Renewable and Sustainable Energy Reviews 100 (2019) 110–126

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

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

Bioinformatics analysis of metagenomics data of biogas-producing microbial T

communities in anaerobic digesters: A review
Le Zhanga, Kai-Chee Loha, , Jun Wei Limb, Jingxin Zhangb
⁎

a
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, S117576 Singapore, Singapore
b
NUS Environmental Research Institute, National University of Singapore, 1 Create Way, Create Tower #15-02, S138602 Singapore, Singapore

ARTICLE INFO ABSTRACT

Keywords: Complex microbial communities in anaerobic digestion (AD) system play a vital role in the production of biogas.
Anaerobic digestion An in-depth understanding of the microbial compositions, diversity/similarity, metabolic networks, functional
Microbial communities gene patterns, and relations between biodiversity and system functions at the genome level could help to op-
Bioinformatics timize microbial productivity and contribute to enhancement of AD process. The study of microbial communities
Metagenomics
has been revolutionized in recent years with the development of high-throughput sequencing technologies.
Pyrosequencing
Artificial neural network
Analysis of high-throughput sequencing data and a suitable bioinformatics analysis approach therefore plays a
very critical role in the investigation of microbial metagenome. The present article reviews the overall procedure
of processing metagenomics data of microbial communities for revealing metagenomics characterization using
bioinformatics approaches. This includes (1) introduction of application case summary, (2) DNA extraction and
high-throughput pyrosequencing, (3) processing metagenomics data using function-based bioinformatics plat-
forms and tools, and (4) several specific bioinformatics analysis of anaerobic microbial communities. Key
findings on anaerobic digestion via bioinformatics analysis are summarized. Limitations and future potential of
bioinformatics approaches for analysis of metagenomics information of microbial communities are also dis-
cussed, with the hope of promoting its further development. Finally, a big-data-based precision fermentation
platform using artificial neural network is proposed for integrating the bioinformatics data of microbial com-
munities with performance of anaerobic digesters to facilitate the usage of huge metagenomics data.

1. Introduction biogas production in AD of organic wastes, it is vital to comprehen-

In recent years, renewable energy sources are receiving increasing
attention globally due to potential energy crisis and increasing en-
vironmental pollution caused by burning of fossil fuels [1]. In 2014, a
new ‘2030 Framework for Climate and Energy’ agreed by the European
Council proposed a 40% cut in greenhouse gas emissions compared to
1990 levels through the use of at least a 27% share of renewable energy
by 2030 [2]. Upgrading the existing biomass feedstock (i.e. food waste,
green wastes, animal manure, crop residues, and algae) to cleaner and
more sustainable energy carriers has unique potential to provide clean
and renewable energy, while simultaneously protecting the environ-
ment [3]. Biogas generated from anaerobic digestion (AD) of organic
wastes through biological functions of substantial anaerobic bacteria
and methanogenic archaea is one such attractive renewable and sus-
tainable energy carrier [4]. The complex microbial communities in
biogas-producing digesters play a significant role in maintaining stable
operation and efficient gas production [5–8]. In order to enhance
sively understand the involved microbial communities in terms of
taxonomic compositions, similarity and diversity, interaction networks,
metabolic networks and relations between operational conditions,
biodiversity, and system functions.
Over the past decade, the development of high-throughput se-
quencing technology and the decrease in its cost greatly facilitated the
wide applications of bioinformatics tools in the studies of metage-
nomics data of microbial communities in anaerobic digesters. This is
evinced by the increasing number of annual journal publications from
2008 to 2017 on “pyrosequencing” and “anaerobic” (based on search
results from Scopus and Web of Science databases) as presented in
Fig. 1.
These studies via bioinformatics tools such as MG-RAST [9], Me-
taVelvet [10], Genovo [11], etc., most of which borrowed from the
fields of data mining, artificial intelligence and statistics, have greatly
enriched our knowledge on microbial compositions and network-based
correlation analyses in biogas digesters under different operating

⁎
Corresponding author.
E-mail address: chelohkc@nus.edu.sg (K.-C. Loh).

https://doi.org/10.1016/j.rser.2018.10.021
Received 10 May 2018; Received in revised form 27 July 2018; Accepted 17 October 2018
Available online 02 November 2018
1364-0321/ © 2018 Elsevier Ltd. All rights reserved.
L. Zhang et al.
Fig. 1. Number of annual journal publications from 2008 to 2017 on “pyr-
osequencing” and “anaerobic” (based on search results from (A) Scopus data-
base and (B) Web of Science database). Trend lines are added via linear fitting
of the data.
conditions. For instance, it is now known that there is a clear correla-
tion between taxonomic and functional genes patterns of anaerobic
microorganisms in biogas-producing digesters [12]. Moreover, it has
been established that both taxonomic and functional patterns can be
influenced by environmental variables such as digester configuration,
feedstock components, temperature, organic loading rate (OLR), hy-
draulic retention time (HRT), and free ammonia concentration [13–15].
Additionally, based on the analysis of functional genes by metage-
nomics studies and network-based approaches, corresponding meta-
bolic pathways can be estimated, consequently pointing to the identi-
fication of the actual dominant metabolic pathways and mechanisms in
the biogas digesters [16,17].
Notwithstanding, how to effectively understand the underlying
biological features of microbial communities in anaerobic digesters
from large-scale sequencing data and how to use this knowledge to
maximize the biogas production are still big challenges, due to the
extremely complex microbial communities, immature gene sequence
analysis technology (e.g. standard procedure, platform and software
tools, storage space, and process speed), costly deep sequencing of
microbial metagenome [18], interpretation of microbial diversity data
containing methodological flaws [19], and fragmented analysis of
various microbial communities. Recently, Ju and Zhang [20] reported a
rigorous experimental design and bioinformatics analysis procedure for
the application of metagenomics in environmental sciences and bio-
technology, with the aim to facilitate more extensive application of
metagenomics in the microbe-environment interactions. Although the
metagenomics sequencing has been conducted on various physical en-
vironments (e.g. soil [21], sea [22], hydrogen bioreactor [23], and
petroleum hydrocarbon contaminated site [24]), the first metagenomics
analysis of microbial communities in biogas digesters was reported in
111
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
Fig. 2. The flow chart of analysis of metagenomics data of microbial commu-
nities in biogas-producing digesters by bioinformatics approaches.
2008, consistent with the statistical results in Fig. 1. Since then,
bioinformatics analysis based on metagenomics sequencing has been
used by many researchers to investigate microbial communities in
anaerobic digesters under various operating conditions. Nevertheless,
much of the related studies are diverse, scattered and not consolidated,
let alone identify the state-of-the-art. An appropriate review in this
topic seeks to fill this gap.
This paper provides a comprehensive technical explanation of the
procedure of using bioinformatics approaches to analyze metagenomics
data of microbial communities in biogas-producing digesters (Fig. 2),
with the hope of improving biogas production. A summary of the ap-
plication case was first presented in Section 2. This was followed by a
general introduction for the DNA extraction and high-throughput se-
quencing methods. A comprehensive review of bioinformatics analysis
of anaerobic microbial communities, including characterization of
taxonomic compositions, alpha and beta diversity analysis, taxonomic
pattern differences and similarities, multivariate statistical analysis and
microbial gene functional patterns analysis follows in Section 3. The
function-based bioinformatics platforms and software tools are also
discussed. Taking advantage of genome sequence information, the
latest progress related to anaerobic digestion operations made with
bioinformatics approaches is reviewed in Section 4. Finally, the lim-
itations and prospects of bioinformatics approaches for analysis of mi-
crobial metagenomics are alluded to in Section 5, which also discusses a
big-data-based artificial intelligence system via artificial neural net-
works for integrating information from various aspects of digester
configuration, operation parameters, feedstock characteristics, process
performance, and microbial communities.
2. Summary of application case and metagenomics data collection
of microbial communities
2.1. Application case summary and analysis
Partial case summary of using bioinformatics methods to investigate
L. Zhang et al.
Fig. 3. Statistical analysis of 82 research papers based on (A) feedstock, (B) country and (C) operation temperature.
microbial diversity and dynamics in biogas-producing digesters is pre-
sented in Table S1 of the Supplemental material. Eighty two typical
research papers in Table S1 in the past decade were statistically ana-
lyzed in terms of feedstock, country and operation temperature, results
of which are presented in Fig. 3.
As shown in Fig. 3A, the most dominant feedstock was food waste,
covering approximately 30% of total feedstocks, followed by sludge
(22.8%), manure (20.3%), agricultural and horticulture waste (15.2%).
Country-based analysis (Fig. 3B) indicated that the most active coun-
tries (> 3%) involving the microbial community analysis in biogas-
producing digesters were China, Denmark, Korea, Germany, USA, UK,
Singapore, Russia and Canada. Meanwhile, analysis on basis of AD
temperature demonstrated that percentage of mesophilic AD was 2.5-
fold higher than that of thermophilic AD (Fig. 3C), which can be at-
tributed to the fact that thermophilic AD consumed more energy for
maintaining higher temperature. However, thermophilic AD was re-
ported to have several advantages, such as preventing foaming by re-
ducing extracellular polymers [25], enhancing easily-degradable matter
digestion [26], and allowing higher OLR and methane productivity
[27]. It is foreseeable that more of thermophilic AD will be conducted
in the future. In addition, it is also noteworthy that several interesting
development trends have been observed over the past decade, from
operating single-stage digesters to two or three-stage digesters [28–30],
from single-country research to multinational cooperative research
[12,31–33], and from simple analysis targets like microbial composi-
tions to complex analysis targets like interaction mechanisms
[16,34–37].
2.2. DNA extraction, verification and storage
DNA extraction is a prerequisite for performing bioinformatics
analysis of metagenomics data of microbial communities. The quality of
DNA would be greatly affected by the different sampling methods [20].
Consequently, the appropriate choice of sampling method of anaerobic
digestate is essential. In order to reduce the experimental errors, it is
recommended to use the same sampling method in the whole experi-
ment. At the same time, repeated trials need to be carried out to obtain
112
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
good repeatability for a digestate sample. Digestate may be rinsed with
phosphate buffer solution and then stored at −20 °C for long-term
storage. Prior to DNA extraction, the digestate could be centrifuged for
10 min and the supernatant should be decanted. Then, a certain amount
(0.3–0.5 g) of wet weight sludge material is used to extract the DNA of
microbial communities. Table 1 summarizes the frequently-used DNA
extraction methods of microbial communities in anaerobic digesters. As
shown in Table 1, the most frequently-used method for DNA extraction
has been using commercial kits, amounting to more than 90% among
all the related studies. CTAB (cetyltrimethylammonium bromide) based
DNA extraction method (6%) is another method for DNA extraction. In
terms of commercial kits, more than 60% products are provided by
MoBio Laboratories (43%, USA) and MP Biomedicals (25%, USA), fol-
lowed by OMEGA (6%, USA), Macherey-Nagel (6%, Germany), Preci-
sion System Science (6%, Japan), and Intron biotechnology (2%,
Korea). Taken together, the USA has the overwhelming share of the
DNA kits market. Upon completion of extraction, the quality of the
extracted DNA can be checked by determining its absorbance at 260 nm
and 280 nm using equipment such as NanoDrop 1000 (Thermo Fisher
Scientific, Waltham, MA) [38]. The typical purity and concentration of
the extracted DNA are ~ 1.9 (Abs 260 nm/Abs 80 nm) and ~ 50 ng/uL,
respectively [39]. Finally, the purified DNA can be stored in TE (Tris-
EDTA) buffer at −20 °C until the next step.
2.3. Frequently-used DNA sequencing methods
Currently, next-generation sequencing (NGS) technologies are able
to quickly and economically perform qualitative and quantitative ana-
lysis of the microbial communities in various AD systems. The most
frequently-used DNA sequencing method is based on the Roche GS FLX
454 pyrosequencing platform [65], amounting in excess of 90% of the
surveyed studies. The rest of the NGS techniques are based on Illumina
(Solexa) sequencing platform [29,30], ABI SOLiD™ short-read DNA
sequencing platform [35], ABI analysis reagents (Big Dye terminator
mix) coupled with Applied Biosystems 3130xl Genetic Analyzer), and
Ion PGM™ Hi-Q™ sequencing kit (Thermo Fisher Scientific) coupled
with Ion PGM™ sequencer operated by Torrent Suite™ software [75].
L. Zhang et al.
Table 1
Frequently-used DNA extraction methods of microbial communities in anaerobic digesters.
DNA extraction methods (percentages)
MoBio PowerSoil DNA extraction kit (43%)
Fast DNA SPIN kit for soil (25%)
E.Z.N.A Soil DNA kit (6%)
NucleoSpin Tissue kit + NucleoSpin soil kit (6%)
Automated nucleic acid extractor (kit) (6%)
CTAB (cetyltrimethylammonium bromide) based DNA extraction method (6%)
I-genomic BYF DNA extraction kit (2%)
DNA extraction kit (2%)
ZR soil microbe DNA kit (2%)
PCR reaction mix (2%)
Currently, although the 454 pyrosequencing technique is increasing in
its use rapidly, the reference data for temporal profiling of anaerobic
microbial communities is still limited [82]. Thus, more related studies
on fundamental analysis of microbial metagenome and establishment of
storage platform should be conducted in detail.
3. Bioinformatics analysis of anaerobic microbial communities
Frequently-used bioinformatics analysis of metagenome of anae-
robic microbial communities are presented in Fig. 4. From this, it can be
seen that bioinformatics analysis of metagenome included several as-
pects, such as pretreatment of raw sequences (Section 3.1), OTUs
clustering analysis (Section 3.2), alpha diversity analysis (Section 3.3),
taxonomic compositions analysis (Section 3.4), microbial difference/
similarity analysis (Section 3.5), multivariate statistical analysis (PCA,
PCoA, NMDS, RDA/CCA) (Section 3.6), and functional gene patterns
prediction (Section 3.7). Each analysis needs corresponding platforms
and tools. Ju and Zhang [20] reported the platforms and software tools
available for the bioinformatics analysis of metagenomes, covering
multiple data processing functions including pretreatment, assembly,
binning and annotation. Similarly, Kumar and Chowdhary [23] re-
ported a variety of software tools coupled with functions and web links
for metagenome data analysis. However, these reported platforms and
software [20,83] had not been totally applied to the anaerobic micro-
bial metagenomics in biogas-producing digesters as they were derived
from various microbial environments such as soil, ocean, wastewater,
bioreactors, and biofilms, etc. In order to provide a more specific in-
troduction of bioinformatics analysis of huge metagenome data, espe-
cially for newcomers to microbial metagenomics, the frequently-used
Fig. 4. Frequently-used bioinformatics analysis of metagenome of anaerobic
microbial communities.
113
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
Product brands Refs.
MoBio Laboratories, CA, USA [29,30,38–56]
MP Biomedicals, Illkirch, France [57]
MP Biomedicals, Australia [58]
MP Biomedicals, Germany [59,60]
MP Biomedicals, USA [61–66]
Q-Bio gene, Australia [67]
Q-Bio gene, Carlsbad, CA, USA [68]
OMEGA, USA [69–71]
Macherey-Nagel, Germany [37,72,73]
Magtration System 6GC, Precision System Science, Chiba, Japan [74–76]
– [34,35,77]
Intron biotechnology, Korea [78]
Felix bio-tech, USA [79]
Zymo Research, Orange, CA, USA [80]
Clontech, Mountain View, CA, USA [81]
bioinformatics platforms and tools in microbial metagenomics-based
studies are summarized in Table 2. Although the surveyed literature
pool might be incomplete and slightly biased, information in Table 2
can still shed light on some general trends in the literature and provide
an example of the usefulness of bioinformatics analysis to analyze a
microbial community.
3.1. Pretreatment of raw sequences
After acquisition of the metagenome data (raw NGS reads), pre-
treatment of raw sequences is a very critical step to attain high quality
reads for downstream analysis. Many bioinformatics platforms and
software tools (Table 2) have been developed in the past decade for this
purpose. The platforms and software tools include Trimmomatic soft-
ware [84], ACE Pyrotag Pipeline (APP) [58], HMMER [54], MG-RAST
[35], ChimeraSlayer [47], RDP tools [100], and UCHIME [70,71,77].
The sequence pretreatment generally includes (i) removing adapters
and linkers, (ii) excluding chimeras and replication, and (iii) demulti-
plexing of barcoded samples and quality control. UCHIME is the most
cited tool to check and remove chimeras from the raw sequences while
MOTHUR and QIIME (http://qiime.org/) are currently the two most
frequently used platforms to denoise the metagenome data. After the
primary processing, the sequencing reads are grouped based on their
unique barcodes, and then the barcodes and primers are removed. For
further analysis, quality control has to be performed. For instance, the
quality control procedure implemented via the RDP Pyrosequencing
Pipeline online tools excludes all sequences that (i) are shorter than
150 bp (the value was adjustable), (ii) have more than one un-
determined nucleotide, (iii) contain any forward primer mismatches
and (iv) contain low quality base scores (Phred quality scores < 25)
before further analysis [100]. Additionally, a “pre-cluster” function is
generally utilized to merge the sequences with 1 bp difference.
3.2. OTUs clustering analysis
The multiple clean sequences are aligned using sequence aligners
such as MOTHUR [48], MUSCLE [85], INFERNAL aligner [100], Py-
NAST [66], and ClustalW [39] coupled with the bacterial and archaeal
database such as SILVA [77]. Subsequently, the aligned sequences are
clustered into operational taxonomic units (OTUs) with average
neighboring clustering algorithm via Usearch software (Usearch-global
command) [91] or various sequence classifiers like RDP Bayesian
Classifier [100], UCLUST-RDP classifier [90], and MEGA/MEGA5
[39,77,89], at usually 97% sequence similarity. Of note, normalization
of sample size can be conducted through the “sub.sample” function
using a software like MOTHUR by resampling the same number of reads
for all samples on the basis of the smallest sample size. OTU-based
L. Zhang et al. Renewable and Sustainable Energy Reviews 100 (2019) 110–126

