Bioresource Technology 341 (2021) 125906

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

Humification process and mechanisms investigated by Fenton-like reaction

and laccase functional expression during composting
Qiuqi Niu , Qingran Meng , Hongxiang Yang , Yiwu Wang , Xiaolan Li , Gen Li , Qunliang Li *
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, PR 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

• The Fenton-like reaction enhanced the

generation of H2O2 and ⋅OH in
composting.
• Laccase induced the HS formation via
the Fenton-like reaction.
• Proteobacteria was main phylum pro­
ducing AA1 containing laccase.
• An optimized method was proposed for
humification in composting.

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

Keywords: This study aims to explore the impacts of the Fenton-like reaction on hydrogen peroxide, hydroxyl radicals,
Composting humic substance (HS) formation, laccase activity and microbial communities during composting to optimize
Fenton-like reaction composting performances. The results indicated that the activity of laccase in the presence of the Fenton-like
Humification mechanisms
reaction (HC) (35.92 U/g) was significantly higher than that in the control (CP) (29.56 U/g). The content of
Laccase
HS in HC (151.91 g/kg) was higher than that in CP (131.73 g/kg), and amides, quinones, aliphatic compounds
Microbial community succession
and aromatic compounds were promoted to form HS in HC by 2D-FTIR-COS analysis. Proteobacteria contributed
most greatly to AA1 at phylum level, Pseudomonas and Sphingomonas abundances increased in HC. Redundancy
analysis indicated that there was a strong positive correlation among the Fenton-like reaction, laccase and HS.
Conclusively, the Fenton-like reaction improved the activity of laccase, promoted the formation of HS and
enhanced the quality of compost.

1. Introduction et al, 2019). Phenols, polyphenols, quinones and aliphatic compounds

Composting is a good option for managing a mass of livestock ma­
nures and agricultural wastes as an economical and eco-friendly tech­
nology (Hartmann et al., 2015). Microorganisms utilize and convert
organic compounds into macromolecular humic substances (HS) for
nourishing soil matrices and adjusting the redox reactions of pollutants.
And the degree of humification determines the quality of compost
products and the potential application of compost (Gao et al., 2019; Xu
that are related to the structural composition of lignin are recognized as
important raw materials for the synthesis of HS in composting, which
are produced by the degradation of lignin (Guo et al., 2019). However,
the recalcitrant lignin as the structural support and barrier of lignocel­
lulose is difficult to be destroyed and attacked by microbes and their
enzymes, which makes the low degradation rate of lignin in traditional
composting (Zhang et al., 2018). Hence, it is vital to find a way to
accelerate the decomposition of lignin and promote the formation of HS

* Corresponding author.
E-mail addresses: liqunliang231@gxu.edu.cn, liqunliang231@163.com (Q. Li).

