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Biochar Combined with Garbage Enzyme Enhances Nitrogen Conservation

during Sewage Sludge Composting: Evidence from Microbial Community and
Enzyme Activities Related to Ammoniation

Article in Agronomy · May 2024

DOI: 10.3390/agronomy14061162

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 agronomy
Article
Biochar Combined with Garbage Enzyme Enhances Nitrogen
Conservation during Sewage Sludge Composting: Evidence
from Microbial Community and Enzyme Activities Related
to Ammoniation
Jishao Jiang 1, *, Huilin Cui 1 , Parag Bhople 2 , Caspar C. C. Chater 3,4 , Fuqiang Yu 5 and Dong Liu 5, *

1 Henan Key Laboratory for Environmental Pollution Control, Key Laboratory for Yellow River and Huai River
Water Environmental Pollution Control, Ministry of Education, School of Environment,
Henan Normal University, Xinxiang 453007, China; [email protected]
2 Crops, Environment, and Land Use Department, Environment Research Centre, Teagasc, Johnstown Castle,
Y35 TC98 Wexford, Ireland; [email protected]
3 Royal Botanic Gardens, Kew, Richmond TW9 3AE, UK; [email protected]
4 Plants, Photosynthesis, and Soil, School of Biosciences, University of Sheffield, Sheffield S10 2TN, UK
5 The Germplasm Bank of Wild Species, Yunnan Key Laboratory for Fungal Diversity and Green Development,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; [email protected]
* Correspondence: [email protected] (J.J.); [email protected] (D.L.)

Abstract: Nitrogen loss is an unavoidable problem during composting processes, and the ammo-
nia oxidation process significantly affects nitrogen transformation. The objective of this study was
to evaluate nitrogen transformation when garbage enzyme (GE), biochar (BC), pelelith (PL) and
combinations thereof were added during sewage sludge composting. Meanwhile, the succession
of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were also explored via quantitative poly-
merase chain reaction and high-throughput sequencing. The results showed that GE + BC and
Citation: Jiang, J.; Cui, H.; Bhople, P.;
GE + PL treatments decreased ammonia (NH3 ) formation by 23.8% and 8.3%, and that of nitrous
Chater, C.C.C.; Yu, F.; Liu, D. Biochar oxide (N2 O) by 25.7% and 26.3% relative to the control, respectively. Simultaneously, the GE, GE + BC,
Combined with Garbage Enzyme and GE + PL treatments boosted the succession of AOA and AOB, and increased the activities of
Enhances Nitrogen Conservation ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) activities and the gene
during Sewage Sludge Composting: copies of AOA and AOB. The AMO activities, NH4 -N, NO3 -N, and C/N, significantly affect AOA
Evidence from Microbial Community and AOB community structures. The network analysis predicted that the AMO and HAO were
and Enzyme Activities Related to secreted mainly by the unclassified_Archaea and norank_Crenarchaeota, whereas it also showed that the
Ammoniation. Agronomy 2024, 14, GE + BC improved microbial associations with AOA, enzymatic activity, and environmental factors.
1162. https://doi.org/10.3390/
Thus, the addition of garbage enzyme and biochar appears to be a promising mitigation strategy to
agronomy14061162
reduce nitrogen losses during the composting process.
Academic Editors: Halyna Kominko
and Grzegorz Izydorczyk Keywords: composting; sewage sludge; biochar; ammonia oxidase; ammoniation

Received: 10 May 2024

Revised: 25 May 2024
Accepted: 28 May 2024
Published: 29 May 2024
1. Introduction
Composting is an effective method of harmless utilization and recycling of organic
solid waste into high-quality environmentally friendly organic fertilizers [1]. As a kind
of fertilizer, nitrogen (N) content has been the main standard to evaluate the quality of
Copyright: © 2024 by the authors. compost, thus N cycling is of central importance among other aspects such as humification
Licensee MDPI, Basel, Switzerland. and greenhouse gases [2–4]. During composting, the N loss, in the form of ammonia (NH3 )
This article is an open access article
via the transformation of ammonium-N (NH4 -N) through ammonification, was generated
distributed under the terms and
during the composting process [5]. However, NH4 -N could strategically be converted into
conditions of the Creative Commons
nitrate (NO3 -N) through nitrification, effectively controlling N loss in the form of NH3
Attribution (CC BY) license (https://
emissions [6]. This nitrification is directly impacted by ammonia oxidizers through the
creativecommons.org/licenses/by/
ammoxidation process, which likely changes the subsequent N cycle and the compost
4.0/).

