Bioresource Technology 341 (2021) 125771

Contents lists available at ScienceDirect

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

Waste-to-energy: Cellulase induced waste activated sludge and paper waste

co-fermentation for efficient volatile fatty acids production and
underlying mechanisms
Jingyang Luo a, b, Yi Li a, b, Yibing Li a, b, Han Li a, b, Xinyang Fang a, b, Yuxiao Li a, b,
Wenxuan Huang a, b, Jiashun Cao a, b, Yang Wu c, *
a
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, 1 Xikang Road, Nanjing 210098, PR
China
b
College of Environment, Hohai University, 1 Xikang Road, Nanjing 210098, PR China
c
State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai
200092, China

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

• Cellulase exhibited positive effects on

WAS and PW co-fermentation.
• Cellulase enhanced solubilization/hy­
drolysis via improving bioavailable
substrates.
• Cellulase enhanced acidification via
stimulating the microbial activities.
• Cellulase enriched the functional an­
aerobes and genetic expressions.

A R T I C L E I N F O A B S T R A C T

Keywords: This study mainly investigated the feasibility of utilizing cellulase to enhance waste activated sludge (WAS) and
Waste activated sludge (WAS) paper waste (PW) co-fermentation for the generation of volatile fatty acids (VFAs). The introduction of cellulase
Paper waste (PW) effectively enhanced the co-fermentation efficiency, and the maximum VFAs generation reached 3014 mg COD/L
Volatile fatty acids (VFAs) production
with 60 mg cellulase/g TSS while it was 1512 mg COD/L in the control reactor. The presence of cellulase
Cellulase
Genetic expressions
evidently improved the concentration of soluble bioavailable substrates (e.g., carbohydrates and proteins) via
inducing the EPS disintegration and PW disruption. More importantly, the functional anaerobes (i.e., Macelli­
bacteroides and Bacteroides) and the microbial activities (i.e., ATP, key acid-forming enzymes, and genetic ex­
pressions) that related with the VFAs biosynthesis were enriched and enhanced due to the stimulation of
cellulase, contributing to the ultimate VFAs promotion. This study provided a novel strategy to recover valuable
products from waste biomass with constructive mechanistic exploration.

1. Introduction biomass “waste-to-energy” concept that transforms waste organics into

renewable energy with high financial potential and eco-friendly bene­
Due to the growing energy depletion and environmental quality fits, has drawn extensive awareness considering the massive generation
deterioration induced by the fossil fuel, the search and preference for of waste biomass (Cao et al., 2020; Yi et al., 2018). Anaerobic fermen­
sustainable energy and fuels have greatly increased. Recently, the tation of organic wastes is a cost-effective and environmentally

* Corresponding author.
E-mail address: [email protected] (Y. Wu).

https://doi.org/10.1016/j.biortech.2021.125771
Received 28 July 2021; Received in revised form 10 August 2021; Accepted 10 August 2021
Available online 14 August 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
J. Luo et al. Bioresource Technology 341 (2021) 125771

