Science of the Total Environment 652 (2019) 709–717

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Science of the Total Environment

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

Characterization of microbial community and main functional groups of

prokaryotes in thermophilic anaerobic co-digestion of food waste and
paper waste
Lu Li, Yu Qin, Zhe Kong, Jing Wu, Kengo Kubota, Yu-You Li ⁎
Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba Ward, Sendai, Miyagi 980-8579, Japan

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Effect of paper waste on microbial com-

munity in thermophilic digestion was
clarified.
• Functional groups of prokaryotes
changed greatly as the paper waste
ratio increased.
• Community of cellulose-degrading bac-
teria changed greatly due to declined
pH value.
• Hydrogenotrophic methanogenesis was
enhanced when the paper waste ratio
≥ 50%.
• The predominant microorganism re-
sponse for each metabolic step was
summarized.

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

Article history: The thermophilic anaerobic co-digestion of food waste and paper waste was successfully operated with a 0% to
Received 19 August 2018 70% fraction of paper waste. The variation of functional microbial community was investigated by 16S rRNA
Received in revised form 17 October 2018 gene analysis. The results indicated that the hydrolyzing bacterial community changed from carbohydrate/
Accepted 21 October 2018
protein-degrading bacteria to cellulose-degrading bacteria when the paper waste ratio was higher than 50%. Sig-
Available online 22 October 2018
nificant changes in the taxon responsible for cellulose degradation were found depending on the paper waste
Editor: Jay Gan fraction. Cellulose-degrading bacteria outcompeted lactic acid bacteria in the degradation of monosaccharide,
resulting in a decline in the proportion of lactic acid bacteria and the absence of an accumulation of lactic acid.
Keywords: At high paper waste ratios, because the cellulose-degrading bacteria, such as Defluviitoga tunisiensis, were
Thermophilic anaerobic co-digestion more likely to degrade monosaccharides directly to acetate and hydrogen rather than to propionate and butyrate,
Food waste the abundance of syntrophs was reduced. The variation of those bacteria with high H2-producing ability signifi-
Paper waste cantly influenced the proportion of hydrogenotrophic archaea. The change in the microbial community as the
Microbial community paper waste fraction increased was illustrated with regard to anaerobic degradation steps.
Cellulose degradation
© 2018 Elsevier B.V. All rights reserved.

1. Introduction

Thanks to its high loading tolerance, energy recovery in the form of

methane and its low generation of biomass, anaerobic digestion has
⁎ Corresponding author. been widely applied in the treatment of the organic fraction of munici-
E-mail address: gyokuyu.ri.a5@tohoku.ac.jp (Y.-Y. Li). pal solid waste (Banks et al., 2011). As a mono-substrate, the digestion