https://doi.org/10.1016/j.biortech.2021.125906
Received 27 July 2021; Received in revised form 29 August 2021; Accepted 3 September 2021
Available online 8 September 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
Q. Niu et al.
during composting.
Some researchers have confirmed that the Fenton-like reaction can
generate highly reactive hydroxyl radicals (⋅OH) causing the surface
oxidation of fiber and attacking the structure of lignin, which can disrupt
the close connection of lignin and hemicellulose and provide more
binding sites for ligninolytic enzymes (Zhang et al., 2018). It was re­
ported that Fenton reagents (FeCl2⋅4H2O and H2O2) can enhance the
decomposition of lignin and improve the degradation efficiency of
lignocellulose (Zeng et al., 2015). The environment of compost is
slightly alkaline, whereas iron in the Fenton reaction only works in the
acidic condition. But copper in the Fenton-like system can work effi­
ciently in near-neutral or neutral condition and can better adapt to
compost (Chen et al., 2019b). Additionally, the circulation rate of Cu+/
Cu2+ is quicker than Fe2+/Fe3+ and relaxes the step of limited rate
(Zhang et al., 2017; Sun et al., 2018). So the Fenton-like reagents (CuCl
and H2O2) are more suitable for compost and the Fenton-like reactions
based on copper is described as follows (Chen et al., 2019b):
1
Cu+ + H2 O2 →Cu2+ + Â⋅OH + OH− , k1 = 1 × 104 M− 1 S−
1
Cu2+ + H2 O2 →Cu+ + HO2 Â⋅ + H+ , k2 = 4.6 × 102 M− 1 S−
Laccase, an important enzyme containing a cluster of four copper
ions, utilizes dioxygen as an electron acceptor and oxidizes phenolic or
non-phenolic compounds related to lignin polymer, generating radical
intermediates that can coupled further with each other to form HS in
composting (de Gonzalo et al., 2016; Sun et al., 2020; Meng et al., 2021).
Moreover, the activity and the stability of free laccase can be reinforced
by Cu2+ of the Fenton-like reaction and laccase also can produce ⋅OH by
catalyzing phenolic substances that lignin can be promoted to produce
by the Fenton-like reaction (Sun et al., 2019; Bissaro et al., 2018). These
researches show that laccase has an inseparable relationship with HS
formation and the Fenton-like reaction during composting. Therefore,
the action process of the Fenton-like reaction during composting can be
that ⋅OH may attack the compact structure of lignin, promote the
decomposition of lignin, which can provide more “land pointing” and
substrates for laccases and then increase the activity of laccases. More
small molecules of humic precursor can be generated by the decon­
struction of lignocellulose, and then can be promoted to form HS by high
activity of laccases, which can improve the humification and the
aromaticity of composting process. Above all, the roles of the Fenton-
like reaction on HS formation and laccases are explored further during
composting in this research, which is to great significance for com­
posting process.
This aim of work is to study systematically the interaction among the
Fenton-like reaction, the formation of HS, the activity of laccases and
microorganisms. To achieve this goal, the specific purposes of the
research included: (1) to identify the effects of the Fenton-like reaction
in composting performances; (2) to analyze the content variation of
H2O2 and ⋅OH after adding CuCl and H2O2; (3) to explore the influences
of the Fenton-like reaction on mechanisms of HS formation by two-
dimensional correlation spectroscopy (2D-COS) FTIR; (4) to establish
the connection among laccases, AA1 and microbial communities; (5) to
assess the relationship between environmental factors and
microorganisms.
2. Materials and methods
2.1. Sampling, composting raw materials and composting experiment set-
up
The raw composting materials contained fresh dairy manure and
sawdust with a ratio of 3.5:1 (wet weight) to achieve the appropriate
moisture content and C/N ratio (Li et al., 2020). The fresh dairy manure
was obtained from the dairy farm (College of Animal Science, Guangxi
University) and the sawdust was gathered from the lumber mill
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Bioresource Technology 341 (2021) 125906
(Nanning, China). The physicochemical properties of the materials are
presented in Table 1. Composting trials were conducted in 40 cm
diameter and 57 cm height of cylindrical plastic buckets without a lid,
and each compost pile weighed about 20 kg. Two treatments were as
follows: CP was set as the control group without Fenton-like reaction
reagents, HC was the treatment group sprayed respectively with
hydrogen peroxide solution (0.2 M, 500 mL) and the cuprous chloride
powder (10.0 g, 97 %). The entire composting process lasted 46 days
(Nov. 4, 2020 to Dec. 20, 2020). The multi-point sampling based on the
composting performances was taken at Day_0 (Nov. 4), Day_4 (Nov. 8),
Day_7 (Nov. 11), Day_12 (Nov. 16), Day_20 (Nov. 24), Day_32 (Dec. 6)
and Day_46 (Dec. 20) to make sure the sampling representative, the
composting materials were turned simultaneously to supply enough
oxygen. Specifically, 300 g composting sample was collected and thor­
oughly mixed from nine different sites of three depths at each sampling