sustainable approach to bio-convert the low-value biomass into high cellulase-stimulated WAS/PW co-fermentation systems remain
value-added chemicals (i.e., volatile fatty acids (VFAs) and methane), as unknown.
well as limited pollution and low carbon footprint (Duan et al., 2020). Therefore, this study mainly investigated the feasibility of utilizing
Currently, the effective disposal of increasing municipal solid waste cellulase to enhance WAS and PW co-fermentation for VFAs production.
(MSW) has become great obstacles to sustainable social development. The impacts of cellulase on the WAS/PW co-fermentation were firstly
For example, approximately 210.3 million tonnes of MSW are generated revealed. Then the variations of bioavailable substrates concentrations
in China annually, and it is estimated to reach 418 million tonnes by were demonstrated, and the physiochemical properties of WAS/PW
2030 (Soomro et al., 2020). Fortunately, approximately 50 ~ 70% of structures were analyzed via SEM analysis. Also, the shift of microbial
MSW were composed of easy-to-degradable organic materials, which community and the microbial activities, such as the critical enzymatic
were commonly regarded as organics fraction of municipal solid waste activities and encoding gene expressions responsible for substrates
(OFMSW), and were considered as ideal feedstocks for resource recovery metabolism and VFAs biosynthesis, were analyzed. This study provided
(Panigrahi and Dubey, 2019). Among them, the paper waste (PW) is the a novel strategy to recover valuable products from waste biomass with
second-largest OFMSW constituting 9–12% (Ranieri et al., 2018). In constructive mechanistic exploration.
particular, over 400 million tonnes of PW were produced, and approx­
imately 50% of them have been recycled because of the various con­ 2. Materials and methods
straints on waste recycling (Soomro et al., 2020). PW contained high
cellulose contents (50–60%), which could convert into fermentable 2.1. WAS and PW used in this study
sugars (Al-Battashi et al., 2019). The utilization of PW as feedstock for
VFAs production might be valuable and feasible for waste management The WAS used in this study was obtained from a municipal waste­
in view of its relatively high availability and low cost (Qin et al., 2018). water treatment plant in Nanjing, China. Before use, the WAS was firstly
However, the biological fermentation demands the involvement of screened with a stainless-steel mesh to remove those undesirable par­
various functional microorganisms. The PW is generally poor in ticulate inorganics, and then was naturally settled and concentrated for
providing sufficient key bacteria for anaerobic fermentation. Moreover, 24 h at 4 ℃. The basic WAS characteristics are: total suspended solids
the imbalance of carbon/nitrogen (C/N) and trace elements in PW, 18075 ± 255 mg/L, volatile suspended solids 9670 ± 170 mg/L, total
which are generally required by the microorganisms, are challenging to carbohydrates 1522 ± 98 mg COD/L, total proteins 8785 ± 172 mg
maintain the process stability for FW mono-fermentation (Gonzalez- COD/L, and pH 6.7 ± 0.2. The C/N ratio (carbon to nitrogen ratio) is 5.6
Estrella et al., 2017a; Gonzalez-Estrella et al., 2017b). One of the most ± 0.1.
essential strategies to reduce process instability and enhance fermen­ The PW was collected from the scrape-offs in the working office. The
tation efficiency is the co-fermentation of two or more substrates. Waste PW was firstly shredded into small pieces by utilizing the mechanical
activated sludge (WAS) is another large generated MSW derived from shredder, and then was soaked with alkaline solutions (pH 10 with
wastewater treatment plants, which has been estimated to be 70 million NaOH adjustment) for 6 h. The treated mixture was finally adjusted to
tons (80% moisture) by 2025 in China (Klatte et al., 2014). It contains neutral condition for further use. The VSS fraction of PW was about
various microorganisms with multiple distinct functions, which is an 80%. The content of cellulose, hemicellulose and lignin in the PW was