https://doi.org/10.1016/j.scitotenv.2018.10.292
0048-9697/© 2018 Elsevier B.V. All rights reserved.
710 L. Li et al. / Science of the Total Environment 652 (2019) 709–717
of paper waste (PW), one of the biggest components of municipal solid
waste, has been shown to be difficult (Jokela et al., 1997; Kamali et al.,
2016). While PW contains a large amount of carbohydrate, it lacks nutri-
ents: an excessive dosage of PW in the anaerobic digestion system in-
creases the C/N ratio. Because of the lack of basic raw materials for the
synthesis of amino acids and proteins (McCarty, 1964), this increase in
the PW ratio significantly inhibits the assimilation of microorganisms.
On the other hand, the carbohydrate in PW is in the form of cellulose
and hemicellulose: unlike starch or glucose, these are referred to as re-
fractory carbohydrates due to their high degree of polymerization
(Leschine, 1995). Besides, lignin, another major component in PW, has
also been classified as a refractory organic matter for degradation.
These factors make it difficult for PW to be degraded anaerobically
and to be consumed by microorganisms.
Conversely, food waste (FW) is classified as a nitrogen-rich biomass
due to its high protein and amino acid content (Banks et al., 2011). It is
therefore reasonable to assume that a supply of essential nitrogen ele-
ment from FW has potential to foster higher effectiveness in the anaer-
obic co-digestion of PW and FW, with a higher harvest than the sole
digestion of PW. As a matter of fact, when collecting municipal waste,
PW and FW are always mixed; for example, a mixture waste of food re-
siduals and tissue is the common garbage from public restaurants. Be-
cause the separation of these two organic wastes is difficult, the direct
co-digestion of these two wastes provides a convenient way to treat
both PW and FW.
Although most previous studies on anaerobic digestion have been
performed under the mesophilic condition of 35 °C (Kong et al., 2018),
attention is increasingly turning towards thermophilic digestion
(Micolucci et al., 2016; Riya et al., 2018). The reported advantages of
thermophilic digestion include the much higher permissible organic
loading rate and a higher methane production than that of mesophilic
digestion (Jang et al., 2016; Labatut et al., 2014). As the degradation of
cellulose is considered the most important point in the anaerobic diges-
tion of PW (Valdez-Vazquez et al., 2005; Yuan et al., 2014), it is well-
understood that to improve the cellulose-degrading rate, the optimum
temperature must be determined. It was recently reported that the
highest hydrogen production activity of microorganisms using cellulose
was at a thermophilic temperature of 55 °C rather than the mesophilic
temperature of 35 °C or a hyper-thermophilic temperature of 70 °C
(Gadow et al., 2016). The results of this comparative study also indi-
cated that the activation energy of cellulose reached its lowest point
when the cellulose-degrading consortium was cultured at a tempera-
ture of 55 °C.
Because of the principal role played by anaerobic microorganisms
in methane fermentation, an investigation of the composition and
variation of microorganisms can lead to a better understanding of
the mechanisms of co-digestion of FW and PW. While the archaeal
communities in anaerobic digestion have been revealed, the specific
functions of bacteria in the co-digestion system of FW and PW re-
main largely unknown. While the variations in the microbial com-
munity of thermophilic digestion with FW or PW feed as the mono-
substrate have been investigated (Kamali et al., 2016; Li et al.,
2011; Zamanzadeh et al., 2016; Zamorano-López et al., 2018), no fur-
ther detailed studies have been published on the variations of bacte-
rial and archaeal communities as the PW ratio in the thermophilic
anaerobic digestion system increases for the co-digestion of high-
strength FW and PW.
In this study, a thermophilic reactor was set up for the co-digestion
of FW and PW. One of the objectives of this work was to investigate
the shift in the structure of the microbial community in response to
the long-term performance of the reactor with the increase in the PW
ratio. Another aim was to seek and identify the functional microorgan-
isms which play important roles in cellulose degradation, protein degra-
dation, lactic acid production and together with the syntrophs,
homoacetogens and methanogens which work in the process of me-
thanogenic degradation. Based on these outcomes, a reasonable
metabolic pathway for the co-digestion of FW and PW in the thermo-
philic anaerobic digestion system was proposed.
2. Materials and methods
2.1. Experimental apparatus and preparation of feedstock
A thermophilic anaerobic digestion system was operated for the
co-digestion of FW and PW. As illustrated in Fig. 1, the system con-
tains a substrate tank in which the substrate was stored at 4 °C,
and a continuously stirred tank reactor with an effective volume of
3 L. The reactor was operated under the thermophilic condition of
55 °C, an organic loading rate of approximately 14.0 g COD L−1 d−1
and a hydraulic retention time of 30 days. Biogas was collected and
measured by a wet tip gas meter (W-NK-0.5B, Shinagawa, Japan).
Water bath circulations were introduced into the system to main-
tain the temperature for the substrate tank and the reactor. Seed
sludge was collected from a local wastewater treatment plant
which deals with municipal wastewater. This system was operated
for 485 days and the PW ratio in substrate was changed at the
127th, 202nd, 279th, 366th and 404th day.
Initially, the reactor was dosed with FW as the sole substrate. When
the monitoring parameters of the system became stable, the PW ratio in
the mixture substrate was elevated to 20% and then to 40%, 50%, 60%,
70% of the total solid (TS) in the following runs. The artificial FW was
prepared monthly and contained the mixture of fruit (30%), vegetables
(36%), staple food (20%), meat and eggs, and all of the organic waste
was crushed into the slurry with a TS of approximately 100 g L−1. The
PW was prepared by mixing office paper, newsprint paper and toilet
paper at a 1:1:1 ratio according to their air-dried weights. The office
paper and newsprint paper had been cut into tatters approximately
2 mm × 10 mm in size by a paper shredder. The mixed PW was stirred
with water to make a bucket of PW slurry with the same TS of 100 g L−1.
In order to maintain the stable growth of microorganisms, FeCl2·H2O,
CoCl2·6H2O and NiCl2·6H2O were added to the substrate to maintain
the trace elements of 10 mg L−1 Fe, 1 mg L−1 Co and 1 mg L−1 Ni
(Qiang et al., 2012).
2.2. Chemical analysis and calculation
The pH was measured by an HM-30R pH meter (TOA-DKK, Japan).
Alkalinity was measured by the titrimetric method and the ammonia ni-
trogen concentration was determined by 7100 Capillary Electrophoresis
(Agilent Technologies, USA). Chemical oxygen demand (COD) was ana-
lyzed according to standard methods (Franson et al., 1994). Carbohy-
drate, protein and lipid concentrations in both the substrate tank and
the reactor were determined with the same method described in a pre-
vious study (Qin et al., 2018). The concentrations of VFAs (acetate, pro-
pionate, butyrate, isobutyrate, valerate, isovalerate and hexanoate) and
lactic acid were measured by a 6890 Series GC system (Agilent, USA). El-
ement components of FW and PW were analyzed by Elementar system
and all the analytical reagents were purchased from Wako Co. Ltd.,
Japan. The volume of generated gas was recorded by a wet gas-meter
and then calculated to the standard condition of 273.15 K, 101325 Pa.
Biogas components were measured by a GC-8A gas chromatograph
(Shimadzu, Japan) with a 0.4 mL injection volume.
Remove efficiency was calculated based on the following equation:
C sub −C rea
Removal efficiency ð%Þ ¼  100% ð1Þ
C sub
where Csub: the concentration of composition in the substrate tank; Crea:
the concentration of composition in the reactor.
 L. Li et al. / Science of the Total Environment 652 (2019) 709–717
Fig. 1. Schematic diagram of lab-scale thermophilic continuously stirred tank reactor (CSTR) system.
Hydrolysis efficiency was calculated based on the following equa-
tion:
 