time: 3 surface sub-samples (1–10 cm depth of pile), 3 middle sub-
samples (around 25 cm depth of pile) and 3 sub-samples from the bot­
tom position of pile (López-González et al., 2015). Samples were stored
at − 20 ℃ and then defrosted at 4 ℃ for follow-up experiments.
2.2. Physicochemical properties analysis
The temperature of compost was measured by a precise digital
thermometer (TP101, China) at 12:00 a.m. each day, meanwhile, the
room temperature was recorded. The moisture content of samples was
depended on the weight loss via drying at 105 ◦ C in a drying oven for 24
h (Niu et al, 2021). The pH was determined using a pH meter (PHS-25,
China) after suspending samples into deionized water with a 1:10 (w/v)
(Wang et al., 2015). The content of organic matter (OM) was calculated
by the loss weight that the dried samples were burned in a muffle
furnace at 550 ℃ (Xu et al., 2019). The total Kjeldahl N content was
measured by a Kjeldahl apparatus (Hanon K9840, Jinan, Shandong,
China) as described by Jiang et al. (2021).
2.3. Reactive oxygen species determinations
The fresh sample and ultrapure water were mixed at a ratio of 1:10
(w/v), which shaken for 1 h at 180 rpm and then centrifuged at 8000
rpm for 8 min (Huang et al., 2019). And the supernatant was filtrated
through a 0.45 μm membrane. The filtrate was tested the content of ⋅OH
and H2O2. The content of ⋅OH was detected by the thiobarbituric acid
(TBA) method based on the procedure by Chen et al. (2021). Subse­
quently, the filtrate (0.5 mL) and 2-deoxy-D-ribose (4 mM, 1 mL) were
mixed and then reacted at 37 ℃ for 60 min. Trichloroacetic acid (0.7 %,
1 mL) and thiobarbituric acid (0.25 %, 1 mL) were added into the
aforementioned mixture. After that, the mixture was heated at 100 ℃
for 15 min. The reductive activity of ⋅OH was determined by an ultra­
violet–visible spectrophotometer (UH5300, China) at 532 nm. The
content of H2O2 was measured according to the documentation of Chen
et al. (2021) and Song et al. (2016). Titanium tetrachloride (20 %, V/V,
0.2 mL) and ammonium hydroxide (0.2 mL) were added in the filtrate
(1 mL) to react until precipitation generation. The supernatant obtained
by centrifugation at 8000 rpm for 8 min was eliminated, and the sedi­
ment was dissolved thoroughly by H2SO4 (2 M, 3 mL). The content of
Table 1
Physiochemical properties of composted raw materials.
Raw Moisture pH OM (%) TKN (%) C/N
materials (%)
Sawdust 5.69 ± 0.21 6.76 ± 82.51 ± 0.75 ± 63.81 ±
0.03 1.09 0.01 0.61
Dairy 80.12 ± 7.86 ± 65.87 ± 1.92 ± 19.93 ±
manure 0.60 0.05 0.95 0.25 0.24
Note: OM = organic matter; TKN = total Kjeldahl nitrogen; C/N = ratio of carbon
to nitrogen.
Q. Niu et al.
H2O2 was assayed by the absorbance of the sample at 410 nm and then
was calculated by the standard curve of H2O2 concentration.
2.4. Humic substance extraction and two-dimensional correlation
spectroscopy of Fourier transform infrared analysis
The method of HS extraction was described by (Niu et al, 2021). The
fresh sample was added into the mixed solution of NaOH (0.1 M) and
Na4P2O7 (0.1 M) at a ratio of 1:10 (w/v), which shook at 180 rpm for 12
h and then was centrifuged at 11000 rpm for 15 min. After that, the
supernatant was filtered through 0.45 μm filter paper and the obtained
filtrate was HS solution. The content of HS was detected by TOC-L CPH
(Shimadzu, Japan).
The HS sample after freeze-drying was mixed with KBr at a ratio of
1:100 (w/w), which was ground well and pressed powder into a tablet.
And the absorbance spectra of HS were scanned from 400 cm− 1 to 4000
cm− 1 by Fourier-transform infrared spectroscopy (IRTracer-100, Shi­
madzu, Japan). Two-dimensional correlation spectroscopy (2D-COS)
distinguishes the sequential order and the relative direction in structural
variations by extending spectra along the second dimension, which is
widely used to study the evolution and interaction mechanisms of
organic matter at the molecular level (Chen et al., 2015). 2D-COS of
FTIR was employed to explore the evolution of functional groups of HS
with composting time as the external perturbation in this research, and
then reveal the molecular composition and formation mechanisms of
HS. The perturbation-induced variation of spectral intensity y(v, t) was
observed between Tmin and Tmax described by Noda and Ozaki (2005)
and Gao et al. (2019), where v and t represent the wavenumber and
perturbation, respectively. The dynamic spectrum y(v, t) was expressed
as follows:
{ recalcitrant organic compounds were left causing microbial metabolism
y(v, t) = y(v, t) − yforTmin ≤ t ≤ Tmax ,
0otherwise
Where y was calculated by y = 1
∫ Tmax
y(v, t)dt.
Tmin − Tmax Tmin
The 2D-COS can be defined as X(v1 , v2 ) = 〈y(v1 , t)Â⋅y(v2 , t ’ )〉, where
X(v1 , v2 ) is the quantitative measure of spectral intensity y(v,t) at v1 and
v2 during a fixed interval. To simplify the numerical calculation, the
function was expressed as follows: X(v1 , v2 ) = ϕ(v1 , v2 ) + iψ (v1 , v2 ). The
synchronous correlation map was made up of auto-peaks at the main
diagonal and cross-peaks located along the off-diagonal, which showed
the degree of molecular structure affected by the external perturbation
and the changes of correlation in functional groups (Zou et al., 2020).
