Accepted Manuscript

Short Communication

Efficient biogas production from cattle manure in a plug flow reactor: A large
scale long term study

Lili Dong, Guangli Cao, Xianzhang Guo, Tianshu Liu, Jiwen Wu, Nanqi Ren

PII: S0960-8524(19)30124-5
DOI: https://doi.org/10.1016/j.biortech.2019.01.100
Reference: BITE 20977

To appear in: Bioresource Technology

Received Date: 2 January 2019

Revised Date: 21 January 2019
Accepted Date: 22 January 2019

Please cite this article as: Dong, L., Cao, G., Guo, X., Liu, T., Wu, J., Ren, N., Efficient biogas production from
cattle manure in a plug flow reactor: A large scale long term study, Bioresource Technology (2019), doi: https://
doi.org/10.1016/j.biortech.2019.01.100

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 Efficient biogas production from cattle manure in a plug flow reactor: A
large scale long term study
Lili Donga, Guangli Caoa, , Xianzhang Guob, Tianshu Liub, Jiwen Wua, Nanqi Rena
a
State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin
Institute of Technology, Harbin 150090, China
b
Chinese Academy of Agricultural Mechanization Sciences, Beijing 100083, China

Abstract

In this study, a large-scale plug flow reactor has been operated at a hydraulic retention time of

25 d with substrate concentration of total solids (7 - 10%) under a temperature of 37 ~ 40°C with a

working volume of 3.85 × 104 m3 and dispose 1504 m3/d of waste for three years. The average

monthly biogas production was 7.45 × 104 m3. At stable stage, the volume biogas, methane

production rate and methane content reached 1.07 m3/m3d, 0.58 m3/m3d and 56.4%, respectively.

Moreover, the specific biogas and methane yields of substrate removal efficiency were up to 0.39

m3/kg VS, 0.22 m3/kg VS and 59%, respectively. Among the bacteria, the genera

Draconibacteriaceae, Ruminofilibacter, Cloacimonetes and vadinBC27, Ruminiclostridium and

Treponema_2 dominated in digestate. The achaeal was dominated by methanogens genera

Methanosarcina and Bathyarchaeota. Long-term operation in large scale demonstrated

technological potential and industrial application of cattle manure treatment for biogas production.

Keywords: cattle manure, biogas production, microbial community, plug flow reactor,

large-scale

Introduction

With the development of international food trade, the demand for the animal source foods is

increasing greatly, leading to the rapid development of large-scale, intensive breeding farms

(Kerssen, 2015). Consequently, huge amounts of animal waste are produced. Thus, it is essential to

find a technologically and economically feasible way to treat large amounts of manure and

wastewater. Anaerobic digestion (AD) is considered one of the most suitable technologies in

manure and wastewater treatment due to the amounts of biogas produced and its economic

*
Corresponding author, Fax: +86 451 86282008. E-mail address: caogl@hit.edu.cn; rennq@hit.edu.cn

1
relevance in addition to the potential to solve the problems of manure waste utilization and

environmental pollution at the same time (Gyuseong et al., 2018). Within the AD process, plug-

flow reactor (PFR), a type of anaerobic digestion process reactor, has been reported to be efficient

for various types of substrate, especially in livestock manure treatment (Batstone et al., 2015).

However, few large-scale applications of PFRs have been reported.

Large-scale biogas production is an established technology in developed countries such as

Sweden, Denmark and the United States (Lönnqvist, 2017). Nevertheless, there is a lack of research

regarding large-scale biogas production systems in developing countries. In general, the types of

reactors used in plants are CSTRs. With the rapid increase in the number of biogas installations on a

large-scale, noticeable challenges have arisen with regards to efficiency, performance, and

construction. Compared to other types of anaerobic reactors, the PFR is usually simpler to operate

due to less internal moving parts. Moreover, start-up for a PFR is typically lower than other kinds of

reactor configurations, and PFR is inexpensive and easy to build which makes it a suitable

technology to improve the livelihoods of farmers. Nevertheless, although many efforts have been

made to carry out biogas production from different substrates in PFRs, most of these studies were

investigated at the lab-scale or small-scale (less than 700 m3), which are not completely suitable for

evaluating large-scale biogas plants (Weiland et al., 2003). Until now, there has been a lack of data

on the long-term operation of large-scale PFR AD plants in terms of the performance and stability,

especially, data on the functional microbial community structure, which is the core of consecutive

conversion of substrate to biogas and has yet to be explored.