Table 2
Frequently-used bioinformatics platforms and tools in microbial metagenomics-based studies.
Platform/software Programme/module Function Refs

Trimmomatic software – Reads trimming [84]

ACE Pyrotag Pipeline (APP) – Reads trimming [58]
HMMER Hmm-search program Reads trimming and alignment [54]
MUSCLE Multiple sequence alignment Aligned sequences [85]
ARB rRNA database BLAST Detected sequence reads [85]
MG-RAST – Quality control [35]
KEGG Metabolic pathways [12,86]
SEED Genomes annotation [12,84,86]
PCoA Principal coordinates analysis [12]
CCA Canonical correspondence analysis [12]
MG-RAST database/server Deposited filtered pyrosequencing data [54,63]
CLC Genomics workbench de novo assembly Genome assembler [84]
MOTHUR RDP classifier Quality control [87]
Bellerophon methods Removed chimera sequences [78]
Chao1, ACE, Shannon, and Simpson index Richness, abundance base coverage and diversity estimation [46,70,78,88]
UniFrac Principal coordinates analysis [88]
– Good's coverage analysis [70]
Against SILVA database Sequences quality check and aligned [48,77]
Sub.sample function Normalization of sample size [77]
FLASH – Merged paired-end reads [87]
Gephi – Topological analysis of interaction network [87]
MetaMIS – Microbial interaction analysis [87]
SILVA NGS server – Taxonomic classification [36]
Ray Meta assembler – Metagenome assembler [36]
AMPHORA2 – Taxonomic profiling [36]
CONCOCT – Contigs binning [36]
MEGAN The lowest common ancestor (LCA) Taxonomic assignment of gene; species richness analysis [42]
algorithm
MEGA/MEGA5 Neighbor-Joining method and Jukes-Cantor Construction of phylogenetic trees or neighbor-joining trees [39,77,89]
model
Ion Reporter NMS ordination Abundance-based difference visualization [75]
UCLUST RDP classifier Clustering OTUs [45,90]
UCHIME – Detected and removed chimeras [70,77]
UPARSE – Clustering OTUs [91]
USEARCH Fastq-filter command Demultiplexed reads trimmed [91]
Cluster-otus command Clustered dereplicated reads [91]
Usearch-global command Reads assigned to OTUs [91]
QIIME – Sequences denoised [80]
BlastTaxonAssigner Sequences assigned to taxonomy [58]
R software package Shannon index, Pielou's index Richness, diversity and evenness estimation [76,92]
PRIMER Bray-Curtis similarity measure Hierarchical cluster analysis and non-metric multidimensional [93]
scaling (NMDS) ordination
PyNAST Against Greengenes core reference set Aligned representative OTUs sequences [47,58,66,91]
Make-phylogeny.py script Built FastTree [91]
RAxML – OTU table and phylogeny construction [66]
PhyML – Phylogenetic inference [47]
BLAST against RDP database – Phylogenetic classification [94]
BLAST against COG database – Functional characterization [34,94]
BLAST2x against KEGG database – Functionally annotated reads [34]
BLASTN/BLASTX against GenBank NT/NR – Classification of sequencing data [42]
databases
BLASTX against NCBI-NR database – Metagenomes align [86]
BLAST2x against SWISSPROT database – Functional annotation of short metagenome contigs [34]
BLASTN against the EzTaxon-e database – Determined taxonomic positions of individual reads [54]
CLUSTAL_X Bio-Edit Multiple alignments of sequences; gaps edit [68]
PAST NMDS Beta diversity metrics visualization [44]
mPUMA and Trinity – Sequences reads process and assembly [95]
ChimeraSlayer – Removed chimeric sequences [47]
ClustalW – Aligned sequences [39]
INFERNAL aligner – Aligned multiple clean sequences [96]
SAMS – Sequences quality assessment [34]
GenDB genome annotation system – Functional annotation of long assembled contigs [34]
Reganor Pipeline – Prediction of coding sequences [34]
RDP (Ribosomal Database Project) RDP Aligner and Classifier; Tree Builder Sequences sorted, trimmed and aligned; biodiversity analysis; [77,97]
taxonomic classification of sequences
Fast UniFrac PCoA Evaluated differences of community structure [41,98]
CANOCO RDA (redundancy analysis) Visualized correlations between community structure and [74,98]
parameters
XLSTAT Canonical correspondence analysis (CCA) Determine correlations between microbial communities and [62,81]
reactors’ performance
STAMP Two-sided Welch's t-test Pairwise statistical comparison of taxonomy between two samples [99]

114
L. Zhang et al. Renewable and Sustainable Energy Reviews 100 (2019) 110–126

2% of the total 231 OTUs. This indicated that the changed parameter
(HRT) was strongly linked to the variation of bacterial dominance.

3.3. Alpha diversity analysis

For investigating the biological richness/diversity of microbial

Fig. 5. Venn diagram showing the total number of OTUs (0.03 distance) and
the number of shared OTUs among four samples in terms of bacteria (Data
adapted from Ref. [52] with permission/license granted by the publisher).
analysis on bacterial and archaeal sequences can then be further per-
formed. For instance, Yun et al. [52] investigated the microbial com-
munity dynamics during mitigation of ammonia inhibition by internal
dilution in high-rate anaerobic digestion of food waste leachate and
established the Venn diagram via R-Venn-Diagram package (Fig. 5). As
shown in Fig. 5, the total number of OTUs and the number of shared
OTUs among four samples (R1, R2, R3 and R4) could be easily observed
from the Venn diagram. Particularly, in terms of bacteria, there were
only five commonly shared OTUs throughout R1 to R4, accounting for
communities in various biogas-producing systems, the OTUs-based
alpha diversity analysis in terms of Chao1 richness estimator (Chao1),
abundance coverage-based estimator of species richness (ACE),
Shannon-Weaver diversity index (Shannon), and Simpson diversity
index (Simpson) can be performed using the MOTHUR package [69], R
software package with VEGAN library [92] and the RDP Pipeline [100].
The corresponding equations for obtaining various indices for alpha
diversity analysis are shown in Table 3. Of those indices, the Chao1,
ACE and Shannon show positive correlation with biodiversity of mi-
crobial communities, while Simpson shows negative correlation with it.
Particularly, the Chao1 and ACE indices can be directly used for cal-
culating the total number of species (abundance) in the microbial
communities while the Shannon and Simpson indices can be used for
estimating the biodiversity. Additionally, the higher the Good's cov-
erage, the more the sequencing results represent the real situation.
For various diversity indices, straightforward calculation using the
given equations to rapidly estimate biological diversity of microbial
communities is a big advantage [106]. However, the diversity indices
like Simpson index and Shannon index combine richness and evenness
components into a simple index through a single measure, leading to a
loss of relative roles of other different variables such as potential eco-
nomic, ecological, and social importance of individual species [107]. To
overcome the shortcomings of the biodiversity indices, complex

Table 3
Equation and index meaning of parameters for alpha diversity analysis.
Index Equation Note Ref.

Chao1 index Schao1 and Sobs are the estimated and observed OUTs number, [101]
respectively; n1 and n2 are the number of OTUs with 1 and 2
n (n 1)
Schao1 = Sobs + 1 1 sequences, respectively.
2(n2+ 1)
ACE index ni is the number of OTUs containing i sequences; Srare is the number [102]
of OTUs containing ‘abund’ number of sequences or less; Sabund is the
S n1
SACE = Sabund + rare + 2
ACE (for ACE 0.8); number of OTUs containing more than ‘abund’ number of sequences;
CACE CACE
‘abund’ is threshold value of dominant OTU.