The asynchronous correlation map represented the variation of diversity
in spectral intensity to distinguish overlapping peaks. The sequential
order of change about the functional group and the direction of spectral
intensity change were analyzed by combining the two correlation maps
(Lasch and Noda, 2017). The data of infrared spectroscopy was analyzed
in 2D-shige and MATLAB2019 was used to plot the 2D-COS map.
2.5. Sample pretreatment and metagenomic sequencing
Based on the procedure presented by Wang et al. (2021), the sample
was required pretreatment to sequence metagenome. The fresh sample
(3.0 g) and sterile distilled water (25 mL) were mixed at room temper­
ature for 15 min, which was centrifuged to obtain microbial cells. The E.
Z.N. A.TM Mag-Blind Soil DNA kit (Omega Bio-Tek, Norcross, GA, U.S.)
was used to extracted total genomic DNA from the preprocessed sample.
DNA extracting solution after concentration and purity was fragmented
to an average size of around 400 bp for paired-end library construction.
Paired-end sequencing was constructed by Illumina NovaSeq/Hiseq
Xten (Illumina Inc., San Diego, CA, USA) at Shanghai Majorbio Bio-
Pharm Technology Co., Ltd. (Shanghai, China).
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Bioresource Technology 341 (2021) 125906
2.6. Statistical analysis
The statistical data was processed and analyzed by SPSS 16.0 and
Microsoft Office Excel 2016, and the figure was made by ORIGIN 9.0 and
R language tool. The significant differences of the research set at p <
0.05 by one-way ANOVA. Representative sequences of non-redundant
gene catalog were annotated by using Diamond 0.8.35 and Hmmscan
against the NCBI NR database and the Carbohydrate-active enzymes
(CAZy) database (http://www.cazy.org/) with an e-value cutoff of 1e-5
for taxonomic annotations and CAZy annotation, which were used to
calculate the abundance of species and CAZy. Redundancy analysis
(RDA) was used to understand the relationship of environmental factors
and microbial community by the R language vegan package.
3. Result and discussion
3.1. Physicochemical properties of the composting matrix
As shown in Fig. 1, the physicochemical properties such as temper­
ature, moisture, pH and C/N were changed significantly at the different
phases of composting, revealing that the performance and efficiency of
composting. After a short time of the heating phase from Day_0 to Day_1,
the composting temperature of the two groups rapidly reached above 50
℃ on Day_2, which was the start of the thermophilic phase (Fig. 1(a)).
The released heat of compost during the heating phase was produced by
the metabolism of mesophilic microorganisms using labile organic
compounds (Wang et al., 2016). The thermophilic phase of CP and HC
remained 12 days from Day_2 to Day_12, illustrating that met the
requirement of killing pathogens (Zhu et al., 2020). The temperature
quickly decreased from Day_13 to Day_25 (cooling phase) because only
in a relative disadvantage as the composting went on (Jiang et al.,
2019). The temperature gradually approached the room temperature on
Day_26, indicating that the compost entered the curing phase. The
moisture content is an effective index of composting process because
microbes conduct biochemical reactions by water as a medium (Guo
et al., 2012). As Fig. 1(b) showed, the moisture content of CP and HC had
a similar trend, and HC was always higher than CP during composting,
which HC might be more beneficial to grow and metabolize for micro­
organisms. In the early stage, the moisture content declined rapidly due
to water vaporization causing by high temperature. The moisture con­
tent of the two groups had a short rise on Day_20, indicating that the
amount of evaporation was less than that of water production by mi­
crobes. The pH firstly increased quickly and then decreased slightly and
it was slightly alkaline during composting (Fig. 1(c)). The increase of pH
in the initial stage of composting was caused by the mineralization of
organic nitrogen, the accumulation of NH3 and the decomposition of
molecular organic acids (Li et al., 2012). The decreased NH3 release and
the dissolution of CO2 in compost made the pH decline from Day_7 to
Day _46. As is shown in Fig. 1(d), the C/N ratio kept decreasing tendency
in CP (from 24.80 to 14.67) and HC (from 25.17 to 14.39), indicating
that the two groups of compost had reached maturity (under 15) (Zhu
et al., 2020).
3.2. Variation in reactive oxygen species
H2O2 is a key reactant of Fenton-like reaction, which initiates the
oxidation reaction of Cu1+ to Cu2+ and generates ⋅OH. Moreover, H2O2
can catalyze and degrade lignocellulose as a co-substrate of enzymes in
composting, such as manganese peroxidase (MnP, EC 1.11.1.13), ver­
satile peroxidase (VP, EC 1.11.1.16), lignin peroxidase (LiP, EC
1.11.1.14) and lytic polysaccharide monooxygenases (AA9-AA11,
AA13-AA16) (Bissaro et al., 2018; Levasseur et al., 2013). Interestingly,
the concentrations of H2O2 in CP and HC presented the same variation
tendency, firstly increased and then decreased, indicating that H2O2 was
generated and consumed during composting, regardless of the impact of
Q. Niu et al. Bioresource Technology 341 (2021) 125906