Therefore, this research assessed a 3-year operation of a large-scale PFR, 3 MW biogas plants,

for biogas production from cattle manure. During the long-time operation of PFR, the accumulative

biogas production, volume of biogas and methane production rates, gas composition, specific

biogas and methane yield, substrate removal efficiency, and the abundance of microbial

communities were monitored under the steady conditions. This study offers important information

2
for the industrial application of biogas production in PFRs using cattle manure as a substrate,

especially from the viewpoint of ‘application’ and ‘operation’.

2. Material and methods

2.1. Substrate and inoculum

Cattle manure was collected from a cowshed located at Shuangcheng in the Northeast of China

by scraping board. The inoculum was obtained from a running 1.5 MW cattle manure disposal

digester (Modern farming group Shangzhi program), which has been operated for five years. That

project was also a PFR anaerobic fermentation process. The inoculum, collected from the liquid

after the secondary solid-liquid separation, was added at 30% (v/v).

2.2. The operation of PFR

Modern farming group of the Shuangcheng 3 MW cattle manure disposal project located in the

northeast of China was built in 2014 and started to run in July 2015. A horizontal PFR for

continuous anaerobic digestion of cattle manure was constructed by reinforced concrete and half the

underground in this project. It consists of sixteen reactors (each reactor side-by-side construction

and each with volume of 2725 m3 and the effective volume 2500 m3), with the total volume of 4.36

× 104 m3, and the effective volume of 3.92 × 104 m3. The single reactor was a length of 99 m, a

width of 6 m, and a height of 4.8 m including 0.3 m for gas space. The PFR could deal with

wastewater of 1920 m3/d in theory and the actual operation of 1504 m3/d with fourteen PFRs. In

order to prevent the substrate accumulation at the inlet and outlet end, the mechanical stirrings at

the inlet and outlet were operated regularly every day. For digesters with a length of 99 m, although

mechanical stirrings with blades of 4 m in diameter were installed before and after the digester, this

would not change the flow state of the substrate. The whole system is divided into four units. The

first one is the manure collection. In this unit, the cattle manure, urine, and back flushing bio-liquid

collected from the cowsheds firstly entered the septic tank and then pumped into the homogenous

pool. In the homogenous pool, temperature and substrate concentration were regulated in the range

3
of 37 ~ 40 °C and 7 ~ 10% total solid (TS), respectively. The second is the main fermentation unit,

which was operated in PFR. The mixed substrate from the first unit of entered the second unit

through the pump. The PFR was operated continuously at hydraulic residence time (HRT) 25 d and

fermentation temperature 37 ~ 40 °C, and the average total organic load rate (OLR) 1.1 × 105 kg

COD/day for three years. During operation, the substrate was discharged through the back end of

the reactor entered to the third unit for post-processing and the biogas from the anaerobic reactor

entered the fourth unit, which was biogas utilization part.

2.3. Analytical methods

The TS and volatile solids (VS) of the manure was detected in triplicate according to standard

methods (American Public Health Association, 1999). The pH and chemical oxygen demand (COD)

were measured by HACH-HQ40d multi and HACH-DRB 200. The total nitrogen (TN) and the total

phosphorus (TP) were assayed by analytikjena multi N/C 2100s and Perkin Elmer Optima 8300.