ideal inoculum source for the anaerobic fermentation of organic matters. respectively 59.3 ± 0.5, 9.3 ± 0.2 and 2.5 ± 0.3%. The C/N ratio of PW
Also, the sufficient trace elements that support the microbial growth, is 90.1 ± 2.0.
and the previously considered drawback of low C/N ratio in WAS is an
excellent makeup for the PW. Moreover, the WAS and PW might be 2.2. Influences of cellulase inducement on the production of VFAs during
mixed with each other at the waste collection and disposal points. WAS/PW co-fermentation
Therefore, the co-fermentation of WAS and PW for VFAs production is
quite applicable and promising. The experiments were operated in 5 identical serum bottles, whose
However, the efficient VFAs generation derived from WAS/PW co- total working volume is 0.6 L. Firstly, 0.3 L of WAS/PW mixtures (with
fermentation might be restricted due to the recalcitrant components in the mass ratio of 75%:25%) was fed into the fermentation reactors with
PW (i.e., cellulose, and lignin) and cemented extracellular polymeric a final concentration of 18 g TSS/L. Then different levels of cellulase, e.
substances (EPS) in WAS, which were not able to be utilized directly and g., 0, 15, 30, 45, and 60 mg/g TSS, were dosed into the corresponding
efficiently by the fermentative bacteria (Chen et al., 2021). Commonly, reactors to investigate the correlations of cellulase dose with VFAs
these fermentation substrates should be firstly solubilized and hydro­ generation derived from WAS/PW co-fermentation. The fermentation
lyzed to simple bioavailable matters for subsequent microbial meta­ reactions were operated in the air-bath shaker with the condition of 180
bolism. Various pre-treatment approaches, including the ultrasonic, rpm and 35℃. The anaerobic environment was maintained by filling
acidic/alkaline treatment and etc., have once been proposed (da Cunha pure nitrogen and sealing the reactors with rubber stoppers. The cor­
et al., 2021). In comparison with the traditional physical and chemical relations of cellulase dose with VFAs generation were based on the
stimulation, the biological approach, such as the enzymatic treatment variation of VFAs concentrations and composition in the fermenters
(Wang et al., 2021b), is also considered as a quick technique to disrupt with different cellulase doses.
the organic wastes structures in a more environmentally friendly and
less energy-consuming way. The enzymatic pre-treatment has previ­ 2.3. Mechanistic insight of VFAs promotion in cellulase-induced WAS/
ously been deployed to pretreat corn stoves and attained an 11–24% PW co-fermentation
improvement for methane production during anaerobic digestion
(Bahreini et al., 2020). However, till now, the study of cellulase-induced To disclose the influences of cellulase on WAS/PW solubilization/
WAS and PW co-fermentation for VFAs production has not been criti­ hydrolysis, the contents of soluble proteins and carbohydrates (domi­
cally explored. Also, the underlying mechanism has yet to be known. For nant organic components) were analyzed at 4 d. In order to further
instance, the anaerobic species, such as the hydrolytic bacteria and demonstrate the changes of organic components caused by cellulase, the
acidogens, played significant roles in the anaerobic fermentation pro­ variations of carbohydrates (i.e., cellulose, hemicellulose, and lignin) in
cess. But the distribution patterns and metabolic activities of functional WAS/FW mixture and soluble microbial products were characterized.
microorganisms involved in VFAs biosynthesis were prone to be Also, the physiochemical structures of WAS/PW mixture were deter­
impacted by the environmental variations, such as the substrates mined via SEM analysis.
composition and exogenous stimulus. How the microbial structure var­ Regarding the correlations of cellulase dosage with the acidification
ied and their associations with the ultimate VFAs generation in the process, the activities of phosphotransacetylase (PTA) and acetate