Fig. 1. Changes of physicochemical parameters during composting (a) Compost piles and room temperatures; (b) Moisture; (c) pH; (d) C/N ratio.

Fenton-like reaction (Fig. 2(a)). The H2O2 content of HC (15.26 mmol/
kg) was significantly higher than that of CP (11.18 mmol/kg) on Day_0
owing to adding H2O2 to composting materials in HC. The content in HC
continued to increase and was always higher than that in CP before
Day_12, indicating that more H2O2 could be produced by enzymes in
HC, such as cellobiose dehydrogenases (AA3_1,), aryl alcohol oxidase
(AA3_2, EC 1.1.3.7), glyoxal oxidase (AA5_1, EC 1.2.3.15) and alcohol
oxidase (AA5_2, EC 1.1.3.13) (Bissaro et al., 2018). And the H2O2 con­
tent of HC obviously declined after Day_20 causing the result that HC
was lower than CP, illustrating that the microbial activity of enzymatic
H2O2 producers weakened and then the amount of H2O2 that was pro­
duced was lower than consumption of H2O2 in later composting.
To explore whether the Fenton-like reaction occurred under H2O2
and CuCl in the composting, the content of ⋅OH was measured, as shown
in Fig. 2(b). ⋅OH, as highly destructive oxygen-derived free radicals, can
fall the molecular weight of lignin and break the internal glycosidic
bonds of hemicellulose and cellulose, which can enhance the penetra­
tion of enzyme molecule and then improve the degradation of ligno­
cellulose in compost (Zhang et al., 2018).The ⋅OH content of HC was
significantly higher than that of CP during the entire composting,
especially, HC (0.051) was twice the value of CP (0.025) on Day_0,
indicating that a large number of hydroxyl free radicals were produced
by the Fenton-like reaction. The ⋅OH concentrations of two groups firstly
increased and then decreased, which could be closely associated with
the content of H2O2 and the activity of laccase because H2O2 was a major
source of ⋅OH and laccase could produce ⋅OH through the oxidation of
phenolic substances (Bissaro et al., 2018). The ⋅OH in HC and CP
continued to rise and reached respectively the peak value of 0.079 and
0.048 on Day_12, illustrating that H2O2 supplied by enzymatic pro­
ducers also could generate ⋅OH through Fenton-type reactions during
the composting (Levasseur et al., 2013). The above results demonstrated
that adding H2O2 and CuCl increased the content of H2O2 and ⋅OH
during the composting, thereby accelerating the depolymerization of the
recalcitrant structure of lignocellulose, improving the formation of HS
and promoting the composting process.
3.3. Effects of Fenton-like reaction on the content and the evolution of
humic substance
Humification, as an important indicator of compost maturity and a
driving factor of compost quality, is the conversion process of organic
compounds by complicated biotic and abiotic reactions. HS is a kind of
stabilized macromolecular organic matter, which can nourish the soil,
improve the carbon fixation ability of soil and mediate the redox re­
actions of pollutants (Rawoteea et al., 2017; Gao et al., 2019). The HS
content of two groups in composting decreased firstly and then kept
growing, revealing that humic substances-like matters were decom­
posed into small-molecule organics during the initial composting and
then these organics were polymerized to form HS through different
pathways as the composting went on (Gao et al., 2019) (Fig. 2(c)). The