Agronomy 2024, 14, 1162. https://doi.org/10.3390/agronomy14061162 https://www.mdpi.com/journal/agronomy

Agronomy 2024, 14, 1162 2 of 15

quality as well [7]. Therefore, it becomes highly important to explore the community
succession pattern of microorganisms related to ammonia oxidation and their convulsive
effects on composting processes.
Bacteria and archaea both are potential ammonia oxidizing microorganisms (ammonia
oxidizing bacteria—AOB, and ammonia-oxidizing archaea—AOA) during the process
of composting. However, to date, the consensus about AOA or AOB regarding their
predominance during composting is inadequate. Zeng et al. [8] reported that gene copies
of AOA were more abundant, while AOB were not detected during the thermophilic and
cooling stages of the composting of agricultural waste. Yan et al. [9] also reported that
AOA outnumber AOB by 2–3 times during cattle manure composting. Contrastingly,
Wu et al. [10] recently reported that AOB played the dominant role in comparison to
the AOA during the composting of agricultural waste when zeolite and biochar were
used as substrates. Previously, Yan et al. [9] had also found similar results during cattle
manure composting. These ambiguities demonstrate that community diversity and the
quantity of AOA and AOB may be differentially impacted by various factors, such as
the raw materials used for composting and the temperature, moisture content, and O2
concentration, warranting further investigation. In addition, ammonia monooxygenase
(AMO) and hydroxylamine oxidoreductase (HAO) were the main enzymes reported during
the ammonia oxidation process [11], which directly determined the final NO3 -N content
of the compost. The NH4 -N is first converted into hydroxylamine (NH2 OH) using AMO,
and then this product may further be oxidated to nitrite by HAO [12]. However, to date,
the quantification and reactivity levels of AMO and HAO during organic solid-waste
composting has been limited.
The N from the compost can be reserved and the quality of the end compost product
could be enhanced by adding appropriate additives. A fermentation solution of waste
vegetables and fruits, along with water and brown sugar, called garbage enzyme (GE), has
been widely used in wastewater treatment, soil fertilizer, and heavy metal ion absorption
research [13], mainly because of its functional capacities to decompose, compose, transform,
and catalyze any chemical reaction [13]. However, due to its low pH (~3.3 units) GE
could potentially slow down the growth of microorganisms and prolong the composting
process [14]. In addition, it is well known that biochar (BC) and pelelith (PL) are widely
used additives during organic waste composting that reduce NH3 emissions and N loss.
Their usage is mainly based on their high total-pore volume and adsorption capacities [15],
which increase porosity and eliminate anaerobic pocket formation [16,17]. Used together,
BC and PL could provide habitat and refuge for bacteria, protect bacteria from low pH
levels, and the generate high temperatures via high specific surface area and total pore
volume [18]. Furthermore, BC contains alkali metal salts and organic functional groups, and
thus has a strong pH-buffering capacity [19]. These advantages are likely to regulate the
succession of ammonia oxidizers and improve the ammonia oxidation process, ultimately
reducing N loss. Furthermore, BC and PL are inexpensive and convenient, which makes
them ideal additives for organic waste composting. However, only a few studies have
attempted to evaluate the effects on composting processes and the successions of AOA and
AOB of GE combined with BC or PL.
Therefore, the objectives of this research were: (i) to gain insights into the effects
of BC and PL combined with GE on N transformation and the activities of AMO and
HAO enzymes; (ii) to quantify gene copies and identify the community composition and
succession patterns of the AOA and AOB during organic composting; and, finally, (iii) to
find the environmental factors driving changes in AOA and AOB community dynamics
during the composting of sewage sludge.