−1
Hydrolysis efficiency g COD L−1 d
ðC −SC sub Þ−ðC rea −SC rea Þ
¼ sub  100%
ðC sub −SC sub Þ
where Csub: the concentration of composition in the substrate tank;
SCsub: the concentration of soluble part composition in the substrate
tank; Crea: the concentration of composition in the reactor; SCrea: the
concentration of soluble part composition in the reactor.
The hydrolysis rate was calculated based on the following equation:
 
−1
Hydrolysis rate g COD L−1 d
ðTCODsub −SCODsub Þ−ðTCODrea −SCODrea Þ
¼  100%
HRT
where TCODsub: total COD of substrate; SCODsub: soluble COD of sub-
strate, TCODrea: total COD of reactor; SCODrea: soluble COD of reactor.
When the hydrolysis rate of carbohydrate and protein was calculated,
the COD was conformed from the corresponding concentration with
the conversion factors of 1.07 and 1.51.
2.3. Microbial population analysis
15 mL of sludge samples were collected from the outlet of the reac-
tor during the stable phase of each run, respectively on Day 97 (Run I),
181 (Run II), 275 (Run III), 350 (Run IV), 396 (Run V) and 453 (Run VI)
for microbial analysis. Samples were centrifuged at 10,000 rpm for
10 min at 4 °C, and 0.15 g of the sediment was used for DNA extraction
by ISOIL for Beads Beating kit (Nippon gene, Japan), then concentrations
and quality were measured with NanoDrop 2000 (Nanodrop Inc., USA)
and diluted to 10 ng μL−1 for PCR. V3-V4 fragment of 16S rRNA gene
were amplified with former primer 341F (5′-CCT AYG GGR BGC ASC
AG-3′) and mixed reverse primer 806R & 806R-P (5′-GGA CTA CHV
GGG THT CTA AT-3′ & 5′-GGA CTA CCA GGG TAT CTA AG-3′ in ratio of
30:1) (Matsubayashi et al., 2017). The PCR condition was as follows:
30 cycles at 94 °C for 5 s, 50 °C for 5 s, 68 °C for 10 s, and a final extension
at 68 °C for 7 min with Low DNA Ex Taq® (TaKaRa, Japan). The purity
and concentration of the amplified DNA was verified by DNA chip
with Bioanalyzer 7500 (Agilent Technologies, USA) and then purified
with Agencourt® AMPure® XP (Beckman Coulter, Inc., USA) according
711
to the manufacturers' instructions. Purified DNA was measured by
Qubit 3.0 ® (Life technologies, USA) and diluted to 4 nmol L−1. Barcode
added PCR products were sequenced by Illumina Miseq platform.
2.4. Sequencing data analysis
ð2Þ
The sequence reads were processed with QIIME (version 1.8.0)
(Caporaso et al., 2010), which is widely used in bioinformatics. Raw
data were filtered to remove poor-quality sequences, including se-
quences which were longer than 480 bp or shorter than 400 bp based
on our primers. Operation taxonomic units (OTUs) were generated
based on 97% similarity, and the clean sequences were aligned against
the most recent vision of the Greengenes database, 13_8, (http://
qiime.org/home_static/dataFiles.html) before removing the Chimeras
with ChimeraSlayer. Singleton OTUs, which only appeared once in all
ð3Þ of the samples, were removed, and sequences were randomly selected
to unify the sequence number of each sample to 26,000 to remove the
bias of sequencing depth. The Chao1 richness index, the Shannon diver-
sity index and the Simpson diversity index were calculated based on
clustered sequence data.
3. Results and discussions
3.1. Variation of parameters and performance of reactor
Based on the results of the elemental analysis, the molecular formu-
las of FW and PW in this study were calibrated as C20H33O12N and
C324H497O308N with a C/N ratio of 17.1 and 277.9, respectively. As the
PW ratio increased, the nitrogen in the substrate decreased correspond-
ingly, resulting in an increase in the C/N ratio from 17.1 to 45.0 as the
PW ratio increased from 0% to 70%. The reactor parameters during the
long-term operation also showed significant variations with the in-
creased PW content. As shown in Table 1, pH decreased from 8.33 to
7.11, and the concentration of total ammonia nitrogen reduced greatly
from 2.25 g N L−1 to 0.11 g N L−1, which resulted in a lowering of the
alkalinity from 8.31 g CaCO3 L−1 to 2.02 g CaCO3 L−1. A gradual increase
in the concentration of COD and carbohydrate in the reactor was noted,
while the concentration of protein decreased. The lipid concentration of
all the samples was under 0.19 g L−1, the VFAs were under 991 mg L−1
and the gradual decrease in VFAs indicated no accumulation. No lactic
acid was detected from the reactor. The total refractory carbohydrate
(including hemicellulose and cellulose) and lignin content accounted
712 L. Li et al. / Science of the Total Environment 652 (2019) 709–717