Srare n1 2
SACE = Sabund + + ACE (for ACE 0.8).
CACE CACE

n1
CACE = 1
Nrare

abund
Nrare = i ni
i= 1

abund
2 Srare i = 1 i(i 1)ni
ACE = max 1, 0
CACE Nrare (Nrare 1)

abund
2 2
Nrare (1 CACE) i = 1 i(i 1)ni
ACE = max ACE 1+ , 0
Nrare (Nrare CACE)
Shannon- Wiener Sobs is actually observed OTUs number; ni is the number of sequences [103]
index Sobs in No. i of OTU; N is total number of sequences.
ni ni
Hshannon = ln
i= 1
N N
Simpson index Sobs is actually observed OTUs number; ni is the number of sequences [104]
Sobs in No. i of OTU; N is total number of sequences.
i=1 ni (ni 1)
Dsimpson =
N(N 1)
Good's coverage n1 is the number of OTUs with 1 sequence; N is total number of [105]
n sequences.
C=1 1
N

115
L. Zhang et al.
Fig. 6. Rarefaction curves for 16 S rRNA genes at different sequence similarities
(Data adapted from Ref. [97] with permission/license granted by the publisher.
For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article).
multivariate analyses such as canonical correspondence analysis
(Section 3.6) should be done to provide more valuable information on
environmental variables associated with the abundance and presence of
the species in a microbial community.
Additionally, alpha rarefaction curve has been mainly used to detect
the adequacy of sequencing. This reflects a relationship between se-
quencing numbers and species abundance, which is generally estab-
lished by randomly subsampling an equal number of sequences across
all OTUs using MOTHUR, QIIME, and R software. For alpha rarefaction
curves, the plateaus reflects adequate sequencing depth while the
monotonically increasing part reflects deficient sequencing depth. For
instance, Smith et al. [97] investigated the microbial community
structure of a pilot-scale thermophilic anaerobic digester treating
poultry litter and compared the rarefaction curves for 16 S rRNA genes
at different sequence similarities (Fig. 6). Results showed that, with
≤ 97% similarity levels, the rarefaction curve was asymptotic, in-
dicating that the sequence dataset had thoroughly sampled diversity in
Fig. 7. Schematic diagram of multi-level species taxonomy of the anaerobic microbial community using Krona [111].
116
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
this analysis and sufficient sequence depth was achieved at these levels.
Nevertheless, the numbers of OTUs at the 99–100% levels were still
rising, signifying much more undetected diversity at this level [97].
3.4. Taxonomic composition analysis
Taxonomic composition analysis is one of the most frequently-used
bioinformatics analysis for anaerobic microbial communities.
Generally, this analysis can be performed through two steps: (i) data-
base comparison and filtration and (ii) taxonomic classification of se-
quences. Particularly, sequences are firstly filtered through a BLAST
search against the sequence database such as SILVA database [36],
EzTaxon-e database [54], GenBank NT/NR database [42], and RDP
database [94], with a common confidence threshold of 80–90%. Sub-
sequently, the satisfactory sequences are phylogenetically assigned to
taxonomic classifications via an RDP naïve Bayesian classifier and
Bergey's taxonomy at a 0.03 distance [108,109]. Additionally, the
phylogenetic composition inference of bacterial communities can be
performed using the CL community software (Chunlab Inc., Seoul,
Korea) [54], PhyML [47], and MetaPhlAn [110]. The taxonomic clas-
sification analysis of microbial communities can be carried out in dif-
ferent levels including domain, phylum, class, order, family, and genus,
which can be visualized in a single diagram of multi-level species tax-
onomy of the whole microbial community through Krona software
[35]. Taking Fig. 7 as an example, microbial community compositions
from the interior to external circle can be presented at domain to order
level, which contributes to rapid analysis of the metagenome samples.
Particularly, within the bacteria domain, phylum Firmicutes, Bacter-
oidetes, and Chloroflexi stood out, while among the phylum Firmicutes,
the order Clostridiales was found in the highest abundance. The num-
bers accompanying the species names showed the corresponding
abundances, respectively.
To date, the analysis of microbial compositions in anaerobic di-
gesters under various operation conditions have been conducted on a
large scale. Therein, the most common results were dominance of
bacteria and archaea based on relative abundance. For instance, Zhang
et al. [30] designed a three-stage anaerobic digester for food waste and
L. Zhang et al. Renewable and Sustainable Energy Reviews 100 (2019) 110–126

Fig. 8. Taxonomic classification of the dominant bacterial groups from a three-stage digester at the genus levels (data adapted from ref. [30] with permission/license
granted by the publisher).

found that the most dominant populations (Fig. 8) in the first stage respectively, indicating that different bacteria were selectively enriched
(hydrolysis), the second stage (acidogenesis) and third third stage in different stages of the anaerobic digestion process.
(methanogenesis) were Trichococcus, Aminobacterium and Levilinea, In this same three-stage digester, co-digestion of food waste and

Fig. 9. Statistical analysis of the differences between relative abundance (Data adapted from ref. [99] with permission/license granted by the publisher).