The gas products (CH4 and CO2) and volatile fatty acids (VFAs) were determined by gas

chromatography (GC, 7890A, Agilent Cooperation, USA).

2.4. DNA extraction and Illumina sequencing

For the analysis of microbial community, samples were taken over the period of 2 years in

PFR, the samples (3-10 mL) were harvested from the middle of the biogas project with a sterilized

sample spoon and stored in a 50 mL sterile plastic test tubes at -80°C before went for DNA analysis.

Microbial DNA was extracted from collected samples using the the bacterial DNA mini kit (Watson

Biotechnologies Co. Ltd., Shanghai, China) according to manufacturer’s protocols. Bacterial V3-V4

region of 16S rRNA gene was amplified using the forward primer 338F (5’-

ACTCCTACGGGAGGCAGCAG-3’). Archaea V4-V5 region of 16S rRNA gene was amplified

using 524F (5' - TGYCAGCCGCCGCGGTAA-3'). Taxonomic classification of each phylotype was

determined using the SILVA rRNA database project with over 97% of sequence similarity.

3. Results and discussion

4
3.1 Properties of substrate

The cattle manure employed in this study was collected via scraping it from the cow barns, and

it contained spilled feed, litters, and water, in addition to feces and urine. The collected cattle

manure and waste were homogeneous mixed in a homogenized tank. Meanwhile, the temperature

was adjusted to around 37 °C, and the concentration was adjusted to 7 - 10% in the homogenizer.

The pH of the substrate was slightly alkaline with the values of 7.85 ± 0.05. The substrate was solid

with an average TS of 7.93% and VS of 6.71%. The COD of the substrate was around 7.89 × 104

mg L-1. The values of the total nitrogen and phosphorus contents and VFA were 741.25 mg·L-1,

675.21 mg·L-1 and 767.30 mg·L-1. As the following operation, these general indicators can be

satisfied the smooth-running anaerobic process.

3.2. Large scale assessment over one year of monitoring

This research assessed a 3-year, long-term operation of a large-scale PFR (with a total volume

of 43600 m3) biogas production project. Here, only 1.5 years of data were analysed. It can be

obtained from Fig. 1 that the PFR could be operated continuously and steadily throughout the year.

Monthly biogas production was in the range of 4.94 - 11.58 × 105 m3/month with an average value

of 7.45 × 105 m3/month. It should be noted that the biogas production was at low levels from the

July to September. It is not difficult to find that the average temperature in these three months is

higher than 26°C. Mader and Davis (2004) have shown that Holstein cows will produce a

nonspecific, physiological heat stress response when the temperature exceeds the upper limit of the

isothermal zone. Although, the TS of the cattle manure increased during heat stress, the nutrients of

cattle manure that tend to be anaerobic reduced, which led a low biogas production of the PFR

everyday. In addition, due to the high temperature, some nutrients in the cattle manure were

decomposed before entering the PFR, resulting in adverse effects on biogas production. Although

the lowest biogas production in August was only 4.94 × 105 m3, the result was equal to the value of

other studies using CSTR and UASB in the lab scale (Angelidaki and Ahring, 1994; Holm-Nielsen

5
et al., 2009). Usually for large-scale anaerobic fermentation plants, the main reactor was adopted

CSTR, e.g. a typical large-scale biogas plant in the German state of Hardegsen, using CSTR as

anaerobic digester, has a total capacity of 2.7 × 104 m3 of biogas per day (Weiland et al., 2003).

Another large biogas plant (DQY 2 MW) in Beijing of China uses medium temperature CSTR

bioreactors with a 6.0 × 105 m3/month biogas production capacity with a substrate of chicken

manure and straw (Pan, 2011). The above results demonstrate that PFR developed in this study

would be another promising approach for continuous biogas production in large-scale plants.