Table 1
Summary of the important operational parameters in thermophilic anaerobic digestion reactor.

Parameters PW ratio (%)

0 20 40 50 60 70

Influent C/N 17.1 20.4 25.8 29.9 35.8 45.0

pH 8.33 ± 0.10 8.29 ± 0.15 7.94 ± 0.20 7.49 ± 0.09 7.42 ± 0.08 7.11 ± 0.12
Ammonia-N (g L−1) 2.25 ± 0.37 1.86 ± 0.12 1.23 ± 0.30 0.52 ± 0.05 0.33 ± 0.10 0.11 ± 0.07
T-Alkalinity (g-CaCO3 L−1) 8.31 ± 0.25 7.94 ± 0.23 5.48 ± 0.59 3.37 ± 0.11 2.82 ± 0.37 2.02 ± 0.28
COD (g L−1) 28.26 ± 1.57 25.50 ± 2.21 27.97 ± 2.98 30.40 ± 1.87 31.10 ± 2.13 33.08 ± 3.82
Carbohydrate (g L−1) 2.42 ± 0.34 3.11 ± 0.51 5.08 ± 0.54 6.65 ± 0.35 7.17 ± 0.95 9.11 ± 1.57
Protein (g L−1) 11.47 ± 0.96 10.66 ± 0.85 12.08 ± 1.11 9.12 ± 0.81 9.05 ± 1.27 8.91 ± 1.21
Lipid (g L−1) 0.17 ± 0.03 0.19 ± 0.05 0.17 ± 0.02 0.16 ± 0.02 N.D.a N.D.
VFA (mg L−1) 991 ± 12 63 ± 3 128 ± 10 12 ± 3 40 ± 16 190 ± 36
GPR (L L−1 d−1) 2.46 ± 0.32 2.27 ± 0.22 2.30 ± 0.58 2.40 ± 0.22 2.25 ± 0.27 2.07 ± 0.23
CH4 content (%) 61.43 ± 2.75 58.38 ± 2.07 56.89 ± 2.26 53.89 ± 2.05 53.58 ± 1.37 51.51 ± 2.10
CO2 content (%) 38.01 ± 2.80 40.72 ± 1.00 43.81 ± 2.40 45.89 ± 0.80 46.42 ± 1.37 48.20 ± 2.54

COD: total chemical oxygen demand; VFA: volatile fatty acids; GPR: gas production rate.
a
N.D. means not detected.