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J. Luo et al. Bioresource Technology 341 (2021) 125771

kinase (ACK), which are responsible for acetic acid biosynthesis, and 2.5. Statistical analysis
succinyl CoA transferase (SCOT) and oxaloacetate transcarboxylase
(OAATC), which are associated with propionic acid, were also evaluated All the testes operated were in triplicates. And the analysis of vari­
(Wang et al., 2019). In addition, adenosine 5′ -triphosphate (ATP) was ance by one-way ANOVA was performed to determine the significance of
analyzed to determine the general activity of anaerobic microorganisms obtained data, and the p < 0.05 was regarded as significant statistical
(Wu et al., 2021b). differences.
Additionally, to reveal the shift in the microbial community and the
related genetic expression that participates in VFA production, the 3. Results and discussion
fermentation samples in the reactors with 0, 15, and 60 mg cellulase/g
TSS were collected at the end of fermentation for Illumina MiSeq 3.1. VFAs promotion from WAS and PW co-fermentation induced by
sequencing. cellulase

As shown in Fig. 1(A), the co-fermentation of WAS and PW exhibited

2.4. Analytical methods advantages over sole WAS fermentation for the VFAs production. The
maximal VFAs accumulation in the sole WAS fermentation system was
The total suspended solids (TSS), volatile suspended solids (VSS), approximately 708 mg COD/L at 10 d while it incremented to 1512 mg
and chemical oxygen demand (COD) analysis were conducted on the COD/L with the introduction of PW. This result indicated the feasibility
basis of standard methods (APHA, 1998). The content of carbohydrates of PW as the resource feedstock for VFAs generation. Meanwhile, the
was determined by the phenol–sulfuric method, while the concentration fermentation efficiency was further accelerated and enhanced with the
of proteins was obtained by the Lowry–Folin method (Luo et al., 2021). addition of cellulase. The highest VFAs production was 1832 mg COD/L
The content of PW (i.e., cellulose and hemicellulose) were detected ac­ with 15 mg cellulase/g TSS, and it further gradually increased along
cording to the method of NREL/TP-510-42618, which referred to the with the rise of cellulase dosage and reached 3014 mg COD/L with 60
previous study (Sluiter et al., 2008). mg cellulase/g TSS. Obviously, the presence of cellulase exhibited pos­
The activities of acid-forming enzymes were analyzed via commer­ itive effects on the generation of VFAs during WAS/PW co-fermentation,
cial enzymatic kits (Sembega biotechnology Co. Ltd., Nanjing, China). and the enhancing effects were directly proportional to the increase of
The ATP content was also tested by ATP assay kit (Beyotime, China). In cellulase dosage.
addition, the characterization of soluble microbial products was per­ Also, the distribution of VFAs composition was slightly affected by
formed by three-dimensional excitation-emission-matrix (EEM) fluo­ the cellulase. In the sole WAS fermentation reactor, the propionic acid
rescence spectrometry (F7000, Hitachi, Japan) (Wawrzynczyk et al., occupied the most (with a ratio of 39.7%), and followed by the butyric
2008). The morphologies of WAS/PW mixture were analyzed by the and valeric acids, whose proportions were 33.4% and 20.7%, respec­
scanning electronic microscopy (SEM, Quanta 450FEG, FEI, USA). The tively. Interestingly, acetic acid only accounted for 6.2%. However, in
C/N ratio was analyzed by the elemental analyzer (Vario EL III, Ele­ the WAS and PW co-fermentation systems, the proportion of butyric and
mentar, Germany). The concentration and composition of VFAs valeric acid was significantly reduced. Correspondingly, the acetic and
(including the acetic, propionic, butyric, and valeric acids) were propionic acids became the predominant VFAs. As exhibited in Fig. 1(B),
detected by the gas chromatography equipped with a flame ionization the acetate distribution in reactors with different cellulase dosages was
detector (Agilent 7820, USA), referring to the previous literature for similar (32.4 ~ 38.5%), while the ratio of propionic acid improved
specific operation conditions (Wu et al., 2021b). gradually. For example, the proportion of propionate was 45.3% in the
The microbial community analysis was conducted via Illumina reactor without cellulase addition, while it gradually increased to 52.7%
MiSeq sequencing by Biozeron Biological Technology Co. Ltd (Shanghai, with 60 mg cellulase/g TSS. The previous publication had demonstrated
China). The extracted DNA by using CTAB/SDS kits was amplified via that the acetic and propionic acids exhibited multifaceted application
the 341F/806R primer set, which targeted the V3-V4 region of bacterial with evident economic benefits, e.g., the desirable carbon source for
16S rDNA (Ikoyi et al., 2018). The cluster of operational taxonomic units nutrients removal in the biological wastewater treatment process (Wang
(OTUs, 97% sequence similarity) was based on the UPARSE package. et al., 2021a). Therefore, the significant VFAs promotion accompanied
The phylogenetic investigation of communities by reconstruction of with the high ratios of acetic and propionic acids in the cellulase-
unobserved states (PICRUSt) was also used to predict the genetic func­ conditioned reactors improved the feasibility and applicable value of
tions related with VFAs biosynthesis based on the Kyoto Encyclopedia of WAS and PW during co-fermentation.
Genes and Genomes (KEGG) categories at different levels (Wu et al., Overall, the above results demonstrated the positive effects of
2021b).

Fig. 1. (A) The variations of total VFAs and (B) proportions of specific VFAs in different reactors during WAS and PW co-fermentation.