3.3. Process performance during the stable stage

To explore the efficiency of PFR in large scale, the PFR performance at the stable stage in

terms of the volume biogas and methane production rate (VBPR and VMPR), biogas composition,

the specific biogas and methane yield (SBY and SMY), and substrate removal efficiency was

evaluated. A period of 50 days (from November 20, 2016 to January 9, 2017) was considered as

reference for steady operational conditions. As shown in Fig. 2a, the VBPR and VMPR were in the

ranges of 0.82 - 1.29 m3/m3d and 0.43 - 0.75 m3/m3d with average values of 1.07 m3/m3d and

0.58 m3/m3d, respectively. It’s obvious that biogas production was kept in a relatively stable stage

during the monitoring period. Generally, cattle manure was considered to be low in conversion

enfficiency compared to other animal manure such as swine and chicken manure. However, the

VMPR in this biogas plant was even slightly higher than the average value (0.56 m3/ m3d) obtained

in a large-scale swine manure biogas plant (Wang et al., 2017), which suggested that the AD

process in PFR was effective in treating cattle manure for methane production. The methane and

carbon dioxide contents were relatively constant at 51.9% to 58.3% and 41.7% to 48.1% with

averages of 54.5% and 45.5% for the monitoring period (Fig. 2b), representing a typical level in

biogas from manure biogas plants (Michael and Gerardi, 2003). The SBY and SMY were observed

in the ranges of 0.32 - 0.50 m3/kg VS and 0.15-0.23 m3/kg VS with the average values of 0.39

m3/kg VS and 0.21 m3/kg VS (Fig. 2c), respectively. According to the methane production yield

6
from cattle manure, it can be obtained that 1 kg of volatile solid can generate 0.39 m3 of biogas and

0.22 m3 of methane. Chynoweth et al. (1993) pointed out that the highest potential methane

production yield from animal waste and water could be achieved 0.2 m3/kg VS. In this regard, the

PFR is undoubtedly an efficient reactor for methane production from cattle manure in large scale. In

addition, an economically feasible biogas production process did not only dependents on high

biogas production efficiency but also on the efficiency of substrate utilization. According to Nielsen

and Angelidak (2008), animal manure especially cattle manure which is rich in lignocellulosic

material, is considered inefficient in substrate conversion. It was reported that only 49% of TS was

removed from cattle manure in an UASB (Abubakar and Ismail, 2012). In this study, more than

41% of the substrate was utilized, and the maximum substrate removal reached 59.9% in operation

(Fig. 2d), indicating the PFR was beneficial for substrate conversion. Moreover, this was the first

time using cattle manure as substrate to investigate the AD process in such large scale.

Overall, based on the aspects of system stability, methane production and substrate utilization,

PFR could be considered a promising efficient and economic configuration to apply on large farms

in the future.

3.4. Establishment of microbial communities during steady anaerobic digestion

To better understand the correlation between performance and microbial community structures

established in PFR, bacterial and archaeal communities were analyzed through Illumina sequencing.

The predominant bacterial phyla in the tested samples (substrate and digestate) were Bacteroidetes,

Firmicutes, Cloacimonetes, Proteobacteria, Spirochaetae, Chloroflexi, and Fibrobacteres (Fig.3a).

Bacteroidete and Firmicutes were two of the main dominant phyla of the digestate. The further

enriched bacteria in the digestate were Spirochaetae, Chloroflexi, and Fibrobacteres, but these phyla

weren’t appeared in the substrate. These bacteria maybe enriched from inoculum, because the

inoculation comes from the same system which was the running homograft. In a recent study,

Bacteroidetes and Firmicutes were suggested to be important in a hydrolytic/acidogenic digester fed