117
L. Zhang et al.
Fig. 10. Ternary plot shows the distribution of bacterial (blue) and archaeal
(red) OTUs with > 1% average abundance (Data adapted from ref. [115] with
permission/license granted by the publisher).
horse manure led to greatly differing dominant bacteria
(Aminobacterium and Proteiniphilum) and archaea (Methanosarcina and
Methanobacterium) compared to those of mono-digestion of food waste,
indicating that different feedstock significantly affected the microbial
compositions [29]. The dominant bacteria and archaea corresponding
to different feedstocks (food waste [68], manure [49], activated sludge
[79], agricultural & horticulture waste [59,60], and industrial bioe-
thanol waste [45,73]) under different operation conditions in anaerobic
digesters are summarized in Table S1. It can be observed from Table S1
that the dominant bacteria and archaea were affected by the different
operation conditions including temperature [67], HRT [76], OLR [63],
additives [93], and pretreatments [43], etc. Moreover, it is worth
mentioning that taxonomic compositions of microbial communities in
similar digesters might also be different, which could be attributed to
the discrepancy in sampling strategies and sequencing methods.
Therefore, analysis of more metagenome samples in the near future is
still necessary to provide statistical supports for the findings of micro-
bial compositions in the previous studies.
3.5. Similarities/differences analysis of microbial taxonomic compositions
On the basis of microbial taxonomic composition analysis, the si-
milarities/differences analysis of different samples are performed and
the results can generally be presented in two ways - statistical analysis
plot and ternary plot. A pairwise statistical comparison of the taxo-
nomies between two samples has also been carried out using STAMP
[112] via two-sided Welch's t-test with the alpha level of 0.05. For in-
stance, Yu et al. [99] compared the relative abundance of microbial
communities from conventional anaerobic digester (CAD) and anae-
robic dynamic membrane bioreactor (AnDMBR) at the genus level and
Table 4
Frequently-used multivariate analysis techniques for microbial communities.
Multivariate analysis Raw-data-based data sets
Linear method
Unconstrained ordinationa PCA
Constrained ordinationb RDA
a
Unconstrained ordination refers to an ordination only using data of species compositions.
b
Constrained ordination refers to an ordination using data of species compositions and environmental parameters.
118
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
obtained the statistical analysis of the differences (Fig. 9). Generally,
the relative abundance is defined as the number of sequences corre-
sponding to a taxon divided by the total number of sequences per
sample (%). The results in Fig. 9 indicated that the AnDMBR contained
more abundant genus Methanosarcina and less abundant genus Metha-
nosaeta than the CAD in a statistically notable way. At the same time,
the total relative abundances of genus Methanosarcina and Methanosaeta
in the AnDMBR were higher than those in the CAD [99].
A ternary plot can also be used to visualize the distribution of the
shared and unique microbial taxonomic compositions in the different
samples [113]. Particularly, only the species existing in all the samples
are kept and imported into R software to construct a ternary plot using
the ‘ternary plot’ function found in the package ‘ggtern’ [114] based on
the relative abundance. The relative abundance of each species in each
sample is defined as the proportion of abundance itself to the total
abundance of that species in all samples. Each species is represented by
a unique point in a ternary plot, and the sizes of the points can be
adjusted according to the corresponding relative abundance. For in-
stance, Lu [115] investigated the distribution of different bacterial and
archaeal communities at the phyla level in the anaerobic granules from
digesters fed with dairy wastewater, brewery wastewater and cannery
wastewater by a ternary plot (Fig. 10), and found that most of high-
abundance bacterial phyla like Chloroflexi, Bacteroidetes and Proteo-
bacteria were shared by all three types of granules. At the same time,
phyla Methanosaeta dominated in both cannery and brewery granules
with more phyla Methanobacterium existing in cannery granules. By
means of the same scheme as Fig. 10, the similarity and specificity of
microbial communities between different types of any other anaerobic
digesters can be distinguished using ternary plots.
3.6. Multivariate statistical analysis
Based on a brief survey of the literature concerning anaerobic mi-
crobial community analysis, the most common multivariate analysis
techniques include principal components analysis (PCA) [58,61,67],
principal coordinate analysis (PCoA) [41,64,88], non-metric multi-di-
mensional scaling (NMDS) [53,56,73], redundancy analysis (RDA)
[39,74], and canonical correspondence analysis (CCA) [54,62,81]. The
similarity of these multivariate techniques is that each one is essentially
regarded as an ordination analysis, with a basic aim of depicting similar
objects near to each other and dissimilar objects further apart from each
other [116]. Generally, these techniques are classified into two groups:
unconstrained ordination analysis (PCA, PCoA and NMDS) and con-
strained ordination analysis (RDA and CCA) based on the types of used
data sets and computing methods (linear versus unimodal) (Table 4).
Previously, Ramette [116] published a good review on the multi-
variate statistical techniques in microbial ecology from the perspective
of methodology. Here, to help readers choose the most appropriate
method from Table 4 for analysis of metagenome of anaerobic micro-
bial communities, special emphasis is placed on the objectives, appli-
cations and features of the techniques. In unconstrained ordination
analysis, a significant difference between PCA and PCoA is that the
starting point matrix for PCoA is distance matrix of similarities or
Distance-based data sets
Unimodal method
– PCoA, NMDS
CCA –
L. Zhang et al.
Fig. 11. (A) Principle component analysis (PCA) ordination representing var-
iation in the composition of microbial communities at genus level. PC1 and PC2
explained 62% and 12% of the total variation, respectively. The location and
length of the arrows indicated organism scores along the respective principal
components (Data adapted from ref. [58] with permission/license granted by
the publisher). (B) Weighted principal coordinates analysis (PCoA) of bacterial
communities during four process stages at stage 1 (day 45, OLR 1.0 g VS/L/d),
stage 2 (day 73, OLR 1.3 g VS/L/d), stage 3 (day 132, OLR 1.5–1.8 g VS/L/d)
and stage 4 (day 289, OLR 0.5–1.0 g VS/L/d), respectively. Symbol designation:
diamond Stage 1; square Stage 2; triangle Stage 3; times Stage 4 (Data adapted
from ref. [81] with permission/license granted by the publisher).
dissimilarities, which is different from the initial data matrix of PCA.
Even so, both PCA and PCoA have found increasing number of appli-
cations in analysis of complex metagenome data. For instance, PCA was
used to compare the difference microbial community structures among
the multiple groups [29,67]. In Fig. 11(A), PCA ordination represented
variations in the compositions of microbial communities at the genus
level with changes of temperature and HRT [58], indicating that PC1
had a strong correlation with temperature (ρ = 0.97, p < 0.001); at
same time, HRT was correlated with PC2 (ρ = -0.85, p < 0.001). PCoA
was frequently used to evaluate the beta diversity of microbial com-
munities using the RDP Pipeline [117], QIIME [64], MOTHUR [88],
and MG-RAST [12]. For instance, PCoA was used to compare popula-
tions of different digesters using the same biosolids stabilization
method and compared the population differences among samples from
the different stabilization methods [41]. In another example as shown
in Fig. 11(B), the changes in bacterial community structure in response
to OLR was analyzed with PCoA, indicating that the triplicate digesters
behaved similarly at each stage [81]. Furthermore, microbial commu-
nity structures significantly shifted from stage 1 to stage 4 with dif-
ferent OLR. Although divergence in community structure between stage
1 and stage 2 was insignificant, the community structure in stage 3 was
significantly altered by the increased OLR. In stage 4, the decreased
OLR pushed the bacterial community structure return to those of stage
1 and 2, but not completely [81]. Notably, it was more difficult for
PCoA to interpret variable contribution due to the fact that PCoA could
not provide a direct link between principal components and environ-
mental variables, and PCoA was recommended over PCA when missing
lots of input data [116].
Nonmetric multidimensional scaling (NMDS), another un-
constrained ordination analysis, is widely used to visualize the diversity
119
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
and similarity of microbial community structures in anaerobic digestion
process over time [74,82], or in multiple digesters under different en-
vironmental parameters such as varied OLR and temperature [39,63]
and additive (e.g., zeolites [118]) using the R package vegan [119].
Essentially, NMDS analysis transforms and condenses the complex
community composition data sets to points in a low dimensional space
where more similar data sets are plotted close together and less similar
ones are plotted with greater ranked distances, showing a clear view of
the relationships of the bacterial and archaeal communities among the
tested samples [116]. In contrast, NMDS and PCoA can use any simi-
larity distance matrix (e.g. Euclidean, Bray-Curtis, and Jaccard) while
PCA can only use the Euclidean distance matrix [120]. However, NMDS
is generally more computer resource intensive than PCoA and PCA
[116], leading to a longer time for NMDS analysis.
In constrained ordination analysis, RDA and CCA are two fre-
quently-used methods in analysis of metagenome data of microbial
communities. These two methods are very similar, except that RDA is
based on linear models whereas CCA is based on unimodal species-
environment relationships [120]. To help users select which method
should be adopted, a procedure [121] using detrended correspondence
analysis (DCA) can firstly be conducted. The procedure mainly includes
four steps i.e. (i) selecting ‘DCA’ for the indirect gradient analysis in the
software (e.g. CANOCO), (ii) selecting detrending by segments, (iii)
selecting the Hill's scaling of ordination scores and (iv) selecting the
other options to run the analysis [121]. By comparing the analysis re-
sults of gradient length with 4.0 and 3.0, the more appropriate method
can be quickly identified. Particularly, unimodal methods such as CCA
should be used when value of gradient length was larger than 4.0;
whereas the linear method is probably a better choice if the value is
shorter than 3.0. In the range between 3 and 4, both RDA and CCA work
reasonably well [121]. Since RDA and CCA are very similar, a brief case
analysis taking RDA as an example was provided. For instance, in Fig.
S1, RDA was used to evaluate the operational and environmental fac-
tors that affected the spatial variability and temporal variability of the
microbial community [39]. The RDA ordination was performed using a
software CANOCO (version 4.5, Wagenignen, The Netherlands). It was
demonstrated that the bacterial community structure was affected by
the digester performance and operational parameters (e.g. pH, HRT,
and temperature) [39]. In a typical CCA plot, the arrows represent
different environmental factors (e.g. operational conditions), and the
longer the ray is, the greater the influence of the environmental factor.
When the angle between the environmental factors is acute, the positive
correlation between these two environmental factors is presented,
whereas an obtuse angle refers to a negative correlation [120]. CCA has
been performed using XLSTAT software to describe the correlations
between microbial communities (bacterial and archaea) and reactors’
performance and operational conditions, including temperature, pH,
alkalinity, and volatile fatty acids (VFAs) [62,81,122]. Another inter-
esting example in the anaerobic digestion systems for treating organic
urban residues is the study of the differences between the bacterial
community composition at class level in sampling times and physico-
chemical parameters of the digesters [123].
3.7. Gene functional annotation and application
To gain insights beyond the taxonomic compositions, functionally
annotation of the metagenomics data can be conducted using bioin-
formatics platforms or tools like BLAST against databases (e.g. SWISS-
PROT [124], COG [34], KEGG [34]), MG-RAST (SEED) [84], GenDB
[125], and IMG/M [126]. Specifically, BLAST against various databases
have been mainly used for the functional annotation of short meta-
genome contigs while GenDB genome annotation system is generally
responsible for functional annotation of long assembled contigs [125].
MG-RAST server is a SEED-based environment that allows users to
upload metagenomes for automated analyses [84]. IMG/M provides
users a genome annotation pipeline and comparative analysis of
L. Zhang et al.
microbial genomes on three dimensions (genes, genomes and functions)
by using several tools like KEGG [127], InterPro [128], Pfam [129], and
Gene Ontology [130]. Hitherto, albeit numerous bioinformatics ap-
proaches have been developed for gene functional annotations, part of
them such as single genome gene prediction tools are not well suited for
complex metagenomics datasets of anaerobic microbial communities
due to the heterogeneous sequence composition, length and errors
[131,132]. Undeniably, powerful gene annotation tools (e.g. IMG/M +
KEGG) can still be widely applied to locate protein coding regions,
which is one of the essential issues in microbial metagenomics studies.
Such an example was the study by Campanaro et al. [84], which made
use of bioinformatics tools (e.g. KEGG, COG and SEED) to profile
functional interactions between microbial species involved in the AD
process and identify key microbial genomes encoding enzymes involved
in the specific metabolic pathways. Moreover, MEGAN can also be used
for functional analysis of multiple metagenomes based on the SEED
hierarchy and the KEGG pathways [86]. Thereby, it is expected that
further studies performed under different operational conditions (e.g.
different substrate and temperature) will allow, in the near future, an
enrichment of the anaerobic microbial genome database and corre-
sponding applications via the increasingly powerful software and al-
gorithms.
4. Key findings on anaerobic digestion via bioinformatics analysis
Many researchers have successfully applied bioinformatics analysis
on microbial metagenomics from anaerobic digesters to identify
dominant bacteria and methanogenic archaea [12,84,133], monitor
microbial community shift [76,98,134], explore methanogenesis
pathway [42,43], and elucidate correlations between digester perfor-
mance, microbial community structures and pertinent environmental
variables [33,135,136]. To help researchers in anaerobic digestion field
quickly know about the latest developments, the progress made mainly
on AD optimization, resilience and robustness, metabolic mechanisms,
microbial modeling and control as well as antibiotic resistance genes
analysis via bioinformatics approaches are summarized.
4.1. Progress on optimized AD processes
Identification of predominant bacteria and methanogenic archaea
was most frequently conducted during the optimized AD processes via
various enhancing strategies such as optimization of operational para-