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cellulase on the WAS and PW co-fermentation for VFAs promotion as WAS/WAS mixture in the control reactor was tight with little porosity,
well as the distribution of generated VFA, and the promoting effects which was not advantageous to the biochemical interactions between
were dose-dependent. the fermentative bacteria and substrates. However, the surface structure
of cellulase-conditioned reactors was much looser with higher porosity.
3.2. Promotion of bioavailable substrates and microbial activities induced Also, the disruptive extent of WAS/PW mixture was associated with
by cellulase during WAS and PW co-fermentation cellulase levels, which was more evident at high dose. This indicated
that the chemical bonds of cellulose-like substances in WAS and PW had
3.2.1. Improvement of bioavailable substrates been effectively ruptured by the cellulase treatment. Meanwhile, these
As is well-known, the abundant bioavailable substrates that could be results were also consistent with the distribution of hemicellulose, and
metabolized by the critical fermentative bacteria is the prerequisite for lignin in cellulase-conditioned reactors. As shown in Fig. 3, the con­
efficient VFAs generation during the anaerobic process (Luo et al., centrations of hemicellulose and lignin were remarkably reduced after
2021). As the main organic components in WAS and PW contained introducing the cellulase. For example, the hemicellulose and lignin
carbohydrates and proteins, the variations of soluble carbohydrates and contents occupied 29.6 and 15.6% in the reactor without cellulase
proteins concentration in different fermenters were correspondingly addition, respectively. However, they were reduced to 21.5 and 15.0%
investigated.
As exhibited in Fig. 2(A), the levels of soluble proteins and carbo­
hydrates were found to be merely 212.9 and 227.7 mg/L in the control
reactor (without cellulase addition), respectively. However, their con­
centrations were both incremented greatly due to the addition of
cellulase, and the promoting effects were also dose-dependent. For
instance, the levels of soluble carbohydrates and proteins were
improved and reached 222.7 and 366.6 mg/L in the WAS/PW fermenter
with 15 mg cellulase/g TSS. They were further increased with the
increment of cellulase dosage and rose to 541.0 and 850.9 mg/L with 60
mg cellulase/g TSS. Meanwhile, the positive effects of cellulase on the
increase of bioavailable substrates could be further confirmed by the
EEM characterization. As shown in Fig. 2(B) ~ 2(F), the major soluble
substrates in fermentation supernatant included tryptophan-like pro­
teins (T1 and T2 positions) and tyrosine-like proteins (B1 and B2 posi­
tions), which are generally considered to be biodegradable by
fermentative bacteria (Han et al., 2021). More importantly, the fluo­
rescence intensities (positively correlated with the content of fluorescent
substrates) were also enhanced with the addition of cellulase, and
showed a dose-dependent upward trend.
The introduction of cellulase might induce EPS disintegration and
Fig. 3. The distribution of hemicellulose and lignin in different reactors during
PW disruption, which resulted in the large release of organic substrates.
WAS/PW co-fermentation.
This assumption was verified by the SEM analysis. The microstructure of

Fig. 2. (A) The soluble carbohydrates and proteins, (B)~(F) The EEM characterization of fermentation supernatant in different reactors during WAS and PW co-
fermentation.