7
with dried hay and straw (Li et al., 2014). Dubinina et al. (2011) found that Spirochetes could use a

variety of carbohydrates including cellulose and hemicellulose. Fibrobacteres is a small bacterial

phylum that includes many of the major rumen bacteria, allowing for the degradation of plant-based

cellulose. Based on the received biogas production results it can be assumed that the bacteria of

these phyla were more important functionally for the digestion of cattle manure wastes in our

experiments. At the genus level, more than 20 types of bacterial genera (relative abundance P≥1%)

were generated in the digestate (Fig. 3c). Among of them, the most dominant genera were

norank_f_Draconibacteriaceae, Ruminofilibacter, unclassified_p_Cloacimonetes, and

vadinBC27_wastewater_sludge_group. Compared with the substrate, the abundance of

norank_f_Draconibacteriaceae, Ruminofilibacter, vadinBC27_wastewater_sludge_group, and

Sedimentibacter were further enriched in the digestate. Draconibacteriaceae (phylum Bacteroidetes)

was identified in the literature as a novel family, which was created to accommodate the newly

described genus Draconibacterium and members of Draconibacteriaceae that can transform sugar

substances into acids during the anaerobic process (Du et al., 2014). Ruminofilibacter (phylum

Firmicutes) and vadinBC27_wastewater_sludge_group (phylum Bacteroidetes) are related to

cellulose degradation. Sedimentibacter has been reported to have a hydrogen-producing acetic acid

bacterium and symbiosis with hydrogen-consuming bacteria, which played the role of linking up

and down from hydrogen production to methane during the transformation of matter

(Cardinalirezende et al., 2009). There was also a lot of Ruminiclostridium (phylum Firmicutes) and

Treponema_2 (phylum Spirochaetaceae) enrichment in the digestate. These group bacteria might be

involved in the AD of cellulose through an extracellular cellulose hydrolysis process and/or the

fermentation of organic substrates originating from cellulose.

The phylum level distribution of the archaeal community for the samples was shown (Fig. 3b).

It was possible to assign more than 99.22% of the sequences, of which more than 99.18% belonged

to the phylum Euryarchaeota in the substrate. The remaining sequences (< 0.04%) were represented

8
by Bathyarchaeota. The Euryarchaeota and Bathyarchaeota were also the dominant phyla, but the

levels of Euryarchaeota decreased to 72.85% and the Bathyarchaeota levels increased to 22.58% in

the digestate. The phylum Euryarchaeota is known to have a methane-metabolism and commonly

exists in anaerobic digesters (Cardinalirezende et al., 2009). Consistent with previous studies, the

Euryarchaeota was the most dominant phylum in this system, but a novel phylum, Bathyarchaeota

presented at a relatively high level and has been recently recognized to have methane metabolism

revealed by genome-centric metagenomics (Evans et al., 2015). These findings indicated that

Bathyarchaeota played an important role in the production of methane. At the genus level (Fig. 3d),

Methanosarcina (phylum Euryarchaeota) was the most dominant genus in the digestate (49.19%),

while this genus represented only 6.41% in the substrate. In contrast, the archaeal community in the

substrate was dominated by Methanobrevibacter (84.31%) (phylum Euryarchaeota). However, the

levels of Methanobrevibacter were low in the digestae (< 5.07%). Sequences belonging to the

acetoclastic methanogen, Methanosaeta, were detected at a low level in all tested samples: 0.37% in

substrate and 8.81% in digestate. The literature reports that Methanosarcina and Methanosaeta

would compete with each other in the same substrate (De et al., 2012). Therefore, a large member of

Methanosarcina existing in the system may lead to a decrease in the number of Methanosaeta.

Methanosarcina is a genus of Euryarchaeote, and as a mixotrophic methanogen, it can metabolize a

wide variety of substrates, such as acetate, hydrogen, carbon dioxide, and methylamines.

Methanosarcina in the digestate was apparently abundant methanogens and should play a

significant role in the biomethanation of substrate in the present study. Norank_p_Bathyarchaeota

(phylum Bathyarchaeota) was another dominant genus in the digestate (22.58%) that only has

0.04% in substrate. According to He et al. (2016), members of Bathyarchaeota have a rich and

diverse metabolic modes that can degrade difficult macromolecules such as aromatic compounds,

chitin, cellulose and proteins. At the same time, small molecular compounds such as acetic acid was

produced, and it can also use H2 and CO2 to obtain energy by self-culture acetic acid production.