for 23.38% of the TS in FW, while it was as high as 88.68% of the TS in the
PW (Table S1). A high gas production rate of 2.46 L L−1 d−1 was ob-
served when the PW ratio was 0%, and this was maintained at above
2.07 L L−1 d−1 when the PW ratio was 70%.
As shown in Fig. 2, the removal efficiency of COD maintained at
around 80% in each PW ratio. Carbohydrate removal efficiency was
high at 95.78% when the PW ratio was 0%, and while it slightly de-
creased with increasing PW ratio, it did not fall below 88.67%. The hy-
drolysis efficiency of COD and carbohydrate slightly fluctuated.
However, the protein removal efficiency, around 50% at first, fell to
just 15% when the PW ratio was 70%. It should be noted that despite
the slight decrease in the removal efficiencies of carbohydrate, the hy-
drolysis rate of carbohydrate rose from 1.96 g COD L−1 d−1 to
2.53 g COD L−1 d−1 along with the increase in the PW ratio.
3.2. Overall analysis of bacterial and archaea community
High-throughput sequencing technology based on an Illumina
MiSeq platform was used to reveal the overall taxonomic composition
of the microbial community. As shown in Table S2, the coverage of
each sample was over 99.3%, exhibiting good coverage in this study.
The Chao1 index, Shannon diversity index and Simpson diversity
index were calculated. Among them, the Shannon diversity index de-
creased from 4.71 to 3.86 with the increase of the PW ratio from 0% to
60% and eventually increased to 4.38 when PW ratio was 70%. The
changes in the Simpson diversity index exhibited the same trend. A
total of 25 phyla (account for 98.7% in total sequence) and 40 classes
(account for 98.7% in total sequence) were obtained and classified
from all the samples: 24 phyla were bacteria while only 1 phylum was
identified as archaea. The relative proportion of archaea ranged be-
tween 3.6% and 7.3%, as shown in Table S3.
3.2.1. Changes in functional bacterial groups
The community variation trend of bacteria differed significantly be-
tween PW40 and PW50, as shown in Fig. 3. Based on the Student's t-test
of these two groups (Group 1 include PW0, PW20 and PW40, Group2
include PW50, PW60 and PW70), the p-value is 0.013, which is b0.05,
indicating a statistically significant difference between the low PW
ratio sample group (b50%) and the high PW ratio sample group
(≥50%). Phylum Firmicutes was overwhelmingly predominant in all
samples, accounting for over 70% in the low PW ratio samples, but rap-
idly reduced to about 40% in high PW ratio samples. A decreasing trend
in the relative abundances of the major orders in Firmicutes was noted,
including Thermoanaerobacterales, MBA08, Clostridiales, Lactobacillales
and OPB54, the exception was SHA98, which increased greatly in high
PW ratio samples. The second predominant phylum, Thermotogae,
which accounted for about 10% in low PW ratio samples increased to
about 20% in high PW ratio samples. These two phyla have been re-
ported to be predominant in thermophilic anaerobic digestion (Jang
et al., 2016). The third predominant candidate phylum, ‘Atribacteria’,
also increased from 5.4% to 24.3% before decreasing to 11.8% in the
last run. The phylum Synergistetes changed from 3.6% to 8.5%, without

Fig. 2. (a) Removal efficiencies of COD, carbohydrate and protein; (b) hydrolysis efficiencies of COD, carbohydrate and protein; (c) hydrolysis rates of COD, carbohydrate and protein.
 L. Li et al. / Science of the Total Environment 652 (2019) 709–717
Fig. 3. Relative proportions of predominant bacterial phyla and predominant orders in phylum Firmicutes.
showing a regular trend. The fifth most abundant phylum in the sam-
ples, Bacteroidetes, increased from 0.3% to 8.7% across the runs. Other
phyla in this study were Acidobacteria, Actinobacteria, Planctomycetes,
Armatimonadetes, Spirochaetes, Caldiserica, Cyanobacteria, Chlorobi,
Fusobacteria, Nitrospirae and some other candidatus phyla. Because all
of these phyla had an abundance of b0.05% in all the sequences, they
were included in the “Others” category.
In order to illustrate the ecological function of the community in
more detail, the OTUs which accounted for N0.5% in each sample (se-
quence number N 130) were selected and aligned with the NCBI data-
base and the Greengenes database. The sum of all of the revealed
genera accounted for N90% in each sample. These bacterial genera
were divided into several functional groups according to the role played
in the hydrolysis, acidogenesis and acetogenesis processes. The details
are listed in Table 2. A total of 9 genera belonging to the phylum
Firmicutes and Thermotogae were classified as cellulose-degrading bac-
teria (CDB) based on a sequence identity of above 95% with the cultured
strains. As shown in Fig. S1(a), the abundance of CDB was at its lowest at
20.0% in PW20 and peaked at 30.5% in PW50. It has been reported that
all of these genera have (hemi) cellulose-degrading ability (Ben Hania
et al., 2012a; Park et al., 2014; Shiratori et al., 2009). Worthy of particu-
lar attention is that the great increase in the population of denovo975
and denovo6440 with the increasing PW ratio, from 0% to 19.4% and
from 4.7% to 10.9% respectively (referencing to Table S4). Denovo975
had an identity of 90% with the species of Caloranaerobacter azorensis,
and denovo6440 had an identity of 91% with the strain Candidatus
OP9 SCG 110722-44 according to the BLAST results. Note that both of
the Caloranaerobacter azorensis species and the strain Candidatus OP9
SCG 110722-44 have been confirmed to have cellulose-degrading abil-
ity (Dodsworth et al., 2013; Wery et al., 2001). Thus, we inferred that
these two OTUs are also responsible for cellulose degradation, and clas-
sified them as putative CDB. Genera of Coprothermobacter in phylum
Firmicutes and Anaerobaculum in phylum Synergistetes were identified
as protein- or peptides-degrading bacteria. With the decline in the pro-
tein concentration in the substrate, the total abundance of protein-
degrading bacteria decreased steadily from 23.6% to 10.5%, as expected.
Genera Lactobacillus and Leuconostoc were identified as lactic acid bac-
teria and their total proportions decreased from 7.7% to 1.0% throughout
the whole process. As an essential group in anaerobic digestion,
syntrophs, which are symbiotic with hydrogenotrophic archaea, play a
role in oxidizing fatty-acid and generating acetate and hydrogen simul-
taneously. These were identified as Pelotomaculum, Syntrophaceticus
and Tepidanaerobacter in this study. Among them, S. schinkii can only
use acetate as an electron donor to generate hydrogen (Westerholm
et al., 2010) while T. syntrophicus can also use both lactic acid and
713
ethanol (Sekiguchi et al., 2006). The total relative proportion of
syntrophs slightly decreased from 2.7% to 0.9%.
3.2.2. Changes of methanogenic archaea
The archaeal community structure was much simpler than the bac-
teria community, since all the sequences were identified as only 6 OTUs
and all of them belonged to phylum Euryarchaeota as given in Table S5.
Among them, denovo3242 was the most predominant OTU, accounting
for N79.6% of the total archaea, and had 100% similarity with the
hydrogenotrophic archaea Methanothermobacter thermautotrophicus
(Wasserfallen et al., 2000). Beside this OTU, there were 4 other OTUs,
Methanothermobacter tenebrarum, Methanoculleus thermophilus,
Methanolinea mesophila and Methanomassiliicoccus luminyensis, which
have been classified as hydrogenotrophic archaea (Dridi et al., 2012;
Nakamura et al., 2013; Sakai et al., 2012; Zhu et al., 2011). The relative
proportion of hydrogenotrophic archaea decreased from 6.0% to 2.9%
at low PW ratios and increased from 2.9% to 6.8% at high PW ratios, as
shown in Fig. S1(b). The predominance of hydrogenotrophic species
in the archaea community in this study is in good agreement with the
results reported from previous studies focused on thermophilic meth-
ane fermentation (Chachkhiani et al., 2004; Hori et al., 2006; Yu et al.,
2014). The second most predominant species was Methanosarcina
thermophila, which can utilize both acetate and methyl compounds
(Aceti and Ferry, 1988). The relative abundance of Methanosarcina
thermophila was stable at each PW ratio and ranged from 0.4% to 0.6%.
3.3. CDB and protein-degrading bacteria responding to the increased PW
ratio
The predominant hydrolyzing groups, which play the important
roles in the hydrolysis step, were the CDB and the protein-degrading
bacteria. The relative abundance of both shifted significantly. The in-
crease of PW not only elevated the amount of carbohydrates in the
substrate, but also raised the refractory proportion of carbohy-
drates, such as cellulose. A 25.6% increase in the relative abundance
of the putative CDB resulting in a 28.2% increase in the total CDB.
Conversely, because the sole contribution of protein was the FW in
this study, when the dosage of FW decreased, the protein concentra-
tion in the substrate also dropped, which led to a 12.7% reduction in
the proteolyzer. The increase in the CDB and decrease in the
proteolyzers resulted in a shift of the metabolism type in the entire
anaerobic digestion system from carbohydrate/protein-degradation
to cellulose-degradation.
714 L. Li et al. / Science of the Total Environment 652 (2019) 709–717