2. Materials and Methods

2.1. Raw Materials, Additives, and Design
A laboratory simulation of the composting experiment was conducted at the School of
Environment, Henan Normal University (Xinxiang, China; N 35◦ 19′ 39.522′′ , E 113◦ 54′ 55.613′′ )
Agronomy 2024, 14, 1162 3 of 15

for 24 days. The raw materials were dewatered sewage sludge (SS) and saw dust (SD),
which were obtained from the Xiaoshangzhuang sewage treatment plant and a local
furniture factory in Xinxiang, respectively. The physicochemical characteristics of the
sewage sludge have been reported in our previous work [20,21]. The GE was prepared
according to the procedure described by Arun and Sivashanmugam [13] using a mixture
of brown sugar, waste fruits, and water. The characteristics of the GE were: pH, 3.1; total
organic carbon (TOC), 3.1 g L−1 ; and total nitrogen (TN), 4.1 g L−1 , respectively. The BC
was purchased form the pingdingshan green source activated carbon Co., Ltd., where the
primary material used was wood pellets. Meanwhile, the BC and PL were shredded to a
size of 3–5 mm particles, with the total pore volume (TPV) of 0.05 and 0.02 cm3 g−1 , and a
BET surface area (SSA) of 14.2 m2 g−1 and 6.5 m2 g−1 , respectively.
The composting experiments consisted of four different treatments. Fresh SS and SD
were mixed in a ratio of 4:1 and used as the control treatment. The second treatment, marked
as GE, received 2% GE solution (on a dry weight basis). The third treatment (BC + GE),
received a 2% GE solution and 10% BC (on a dry weight basis). Finally, the fourth treatment
(BC + PL) was composed of a 2% GE solution and 10% PL. The fermentation process
refers to a similar procedure to the one described by Jiang et al. [20,21]. During the
composting process, sufficient O2 was supplied by periodical turning and ventilating of
the composting pile: 4 instances of ventilating and 2 of turning, during the first 12 days;
with only 2 instances of ventilating during the final 13–24 days.

2.2. Sampling and Determination of Physicochemical Properties

The compost was sampled on days 0, 4, 8, 13, and 24 according to temperature changes.
Before sampling, the composting mixture needed to be overturned evenly. Three samples
from each treatment were collected and stored separately. Each sample was further divided
into four parts of which one part was used for the analysis of the total nitrogen (TN) after
air-drying. The second part was used to measure physicochemical indexes and enzymatic
activities, and was stored at a cool temperature of 4 ◦ C until analysis. While the fourth part
was immediately frozen at −80 ◦ C for microbial assays. The NH3 and N2 O emissions were
measured through a portable gas detector [22,23]. A compost extract was obtained from
the mixture of compost and deionized water with a ratio of 1:10 (w/v). After centrifugation
and filtration, the pH, EC, NO3 -N, and NH4 -N were measured by following standard
laboratory methods [24]. The TN concentration of the pulverized samples was analyzed by
an elemental analyzer (Elemental Vario EL, Frankfurt, Germany) [23].

2.3. Enzyme Activity Analysis

Quantification with fluorescein diacetate as a substrate: Fluorescein diacetate hydro-
lase (FDA) activity was measured at 620 nm using a colorimetric method. The AMO activity
of the composting samples was determined using the AMO ELISA kit, and calculated at
420 nm using a colorimetric method [10]. To measure HAO activity, the samples were sub-
jected to a reaction system, which consisted of compost extract, K3 [Fe(CN)6 ] (0.01 mol/L),
EDTA (0.04 mmol/L), Tris-HCl (10 mmol/L), and HONH3 Cl-N (15 mg/L). The system
was placed in a water bath at 25 ◦ C for 5 min, and the decrease in K3 Fe(CN)6 per minute
was measured to quantify HAO activity [25].