meters [137], supplementation of additives [138], pretreatment of
substrates [139] and structure optimization of digesters [29,30], etc.
The identification results contributed a lot to explanation of the en-
hancing mechanisms of the diverse techniques. For instance, during
activated carbon enhanced anaerobic digestion of food waste, Zhang
et al. [138] concluded that operation stability of anaerobic digestion
process in lab- and pilot-scale digesters were greatly enhanced by ac-
tivated carbon supplementation, and demonstrated that the abundance
of the predominant phyla Firmicutes, Elusimicrobia and Proteobacteria
were selectively enhanced by 1.7–2.9 times due to supplementation of
activated carbon using pyrosequencing of 16 S rRNA gene. In another
example of optimized anaerobic digestion of oil palm biomass by op-
timizing of total solids (TS) contents, feedstock to inoculum (F:I) ratios
and carbon to nitrogen (C:N), Suksong et al. [137] demonstrated that
the highest methane yield was achieved at TS content of 16%, C:N ratio
of 30:1 and F:I ratio of 2:1. The enhancement of AD process was at-
tributed to the selective enrichment of bacteria Ruminococcus sp. and
Clostridium sp. and enriched archaea Methanoculleus sp. [137]. Re-
cently, by pyrosequencing analysis, it was also found that different
microbial communities in terms of hydrolyzing bacteria, acidogenic
bacteria and methanogenic archaea, corresponding to hydrolysis,
acidogenesis and methanogenesis, respectively, were selectively en-
riched in the three separate chambers of the three-stage anaerobic di-
gesters [29,30]. Additionally, the high throughput 16S rRNA amplicon
120
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
sequencing was recently used to investigate the methanogens and
syntrophic bacterial species that were difficult to isolate and cultivate,
among microbial communities in biological biogas upgrading systems
with external hydrogen [140–143]. It has been found that addition of
external hydrogen can selectively enrich both hydrogenotrophic me-
thanogens and homoacetogenic species such as Acetobacterium woodii
and Moorella thermoacetica that produce acetate from H2 and CO2
[144,145]. More similar examples of using bioinformatics methods to
investigate the microbial dynamics were listed in Table S1 in the
Supplementary material. In each case, the feedstock, AD operation
conditions, reactor performance and major findings related to the mi-
crobial communities were provided.
4.2. Progress on understanding of microbial function and connectivity
Well understanding of the microbial function and connectivity is a
prerequisite to control and engineer the AD process with the aim to
optimize its efficiency [12]. In recent years, lots of progress towards
functional characterization and interactions of key microbial species in
biogas-producing digesters has been made with the help of bioinfor-
matics platforms and tools (Section 3.7). For instance, metagenomics
analysis and functional characterization of the biogas microbiome using
high throughput shotgun sequencing and a novel binning strategy were
performed to disclose nearly one million genes and extract 106 mi-
crobial genomes [84]. As a result, several key microbial genomes en-
coding enzymes involved in metabolic pathways including amino acids
fermentation, fatty acids degradation, carbohydrates utilization and
syntrophic acetate oxidation were identified [84]. Utilizing functional
annotation methodology, consolidated with the KEGG Orthology da-
tabase, a new uncultured archaeon associated with Methanomassilii-
coccales was identified and was putatively having a methylotrophic
methanogenic pathway [84]. Similarly, Campanaro et al. [146] eluci-
dated the microbial ecology in twelve thermophilic and mesophilic full-
scale biogas plants via a genome-centric metagenomic approach. In this
study, 132 Metagenome-Assembled Genomes (MAGs) were identified
using Prodigal (v2.6.2) software and their abundance profiles in dif-
ferent samples were analyzed with FOAM software [146]. The anno-
tation study provided a clue for the identification of the most abundant
core members of the anaerobic digestion microbiome [84]. The net-
work analysis shed light on species-species associations and allowed
investigation of syntrophic interactions between core members
[16,146]. Furthermore, by investigating the comprehensive biochem-
ical pathways in multiple samples and its link with different aspects of
information (e.g. environmental factors, genomes abundance profile
and predicted functional pathways), this study [146] revealed im-
portant relationships existing between the high-abundant core species
(e.g. acetate utilizers) of the thermophilic digesters, their metabolic
pathways and the crucial operational parameters including feedstock,
temperature, VFA concentration and methane yield, thus provided a
promising widely used strategy for the future studies to predict the
functional role of microbes participating in anaerobic decomposition of
organic wastes.
In terms of microbial connectivity, progress in connectivity of mi-
crobial communities during the anaerobic digestion process was re-
cently reviewed by De Vrieze and Verstraete [147], who concluded that
a richer knowledge base on microbial communication in anaerobic di-
gesters through quorum sensing and nanowires should be further es-
tablished. To this end, methods to detect centers of concentrated ac-
tivity and the highly active and well-connected species with a central
role in the anaerobic digestion process have to be firstly optimized
[147]. In addition, light sheet fluorescence microscopy was recently
reported as a promising tractable tool for examining microbial com-
munities [148]. However, most of the current studies are based on
eukaryotic cell tracking experiments; the further studies on examining
more and different microbial species and engineered strains that allow
delineation of how specific genetic factors affect microbial connectivity
L. Zhang et al.
remain to be conducted.
4.3. Progress on exploration of methanogenesis pathway
Sufficient understanding of methanogenesis pathways could sig-
nificantly contribute to treating wastes and producing biogas with high
efficiency through AD process. In this aspect, metagenomic sequencing
analysis coupled with radioisotopic analysis has been successfully ap-
plied to reveal the dominant methanogenic pathways in biogas reactors
treating either sludge or manure [12]. The results exemplified a dis-
crepancy between the metabolic patterns revealed by metagenomics
analysis and metabolic pathways determined by radioisotopic analysis.
Specifically, the aceticlastic methanogenic pathway was dominant in
radioisotopic experiments, while the hydrogenotrophic methanogenic
pathway was dominant in metagenomics analysis [12]. Similarly, stu-
dies in metabolic pathways and networks of microbial communities
using bioinformatics analysis are available in the optimized AD sys-
tems. Zhang et al. [16] tracked the changes in biometabolic pathways
and the microbial network using the KEGG pathway analysis and
taxonomic tree analysis in an optimized anaerobic digester for food
waste by incorporating activated carbon. The KEGG pathway analysis
revealed that the propanoate metabolic pathway that converted pro-
panoic acid to acetic acid became more prominent in activated carbon
enhanced AD digesters. The biometabolic pathways of lipid metabolism
and methane metabolism were also found to enhance [16]. Microbial
network analysis revealed that activated carbon promoted the pro-
liferation of syntrophic microbial species like Geobacter and Methano-
saeta, forming a highly intensive syntrophic microbial network [16].
Recently, Jiang et al. [149] examined how a pyrosequencing technique
using a fragment of the methyl Co-A reductase gene (mcrA) and a
radiolabelling technique using 14C labelled sodium acetate can be ap-
plied to investigate the relationship between total ammonia nitrogen
(TAN) and the dominant methanogen pathway by quantifying the
percentage of methane formation through acetoclastic and syntrophic
acetate oxidation pathways in anaerobic digesters. The ratio between
14
CO2 and 14CH4 in high TAN (11.1 g/kg wet weight) digesters ranged
from 2.1 to 3.0, showing 68–75% of methane was produced via the
hydrogenotrophic route; whereas in low ammonia (0.2 g/kg wet
weight) digesters the ratio was 0.1–0.3, revealing 9–23% of methane
was produced by the hydrogenotrophic route [149]. A quantitative
correlation between TAN and the dominant methanogen pathway can
be further optimized and utilized to manipulate the acetoclastic and
syntrophic methanogenesis. During the acclimation to extremely high
ammonia levels (10 g NH4+-N /L) in continuous AD process, 16 S rRNA
gene sequencing revealed a dramatic microbiome change throughout
the ammonia acclimation process [150]. A critical point of NH4+-N
concentration for a clear shift from aceticlastic to complex and flexible
pathways was found to be 5 g/L [151]. At extreme ammonia levels
(> 7 g NH4+-N /L), Clostridium ultunense, a syntrophic acetate oxi-
dizing bacteria, alongside with hydrogenotrophic methanogen Metha-
noculleus spp. increased significantly, indicating strong hydro-
genotrophic methanogenic activity [150]. Additionally, effect of
temperature (55–65 °C) and hydraulic retention time (2–4 days) on
methanogenesis pathways was studied in anaerobic digesters treating
with waste activated sludge [58]. Stable isotopic signatures (δ13C)
analysis showed that elevated temperature stimulated syntrophic
acetate oxidation [58]. Community analysis using 16S rRNA pyr-
osequencing evidenced the dominance of Methanosarcina at 55–60 °C,
whereas a further increase to 65 °C led to loss of Methanosarcina,
alongside with reduced methane yield and an accumulation of VFAs
[58]. And the similar trend was also applied to the case of reducing the
HRT to 2 days [58].
4.4. Progress on modeling and control of microbial community dynamics
In recent years, there has also been some progress made towards
121
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
community dynamics modeling and how the physicochemical and op-
erational parameters controlled these dynamics. For instance, to facil-
itate rational control and intervention in microbial communities,
Hanemaaijer et al. [152] introduced systematically the modeling ap-
proaches for microbial community studies from metagenomics to in-
ference of the community structure. They focused on two types of
models, a mathematical model that could integrate existing physiolo-
gical, genomic, and physicochemical information with metagenomics
data and a simpler coarse-grained model that could solve the inference
problem from the experimental data [152]. The former model has been
mainly studied and used due to its capability of maximizing information
content and predictive power. The latter model has received re-
markably little attention, albeit it can potentially be optimized to be
more detailed genome-scale stoichiometric models that can act as het-
erogeneous data integrators. Similarly, Succurro et al. [153] provided
an overview of mathematical models of natural microbial ecosystems
and emphasized that the choice of specific problem scale was a critical
point in the development of a theoretical description of a microbial
community. A recent expert consensus is built that it is time to develop
more extensively hypothesis-driven methods, metagenomic microbial
interaction simulators (e.g. MetaMIS [154]) and mathematical in-
tegration tools (e.g. MetaTopics [155]) to advance the field of commu-
nity dynamics modeling from a purely descriptive representation to a
sound biogeochemical theory [153,156–159]. More recently, a critical
review on mathematical modeling of microbial ecosystems was pub-
lished by Succurro and Ebenhöh [160], who focused on two major
classes of mathematical methods (constraint-based stoichiometric
models and differential equation models) and introduced their recent
applications to the study of microbial communities. The advantage of
these two models is that they are deterministic approaches that can
effectively provide a macroscopic representation of biological systems
from microscopic behaviors [160]. To go a step further, an integration
modeling method was illustrated in order to understand emergent
patterns in microbial systems and their dynamics regulated by diverse
spatio-temporal phenomena [160]. Taken together, it was re-
commended that modelers and experimentalists should work together
from the conceptual phases of the experimental design to guarantee
exact integration of theory and experiments [153]. In this way, more
common interdisciplinary languages can be developed that can help
unravel the mechanisms among the complex microbial interactions.
Knowledge obtained from the modeling of microbial communities
provides the possibility of optimizing the AD process by regulating the
microorganisms. In this light, some progress in microbial community
structure regulation has been made. Feedstock composition, digester
configuration, operating parameters (e.g. temperature), and environ-
ment conditions are the leading driving factors for community structure
changes [161]. For instance, in the anaerobic digesters of pig manure,
the major genus at pH 7.0 was Methanocorpusculum whereas that was
Methanosarcina at both pH 6.0 and 8.0, indicating that predominant
species in a microbial community can be controlled through pH [162].
Moreover, there has also been study focusing on developing early traffic
light system to avoid system failure through using alkalinity as an in-
dicator of process stability in an anaerobic digestion system [163–165].
However, as imaging the complex three-dimensional multi-species
communities still presents considerable technical challenges, how such
information can be used sufficiently for current AD applications is still
under development.
4.5. Progress on identification and analysis of antibiotic resistance genes
In order to properly define the risks posed by land application of
anaerobic digestate, high-throughput sequencing-based metagenomics
approaches have been successfully utilized to detect the antibiotic re-
sistance genes (ARGs) in biogas digesters. Lee et al. [166] characterized
the diversity and amounts of ARGs in representative organic wastes
(manure, sludge and food waste-recycling wastewater) and found that
L. Zhang et al.
Fig. 12. A big-data-based precision fermentation platform using artificial neural network.
total relative amount of ARGs were highest in manure, followed by
sludge and food waste-recycling wastewater. Diversity and mechanisms
of ARGs were considerably different between substrates [166]. In a
recent study, Luo et al. [33] found that the total abundance of ARGs in
digesters fed with manure and industrial wastes varied from 7 × 10-3 to
1.1 × 10-1 copy of ARGs/copy of 16 S rRNA gene. The samples obtained
from thermophilic biogas digesters exhibited a lower total abundance of
ARGs [33], which showed the superiority of thermophilic AD for ARGs
removal [167,168]. More recently, by using Illumina MiSeq sequencing
and high throughput fluorescent quantitative PCR, antibiotic resistant
bacteria (ARB) and ARGs from pig manure to fields were monitored
[169]. The results indicated that 83 ARGs and 3 transposons genes were
detected. Relative abundance of Macrolide-Lincosamide-Streptogramin
and tetracycline resistance genes reduced after anaerobic digestion.
Nevertheless, the relative abundance of aminoglycoside, sulfa, florfe-
nicol, amphenicol and chloramphenicol resistance genes were enriched
by 52–270 times [169]. Long-term application of biogas residue con-
taminated with antibiotics in fields increased the number of ARB and
the relative abundance of ARGs in the vegetable soil [169]. Hence, to