3.4. Variation of CDB effecting on the lactic acid bacteria and syntrophs
Optimum pH

6.0–7.7

5.7–6.1
7.3–7.5
7.0–9.0

6.6–7.3
Unlike previous studies on the mono-digestion of cellulose or lignin

6.9

6.5

7.5

7.8
(Callaghan et al., 2002; Yen and Brune, 2007), stable operation was

7
achieved in the co-digestion of PW and FW in this study with a high car-
Ethanol

bohydrate removal efficiency above 90.0% even at a high organic load-

ing rate of 14 g L−1 d−1. The high removal efficiency of carbohydrate
+

+
+
+
+
+

+
was mainly attributed to the overwhelmingly predominance of CDB in
Acetate

the bacterial community. As shown in Fig. S1(a), the relative abundance

of CDB and putative CDB increased by 28.2% when the PW ratio in-
+
+
+
+
+
+
+

+
+

+
+

+
creased from 0% to 70%. The high occupancy of CDB ensured the hydro-
H2 + CO2

lysis of carbohydrate, enabling a stable carbohydrate removal efficiency

during the entire operation. Apart from the increase in the total abun-
dance of CDB, other significant changes in specific species when the
+
+
+
+
+
+
+

+
+

+
+
+
PW ratio was elevated from 40% to 50% greatly influenced the following
Lactic acid

acidogenesis and acetogenesis processes. As shown in Fig. 4, while

Products

Defluviitoga tunisiensis, Candidatus OP9 SCG 110722-44, Clostridium

+

+
+

+
+
putrefaciens, Ruminiclostridium sp., Herbinix hemicellulosilytica and
Defluviitalea sp. were predominant at low PW ratios, the predominant
Function of closest relatives