9
 Compared with CSTR anaerobic digestion process using cow manure and/or biomass as

substrate. The reported five species bacterial phyla including Bacteroidetes, Proteobacteria,

Firmicutes, Fibrobacteres, and Spirochaetes (Hu et al., 2017) were retrieved, while Cloacimonetes

and Chloroflexi were appeared in this study. Meanwhile, Bacteroidetes and Firmicutes tended to

form dominant populations in PFR process. In addition, the archaeal in CSTR was Euryarchaeota,

but there was another dominant genus Bathyarchaeota in this study. From the analyses of both the

phylum and genus levels of the bacterial and archaeal communities, the PFR anaerobic digestion

process could promote the enrichment of functional microbial population, and these communities

tended to be abundant and complementary, made the system high effective and stable.

Conclusions

The PFR operated successfully for continuous and stable biogas production using cattle manure

as substrate at 7~10% TS, HRT 25 d, and temperature 37 ~ 40 oC for a long period of time. The

maximum monthly biogas production achieved 11.6 × 105 m3. During operation, an abundance of

lignocellulose decomposing bacteria (Bacteroidete, Firmicutes, Cloacimonetes, Fibrobacteres) and

methane-forming Methanosarcina and Bathyarchaeota were enriched, making the PFR come up to

a methane theoretical yield of nearly 50% and the substrate removal of 59%. These results showed

that the AD process via PFR in large scale has great potential for practice application.

ACKNOWLEDGEMENTS

This research was supported by Special Fund for Scientific and Technological Innovation of

Harbin (No. JJ20170385) and State Key Laboratory of Urban Water Resource and Environment

(Harbin Institute of Technology ) (No . 2018TS02).

REFERENCES

[1] Abubakar, B.S.U.I., Ismail, N., 2012. Anaerobic digestion of cow dung for biogas production.

ARPN-JEAS. 7(2), 169-172.

[2] Angelidaki, I., Ahring, B.K., 1994. Anaerobic thermophilic digestion of manure at different

10
ammonia loads: effect of temperature. Water Res. 28, 727-731.

[3] Batstone, D.J., Puyol, D., Flores-Alsina, X., Rodríguez, J., 2015. Mathematical modelling of

anaerobic digestion processes: applications and future needs. Rev Environ Sci Bio. 14 (4), 595-613.

[4] Cardinalirezende, J., Debarry, R.B., Colturato, L.F., Carneiro, E.V., Chartone-Souza, E.,

Nascimento, A., 2009. Molecular identification and dynamics of microbial communities in reactor

treating organic household waste. Appl Microbiol Biot. 84(4), 777-789.

[5] Chynoweth, J.N., Owen., 1993. Biochemical methane potential of municipal solid waste

components. Waterence & Technology. 27(2), 1-14.

[6] De, Vrieze, J., Hennebel, T., Boon, N., 2012. Methanosarcina: The rediscovered methanogen for

heavy duty biomethanation. Bioresour. Technol. 112, 1-9.

[7] Du, Z.J., Wang, Y., Dunlap, C., Rooney, A.P., Chen, G.J., 2014. Draconibacterium orientale gen.

nov., sp. nov., isolated from two distinct marine environments, and proposal of Draconibacteriaceae

fam. nov. Int J Syst Evol Micr. 64(5); 1690-1696.

[8] Dubinina, G., Grabovich, M., Leshcheva, N., A. Rainey, F., Gavrish, E., 2011. Spirochaeta

perfilievii sp. nov., an oxygen-tolerant, sulfide-oxidizing, sulfur- and thiosulfate-reducing

spirochaete isolated from a saline spring. Int J Syst Evol Micr. 61, 110-117.