2.4. Microbial Analysis

Composting samples on days 4, 13, and 24 were selected to study the structure and
quantity of the AOA and AOB. Total genomic DNA was extracted from a 0.5 g com-
post sample according to the instructions of the MoBioPowerSoil® DNA Isolation Kit
(12888) (MO BIO Laboratories, Inc., Carlsbad, CA, USA). The quality of genomic DNA
was determined by 1% agarose gel electrophoresis, followed by NanoDrop2000 (Thermo
Scientific Company, Waltham, MA, USA) to determine the DNA concentration and pu-
rity. The AOB and AOA gene copies were detected by q-PCR using an ABI 7300 fluo-
rescence quantitative PCR instrument (Applied Biosystems, Waltham, MA, USA) with
Agronomy 2024, 14, 1162 4 of 15

the primers Arch-amoAF (STAATGGTCTGGCTTAGACG)/Arch-amoAR (GCGGCCATC-

CATCTGTATGT) and AmoA-1F (GGGGTTTCTACTGGTGGT)/AmoA-1R (CCCCTCK-
GSAAAGCCTTCTTC), respectively [10]. The OD260 values of AOA and AOB plasmids
were measured with a UV spectrophotometer (NanoDrop2000, Thermo Fisher Scientific,
USA) and then converted into a copy number to draw a standard curve. The linearity (R2 )
of the standard curves of AOA and AOB genes was 0.9986 and 0.9999, respectively.
Using the previously extracted DNA as a template, PCR amplification of the archaeal
and bacterial 16S rRNA gene was performed with the same primers as stated above. The
thermal cycling steps for PCR amplification of AOA and AOB were: pre-deformation at
95 ◦ C for 3 min, 1 cycle; deformation at 95 ◦ C for 30 s; annealing at 55 ◦ C for 30 s and
extension at 72 ◦ C for 45 s for a total of 27 cycles; and final extension at 72 ◦ C for 10 min
(PCR instrument: ABI GeneAmp® 9700, Foster City, CA, USA). The 16S rDNA sequences
of the AOA and AOB genes were analyzed using the Illumina MiSeq paired-end 300 bp
protocol (Illumina, Inc., San Diego, CA, USA).

2.5. Statistical Analyses

All indicator data were collected in triplicate. IBM SPSS Statistics 20.0 was used to
calculate the mean ± standard deviation of the physicochemical indexes and enzymes.
The differences in enzymatic activities were evaluated by principal component analysis
(PCA) using CANOCO 5.0. While the online platform i-Sanger (http://www.i-sanger.com/,
accessed on 1 June 2023) was used to conduct AOA and AOB microbial community diversity
analyses. Correlations between the environmental factors, enzyme activities, and microbial
community composition of AOA and AOB were assessed by redundancy analysis (RDA)
using CANOCO 5.0. However, many environmental factors can affect the distribution
of the microbial structure during the process of composting, but many of them have a
strong collinearity. Therefore, the driving environmental factor should be screened prior to
environmental factor analysis in order to remove the factors that have a strong collinearity.
In the present study, the variance inflation factor (VIF) was measured and the environmental
factors with a VIF > 20 were removed from the subsequent analysis. Based on the results of
the VIF values (Table S1), pH, TOC, TN, and HAO were removed. Therefore, only the EC,
MC, NH4 -N, NO3 -N, C/N, FDA, and AMO were used for the redundancy analysis (RDA)
using Canoco 5.0 software.