4
Q. Niu et al.
Fig. 2. Changes of reactive oxygen species and HS during composting (a) H2O2 content; (b) ⋅OH content; (c) HS content.
HS content of HC increased by 50.52 % until the end of composting,
which was obviously higher than CP (23.52 %). It was reasonable to
suppose the HS formation could be promoted and the compost quality
could be improved under the Fenton-like reaction according to the
above phenomena.
To explore the process of HS formation and the transformation
sequence of HS functional groups, the 2D-FTIR-COS of HS was analyzed
in the regions of 800–1200 cm− 1 and 1300–1750 cm− 1 (Fig. 3). The
synchronous maps showed 7 auto-peaks at 890, 930, 1090, 1200, 1430,
1490 and 1650 cm− 1 in CP, and 8 auto-peaks at 890, 930, 1090,
1120–1145, 1200, 1430, 1577 and 1650 cm− 1 in HC. Moreover, the
signs of auto-peaks in synchronous maps were positive, indicating that
the functional groups changed simultaneously with composting
fermentation. The positive cross-peaks were found at (1090, 890),
(1200, 890), (1200, 1090), (1650, 1430) and (1490, 1430) in CP syn­
chronous maps, were found at (1090, 890), (1200, 890), (1120–1145,
890), (1200, 1090), (1577, 1430), (1650, 1430) and (1650, 1577) in HC
synchronous maps, and the which suggested that the peak intensity of
functional groups was added as composting time went on. The most
significant changes of CP were observed at 1430 and 1490 cm− 1, cor­
responding to the C = O stretching of carboxylate and the C = C
stretching of aromatic ring, which indicated that the formation of HS in
CP was the most relevant to the enrichment of carboxylic acids and
aromatic structures (Liu et al., 2020). However, the peaks of 1577 and
1650 cm− 1 that attributed to the C = C stretching of imidazole, aromatic
5
Bioresource Technology 341 (2021) 125906
ring modes and the C = O stretching of quinone, ketone or amide I were
changed most obviously in HC, suggesting that the increase of amides,
quinones, aromatic heterocycle compounds and aromatic compounds
contributed most to the HS formation of HC (Gao et al., 2019).
Furthermore, the auto-peak at 1120–1145 cm− 1 only appeared in HC,
which was associated with the formation of the C-O stretching of
aliphatic OH (Li et al., 2011; Liu et al., 2020). According to cross-peaks
in asynchronous maps, the sequence of HS structural variation in CP was
as follows: 1200 > 890 > 1090 cm− 1 and 1430 > 1650 > 1490 cm− 1,
corresponding to the C-O stretching of aromatic acid, aliphatic acid
ester > the C–H deformation and vibration > the C-O stretching of
polysaccharides or polysaccharide-like substances and the C = O
stretching of carboxylate > the C = O stretching of quinone, ketone or
amide I > the C = C stretching of aromatic ring. While the order of
structural change of HS in HC was as followed: 1120–1145 > 890 >
1200 > 1090 cm− 1 and 1650 > 1577 > 1430 cm− 1. This translated to the
C-O stretching of aliphatic OH > the C–H deformation and vibration >
the C-O stretching of aromatic acid, aliphatic acid ester > the C-O
stretching of polysaccharides or polysaccharide-like substances and the
C = O stretching of quinone, ketone or amide I > the C = C stretching of
imidazole, aromatic ring modes > the C = O stretching of carboxylate.
Compared with CP, it was noteworthy that amides, quinones, aliphatic-
like substances and aromatic compounds were firstly formed, and car­
boxylic acids and aromatic acids were then formed in HS of HC. Taken
together, the Fenton-like reaction changed the formation sequence of
Q. Niu et al. Bioresource Technology 341 (2021) 125906