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in the reactor with 15 mg cellulase/g TSS, and further dropped to 0.8% synchronously during the co-fermentation of WAS and PW, thereby
and 12.3% in the reactor with 60 mg cellulase/g TSS. Thus, the decre­ attaining a satisfactory VFA production performance.
ment in the hemicellulose and lignin indicated that the cellulose-like
substances in WAS and PW had been indeed decomposed by cellulase,
thereby promoting the concentration of soluble substrate. 3.3. Variations of microbial community and metabolic genetic functions
Generally, the evident increase of bioavailable substrates in induced by cellulase
cellulase-induced WAS/PW fermenters indicated the acceleration of
solubilization and hydrolysis steps, which could contribute to the VFAs During the fermentation process, the microorganisms played an
promotion in the process of anaerobic fermentation. essential role in VFAs production (Luo et al., 2020b). Therefore, the
variation of microbial communities induced by cellulase might influence
3.2.2. Enhancement of microbial activities the ultimate VFAs generation. In this study, the 16 s RNA amplicon high-
In addition to sufficient bioavailable substrates, VFAs generation in throughput sequencing method was applied to determine the microbial
the acidification process was also highly dependent on microbial ac­ composition in different cellulase-conditioned reactors. The coverage
tivities (Luo et al., 2020a). The level of ATP, which was an essential estimators of all samples were higher than 0.97, indicating that the
bioenergy for VFAs production, had been frequently considered as the microbial community was well represented by the collected gene se­
direct indicator of general microbial activities (Wu et al., 2021a). As quences (Table 1). Meanwhile, the OTU and Chao index were 1953 and
exhibited in Fig. 4, the level of ATP in the reactor without cellulase 2679 in the reactor without cellulase addition. However, they were
addition was merely 0.45 uM/L. Nevertheless, it was observed to be decreased to 1480 and 2107 in the reactor with 15 mg cellulase/g TSS,
enhanced by the introduction of cellulase during WAS and PW co- and further dropped to 1055 and 1561 in the reactor with 60 mg
fermentation. For instance, the level of ATP was 0.62 uM/L in the cellulase/g TSS. Therefore, the introduction of cellulase decreased the
reactor with 15 mg cellulase/g TSS, while it further improved to the 1.38 microbial richness, which might be conducive to enrich functional mi­
uM/L in the reactor with 60 mg cellulase/g TSS. croorganisms for VFAs biosynthesis.
In addition, the activities of several critical acid-forming enzymes Meanwhile, as illustrated in Fig. 5 (A), the composition of the mi­
related with the VFAs variations (particularly the acetic and propionic crobial populations at the phylum level shifted in the presence of
acids, which were the predominant VFAs in this study) also exhibited a cellulase. The predominant bacteria in the control reactor was Bacter­
similar trend. For example, compared with the reactor without cellulase oidetes (26.5%), followed by Proteobacteria (19.3%), Firmicutes (17.5%)
addition (100% of control), the activities of acetate kinase (AK) and and Spirochaetes (14.3%). However, the relative abundance of Bacter­
phosphotransacetylase (PTA), which were vital enzymes regulating the oidetes was observed to be remarkably increased to 49.9% in the reactor
acetate production (Wang et al., 2019), were increased to 115.5 and with 15 mg cellulase/g TSS, and 45.0% in the reactor with 60 mg
106.8% in the reactor with 15 mg cellulase/g TSS, and further improved cellulase/g TSS. The previous publication had indicated that the Bac­
to 148.4 and 141.8% with 60 mg cellulase/g TSS, respectively. More­ teroidetes was a typical VFAs producer, containing various functional
over, the enzymatic activities of oxaloacetate transcarboxylase (OAATC)
and CoA transferase (CoAT), which took part in the formation of pro­ Table 1
pionic acid, were 112.5 and 108.6% in the reactor with 15 mg cellulase/ α biodiversity of fermentation reactor with different cellulase addition.
g TSS, and further increased to 139.1 and 133.7% with the dosage of 60 Sample α = 0.03
mg cellulase/g TSS, respectively. These results agreed with the VFA
OTU Chao Coverage Shannon Simpson
composition variations (increased acetate and propionate proportions)
in cellulase-conditioned reactors. 0 mg cellulase/g TSS 1953 2679 0.9804 5.45 0.0155
15 mg cellulase/g TSS 1480 2107 0.9809 4.34 0.0929
Overall, the presence of cellulase exhibited positive effects on the
60 mg cellulase/g TSS 1055 1561 0.9703 4.39 0.0601
enhancement of bioavailable substrates and microbial activities

Fig. 4. The key enzymatic activities in different reactors during WAS and PW co-fermentation.

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Fig. 5. The microbial populations at (A) phylum level and (B) genus level in different reactors during WAS and PW co-fermentation.