3. Results and Discussion

3.1. Dynamic Changes in the NH3 and N2 O Emissions
NH3 and N2 O emissions are the main sources of N loss during a well-controlled
composting process [26,27]. As shown in Figure 1a, the NH3 emitted during the days 3–12
made up approximately 82.0–88.2% of the total gas emissions. With rapid decomposition
of the organic matter (OM), the NH3 emissions increased quickly at the initial stage and
reached to the maximums on day 6 for all treatments except the GE + BC treatments on day
8. The maximum emissions of NH3 occurred at levels of 81.4, 43.9, 70.5, and 71.3 mg/d
in the CK, GE, GE + BC, and GE + PL treatments, respectively. After the 8th day of the
composting process, the NH3 emissions were stable and similar to those observed on
day 1. During the whole process, the cumulative NH3 emissions in the treatments of the
CK, GE, GE + BC, and GE + PL treatments were 528, 307, 402, and 485 mg, respectively
(Figure 1a). Compared to CK, the NH3 emissions in the GE, GE + BC, and GE + PL
treatments reduced by 41.8%, 23.8%, and 8.3%, respectively. These results indicate that
additives were effective in compensating the loss of N via NH3 emissions. These results
are in line with previous studies, which suggested that the low-pH and enzyme-rich
activities of GE and the adsorption capacity of BC and PL are major sources of reduced N
mineralization in composting processes where GE and BC or PL were used as a process
catalyst and additives, respectively [17]. However, in our study, the values in the GE + BC
and GE + PL treatments were significantly higher than the GE treatments. It can be said
that the benefits of adding BC and PL may have been underestimated in previous studies
 previous studies, which suggested that the low-pH and enzyme-rich activities of GE and
the adsorption capacity of BC and PL are major sources of reduced N mineralization in
composting processes where GE and BC or PL were used as a process catalyst and addi-
Agronomy 2024, 14, 1162 tives, respectively [17]. However, in our study, the values in the GE + BC and GE5+ofPL 15
treatments were significantly higher than the GE treatments. It can be said that the benefits
of adding BC and PL may have been underestimated in previous studies and that such
additions
and are far
that such more advantageous
additions are far morefor the growth offor
advantageous microorganisms,
the growth of strengthening
microorganisms, the
ammonification
strengthening theprocess comparedprocess
ammonification to the GE-only
comparedtreatments with no
to the GE-only additiveswith
treatments [28].no
In
additives
addition, [28]. In addition,
the SSA and TPVthe SSAduring
of BC and TPV of BC during
different different
composting composting
processes were allprocesses
greater
were all greater
than the PL duringthan
thethe PL during
entire the entire
composting composting
process process (Table
(Table 1); therefore, the GE1);+therefore,
BC treat-
the GE + BC treatments may further adsorb additional
ments may further adsorb additional NH3 compared to the GE NH compared to the
3 + PL treatments and,GE +ulti-
PL
treatments and, ultimately, may result in
mately, may result in reduced NH3 emissions. reduced NH 3 emissions.

120 12 5
600
a 50
b c

Cumulative NO2 emissions (mg)

Cumulative NH3 emissions (mg)

CK 500
GE 40
100 10
GE+BC 400
30
NH3 emission (mg d−1)

GE+PL 4
NO2 emission (mg d−1)
300
80

NH4−N (mg kg−1)

200
8 20

100 10
60 6
0 0 3
CK GE GE+BC GE+PL CK GE GE+BC GE+PL

40 4

2
20 2

0 0
1
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
1.4 3.5 30

1.2 d ee f
3.0 28
1.0
NO3−N (mg kg−1)

2.5 26

TN (mg kg−1)
0.8
INB

0.6 2.0 24

0.4
1.5 22
0.2
1.0 20
0.0

0.5 18
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days) Time (days)

Figure 1.
1. NH
NH3 (a), N2O (b) emissions, NH4−N (c), NO3−N (d), the INB index (e), and TN (f).
Figure 3 (a), N2 O (b) emissions, NH4 -N (c), NO3 -N (d), the INB index (e), and TN (f).

