly different, ranging from 38.4% to 45.2%. For the VS reduction of WS/DM, ratio 1:9
was 50.0% more than that of WS/DM 9:1(p < 0.05). As it is known,
TS of lignocellulose is mainly composed of VS and ash (Soest et al.,
1991). And the results indicated that there might be more ash content in the WS, causing the TS reduction trend to disagree with that
of the VS. Unlike RS/DM and WS/DM, the TS reduction of CS/DM
1:9 was signicantly different from that of CS/DM 7:3, with an increase of 40.2% (p < 0.05). And for VS reduction, CS/DM 1:9 was signicantly different from that of 7:3 and 9:1, increasing 35.0% and
35.5%, respectively (p < 0.05). In sum, SRs/DM 1:9 performed best
in the organic solids destruction, and rather than those of RS/DM
and WS/DM, different ratios of CS/DM led to different substrate
reduction results.
The ANOVA also showed that the means of three straws were
not signicantly different from each other in the VS and TS reductions. However, Fig. 5 C differed as the biogas productivity of WS/
DM 3:7 reached 198 mL g 1 VS, 9.6% and 16.7% more than that of
RS/DM 3:7 and CS/DM 3:7, respectively (p < 0.05). Compared to
RS/DM 5:5, the biogas productivity of WS/DM 5:5 was better,
reaching 209 mL g 1 VS, which also was the best of all the SRs/
DM ratios (p < 0.05). These results disagreed with those of Wu
et al. (2010), who found that WS demonstrated a lower biogas productivity than CS and oat straw even it had a higher carbon content

than the latter two residues. But for the biogas production from VS
mass in this work, WS had a better performance than RS and CS.
3.3. Elements analysis using a PCA
Generally speaking, the C/N ratios of different substrates mixtures in AD greatly inuence biogas production (Kayhanian,
1999; Wang et al., 2012). Results of the element analysis showed
that the feedstock of SRs/DM in the ve mixing ratios had a C/N ratio of about 7.8, 10.7, 15, 21 and 33, respectively. However, with
the increasing of the C/N, there was no obvious trend for the biogas
production change in the ve mixing ratios. And with the semicontinuous co-digestions going on, the biogas production came
to a stop.
To better understand the processes in play during the biogas
production, this work tested not only C and N contents, but also
P, K, S, Fe, Co, and Ni in all the feedstocks and efuent samples at
the end of the digestion. And PCA was used to determine the
importance of each element.
In this study, PCA showed a smaller partial correlation
(KMO = 0.931) and a high dependence (P < 0.001) (Fig. 6). In general, the KMO value is higher than 0.9 and the P value is lower than
0.001 can be viewed as very suitable for PCA. The PCA of all the
samples yielded two PC factors (PC1 and PC2) that accounted for
84.69% of the variance. The plots of PCA factor scores vs. elements
provided an indication of the element types of each PC and therefore identied the elements that contributed to the variance in the
specic SRs/DM ratio, which may explain the biogas production
differences (Fig. 6). Eight elements could be identied by PCA.
PC1 (64.51% of the variance) was mainly related with TN, TP, Tsu,
Tfe, Tco and Tni, which were indicative of ammonia, phosphate
and trace element. PC2 (20.18% of the variance) was associated
with a primary element, carbon, which was indicative of carbohydrate, including hemicellulose (ve carbon polymers), cellulose
(six carbon polymers) and lignin (phenol polymers), which were
mainly in straws (Bauer et al., 2009). TK had comparative scores
in both of the two PC plots.
In Fig. 6, ve regions could be drawn. Region I, including three
efuent samples (outCS5:5, outWS5:5, and outWS1:9), clustered
with higher second (>0.6) and moderate rst (<0.4) PCA loading,
indicated that element C and K were dominant in the three efuent

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J. Li et al. / Bioresource Technology 156 (2014) 307313

Fig. 5. Substrate utilization of the semi-continuous co-digestion of SRs and DM under ve mass mixing ratios: (A) VS reduction (B) TS reduction (C) Biogas production. Each
data point is the average of three independent replications. Vertical bars represent standard deviations. The ANOVA test was conducted to determine the differences between
ratios. Values with the same letters indicate no signicant difference at p < 0.05.