microorganisms that participate the transformation of organics into conditioned reactors. For example, in comparison with the control re­
VFAs (Luo et al., 2020b). Moreover, Spirochaetes had been accepted as actors, the relative abundance of glucokinase [EC:2.7.1.2], which cata­
the acetate-oxidizing bacteria, which could utilize acetate to produce lyzed the first step of glycolysis (Wu et al., 2021b), was improved 1.1
hydrogen during the fermentation system (Si et al., 2016). The relative and 1.5 folds in the reactors with 15 and 60 mg cellulase/g TSS,
abundance of Spirochaetes was 14.3% in the reactor without cellulase respectively. Meanwhile, the relative abundance of 6-phosphofructoki­
addition, while it was reduced to 7.9 and 4.1% in the reactor with 15 and nase [EC:2.7.1.11], which was also key enzyme involved in glycolysis
60 mg cellulase/g TSS, respectively. Therefore, the increase of Bacter­ and catalyzed the ATP-dependent phosphorylation of several hexoses to
oidetes abundance but with the decrease of Spirochaetes in cellulase- hexose-6-phosphate (Gonzalez-Gil et al., 2019), was 0.68‰ in the con­
conditioned reactors were conducive to both the WAS/PW hydrolysis trol reactor, while increased to 0.97‰ in the reactor with 15 mg cellu­
and acidification processes, which was well consistent with the high lase/g TSS, and further improved to 1.06‰ with 60 mg cellulase/g TSS.
VFAs production in the corresponding fermentation reactors. The upregulation of these functional genes would absolultely enhance
The variation of microbial population caused by cellulase was the substrates metabolism in WAS/PW for the subsequent VFAs
further demonstrated at the genus level. As demonstrated in Fig. 5, generation.
various functional bacteria that participated in VFAs generation were Moreover, several gene expressions involved in VFA biosynthesis (e.
found in different WAS/PW fermenters but with quite different abun­ g., acetyl-CoA acyltransferase [EC:2.3.1.16], pyruvate carboxylase sub­
dances. The dominant functional VFAs producers in the control reactor unit B [EC:6.4.1.1], acetyl-CoA hydrolase [EC:3.1.2.1], etc.) were all
included Macellibacteroides (6.4%), Ruminococcus (5.4%), Treponema significantly enhanced in the cellulase-conditioned reactor. Besides, the
(2.8%), Bacteroides (1.3%), Saccharofermentans (1.3%), and etc. (Lay relative abundance of acetate kinase [EC:2.7.2.1], which played an
et al., 2012; Li et al., 2019). But the predominant VFAs producers in essential role in acetate production (Wang et al., 2021b), was 0.51‰ in
cellulase-conditioned reactors had been shifted to Macellibacteroides and the control, while increased to 0.58‰ in the reactor with 15 mg cellu­
Bacteroides. The relative abundance of Macellibacteroides and Bacteroides lase/g TSS, and further improved to 0.82‰ in the reactor with 60 mg
was 4.2 and 13.2% in the reactor with 15 mg cellulase/g TSS, while 15.2 cellulase/g TSS. These results were also consistent with the enzymatic
and 5.0% in the reactor with 60 mg cellulase/g TSS. Xin et al. had re­ activity of AK, which was one of the essential reasons responsible for the
ported that Macellibacteroides could effectively degrade various re­ VFAs promotion.
fractory organic substances (e.g., cellulose, aromatics) to produce VFAs Generally, the cellulase conditioning enriched the functional mi­
(Xin et al., 2021), while Li et al. had indicated that Bacteroides could also croorganisms and enhanced the related gene expressions, thereby pro­
convert waste wood (mainly composed of cellulose, hemicellulose, and moting the co-fermentation efficiency of WAS and PW.
lignin) into VFAs (Li et al., 2019). Hence, the enrichment of Macelli­
bacteroides and Bacteroides in the cellulase-conditioned reactor contrib­ 4. Conclusion
uted to the degradation of PW, and subsequently promoting the ultimate
VFAs production. This study demonstrated the dose-dependent positive effects of
In addition, in order to further explore the underlying mechanisms cellulase on the WAS/PW co-fermentation for VFAs promotion with
for the VFAs promotion driven by cellulase during the WAS/PW co- ideal individual VFA distribution. The cellulase induced the WAS and
fermentation, the relative abundance of metabolic pathway and func­ PW disruption that resulted in the improved bioavailable substrates.
tional gene expressions participated in VFAs generation were predicted Moreover, the cellulase conditioning was conducive to the enrichment
by PICRUSt. As illustrated in Fig. 6(A), the related metabolic pathway of functional fermentative microorganisms. The general microbial ac­
involved in VFAs generation were observed to be enhanced in the tivities related with VFAs biosynthesis, including the ATP level, activ­
cellulase-conditioned reactors. For instance, the relative abundance of ities of VFAs formation related enzymes, and expressions of critical
the metabolism of carbohydrate and amino acid, which were two main encoding genes were improved. Overall, it was the synchronous
components in WAS and PW, was 9.3 and 9.9% in the control reactor. enhancement of metabolic substrates and microbial activity that caused
However, their abundance was increased to 10.0 and 10.1% in the the significant VFAs promotion derived from WAS/PW co-fermentation.
reactor with 15 mg cellulase/g TSS, and further slightly improved to
10.2 and 10.3% in the reactor with 60 mg cellulase/g TSS. Moreover, CRediT authorship contribution statement
various functional gene expressions related with substrates metabolism
and VFAs biosynthesis were also found to be enriched in the cellulase- Jingyang Luo: Conceptualization, Methodology, Investigation, Data