reduce the spreading of ARGs in the agricultural environment, more
effective prevention and control measures remain to be explored. In
another research performed in anaerobic digesters fed with microwave-
pretreated sewage sludge, most ARGs concentration tended to decrease
in pretreatment but enriched after AD [170]. Therefore, the microwave
pretreatment is a very promising technology that can reduce the
abundance of ARGs in bio-fertilizers made from anaerobic digestate. In
addition, for enhancement characterization and quantification of ARGs
in environmental metagenomes, an open online analysis pipeline,
ARGs-OAP, was developed consisting of a database termed SARG ver-
sion 2.0 [171]. The new SARG database contains sequences not only
from CARD and ARDB databases, but also sequences from the latest
protein collection of the NCBI-NR database [171]. ARGs-OAP and the
database can be accessed through http://smile.hku.hk/SARGs, which
offer huge potential in respect to more comprehensive identification
and analysis of ARGs.
5. Limitations and prospects of bioinformatics analysis on
microbial metagenomics
Metagenome analyses and metatranscriptome analyses of biogas-
producing microbial communities are rapidly developing in many
countries (Fig. 3B). However, there are several technical bottlenecks
that need to be addressed before bioinformatics analysis on microbial
metagenomics become a welcome tool.
Firstly, the rapid development of sequencing techniques has
122
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
resulted in the increase in depth of sequencing, which has led to a
critical issue about metagenomics data storage. For instance, the 454
pyrosequencing technology can generally produce up to 1000,000 reads
per sequencing run (equivalent to around 0.7 GB), while HiSeq. 2500
can generate a much greater output (600–1000 GB per run) [172,173].
Further analysis of raw sequences will enlarge the amount of data by
ten to twenty times each analysis time [173]. Such a huge amount of
data has become a challenge for bioinformatics analysis [132]. More-
over, the NGS-based metagenome research on anaerobic microbial
communities is still in its infancy. Analysis of more microbial meta-
genomes newly sequenced is still required to provide statistical support
for the findings derived from the previous studies, which also leads to
the necessity for a larger storage space. Thus, a standard storage plat-
form should be established and used to investigate metadata of anae-
robic microbial communities in the future research.
Secondly, many bioinformatics tools and methods for data proces-
sing have been borrowed from the fields of data mining, artificial in-
telligence and statistical methods [174]. However, the characteristics of
microbial genomic data may differ significantly from those of the ori-
ginal data for which the tools were developed [174]. Though many
computational methods have been successfully applied for genomic
data analysis, some challenges still exist. Currently, assembly and bin-
ning of super-large scale of sequences remain an insurmountable hurdle
[83], which can be attributed to the limited computing capability and
specific internal storage capability. Furthermore, there exists a paradox
between data processing speed and accuracy due to the fact that
taxonomic binning and assembly generally need several hours to sev-
eral days [173]. Thus, on the premise of high enough accuracy, more
efforts are needed to develop new powerful bioinformatics methods and
effectively integrate supercomputing technology into current proces-
sing tools to rapidly complete the analysis on microbial genomic data.
Thirdly, the relatively high cost of sequencing has led to the fact
that some metagenomics studies were performed lacking repeated trials
[20], which could compromise the data reproducibility, making it hard
to confirm whether the observed differences were significant or merely
artefacts of the analytical techniques. Therefore, sequencing in tripli-
cate is recommended for a more reliable data analysis. According to a
recent study from Campanaro et al. [175], reliability of the 16S rRNA
amplicon sequencing results could be biased by two main effects, which
were the failure of universal primers to match all the 16S rRNA targets
and the limited number of hypervariable regions (e.g. V4) studied.
Thus, to improve the evaluation of abundance for crucial taxonomic
groups, use of multiple marker genes and analysis at transcriptional
level are recommended [175]. Moreover, to date, most bioinformatics
analysis depended on sequence comparison against reference databases.
L. Zhang et al.
However, practices to use current databases for bioinformatics analysis
can be problematic considering the potential incompleteness of data-
bases. Therefore, updated databases with better quality data are needed
to support stable and effective analysis of anaerobic microbial meta-
genomics information.
Fourthly, metagenomics techniques provide deeper insights into
microbial communities, but have been generally used for descriptive
and explanatory approaches to reply the initial ‘who is there?’ question
[132]. The challenges that exist between lab-, pilot- and field-scale data
and extrapolation are still existing. In fact, there is still a major gap
between what we can observe from the microbial communities via
bioinformatics approaches and what species and gene functions we can
manipulate, due to the fact that a microbial ecosystem is an extremely
complex network of dynamic spatio-temporal interactions among mi-
croorganisms as well as between microorganisms and the ambient en-
vironment [176]. To fill this gap, more insights should be provided into
how the genetic properties and the dynamic connectivity between in-
dividual microorganisms generate their dynamic activities. Further-
more, concentrations and fluxes of nutrients within the microbial
communities should also be investigated thoroughly.
Last but not least, the current depth of mining on metagenomics
data of anaerobic microorganisms, mainly focusing on straightforward
sequence similarity searches, might not be satisfactory since microbial
genomes ought to be the fundamental basis for microbial ecology
[177]. For deeper data mining, the combination of metagenomics-based
bioinformatics methods with key approaches in biotechnology (e.g. T-
RFLP, FISH, PCR-DGGE and qPCR [178]) is highly recommended
[5,23]. Even so, there still may be a long way to reach the ideal point
where the digester performance can be finely regulated based on real-
time analysis of metagenomics information of anaerobic microbial
communities. To fulfill such endeavors and continue to provide in-
sightful solutions, a big-data-based precision fermentation platform
using artificial neural network is proposed (Fig. 12). Through an arti-
ficial intelligence (AI) workflow, the huge data (e.g. elemental com-
ponents of feedstock, operational parameters, and metagenomics data
of microbial communities) derived from substantial previous AD prac-
tices can be formulated into convolutional neural networks, which can
be further optimized into valuable networks such as deep Q network
using the experimental data as training information. In the valuable
network, with new feedstock components input, a best combination of
controlling parameters, digester type and microbial community in-
formation can be recommended to facilitate the practical AD opera-
tions. Although there are only a few research reports [179–181] based
on the implementation of AI approaches in the past decade, the im-
plementation of AI in the simulation, control and optimization of
anaerobic digestion possesses great potential in the near future, parti-
cularly with the fast development of novel and hybrid AI approaches.
For instance, a novel algorithm for designing minimal microbial com-
munities with desired metabolic capacities was developed [182]. Spe-
cifically, this algorithm can help researchers identify minimal sets of
microbial species that can collectively provide the enzymatic capacity
required to synthesize a group of desired target metabolites from a
predefined set of available substrates [182]. This finding may present a
first step towards the goal that various microbial communities can be
manipulated for the anaerobic digesters’ optimization and industrial
applications.
6. Conclusions
In the past decade, considerable research on metagenomics of
biogas-producing microbial communities has provided insights into
their diversity and shift. Many new applications of bioinformatics
technology on anaerobic digestion have emerged in the recent years,
which offer huge potential in the optimized biogas industries with
higher efficiency and sustainability. There are still several challenges
(e.g. data storage, data processing technique, data reliability and data
123
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
application) that should be overcome before microbial communities can
be finely regulated for the enhanced and commercial biogas production
process. The present review indicates that most of the current chal-
lenges can be overcome by substantial collaboration among scholars
from fields of biological sciences, environmental engineering, chemical
engineering and computer science. Nevertheless, more research ded-
ication is needed to integrate these methods and tools as an essential
strategy for the development of molecular microbiology for industrial
applications. Herein, a big-data-based precision fermentation platform
using artificial neural network, specifically, is likely to be at the fore-
front of these developments. Hence, it is possible to maximize the
methane yield in a commercial biogas plant using the precision fer-
mentation platform coupling with versatile bioinformatics approaches
in the near future.
Acknowledgements
We greatly thank the reviewers for their review and constructive
comments on this manuscript. This research project was funded by the
National Research Foundation, Prime Minister's Office, Singapore under
its Campus for Research Excellence and Technological Enterprise
(CREATE) Programme. The authors greatly thank Associate Professor
Yen Wah Tong from NUS for his help in facilitating some of the re-
search. Le Zhang would like to thank the China Scholarship Council
(CSC) for financial support. For all copyrighted figures including
Figs. 5, 6, 8, 9, 10 and 11, the permission attained from the publishers
for adaptation and reuse in the present work has been acknowledged.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.rser.2018.10.021.
References
[1] Kougias PG, Angelidaki I. Biogas and its opportunities-a review. Front Environ Sci
Eng 2018;12:1–12.
[2] European Commission, Energy Strategy and Energy Union. A policy framework for
climate and energy in the period from 2020 to 2030 [COM(2014) 15], 〈https://
eur-lex.europa.eu/legal-content/EN/TXT/?Uri=COM%3A2014%3A15%3AFIN〉;
2014 [Accessed 20 July 2018].
[3] Tabassum MR, Xia A, Murphy JD. Potential of seaweed as a feedstock for renew-
able gaseous fuel production in Ireland. Renew Sustain Energy Rev
2017;68:136–46.
[4] Anyaoku CC, Baroutian S. Decentralized anaerobic digestion systems for increased
utilization of biogas from municipal solid waste. Renew Sustain Energy Rev
2018;90:982–91.
[5] Ju F, Lau F, Zhang T. Linking microbial community, environmental variables, and
methanogenesis in anaerobic biogas digesters of chemically enhanced primary
treatment sludge. Environ Sci Technol 2017;51:3982–92.
[6] Wang P, Wang H, Qiu Y, Ren L, Jiang B. Microbial characteristics in anaerobic
digestion process of food waste for methane production-a review. Bioresour
Technol 2018;248:29–36.
[7] Hassa J, Maus I, Off S, Pühler A, Scherer P, Klocke M, et al. Metagenome, meta-
transcriptome, and metaproteome approaches unraveled compositions and func-
tional relationships of microbial communities residing in biogas plants. Appl
Microbiol Biotechnol 2018:1–19.
[8] Kim E, Lee J, Han G, Hwang S. Comprehensive analysis of microbial communities
in full-scale mesophilic and thermophilic anaerobic digesters treating food waste-
recycling wastewater. Bioresour Technol 2018;259:442–50.
[9] Keegan KP, Glass EM, Meyer F. MG-RAST, a metagenomics service for analysis of
microbial community structure and function. Microb Environ Genom
2016:207–33.
[10] Namiki T, Hachiya T, Tanaka H, Sakakibara Y. MetaVelvet: an extension of Velvet
assembler to de novo metagenome assembly from short sequence reads. Nucleic
Acids Res 2012;40:e155.
[11] Laserson J, Jojic V, Koller D. Genovo: de novo assembly for metagenomes. J
Comput Biol 2011;18:429–43.
[12] Luo G, Fotidis IA, Angelidaki I. Comparative analysis of taxonomic, functional, and
metabolic patterns of microbiomes from 14 full-scale biogas reactors by metage-
nomic sequencing and radioisotopic analysis. Biotechnol Biofuels 2016;9:51.
[13] Rincón B, Borja R, González J, Portillo M, Sáiz-Jiménez C. Influence of organic
loading rate and hydraulic retention time on the performance, stability and mi-
crobial communities of one-stage anaerobic digestion of two-phase olive mill solid
residue. Biochem Eng J 2008;40:253–61.
L. Zhang et al.
[14] De Vrieze J, Saunders AM, He Y, Fang J, Nielsen PH, Verstraete W, et al. Ammonia
and temperature determine potential clustering in the anaerobic digestion mi-
crobiome. Water Res 2015;75:312–23.
[15] Regueiro L, Spirito CM, Usack JG, Hospodsky D, Werner JJ, Angenent LT.
Comparing the inhibitory thresholds of dairy manure co-digesters after prolonged
acclimation periods: Part 2–correlations between microbiomes and environment.
Water Res 2015;87:458–66.
[16] Zhang J, Mao L, Zhang L, Loh K-C, Dai Y, Tong YW. Metagenomic insight into the
microbial networks and metabolic mechanism in anaerobic digesters for food
waste by incorporating activated carbon. Sci Rep 2017;7:11293.
[17] Ye L, Zhang T, Wang TT, Fang ZW. Microbial structures, functions and metabolic
pathways in wastewater treatment bioreactors revealed using high-throughput
sequencing. Environ Sci Technol 2012;46:13244–52.
[18] Genitsaris S, Monchy S, Denonfoux J, Ferreira S, Kormas KA, Sime-Ngando T, et al.
Marine microbial community structure assessed from combined metagenomic
analysis and ribosomal amplicon deep-sequencing. Mar Biol Res 2016;12:30–42.
[19] Krakat N, Anjum R, Demirel B, Schröder P. Methodological flaws introduce strong
bias into molecular analysis of microbial populations. J Appl Microbiol
2017;122:364–77.
[20] Ju F, Zhang T. Experimental design and bioinformatics analysis for the application
of metagenomics in environmental sciences and biotechnology. Environ Sci
Technol 2015;49:12628–40.
[21] Alvarez-Silva MC, Álvarez-Yela AC, Gómez-Cano F, Zambrano MM, Husserl J,
Danies G, et al. Compartmentalized metabolic network reconstruction of microbial
communities to determine the effect of agricultural intervention on soils. PloS One
2017;12:e0181826.
[22] Badhai J, Ghosh TS, Das SK. Taxonomic and functional characteristics of microbial
communities and their correlation with physicochemical properties of four geo-
thermal springs in Odisha, India. Front Microbiol 2015;6:1166.
[23] Kumar GR, Chowdhary N. Biotechnological and bioinformatics approaches for
augmentation of biohydrogen production: a review. Renew Sustain Energy Rev
2016;56:1194–206.
[24] Mukherjee A, Chettri B, Langpoklakpam JS, Basak P, Prasad A, Mukherjee AK,
et al. Bioinformatic approaches including predictive metagenomic profiling reveal
characteristics of bacterial response to petroleum hydrocarbon contamination in
diverse environments. Sci Rep 2017;7:1108.
[25] Suhartini S, Heaven S, Banks CJ. Comparison of mesophilic and thermophilic
anaerobic digestion of sugar beet pulp: performance, dewaterability and foam
control. Bioresour Technol 2014;152:202–11.