Fig. 6. PCA of the semi-continuous co-digestion of SRs and DM under ve mass

mixing ratios. Sample inCS, inRS and inWS stand for the feedstock of co-digestion of
SRs and DM. Sample outCS, outRS and outWS stand for the efuent of co-digestion
of SRs and DM at the end of the digestion.

samples. As Fig. 5C showed, WS/DM 5:5 obtained the highest biogas productivity per gram of VS. PCA here may give reason for this,
that was, compared with other out samples, outWS5:5 had the
highest TC and TK content, and contained moderate TN, TP, and

trace element amount, which was suitable for methanogenesis.

However, biogas yield of WS5:5 was not signicant different from
that of CS/DM 5:5. This indicated that good biogas production also
needed suitable elements content as SRs/DM 5:5, not only focusing
on the C/N ratio. Region III, including outCS7:3 and outWS3:7, obtained moderate PC2 (>0.1) and lower PC1 (< 0.1) loading, which
suggested trace elements were lacking in the three samples. That
maybe why CS/DM 7:3 had the worst result for TS and VS removal,
while CS/DM 1:9, RS/DM 3:7and WS/DM 1:9 had the best (outCS
1:9 and outRS 3:7 gathered together in Fig. 6 Region II with PC 1
loading >0.2). Similarly, region IV and V were also lack of trace elements. Whats worse, region V had the least content of the eight
elements (PC 1 loading < 0.15, PC 2 loading < 0.4). As a result,
mixtures in this region (WS/DM 9:1 and CS/DM 9:1) had a bad performance in the gas production. Furthermore, the similarity of PCA
loading of CS/DM 9:1, WS/DM 9:1, RS/DM 7:3and WS/DM 7:3 in
Fig. 6 region V was consistent with the pH trend of them in the
co-digestion as shown in Fig. 4. Ratios 7:3 and 9:1 had more straw
mass, which meant more carbon and potassium contents. This was
seemingly contradictory with the PCA loading in region V, and further investigation will be needed to better understand the results.
Overall, from PCA and biogas production results, it showed that
compared with region III, IV, and V, region I and II had more N, P,
and trace elements contents, and got better AD performance. It
was known that methanogens need trace elements, especially Fe,
Co, Ni, to initiate methane production (Gustavsson et al., 2011;
Uemura, 2010; Zhang and Jahng, 2012; Zitomer et al., 2008). As a
result, ratios in region I and II with more Fe, Co, Ni and appropriate
element composition obtained higher biogas production rate and
yield than the others.
PCA also showed that with ratios changing from 1:9 to 9:1, i.e.
straws became more and more in the feedstock, the PC1 loading of

J. Li et al. / Bioresource Technology 156 (2014) 307313

the feedstock samples became lower and lower, but the PC2 loading varied little, indicating it were N, P, and trace elements in PC1
that determined the differences of the element composition of the
feedstocks. In other words, if there was much more straw in the
substrate, there was less N, P, and trace elements, which leaded
to less biogas yield as this work showed. In general, these PCA results suggested that co-digestion of SRs and DM with suitable N, P,
and trace elements content as ratio 5:5 was an effective way to
prolong the period of the highest gas production and improve biogas yield.
4. Conclusion
This study examined the mechanisms underlying the biogas
production of anaerobic semi-continuous co-digestion using three
types of straws and ve mixing ratios. First, it was found the AD
could be divided into four periods, with the highest biogas production rate occurring in the rst stage of stabilization, except for SRs/
DM 9:1. CS/DM 5:5 showed the highest nal cumulative biogas
production. SRs/DM 1:9 increased fast in methane content. From
PCA, it was suggested good biogas production needed suitable N,
P, and trace elements contents. Overall, except SRs/DM 9:1, the
other four ratios had a high potential for anaerobic semi-continuous co-digestion.
Acknowledgements
The authors gratefully thank Dr. Liette Vasseur (Minjiang scholar at Fujian Agriculture and Forestry University) for the English
scientic editing. The authors would like to acknowledge nancial
support from the National Science & Technology Pillar Program
during the Twelfth Five-year Plan Period (2011BAD15B03).
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