Monosaccharide-degrading

species shifted to Defluviitoga tunisiensis, Candidatus OP9 SCG 110722-

44, Caloranaerobacter sp. and Ruminiclostridium thermocellum at high
Lactic acid bacteria
Lactic acid bacteria
Lactic acid bacteria
Lactic acid bacteria
Lactic acid bacteria
Lactic acid bacteria

PW ratios. Those CDB which were predominant in high PW ratios

Protein-degrading
Protein-degrading

have been reported to grow at an optimum pH at a neutral condition

b7.0 (Ben Hania et al., 2012b; Johnson et al., 1982; Wery et al., 2001),
Syntrophs
Syntrophs
Syntrophs

while at low PW ratios, the optimum pH for the predominant CDB has
been reported to be relative alkalinity, and over 7.0 (Bouanane-
CDB
CDB
CDB
CDB
CDB
CDB
CDB
CDB
CDB

Darenfed et al., 2011). This is completely different from that in high

PW ratios. Therefore, the variation in the CDB species was considered
Identity (%)
Variation of relative proportion, phylogenetic classification and metabolism descriptions of functional bacteria at different paper waste ratios.

to correspond with the decrease in the pH value from 8.3 to 7.1 in this
study.
100

100

100

100
100
100
100
100

100
95

99
95
97
96
98
95

95

98

99
97
96

The variation of the CDB affected the abundance of lactic acid bacte-
ria. All the lactic acid bacteria in this study were reported to use mono-
Accession number

saccharide as the sole carbon source, while the CDB in this study were
all reported to have monosaccharide-degrading ability. The increase in
MH298782

MG825725

MG674712
KM036189

MF179627

MF093231
MF443391
NR125455

NR136763

NR108234
NR074653

NR074997

NR041320
NR116297
NR040966
LN881581
KT274710

CP016502

CP016502
KF766957

the CDB led to competition with lactic acid bacteria because the CDB
AB10748

consumed the majority of monosaccharide, leaving little monosaccha-

ride for the lactic acid bacteria. Also, with the increase in the PW ratio,
cellulose gradually became the main carbohydrate component rather
Coprothermobacter proteolyticus
Ruminiclostridium thermocellum

Tepidanaerobacter syntrophicus
Ruminiclostridium stercorarium

Pelotomaculum isophthalicicum

than the monosaccharide in the substrate. This greatly reduced the

Defluviitalea isophthalicicum
Ruminiclostridium caenicola
Herbinix hemicellulosilytica

availability of monosaccharide for lactic acid bacteria, resulting eventu-

Fervidobacterium riparium
Hydrogenispora ethanolica

Syntrophaceticus schinkii
Clostridium putrefaciens

CDB: cellulose-degrading bacteria. The sequence number of each sample was unified to 26,000.

ally in a sharp decline in lactic acid bacteria occupancy. Lactic acid is

Lactobacillus helveticus
Defluviitoga tunisiensis

Anaerobaculum mobile
Herbivorax saccincola

Lactobacillus hilgardii

Leuconostoc gelidum
Lactobacillus rossiae

usually reported as one of the organic acids produced in anaerobic di-

Lactobacillus brevis
Lactobacillus sakei
Closest relatives

gestion. The accumulation of lactic acid further acidizes the system

and inhibits methanogenesis. The lactic acid bacteria were also reported
to generate bacteriocins which inhibit the growth of other bacteria
(Noike et al., 2002). In this regard, the decrease in the lactic acid bacteria
inversely benefited the growth of CDB and other bacteria.
The CDB also reduced the proportion of the syntrophs. The CDB at
4622

1088

1909
204

296

832

193

135
70

10

10
77

71

86

13

the high PW ratios, such as Defluviitoga tunisiensis, were more likely to

0

4
0

1
0
1

degrade monosaccharide directly to acetate and hydrogen without pro-

5222

1108

1036
1768

ducing propionate and butyrate (Ben Hania et al., 2012a) than the CDB
196

203
60

12

33
36
48
4

2
6
0

3
0
6

0
0
0

at low PW ratios. The decrease in the VFA concentration also resulted in

the decline in the abundance of syntrophs which utilize VFAs as a sub-
5387

1065
1334

3964
2164
119

175

119
156
232
50

34

64

65

strate to generate acetate and hydrogen. The relative proportion of

8

0
0

1
0
0

syntrophs at high PW ratios decreased to less than half that at low PW

1989

1330

1824
1410

4222
1341
190

278

915

200
147

384

ratios.
40

87
85

75

68
0
0

4
0
0
2736

3855
1736
1055

3.5. Hydrogen production and consumption

PW ratio (%)