[26] Riggio S, Hernandéz-Shek M, Torrijos M, Vives G, Esposito G, Van Hullebusch E,
et al. Comparison of the mesophilic and thermophilic anaerobic digestion of spent
cow bedding in leach-bed reactors. Bioresour Technol 2017;234:466–71.
[27] Munk B, Guebitz GM, Lebuhn M. Influence of nitrogen-rich substrates on biogas
production and on the methanogenic community under mesophilic and thermo-
philic conditions. Anaerobe 2017;46:146–54.
[28] Li W, Loh K-C, Zhang J, Tong YW, Dai Y. Two-stage anaerobic digestion of food
waste and horticultural waste in high-solid system. Appl Energy 2017;209:400–8.
[29] Zhang J, Loh K-C, Lee J, Wang C-H, Dai Y, Tong YW. Three-stage anaerobic co-
digestion of food waste and horse manure. Sci Rep 2017;7:1269.
[30] Zhang J, Loh K-C, Li W, Lim JW, Dai Y, Tong YW. Three-stage anaerobic digester
for food waste. Appl Energy 2017;194:287–95.
[31] Luo G, Angelidaki I. Analysis of bacterial communities and bacterial pathogens in a
biogas plant by the combination of ethidium monoazide, PCR and Ion Torrent
sequencing. Water Res 2014;60:156–63.
[32] Luo G, De Francisci D, Kougias PG, Laura T, Zhu X, Angelidaki I. New steady-state
microbial community compositions and process performances in biogas reactors
induced by temperature disturbances. Biotechnol Biofuels 2015;8:3.
[33] Luo G, Li B, Li L-G, Zhang T, Angelidaki I. Antibiotic resistance genes and corre-
lations with microbial community and metal resistance genes in full-scale biogas
reactors as revealed by metagenomic analysis. Environ Sci Technol
2017;51:4069–80.
[34] Schlüter A, Bekel T, Diaz NN, Dondrup M, Eichenlaub R, Gartemann K-H, et al. The
metagenome of a biogas-producing microbial community of a production-scale
biogas plant fermenter analysed by the 454-pyrosequencing technology. J
Biotechnol 2008;136:77–90.
[35] Wirth R, Kovács E, Maróti G, Bagi Z, Rákhely G, Kovács KL. Characterization of a
biogas-producing microbial community by short-read next generation DNA se-
quencing. Biotechnol Biofuels 2012;5:41.
[36] Güllert S, Fischer MA, Turaev D, Noebauer B, Ilmberger N, Wemheuer B, et al.
Deep metagenome and metatranscriptome analyses of microbial communities af-
filiated with an industrial biogas fermenter, a cow rumen, and elephant feces re-
veal major differences in carbohydrate hydrolysis strategies. Biotechnol Biofuels
2016;9:121.
[37] Sträuber H, Lucas R, Kleinsteuber S. Metabolic and microbial community dynamics
during the anaerobic digestion of maize silage in a two-phase process. Appl
Microbiol Biotechnol 2016;100:479–91.
[38] Bareither CA, Wolfe GL, McMahon KD, Benson CH. Microbial diversity and dy-
namics during methane production from municipal solid waste. Waste Manag
2013;33:1982–92.
[39] Lee S-H, Kang H-J, Lee YH, Lee TJ, Han K, Choi Y, et al. Monitoring bacterial
community structure and variability in time scale in full-scale anaerobic digesters.
J Environ Monit 2012;14:1893–905.
[40] Zhang H, Banaszak JE, Parameswaran P, Alder J, Krajmalnik-Brown R, Rittmann
BE. Focused-Pulsed sludge pre-treatment increases the bacterial diversity and re-
lative abundance of acetoclastic methanogens in a full-scale anaerobic digester.
Water Res 2009;43:4517–26.
124
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
[41] Bibby K, Viau E, Peccia J. Pyrosequencing of the 16S rRNA gene to reveal bacterial
pathogen diversity in biosolids. Water Res 2010;44:4252–60.
[42] Li A, Chu Y, Wang X, Ren L, Yu J, Liu X, et al. A pyrosequencing-based metage-
nomic study of methane-producing microbial community in solid-state biogas re-
actor. Biotechnol Biofuels 2013;6:3.
[43] Wong MT, Zhang D, Li J, Hui RKH, Tun HM, Brar MS, et al. Towards a metage-
nomic understanding on enhanced biomethane production from waste activated
sludge after pH 10 pretreatment. Biotechnol Biofuels 2013;6:38.
[44] Ghanimeh SA, Saikaly PE, Li D, El-Fadel M. Population dynamics during startup of
thermophilic anaerobic digesters: the mixing factor. Waste Manag
2013;33:2211–8.
[45] Röske I, Sabra W, Nacke H, Daniel R, Zeng A-P, Antranikian G, et al. Microbial
community composition and dynamics in high-temperature biogas reactors using
industrial bioethanol waste as substrate. Appl Microbiol Biotechnol
2014;98:9095–106.
[46] Sun R, Xing D, Jia J, Zhou A, Zhang L, Ren N. Methane production and microbial
community structure for alkaline pretreated waste activated sludge. Bioresour
Technol 2014;169:496–501.
[47] Martínez MA, Romero H, Perotti NI. Two amplicon sequencing strategies revealed
different facets of the prokaryotic community associated with the anaerobic
treatment of vinasses from ethanol distilleries. Bioresour Technol
2014;153:388–92.
[48] Lu X, Rao S, Shen Z, Lee PK. Substrate induced emergence of different active
bacterial and archaeal assemblages during biomethane production. Bioresour
Technol 2013;148:517–24.
[49] Tuan NN, Chang Y-C, Yu C-P, Huang S-L. Multiple approaches to characterize the
microbial community in a thermophilic anaerobic digester running on swine
manure: a case study. Microbiol Res 2014;169:717–24.
[50] Ghasimi DS, Lier JB, Zandvoort MH, Kreuk M, Tao Y. Microbial population dy-
namics during long-term sludge adaptation of thermophilic and mesophilic se-
quencing batch digesters treating sewage fine sieved fraction at varying organic
loading rates. Biotechnol Biofuels 2015;8:171.
[51] Cho S-K, Kim D-H, Quince C, Im W-T, Oh S-E, Shin SG. Low-strength ultra-
sonication positively affects methanogenic granules toward higher AD perfor-
mance: implications from microbial community shift. Ultrason Sonochem
2016;32:198–203.
[52] Yun YM, Kim DH, Cho SK, Shin HS, Jung KW, Kim HW. Mitigation of ammonia
inhibition by internal dilution in high‐rate anaerobic digestion of food waste lea-
chate and evidences of microbial community response. Biotechnol Bioeng
2016;113:1892–901.
[53] Westerholm M, Crauwels S, Houtmeyers S, Meerbergen K, Van Geel M, Lievens B,
et al. Microbial community dynamics linked to enhanced substrate availability and
biogas production of electrokinetically pre-treated waste activated sludge.
Bioresour Technol 2016;218:761–70.
[54] Azizi A, Kim W, Lee JH. Comparison of microbial communities during the anae-
robic digestion of Gracilaria under mesophilic and thermophilic conditions. World
J Microbiol Biotechnol 2016;32:158.
[55] Westerholm M, Crauwels S, Van Geel M, Dewil R, Lievens B, Appels L. Microwave
and ultrasound pre-treatments influence microbial community structure and di-
gester performance in anaerobic digestion of waste activated sludge. Appl
Microbiol Biotechnol 2016;100:5339–52.
[56] Li N, Xue Y, Chen S, Takahashi J, Dai L, Dai X. Methanogenic population dynamics
regulated by bacterial community responses to protein-rich organic wastes in a
high solid anaerobic digester. Chem Eng J 2017;317:444–53.
[57] Yu Z, Wen X, Xu M, Huang X. Characteristics of extracellular polymeric substances
and bacterial communities in an anaerobic membrane bioreactor coupled with
online ultrasound equipment. Bioresour Technol 2012;117:333–40.
[58] Ho D, Jensen P, Batstone D. Effects of temperature and hydraulic retention time on
acetotrophic pathways and performance in high-rate sludge digestion. Environ Sci
Technol 2014;48:6468–76.
[59] Ziganshin AM, Liebetrau J, Proeter J, Kleinsteuber S. Microbial community
structure and dynamics during anaerobic digestion of various agricultural waste
materials. Appl Microbiol Biotechnol 2013;97:5161–74.
[60] Lebuhn M, Hanreich A, Klocke M, Schlüter A, Bauer C, Pérez CM. Towards mo-
lecular biomarkers for biogas production from lignocellulose-rich substrates.
Anaerobe 2014;29:10–21.
[61] Solli L, Håvelsrud OE, Horn SJ, Rike AG. A metagenomic study of the microbial
communities in four parallel biogas reactors. Biotechnol Biofuels 2014;7:146.
[62] Razaviarani V, Buchanan ID. Anaerobic co-digestion of biodiesel waste glycerin
with municipal wastewater sludge: microbial community structure dynamics and
reactor performance. Bioresour Technol 2015;182:8–17.
[63] Ziganshina EE, Belostotskiy DE, Ilinskaya ON, Boulygina EA, Grigoryeva TV,
Ziganshin AM. Effect of the organic loading rate increase and the presence of
zeolite on microbial community composition and process stability during anae-
robic digestion of chicken wastes. Microb Ecol 2015;70:948–60.
[64] Belostotskiy DE, Ziganshina EE, Siniagina M, Boulygina EA, Miluykov VA,
Ziganshin AM. Impact of the substrate loading regime and phosphoric acid sup-
plementation on performance of biogas reactors and microbial community dy-
namics during anaerobic digestion of chicken wastes. Bioresour Technol
2015;193:42–52.
[65] Sun L, Liu T, Müller B, Schnürer A. The microbial community structure in in-
dustrial biogas plants influences the degradation rate of straw and cellulose in
batch tests. Biotechnol Biofuels 2016;9:128.
[66] Oosterkamp MJ, Méndez-García C, Kim C-H, Bauer S, Ibáñez AB, Zimmerman S,
et al. Lignocellulose-derived thin stillage composition and efficient biological
treatment with a high-rate hybrid anaerobic bioreactor system. Biotechnol Biofuels
L. Zhang et al.
2016;9:120.
[67] Pervin HM, Dennis PG, Lim HJ, Tyson GW, Batstone DJ, Bond PL. Drivers of mi-
crobial community composition in mesophilic and thermophilic temperature-
phased anaerobic digestion pre-treatment reactors. Water Res 2013;47:7098–108.
[68] Cho SK, Im WT, Kim DH, Kim MH, Shin HS, Oh SE. Dry anaerobic digestion of food
waste under mesophilic conditions: performance and methanogenic community
analysis. Bioresour Technol 2013;131:210–7.
[69] Su H, Liu L, Wang Q, Wang S, Tan T, Hou X, et al. Semi-continuous anaerobic
digestion for biogas production: influence of ammonium acetate supplement and
structure of the microbial community. Biotechnol Biofuels 2015;8:13.
[70] Li L, He Q, Ma Y, Wang X, Peng X. Dynamics of microbial community in a me-
sophilic anaerobic digester treating food waste: relationship between community
structure and process stability. Bioresour Technol 2015;189:113–20.
[71] Li L, He Q, Wang X, Peng X, Ma Y. A mesophilic anaerobic digester for treating
food waste: process stability and microbial community analysis using pyr-
osequencing. Microb Cell Fact 2016;15:65.
[72] Jang HM, Ha JH, Kim M-S, Kim J-O, Kim YM, Park JM. Effect of increased load of
high-strength food wastewater in thermophilic and mesophilic anaerobic co-di-
gestion of waste activated sludge on bacterial community structure. Water Res
2016;99:140–8.
[73] Leite AF, Richnow H-H, Harms H, Janke L, Nikolausz M. Lessons learned from the
microbial ecology resulting from different inoculation strategies for biogas pro-
duction from waste products of the bioethanol/sugar industry. Biotechnol Biofuels
2016;9:144.
[74] Lee J, Han G, Shin SG, Koo T, Cho K, Kim W, et al. Seasonal monitoring of bacteria
and archaea in a full-scale thermophilic anaerobic digester treating food waste-
recycling wastewater: correlations between microbial community characteristics
and process variables. Chem Eng J 2016;300:291–9.
[75] Han G, Shin SG, Lee J, Shin J, Hwang S. A comparative study on the process
efficiencies and microbial community structures of six full-scale wet and semi-dry
anaerobic digesters treating food wastes. Bioresour Technol 2017;245:869–75.
[76] Cho K, Shin SG, Kim W, Lee J, Lee C, Hwang S. Microbial community shifts in a
farm-scale anaerobic digester treating swine waste: correlations between bacteria
communities associated with hydrogenotrophic methanogens and environmental
conditions. Sci Total Environ 2017;601–602:167–76.
[77] Maspolim Y, Zhou Y, Guo C, Xiao K, Ng WJ. Determination of the archaeal and
bacterial communities in two-phase and single-stage anaerobic systems by 454
pyrosequencing. J Environ Sci 2015;36:121–9.
[78] Kim S, Bae J, Choi O, Ju D, Lee J, Sung H, et al. A pilot scale two-stage anaerobic
digester treating food waste leachate (FWL): performance and microbial structure
analysis using pyrosequencing. Process Biochem 2014;49:301–8.
[79] Yu B, Lou Z, Zhang D, Shan A, Yuan H, Zhu N, et al. Variations of organic matters
and microbial community in thermophilic anaerobic digestion of waste activated
sludge with the addition of ferric salts. Bioresour Technol 2015;179:291–8.
[80] Garcia SL, Jangid K, Whitman WB, Das K. Transition of microbial communities
during the adaption to anaerobic digestion of carrot waste. Bioresour Technol
2011;102:7249–56.
[81] Chen S, Cheng H, Wyckoff KN, He Q. Linkages of Firmicutes and Bacteroidetes
populations to methanogenic process performance. J Ind Microbiol Biotechnol
2016;43:771–81.
[82] Lee J, Hwang B, Koo T, Shin SG, Kim W, Hwang S. Temporal variation in me-
thanogen communities of four different full-scale anaerobic digesters treating food
waste-recycling wastewater. Bioresour Technol 2014;168:59–63.
[83] Wei ZY, Jin DC, Deng Y. Bioinformatics tools and applications in the study of
environmental microbial metagenomics. Microbiol China 2015;42:890–901. [In
Chinese].
[84] Campanaro S, Treu L, Kougias PG, Francisci D, Valle G, Angelidaki I. Metagenomic
analysis and functional characterization of the biogas microbiome using high
throughput shotgun sequencing and a novel binning strategy. Biotechnol Biofuels
2016;9:26.
[85] Krause L, Diaz NN, Edwards RA, Gartemann K-H, Krömeke H, Neuweger H, et al.
Taxonomic composition and gene content of a methane-producing microbial
community isolated from a biogas reactor. J Biotechnol 2008;136:91–101.
[86] Mitra S, Rupek P, Richter DC, Urich T, Gilbert JA, Meyer F, et al. Functional
analysis of metagenomes and metatranscriptomes using SEED and KEGG. BMC
Bioinforma 2011;12:S21.
[87] Shaw GT-W, Liu A-C, Weng C-Y, Chou C-Y, Wang D. Inferring microbial interac-
tions in thermophilic and mesophilic anaerobic digestion of hog waste. PloS One
2017;12:e0181395.
[88] Guo X, Wang C, Sun F, Zhu W, Wu W. A comparison of microbial characteristics
between the thermophilic and mesophilic anaerobic digesters exposed to elevated
food waste loadings. Bioresour Technol 2014;152:420–8.
[89] Kröber M, Bekel T, Diaz NN, Goesmann A, Jaenicke S, Krause L, et al. Phylogenetic
characterization of a biogas plant microbial community integrating clone library
16S-rDNA sequences and metagenome sequence data obtained by 454-pyr-
osequencing. J Biotechnol 2009;142:38–49.
[90] Pope PB, Vivekanand V, Eijsink VG, Horn SJ. Microbial community structure in a
biogas digester utilizing the marine energy crop Saccharina latissima. 3 Biotech
2013;3:407–14.
[91] Wilkins D, Rao S, Lu X, Lee PK. Effects of sludge inoculum and organic feedstock
on active microbial communities and methane yield during anaerobic digestion.
Front Microbiol 2015;6:1114.
[92] Oksanen J, Kindt R, Legendre P, O’Hara B, Stevens MHH, Oksanen MJ, et al. The
vegan package. Commun Ecol Package 2007;10:631–7.
[93] Eduok S, Ferguson R, Jefferson B, Villa R, Coulon F. Aged-engineered nano-
particles effect on sludge anaerobic digestion performance and associated
125
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
microbial communities. Sci Total Environ 2017;609:232–41.
[94] Rademacher A, Zakrzewski M, Schlüter A, Schönberg M, Szczepanowski R,
Goesmann A, et al. Characterization of microbial biofilms in a thermophilic biogas
system by high-throughput metagenome sequencing. FEMS Microbiol Ecol
2012;79:785–99.
[95] Town JR, Links MG, Fonstad TA, Dumonceaux TJ. Molecular characterization of
anaerobic digester microbial communities identifies microorganisms that correlate
to reactor performance. Bioresour Technol 2014;151:249–57.
[96] Jang HM, Kim M-S, Ha JH, Park JM. Reactor performance and methanogenic ar-
chaea species in thermophilic anaerobic co-digestion of waste activated sludge
mixed with food wastewater. Chem Eng J 2015;276:20–8.
[97] Smith AM, Sharma D, Lappin-Scott H, Burton S, Huber DH. Microbial community
structure of a pilot-scale thermophilic anaerobic digester treating poultry litter.