Table 1.
Table 1. Changes
Changes in
in specific
specific surface
surface areas
areas (SSA)
(SSA) and
and total
total pore
pore volume
volume (TPV)
(TPV) of
of PL
PL and
and BC
BC during
during
the composting process.
the composting process.
Composting BC PL
Composting BC PL
Time (Days) SSA (m2 g−1) TPV (cm g ) 3 −1 SSA (m 2 g−1) TPV (cm3 g−1) 3 −1
Time (Days) SSA (m2 g−1 ) TPV (cm3 g−1 ) SSA (m2 g−1 ) TPV (cm g )
0 14.19 ± 0.34 0.050 ± 0.001 6.53 ± 0.26 0.019 ± 0.001
4 0 ± 0.11
7.23 14.19
0.039± ±0.34
0.002 0.050 ± 3.49
0.001± 0.11 6.53 ± 0.26 0.021 ± 0.019
0.003± 0.001
4 7.23 ± 0.11 0.039 ± 0.002 3.49 ± 0.11 0.021 ± 0.003
8 6.54
8 ± 0.38 0.038
6.54 ± 0.003
± 0.38 0.038 ± 2.35
0.003± 0.23 2.35 ± 0.23 0.022 ± 0.022
0.002± 0.002
13 6.13
13± 0.64 0.034 ±
6.13 ± 0.640.001 4.00
0.034 ± 0.001± 0.88 4.00 ± 0.88 0.023 ± 0.009
0.023 ± 0.009
18 18± 0.40
6.11 6.11 ± 0.40
0.035 ± 0.001 0.035 ± 2.80
0.001± 0.30 2.80 ± 0.30 0.017 ± 0.017
0.006± 0.006
24 24± 0.35
5.34 5.34 ± 0.35
0.035 ± 0.005 0.035 ± 3.16
0.005± 0.15 3.16 ± 0.15 0.015 ± 0.015
0.001± 0.001

Similar to
to the
the NH
NH33 emissions, the intensity
intensity of
of NN22O emissions was higher during the the
initial thermophilic period (days 1–6) (Figure 1b), and similar results were
initial thermophilic period (days 1–6) (Figure 1b), and similar results were also found also found that
by Awasthi
Awasthi etet al.
al. [15]
[15] during
during composting.
composting. TheThe peak
peak values
values of
of N
N22O emissions appeared on
day 11 with
with the
the values
values of
of 9.6,
9.6, 8.0,
8.0, and
and6.9 mgdd−−11 in
6.9mg in the CK, GE, and GE + PL treatments,
respectively, −1
respectively, while
while the
the GE
GE ++ BCBC treatments
treatments onon day
day33hadhada avalue
valueofof5.5
5.5mg
mgd d−1. . The
The
cumulative N2 O emissions for the four treatments were 47.3, 40.3, 33.5, 33.1 mg, respectively.
The three additive treatments showed reduced emissions of N2 O by 10.4%, 25.7, and 26.3%
relative to the control treatment. Geng et al. [29] reported that biochar could reduce
cumulative N2 O releases by 47.7% during kitchen waste composting. The BC and PL
not only have a large SSA that can adsorb the emitted N2 O, but also may increase the
TPV of the composting pile and impede the formation of an anaerobic environment, thus
Agronomy 2024, 14, 1162 6 of 15

further decreasing the N2 O emissions caused by the denitrification process and mediated
by denitrifying microbial communities.