178
456

250

926
513

491

132

153
20

69

43

18

84

49
0

High H 2 -producing ability has been reported at a high C/N

3170
1071

5197

1162
599
421
212
103

850

489
258

136
296
276
32

31
26
15

67
16

ratio of 47:1 (Lin, 2004) as opposed to the ratio of 25:1 in nor-

0

mal anaerobic digestion (Parkin and Owen, 1986). This was in

denovo6151
denovo2999

denovo3430
denovo3241
denovo5581
denovo1702

denovo6439

denovo3720
denovo4182
denovo4667
denovo5881
denovo4772

denovo4262

denovo4664
denovo942
denovo473

denovo433

denovo471

denovo623

denovo474

accordance with a C/N ratio of 45:1 when PW was 70% in this

denovo6

research.
OTU ID
Table 2

As explained, the CDB at high PW ratios were capable of directly

degrading monosaccharide to acetate rather than propionate and
 L. Li et al. / Science of the Total Environment 652 (2019) 709–717 715

Fig. 4. Variation of the main identified bacterial and archaea species along with the increase in the paper waste ratio.

butyrate, thus enabling them to produce more hydrogen. This is based perspectives, both the H2-producing and H2-consuming processes
on the following equation: were enhanced at high PW ratios.

C 6 H12 O6 þ 2H 2 O→2CH3 COOH þ 4H2 þ 2CO2 ð4Þ 3.6. Variation of metabolic pathways

C 6 H12 O6 þ 2H 2 →C 2 H 5 COOH þ 2H 2 O
C 6 H12 O6 →C 3 H7 COOH þ 2H2 þ 2CO2
This was especially relevant to Defluviitoga tunisiensis, which has been
reported to have high H2-producing ability when fed with cellulose
(Maus et al., 2015). With the increase in the total number of H2-
producing bacteria, the hydrogen production capacity of the system was
increased, and the hydrogenotrophic archaea increased correspondingly.
It was reported that with the increasing content of H2 in the anaero-
bic digestion system, the metabolic pathway from propionate to acetate
becomes inhibited, leading to VFA accumulation (Lin, 2004). In this re-
search, the increasing abundance of hydrogenotrophic archaea elimi-
nates the adverse effects of excessively produced H2 from cellulose,
ensuring that the operation of the system remains stable. From these
ð5Þ In this study, great differences were found in the metabolic path-
ways for low PW ratios and high PW ratios due to the change of the cor-
ð6Þ responding functional bacteria and archaea. The details are provided in
Fig. 5, where the microorganisms which increased in abundance are
marked in red and those which decreased in abundance are marked in
green. Note that the abundance of the microorganisms is roughly indi-
cated by the size of the wording. As the condition changed from a low
PW ratio to a high PW ratio, bacteria evolved either in the direction of
degrading cellulose rather than carbohydrate and protein, or by
degrading monosaccharides directly to acetate and hydrogen. The ar-
chaea community changed along with the H2-producing bacteria. That
is, due to the formation of a new colony structure, when the PW ratio
≥ 50%, the thermophilic anaerobic digestion system obtained apprecia-
ble stability and high cellulose tolerance. The microbial community
has been reported to maintain a functional redundancy which can

Fig. 5. Functional groups of microorganisms in the hydrolysis, acidogenesis, acetogenesis and methanogenesis processes. The microorganisms which increased in abundance are marked in
red and those which decreased in abundance are marked in green.
716 L. Li et al. / Science of the Total Environment 652 (2019) 709–717
continue to conduct normal functions even when the community struc-
ture is forced to change due to environmental stress (Berga et al., 2012).
The variations of the functional microorganisms in this system and the
main metabolic pathways illustrate the stability and high cellulose tol-
erance the of thermophilic anaerobic co-digestion system.
4. Conclusions
The change in the functional bacteria and archaea community
during thermophilic anaerobic co-digestion of food waste and paper
waste was investigated by 16S rRNA gene analysis. The hydrolysis bac-
teria shifted from carbohydrate/protein-degrading bacteria to cellulose-
degrading bacteria. The evolution of CDB was observed from low to high
paper waste ratios, enhancing the cellulose tolerance and helping to
maintain the stability of thermophilic co-digestion of food waste and
paper waste. CDB outcompeted lactic acid bacteria in the degradation
of monosaccharides, reducing the proportion of lactic acid bacteria.
CDB in high paper waste ratios directly hydrolyzed monosaccharide to
acetate and hydrogen, avoiding the accumulation of propionate and bu-
tyrate. The increase in CDB, which obtained high H2-producing ability,
eventually enhanced H2-production, also resulting in the predominant
abundance of hydrogenotrophic archaea at a high paper waste ratio.
Acknowledgements
This work was also supported by Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant No. 18J11397. We gratefully acknowl-
edge the financial support from the Japan Science and Technology
Agency (JST) in the Japanese-Chinese Research Cooperative Program
on “Research and Development to Find Solutions to Environmental
and Energy Issues in Urban Areas” (16769220A) and a Grant in-Aid of
Tohoku University Division for Interdisciplinary Advanced Research
and Education.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.10.292.
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