Appl Microbiol Biotechnol 2014;98:2321–34.
[98] Li J, Rui J, Pei Z, Sun X, Zhang S, Yan Z, et al. Straw-and slurry-associated pro-
karyotic communities differ during co-fermentation of straw and swine manure.
Appl Microbiol Biotechnol 2014;98:4771–80.
[99] Yu H, Wang Z, Wu Z, Zhu C. Enhanced waste activated sludge digestion using a
submerged anaerobic dynamic membrane bioreactor: performance, sludge char-
acteristics and microbial community. Sci Rep 2016;6:20111.
[100] Cardinali-Rezende J, Rojas-Ojeda P, Nascimento AM, Sanz JL. Proteolytic bacterial
dominance in a full-scale municipal solid waste anaerobic reactor assessed by 454
pyrosequencing technology. Chemosphere 2016;146:519–25.
[101] Chao A. Nonparametric estimation of the number of classes in a population. Scand
J Stat 1984:265–70.
[102] Chao A, Chiu CH. Species richness: estimation and comparison. Wiley StatsRef:
Stat Ref Online 2016. https://doi.org/10.1002/9781118445112.stat03432.pub2.
[103] Keylock C. Simpson diversity and the Shannon–Wiener index as special cases of a
generalized entropy. Oikos 2005;109:203–7.
[104] Simpson EH. Measurement of diversity. Nature 1949;163:688.
[105] Good IJ. The population frequencies of species and the estimation of population
parameters. Biometrika 1953;40:237–64.
[106] Morris EK, Caruso T, Buscot F, Fischer M, Hancock C, Maier TS, et al. Choosing
and using diversity indices: insights for ecological applications from the German
Biodiversity Exploratories. Ecol Evol 2014;4:3514–24.
[107] Barrantes G, Sandoval L. Conceptual and statistical problems associated with the
use of diversity indices in ecology. Rev Biol Trop 2009;57:451–60.
[108] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid as-
signment of rRNA sequences into the new bacterial taxonomy. Appl Environ
Microbiol 2007;73:5261–7.
[109] Xie Z, Wang Z, Wang Q, Zhu C, Wu Z. An anaerobic dynamic membrane bioreactor
(AnDMBR) for landfill leachate treatment: performance and microbial community
identification. Bioresour Technol 2014;161:29–39.
[110] Xu Z, Hansen MA, Hansen LH, Jacquiod S, Sørensen SJ. Bioinformatic approaches
reveal metagenomic characterization of soil microbial community. PloS One
2014;9:e93445.
[111] Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a
Web browser. BMC Bioinforma 2011;12:385.
[112] Parks DH, Tyson GW, Hugenholtz P, Beiko RG. STAMP: statistical analysis of
taxonomic and functional profiles. Bioinformatics 2014;30:3123–4.
[113] Bulgarelli D, Rott M, Schlaeppi K, van Themaat EVL, Ahmadinejad N, Assenza F,
et al. Revealing structure and assembly cues for Arabidopsis root-inhabiting bac-
terial microbiota. Nature 2012;488:91–5.
[114] Hamilton N. ggtern: an extension to “ggplot2”, for the creation of ternary dia-
grams. R package Version 2.1.4; 2016 〈http://CRAN.R-project.org/package=
ggtern〉.
[115] Lu Y. Microbial ecology of fermentative microbes in anaerobic granules [Ph.D.
thesis]. School of Chemical Engineering, The University of Queensland; 2014.
https://doi.org/10.14264/uql.2015.21.
[116] Ramette A. Multivariate analyses in microbial ecology. FEMS Microbiol Ecol
2007;62:142–60.
[117] Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal database
Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res
2013;42:D633–42.
[118] Ziganshina EE, Ibragimov EM, Vankov PY, Miluykov VA, Ziganshin AM.
Comparison of anaerobic digestion strategies of nitrogen-rich substrates: perfor-
mance of anaerobic reactors and microbial community diversity. Waste Manag
2017;59:160–71.
[119] Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci
2003;14:927–30.
[120] Paliy O, Shankar V. Application of multivariate statistical techniques in microbial
ecology. Mol Ecol 2016;25:1032–57.
[121] Lepš J, Šmilauer P. Multivariate analysis of ecological data using CANOCO.
Cambridge University Press; 2003.
[122] Shi J, Wang Z, Stiverson JA, Yu Z, Li Y. Reactor performance and microbial
community dynamics during solid-state anaerobic digestion of corn stover at
mesophilic and thermophilic conditions. Bioresour Technol 2013;136:574–81.
[123] Alcántara-Hernández RJ, Taş N, Carlos-Pinedo S, Durán-Moreno A, Falcón LI.
Microbial dynamics in anaerobic digestion reactors for treating organic urban
residues during the start-up process. Lett Appl Microbiol 2017;64:438–45.
[124] Boeckmann B, Bairoch A, Apweiler R, Blatter M-C, Estreicher A, Gasteiger E, et al.
The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003.
Nucleic Acids Res 2003;31:365–70.
[125] Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, et al.
GenDB—an open source genome annotation system for prokaryote genomes.
Nucleic Acids Res 2003;31:2187–95.
L. Zhang et al.
[126] Markowitz VM, Chen IMA, Chu K, Szeto E, Palaniappan K, Pillay M, et al. IMG/M 4
version of the integrated metagenome comparative analysis system. Nucleic Acids
Res 2013;42:D568–73.
[127] Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, et al. KEGG for
linking genomes to life and the environment. Nucleic Acids Res 2007;36:D480–4.
[128] Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, et al. InterPro:
the integrative protein signature database. Nucleic Acids Res 2008;37:D211–5.
[129] Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam:
the protein families database. Nucleic Acids Res 2013;42:D222–30.
[130] Consortium GO. The Gene Ontology (GO) database and informatics resource.
Nucleic Acids Res 2004;32:D258–61.
[131] Hoff KJ. The effect of sequencing errors on metagenomic gene prediction. BMC
Genom 2009;10:520.
[132] Jünemann S, Kleinbölting N, Jaenicke S, Henke C, Hassa J, Nelkner J, et al.
Bioinformatics for NGS-based metagenomics and the application to biogas re-
search. J Biotechnol 2017;261:10–23.
[133] Bassani I, Kougias PG, Treu L, Angelidaki I. Biogas upgrading via hydro-
genotrophic methanogenesis in two-stage continuous stirred tank reactors at me-
sophilic and thermophilic conditions. Environ Sci Technol 2015;49:12585–93.
[134] Jang HM, Kim JH, Ha JH, Park JM. Bacterial and methanogenic archaeal com-
munities during the single-stage anaerobic digestion of high-strength food was-
tewater. Bioresour Technol 2014;165:174–82.
[135] Koo T, Shin SG, Lee J, Han G, Kim W, Cho K, et al. Identifying methanogen
community structures and their correlations with performance parameters in four
full-scale anaerobic sludge digesters. Bioresour Technol 2017;228:368–73.
[136] Hidaka T, Tsushima I, Tsumori J. Comparative analyses of microbial structures
and gene copy numbers in the anaerobic digestion of various types of sewage
sludge. Bioresour Technol 2018. https://doi.org/10.1016/j.biortech.2017.12.097.
[137] Suksong W, Kongjan P, Prasertsan P, Imai T, Sompong O. Optimization and mi-
crobial community analysis for production of biogas from solid waste residues of
palm oil mill industry by solid-state anaerobic digestion. Bioresour Technol
2016;214:166–74.
[138] Zhang L, Zhang J, Loh K-C. Activated carbon enhanced anaerobic digestion of food
waste-Laboratory-scale and pilot-scale operation. Waste Manag 2018;75:270–9.
[139] Zhang J, Li W, Lee J, Loh K-C, Dai Y, Tong YW. Enhancement of biogas production
in anaerobic co-digestion of food waste and waste activated sludge by biological
co-pretreatment. Energy 2017;137:479–86.
[140] Agneessens LM, Ottosen LDM, Voigt NV, Nielsen JL, de Jonge N, Fischer CH, et al.
In-situ biogas upgrading with pulse H2 additions: the relevance of methanogen
adaption and inorganic carbon level. Bioresour Technol 2017;233:256–63.
[141] Bassani I, Kougias PG, Treu L, Porté H, Campanaro S, Angelidaki I. Optimization of
hydrogen dispersion in thermophilic up-flow reactors for ex situ biogas upgrading.
Bioresour Technol 2017;234:310–9.
[142] Kougias PG, Treu L, Benavente DP, Boe K, Campanaro S, Angelidaki I. Ex-situ
biogas upgrading and enhancement in different reactor systems. Bioresour
Technol 2017;225:429–37.
[143] Mulat DG, Mosbæk F, Ward AJ, Polag D, Greule M, Keppler F, et al. Exogenous
addition of H2 for an in situ biogas upgrading through biological reduction of
carbon dioxide into methane. Waste Manag 2017;68:146–56.
[144] Schuchmann K, Müller V. Autotrophy at the thermodynamic limit of life: a model
for energy conservation in acetogenic bacteria. Nat Rev Microbiol
2014;12:809–21.
[145] Angelidaki I, Treu L, Tsapekos P, Luo G, Campanaro S, Wenzel H, et al. Biogas
upgrading and utilization: current status and perspectives. Biotechnol Adv
2018;36:452–66.
[146] Campanaro S, Treu L, Kougias PG, Luo G, Angelidaki I. Metagenomic binning
reveals the functional roles of core abundant microorganisms in twelve full-scale
biogas plants. Water Res 2018;140:123–34.
[147] De Vrieze J, Verstraete W. Perspectives for microbial community composition in
anaerobic digestion: from abundance and activity to connectivity. Environ
Microbiol 2016;18:2797–809.
[148] Parthasarathy R. Monitoring microbial communities using light sheet fluorescence
microscopy. Curr Opin Microbiol 2018;43:31–7.
[149] Jiang Y, Banks C, Zhang Y, Heaven S, Longhurst P. Quantifying the percentage of
methane formation via acetoclastic and syntrophic acetate oxidation pathways in
anaerobic digesters. Waste Manag 2018;71:749–56.
[150] Tian H, Fotidis IA, Mancini E, Treu L, Mahdy A, Ballesteros M, et al. Acclimation to
extremely high ammonia levels in continuous biomethanation process and the
associated microbial community dynamics. Bioresour Technol 2018;247:616–23.
[151] Yang Z, Wang W, He Y, Zhang R, Liu G. Effect of ammonia on methane production,
methanogenesis pathway, microbial community and reactor performance under
mesophilic and thermophilic conditions. Renew Energy 2018;125:915–25.
[152] Hanemaaijer M, Röling WF, Olivier BG, Khandelwal RA, Teusink B, Bruggeman FJ.
Systems modeling approaches for microbial community studies: from metage-
nomics to inference of the community structure. Front Microbiol 2015;6:213.
[153] Succurro A, Moejes FW, Ebenhöh O. A diverse community to study communities:
integration of experiments and mathematical models to study microbial consortia.
J Bacteriol 2017;199. [e00865-16].
[154] Shaw GTW, Pao YY, Wang D. MetaMIS: a metagenomic microbial interaction
126
Renewable and Sustainable Energy Reviews 100 (2019) 110–126
simulator based on microbial community profiles. BMC Bioinforma 2016;17:488.
[155] Yan J, Chuai G, Qi T, Shao F, Zhou C, Zhu C, et al. MetaTopics: an integration tool
to analyze microbial community profile by topic model. BMC Genom 2017;18:962.
[156] Hanemaaijer M, Olivier BG, Röling WF, Bruggeman FJ, Teusink B. Model-based
quantification of metabolic interactions from dynamic microbial-community data.
PloS One 2017;12:e0173183.
[157] Cardona C, Weisenhorn P, Henry C, Gilbert JA. Network-based metabolic analysis
and microbial community modeling. Curr Opin Microbiol 2016;31:124–31.
[158] Song HS, Cannon WR, Beliaev AS, Konopka A. Mathematical modeling of micro-
bial community dynamics: a methodological review. Processes 2014;2:711–52.
[159] Larsen PE, Gibbons SM, Gilbert JA. Modeling microbial community structure and
functional diversity across time and space. FEMS Microbiol Lett 2012;332:91–8.
[160] Succurro A, Ebenhöh O. Review and perspective on mathematical modeling of
microbial ecosystems. Biochem Soc Trans 2018;46:403–12.
[161] Chen Y, Lan S, Wang L, Dong S, Zhou H, Tan Z, et al. A review: driving factors and
regulation strategies of microbial community structure and dynamics in waste-
water treatment systems. Chemosphere 2017;174:173–82.
[162] Zhou J, Zhang R, Liu F, Yong X, Wu X, Zheng T, et al. Biogas production and
microbial community shift through neutral pH control during the anaerobic di-
gestion of pig manure. Bioresour Technol 2016;217:44–9.
[163] Argyropoulos A. Soft sensor development and process control of anaerobic di-
gestion [Ph.D. thesis]. University of Exeter; 2013http://hdl.handle.net/10871/
15068.
[164] Bai X, Li Z, Wang X, He X, Cheng S, Bai X, et al. Online measurement of alkalinity
in anaerobic co-digestion using linear regression method. Int J Agric Biol Eng
2017;10:176–83.
[165] Amha YM, Anwar MZ, Brower A, Jacobsen CS, Stadler LB, Webster TM, et al.
Inhibition of anaerobic digestion processes: applications of molecular tools.
Bioresour Technol 2018;247:999–1014.
[166] Lee J, Shin SG, Jang HM, Kim YB, Lee J, Mo Y. Characterization of antibiotic
resistance genes in representative organic solid wastes: food waste-recycling
wastewater, manure, and sewage sludge. Sci Total Environ 2017;579:1692–8.
[167] Tian Z, Zhang Y, Yu B, Yang M. Changes of resistome, mobilome and potential
hosts of antibiotic resistance genes during the transformation of anaerobic diges-
tion from mesophilic to thermophilic. Water Res 2016;98:261–9.
[168] Jang HM, Shin J, Choi S, Shin SG, Park KY, Cho J, et al. Fate of antibiotic re-
sistance genes in mesophilic and thermophilic anaerobic digestion of chemically
enhanced primary treatment (CEPT) sludge. Bioresour Technol 2017;244:433–44.
[169] Pu C, Liu H, Ding G, Sun Y, Yu X, Chen J, et al. Impact of direct application of
biogas slurry and residue in fields: In situ analysis of antibiotic resistance genes
from pig manure to fields. J Hazard Mater 2018;344:441–9.
[170] Tong J, Liu J, Zheng X, Zhang J, Ni X, Chen M, et al. Fate of antibiotic resistance
bacteria and genes during enhanced anaerobic digestion of sewage sludge by
microwave pretreatment. Bioresour Technol 2016;217:37–43.
[171] Yin X, Jiang XT, Chai B, Li L, Yang Y, Cole JR, et al. ARGs-OAP v2.0 with an
expanded SARG database and hidden Markov models for enhancement char-
acterization and quantification of antibiotic resistance genes in environmental
metagenomes. Bioinformatics 2018:1–8.
[172] Oulas A, Pavloudi C, Polymenakou P, Pavlopoulos GA, Papanikolaou N, Kotoulas
G, et al. Metagenomics: tools and insights for analyzing next-generation sequen-
cing data derived from biodiversity studies. Bioinform Biol Insights 2015:9.
[BBI.S12462].
[173] Dröge J, McHardy AC. Taxonomic binning of metagenome samples generated by
next-generation sequencing technologies. Brief Bioinform 2012;13:646–55.
[174] Tabish M, Azim S, Hussain MA, Rehman SU, Sarwar T, Ishqi HM. Bioinformatics
approaches in studying microbial diversity. Management of microbial resources in
the environment. Dordrecht: Springer; 2013. p. 119–40.
[175] Campanaro S, Treu L, Kougias PG, Zhu X, Angelidaki I. Taxonomy of anaerobic
digestion microbiome reveals biases associated with the applied high throughput
sequencing strategies. Sci Rep 2018;8:1926.
[176] Widder S, Allen RJ, Pfeiffer T, Curtis TP, Wiuf C, Sloan WT, et al. Challenges in
microbial ecology: building predictive understanding of community function and
dynamics. ISME J 2016;10:2557–68.
[177] Hiraoka S, Yang C, Iwasaki W. Metagenomics and bioinformatics in microbial
ecology: current status and beyond. Microbes Environ 2016;31:204–12.
[178] Tolvanen KE, Karp MT. Molecular methods for characterizing mixed microbial
communities in hydrogen-fermenting systems. Int J Hydrogen Energy
2011;36:5280–8.
[179] Palaniswamy D, Ramesh G, Sivasankaran S, Kathiravan N. Optimising biogas from
food waste using a neural network model. In: Proceedings of the institution of civil
engineers-municipal engineer. Thomas Telford Ltd; 2016. p. 221–9.
[180] Nair VV, Dhar H, Kumar S, Thalla AK, Mukherjee S, Wong JW. Artificial neural
network based modeling to evaluate methane yield from biogas in a laboratory-
scale anaerobic bioreactor. Bioresour Technol 2016;217:90–9.
[181] Antwi P, Li J, Boadi PO, Meng J, Shi E, Deng K, et al. Estimation of biogas and
methane yields in an UASB treating potato starch processing wastewater with
backpropagation artificial neural network. Bioresour Technol 2017;228:106–15.
[182] Eng A, Borenstein E. An algorithm for designing minimal microbial communities
with desired metabolic capacities. Bioinformatics 2016;32:2008–16.