Fig. 3. Synchronous and asynchronous maps of HS via two-dimensional FTIR correlation spectra (2D-FTIR-COS) (a) CP in the region of 800–1200 cm− 1; (b) HC in the
region of 800–1200 cm− 1; (c) CP in the region of 1300–1700 cm− 1; (d) HC in the region of 1300–1700 cm− 1. The color from blue to red represents the correlation
intensity from negative to positive. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

6
Q. Niu et al.
the functional group of HS and promoted the formation of amides,
quinones, aliphatic-like substances and aromatic compounds in HS, thus
heightening the degree of humification and aromaticity.
3.4. Analysis of auxiliary activity family 1 based on metagenome during
composting
The CAZy auxiliary activity family 1 (AA1) as multicopper oxidases
includes three subfamilies, laccases (AA1_1, EC 1.10.3.2), ferroxidases
(AA1_2) and the laccase-like multicopper oxidases (AA1_3) (Levasseur
et al., 2013). These enzymes employ phenols and related substances as
donors with oxygen as electron acceptors, and are widely distributed in
nature from fungi, bacteria, plants and insects. Laccases can not only
catalyze high-molecular lignin to decompose but also catalyze low-
molecular phenols, amides, aliphatics and their various derivatives to
polymer the stable HS and improve the humification degree (Munk
et al., 2015; Sun et al., 2020). Additionally, laccase might be involved
synergistically with AA3_2 to generate ⋅OH to attack recalcitrant
lignocellulose (Levasseur et al., 2013). The laccase activity of the
different treatments increased slowly at the start of composting, which
could be associated with the reduction of HS content causing by the
degradation of humic precursors through laccases (Fig. 4(a)). The lac­
case activity increased rapidly on Day_4, HC reached the maximum
value (35.92 U/g) on Day_12, and CP reached the highest point (29.56
U/g) on Day_20, indicating that the presence of copper ions could pro­
mote the activity of laccase and a mass of HS precursors could be
recombined to form macromolecular HS by laccase-mediated humifi­
cation reactions in the meantime (Chen et al., 2019a). Subsequently, the
laccase activity began to decrease gradually, and the rate of descent of
HC was lower than that of CP, suggesting that humic acid produced with
the composting fermentation inhibited the activity of laccase but copper
ions could restore the deactivated laccases (Keum and Li, 2004).
Therefore, laccase containing four copper ions could extract an electron
from unstable Cu1+ to form radical intermediates and then could un­
dergo the next polymerization reaction or decomposition reaction, and
more “landing points” were provided for laccase after the Fenton-like
reaction modifying lignin, which increased the activity of laccase (Sun
et al., 2020; Zhang et al., 2018). Ferroxidases can reduce the toxic level
of hydroxyl radical and adjust the Fenton reaction (Kersten and Cullen,
2007). The laccase-like multicopper oxidases can degrade lignin
(Levasseur et al., 2010). The change trends of AA1 abundance in HC and
CP were showed in Fig. 4(b), rase first and then fell, which were
consistent with the laccase activity. And the abundance of AA1 reached
the peak value in CP (15582) on Day_20 and in HC (20728) on Day_12,
and the abundance of CP was always lower than HC during composting.
Hence, the Fenton-like reaction regulates the genes encoding of laccases,
ferroxidases and the laccase-like multicopper oxidases to increase the
abundance of AA1.
AA2 including MnP, VP and LiP also could synergistically attack and
degrade lignin in composting (Munk et al, 2015), and its abundance
showed a trend of initial increase and then decrease in HC and CP. The
AA2 abundance of HC reached the highest point (5272) on Day_12, CP
reached the peak (5152) on Day_20, and the abundance of HC was
significantly higher than that of CP before Day_20. Moreover, AA2
needed to use H2O2 as the final electron acceptor for modifying lignin,
and the variation tendency of AA2 was similar to the content of H2O2,
suggesting that AA2 and H2O2 were deeply interconnected during the
composting. The abundance of lytic polysaccharide monooxygenases
(AA9, AA11, AA15 and AA16) gradually increased with the composting
fermentation, which might be one of the factors that led the decrease of
H2O2 content.
To better understand the influence of the Fenton-like reaction on
AA1 and related encoding genes, the species contribution of microor­
ganisms for AA1 at the phylum level and at the genus level was estab­
lished based on metagenomics analysis, as shown in Fig. 4(c) and (d).
The relative abundance of Proteobacteria was the highest in AA1
7
Bioresource Technology 341 (2021) 125906
contribution at the phylum level, followed by Actinobacteria and Acid­
obacteria. And Proteobacteria was the most dominant microorganism of
laccase production according to the research of Jiang et al. (2021) and
the abundance of HC was higher than CP during composting, revealing
that the Fenton-like reaction could enhance Proteobacteria to express
laccase. Proteobacteria was a low relative abundance on Day_0 and
reached the highest on Day_4 in CP (48.74 %) and HC (54.67 %), which
could be caused by Proteobacteria utilizing rapidly the labile organic
molecules in the initial composting (Li et al., 2020). The relative
abundance of Actinobacteria was highest on Day_0, began to decline on
Day_4, and then increased on Day_20 in CP and HC, suggesting that
Actinobacteria could spore to stand the high temperature and just a few
Actinobacteria could work during the thermophilic phase (Jiang et al.,
2019). The contribution of Acidobacteria to AA1 in CP and HC firstly
increased and then decreased, and the relative abundance of HC was
lower than CP on Day_12, revealing that the Fenton-like reaction could
inhibit the AA1 genetic expression of Acidobacteria at the same time.
Moreover, Ascomycota was the most dominant fungus that contributed
to AA1, and AA1_3 which played an important role in lignin degradation
was usually found in Ascomycota (Levasseur et al., 2013). As indicated
above, the Fenton-like reaction during the composting regulated the
encoding genes of AA1 and optimized the metabolic niche of Proteo­
bacteria, which could promote Proteobacteria and other microbes to
produce AA1 and create the composting habitat of improving the
degradation of lignin and the formation of HS.
Luteimonas, Pseudoxanthomonas, Corynebacterium, Streptomyces, Sol­
irubrobacter and Pseudomonas were dominant genera that contributed
the most to AA1. Luteimonas and Pseudoxanthomonas were genera of
Proteobacteria, which made Proteobacteria contribute the most to AA1.
The relative abundance of Luteimonas increased on Day_4, maintained a
similar abundance in later stages and HC was higher than CP from Day_4
to Day_46. Luteimonas was involved in the decomposition of poly­
saccharides, implying that the degradation of lignocellulose could be
faster on Day_4 and the result of the lignocellulose decomposition in HC
might be better (Xu et al., 2019). The abundance of Pseudoxanthomonas
firstly increased and then decreased, which was closely associated with
the degradation of benzene series and cellulose for the formation of
humic precursors (Kumar et al., 2015). The potentially pathogenic
Corynebacterium was the most abundant on Day_0 and plummeted as the
composting progress, indicating that compost could reduce the patho­
genic of dairy manure and improve the applied value of manure in
fertilizer (Niu et al., 2021). It was widely recognized Streptomyces
belonging to Actinobacteria could secret bacterial laccases to assist in
lignin modification and take part in the hydrolytic and oxidative attack
of recalcitrant lignocellulose (Fernandes et al., 2014; de Gonzalo et al.,
2016). Accordingly, the Streptomyces AA1 genes increased in the initial
phase of composting on account of the relatively high degradation rate
of organic compounds. It was worth noting that the observed rise of
Pseudomonas and Sphingomonas on Day_4 was closely related to the in­
crease of laccase activity because Pseudomonas and Sphingomonas had a
key role in generating laccases (Sonoki et al., 2009; Santos et al., 2013).
Furthermore, Pseudomonas and Sphingomonas abundances in HC were
higher than in CP during the composting, especially Sphingomonas,
verifying that the activity of laccases was higher after adding H2O2 and
CuCl. It’s remarkable that the highest abundance of Bacillus was in the
thermophilic phase, suggesting that laccases from Bacillus could tolerate
high temperature and then the contribution of Bacillus to AA1 was
improved on Day_4 (de Gonzalo et al., 2016). In general, the results
confirmed that the microbial species contribution of AA1 was changed,
and the secretion of laccase was promoted by the Fenton-like reaction,
which might enhance the ability of HS formation and improve the effi­
ciency of composting.
Q. Niu et al. Bioresource Technology 341 (2021) 125906

Fig. 4. Variations of laccase activity (a) and AA1 abundance (b) at the different composting periods. Succession of microbial community contributing to AA1 (c)
Relative abundance of microbial community at the phylum level; (d) Relative abundance of microbial community at the genus level.