[9] Evans, P.N., Parks, D.H., Chadwick, G.L., Robbins, S.J., Orphan, V.J., Golding, S.D., 2015.

Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric

metagenomics. Science. 350(6259), 434.

[10] Gyuseong, H., Seung, G.S., Kyungjin, C., Joonyeob, L., Woong, K., Seokhwan, H., 2018.

Temporal variation in bacterial and methanogenic communities of three full-scale anaerobic

digesters treating swine wastewater. Environ Sci Pollut R. 1, 1-10.

[11] He, Y., Li, M., Perumal, V., 2016. Genomic and enzymatic evidence for acetogenesis among

multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine sediments. Nature

Microbiology. 35.

11
[12] Holm-Nielsen, J.B., Seadi, T.A., Oleskowicz-Popie, P., 2009. The future of anaerobic digestion

and biogas utilization. Bioresour. Technol. 100, 5478-5484.

[13] Hu, J., Jing, Y., Zhang, Q., Guo, J., Lee, D., 2017. Enzyme hydrolysis kinetics of microgrinded

maize straws. Bioresour. Technol. 240, 177-180.

[14] Kerssen, T.M., 2015. Food sovereignty and the quinoa boom: challenges to sustainable re-

peasantisation in the southern Altiplano of Bolivia. Third World Quarterly. 36(3), 489-507.

[15] Li, Y.F., Chen, P.H., Yu, Z., 2014. Spatial and temporal variations of microbial community in

a mixed plug-flow loop reactor fed with dairy manure. Microb Biotechnol. 7, 332-346.

[16] Lönnqvist, T., 2017. Biogas in Swedish transport- a policy-driven systemic transition. PhD

Thesis. Royal Institute of Technology – KTH.

[17] Mader, T.L., Davis, M.S., 2004. Effect of management strategies on reducing heat stress of

feedlot cattle: feed and water intake. J Anim Sci Biotechno. 82(10), 3077-3087.

[18] Michael, H., Gerardi, A.J., 2003. The microbiology of anaerobic digesters. Wiley-Interscience,

A John Wiley & Sons, Hoboken, New Jersey.

[19] Nielsen, H.B., Angelidaki, I., 2008. Strategies for optimizing recovery of the biogas process

following ammonia inhibition. Bioresour. Technol. 99, 7995-8001.

[20] Pan, W.Z., 2011. Analysis of the large farms biogas projects-take the Beijing Deqingyuan

biogas project as an example. Engineering Sciences. 02.

[21] Weiland P., Rieger, C.H., Ehrmann, T.H., 2003. Evaluation of the newest biogas plants in

Germany with respect to renewable energy production, green house gas reduction and nutrient

management. Proceedings of the future of biogas in Europe-II. Denmark Esbjerg: University of

Southern Denmark.

[22] Wang, Y., Zhang, W., Dong, H., Zhu, Z., Li, B., 2017. Performance evaluation of a large-scale

swine manure mesophilic biogas plant in China. American Society of Agricultural and Biological

Engineers. 60(5), 1713-1720.

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Figure 1 Biogas production in PFR from January 2016 to March 2017 per month and the cumulative
biogas production

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Figure 2 Performance of PFR during the stable stage.(a) Volume biogas production rate and
Volume methane production rate; (b ) Variation of methane and carbon dioxide content in produced
biogas from PFR; (c) Variation of specific biogas and methane yield on per kg VS; (d) Variation of
chemical oxygen demand removal efficiency based on the in and out total chemical oxygen
demand during the anaerobic digestion process on cattle manure substrate

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Figure 3 Phylum level distribution of bacterial (a) and archaeal (b) community and genus level of
bacterial (c) and archaeal (d) community in substrate and digestate: the species, whose sums of
percentage in the samples are less than 1%, are classified as ‘the others’

15


 A novel approach for producing biogas in large scale from cattle manure was provided

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17