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J. Luo et al. Bioresource Technology 341 (2021) 125771

Fig. 6. The relative abundance of (A) KEGG pathway and (B) functional gene expressions in different reactors during WAS and PW co-fermentation.

curation, Formal analysis, Writing – original draft, Visualization, Vali­ Formal analysis, Funding acquisition, Writing - review & editing, Su­
dation. Yi Li: Data curation, Formal analysis, Writing – original draft, pervision, Visualization.
Visualization, Validation. Yibing Li: Methodology, Investigation, Visu­
alization. Han Li: Methodology, Investigation, Visualization. Xinyang Declaration of Competing Interest
Fang: Writing - review & editing, Methodology. Yuxiao Li: Writing -
review & editing, Methodology. Wenxuan Huang: Writing - review & The authors declare that they have no known competing financial
editing, Methodology. Jiashun Cao: Investigation, Data curation. Yang interests or personal relationships that could have appeared to influence
Wu: Conceptualization, Methodology, Investigation, Data curation, the work reported in this paper.

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J. Luo et al. Bioresource Technology 341 (2021) 125771

Acknowledgements Lay, C.-H., Lin, H.-C., Sen, B., Chu, C.-Y., Lin, C.-Y., 2012. Simultaneous hydrogen and
ethanol production from sweet potato via dark fermentation. J. Cleaner Prod. 27,
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The work is financially supported by the “National Natural Science Li, D., Yin, F., Ma, X., 2019. Achieving valorization of fermented activated sludge using
Foundation of China (No.52070069)”, “Fundamental Research Funds pretreated waste wood feedstock for volatile fatty acids accumulation. Bioresour.
for the Central Universities (No: B200202099)”, “China Postdoctoral Technol. 290, 121791.
Luo, J., Huang, W., Guo, W., Ge, R., Zhang, Q., Fang, F., Feng, Q., Cao, J., Wu, Y., 2020a.
Science Foundation (No. 2021M692423)”, “Shanghai Post-doctoral Novel strategy to stimulate the food wastes anaerobic fermentation performance by
Excellence Program (No. 2020419)”, “State Key Laboratory of Pollu­ eggshell wastes conditioning and the underlying mechanisms. Chem. Eng. J. 398,
tion Control and Resource Reuse Foundation (No. PCRRF20005)” and 125560.
Luo, J., Huang, W., Zhang, Q., Guo, W., Wu, Y., Feng, Q., Fang, F., Cao, J., Su, Y., 2020b.
the “Priority Academic Program Development of Jiangsu Higher Edu­ Effects of different hypochlorite types on the waste activated sludge fermentation
cation Institutions (PAPD), China”. from the perspectives of volatile fatty acids production, microbial community and
activity, and characteristics of fermented sludge. Bioresour. Technol. 307, 123227.
Luo, J., Wang, F., Cheng, X., Huang, W., Zhang, Q., Fang, F., Cao, J., Wu, Y., 2021.
Appendix A. Supplementary data Metatranscriptomic insights of the metabolic process enhancement during food
wastes fermentation driven by linear alkylbenzene sulphonates. J. Cleaner Prod.
Supplementary data to this article can be found online at https://doi. 315, 128145.
Panigrahi, S., Dubey, B.K., 2019. A critical review on operating parameters and strategies
org/10.1016/j.biortech.2021.125771. to improve the biogas yield from anaerobic digestion of organic fraction of municipal
solid waste. Renewable Energy 143, 779–797.
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