8
Q. Niu et al. Bioresource Technology 341 (2021) 125906

3.5. Redundancy analysis among environmental factors, auxiliary

activity family 1 and microbial community

The relationships of environmental factors, AA1 and microbial

community were analyzed by RDA, which was beneficial to optimize
environmental parameters for achieving the ideal performances of
compost. In Fig. 5(a), RDA1 and RDA2 respectively explained 85.51 %
and 7.06 % of the succession of microorganisms of AA1. The relevant
order between environmental factors and related microorganisms of
AA1 at the phylum level was Laccase activity > H2O2 > C/N > ⋅OH > HS
> pH > Moisture > Temperature. The laccase activity had a greater
effect on the microbes producing AA1, indicating that microorganisms
related to AA1 could generate laccase and enhance the activity of lac­
case. The HS content was positively correlated to laccase activity, H2O2
and ⋅OH, but was negatively correlated to C/N and Moisture. Especially,
laccase activity, H2O2 and HS had a strong correlation, revealing that the
substrates attacked by H2O2 were promoted to form HS through the
polymerization of laccase. The AA1 community compositions of HC and
CP were similar on Day_0, whereas they had large microbial differen­
tiations on Day_4 and Day_12. Compared with CP, HC on Day_12 and
Day_20 was more positively related to laccase activity, H2O2 and ⋅OH. It
unambiguously demonstrated that the Fenton-like reaction could have
an obvious impact on the microbial species contribution of AA1 and
enhance the interaction among laccase activity, H2O2 and ⋅OH. In
addition, Proteobacteria was greatly influenced by environmental factors
according to analyzing phylum level, and they had a significantly pos­
itive relationship with ⋅OH, laccase activity and HS, which indicated that
abundant Proteobacteria could secrete more laccases and then promote
the formation of HS. And beyond that, the temperature had the greatest
relationship with Actinobacteria, demonstrating that high temperature
affected more easily the abundance of Actinobacteria.
The correlation between environmental factors and AA1 was as fol­
lows: Temperature > C/N > HS > Moisture > Laccase activity > pH >
H2O2 > ⋅OH, and RDA1 and RDA2 respectively accounted for 98.61 %
and 0.05 % of the change of AA1 (Fig. 5(b)). And the abundance of AA1
was found to have a significant positive correlation with laccase activity,
H2O2, ⋅OH, HS, pH and temperature, but was negatively related to C/N
and moisture. And the results confirmed that the substrates that AA1
need were lignocellulose deconstructed by H2O2 and ⋅OH and AA1
played an important role in the formation of HS. It was noteworthy that
there was a strong correlation between AA1 and laccase activity, indi­
cating that the abundance of AA1 gene was closely related to the activity
of laccase and a high abundance of AA1 could improve the activity of
laccase. As can be seen in Fig. 5(b) and (a), H2O2 was positively corre­ Fig. 5. Redundancy analysis assessing the relationship between environmental
lated to ⋅OH, demonstrating that the H2O2 was a major source of ⋅OH. To factors and microbial community (a), environmental factors and AA1 (b).
sum up, it was reasonable to speculate that the interactions of H2O2,
⋅OH, microbes producing AA1 and laccase activity were influenced by Investigation, Writing – original draft. Qingran Meng: Data curation,
the Fenton-like reaction, and thus affected the process of humic pre­ Investigation, Formal analysis. Hongxiang Yang: Data curation,
cursors to form HS. Investigation. Yiwu Wang: Data curation, Investigation. Xiaolan Li:
Data curation, Formal analysis. Gen Li: Validation, Formal analysis.
4. Conclusion Qunliang Li: Conceptualization, Supervision, Resources, Writing – re­
view & editing.
The Fenton-like reaction during composting habitats facilitated to
generate H2O2 and ⋅OH to attack lignocellulose, and improved the ac­ Declaration of Competing Interest
tivity of laccases, releasing more amides, quinones, aliphatic compounds
and aromatic compounds to form HS. The abundance of AA1 including The authors declare that they have no known competing financial
laccases was enhanced in HC, in which Proteobacteria was the most interests or personal relationships that could have appeared to influence
dominant phylum for producing AA1. RDA revealed that the significant the work reported in this paper.
interaction occurred among the Fenton-like reaction, laccases activity
and HS formation. Overall, these findings provided new insights into Acknowledgements
understanding the humification process and mechanisms in composting
mediated by the Fenton-like reaction. This work was supported by the National Natural Science Foundation
of China (No.21878057). The authors thank other members of labora­
CRediT authorship contribution statement tory for providing scientific assistance and helpful discussion.

Qiuqi Niu: Conceptualization, Methodology, Data curation,

9
Q. Niu et al. Bioresource Technology 341 (2021) 125906

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