3.2. Dynamic Changes in NH4 -N, NO3 -N, and TN Concentrations

NH4 -N is the substrate directly acted upon by the ammonia-oxidizing microorganisms
and, therefore, has a significant influence on subsequent nitrification processes [30]. The
NH4 -N content increased sharply (Figure 1c) because of the intense decomposition of
nitrogen from the organic substrate where all treatments showed maximum values on
day 4, in a range of 3.71, 3.62, 4.20, and 4.01 mg kg−1 . The values for GE + BC and GE + PL
were greater than those for the CK and GE treatments, which might be attributed to the low
pH of GE, which may slightly inhibit the decomposition of organic matter [23]. Meanwhile,
the BC and PL alleviated the inhibition of GE, promoted the degradation of organic matter,
and effectively absorbed the emitted NH3 [17]. Following this, the NH4 -N contents in
the GE + BC and GE + PL treatments showed an obvious decrease certainly due to the
intense nitrification and, therefore, perhaps not greatly different within the CK and GE
treatments. At the end of the composting process, the NH4 -N contents for the GE + BC and
GE + PL treatments (2.65 and 2.55 mg kg−1 ) were less than those for CK and GE (3.65 and
3.54 mg kg−1 ).
Similarly, the NO3 -N contents firstly increased during days 0–8 and then decreased
until the end of the composting period (Figure 1d). This is in line with the previous study
conducted on SS composting [17]. The difference was that the maximum NO3 -N contents
appeared on day 8, which lagged behing the peak values of the NH4 -N contents. Except
for days 0 and 4, the NO3 -N contents for the GE + BC and GE + PL treatments were higher
than those for the CK and GE treatments. At the end of composting period, the NO3 -N
contents for the four treatments were 0.21, 0.28, 0.47, and 0.41 mg/kg, respectively, and
in the order of GE + BC > GE + PL > GE treatment (123.8%, 95.2%, 32.9%) relative to the
control treatment. Together with those of NH4 -N, the overall results showed the GE + BC
and GE + PL treatments enhanced ammonification and nitrification during the composting
process, thus improving the NO3 -N content of the end compost.
The index of the N balance (INB) was used to evaluate the total variation in dissolved
inorganic N in the incubation system, such as the soil incubation and wastewater treatment,
which was calculated based on the previous study [31]. As shown in Figure 1e, the INB
in all treatments sharply increased during days 0–4 in all treatments, and then began to
decrease gradually during the latter 4–8 days. The values of INB during days 0–4 ranged
from 0.81 to 2.98, suggesting that the N loss was a result of the ammonification of the
organic N during this stage [31]. The NH3 emissions via ammonification during days 0–4
could be explained by this phenomenon (Figure 1a). And then, the IBN became almost
near 1.0, which indicates no remarkable amount of DIN production from organic matter
and no N loss emitted from NH3 and N2 O during this stage [32].
Figure 1f shows the TN content for all four treatments with a descending trend during
the whole composting process. The rapid decrease during days 0–12 was likely because of
the intense emissions of NH3 and N2 O (Figure 1a,b). The TN contents of the end compost
in the CK, GE, GE + BC, and GE + PL treatments therefore reduced by 30.9%, 22.6%, 23.4%,
and 21.9% relative to the initial values. This indicated that the BC and PL were correlate
with GE and effectively reduced the N loss from NH3 and N2 O emissions, especially within
the GE + BC treatment.

3.3. Dynamic Changes in FDA, AMO, and HAO Activities

Figure 2a shows that the additives (in the GE, GE + BC, and GE + PL treatments)
significantly increased the fluorescein diacetate hydrolase (FDA) activities at the initial
stage relative to the control treatment, and the activity of FDA for the GE + BC treatments
were always highest among the four treatments during the whole composting process.
During the whole stage, the FDA activities of the four treatments declined. Although, these
results were inconsistent with our previous study [17], showing maximum FDA activities in
 3.3. Dynamic Changes in FDA, AMO, and HAO Activities
Figure 2a shows that the additives (in the GE, GE + BC, and GE + PL treatments)
significantly increased the fluorescein diacetate hydrolase (FDA) activities at the initial
Agronomy 2024, 14, 1162 stage relative to the control treatment, and the activity of FDA for the GE + BC7treatments
of 15
were always highest among the four treatments during the whole composting process.
During the whole stage, the FDA activities of the four treatments declined. Although,
these results were inconsistent with our previous study [17], showing maximum FDA ac-
the similar treatment of additives on day 4. However, an intense decrease in FDA activities
tivities in the similar treatment of additives on day 4. However, an intense decrease in
in this study in the GE, GE + BC, and GE + PL treatments was detected during days 0–4.
FDA activities in this study in the GE, GE + BC, and GE + PL treatments was detected
This can be attributed to the fact that, during this stage, the OM contents decreased rapidly
during days 0–4. This can be attributed to the fact that, during this stage, the OM contents
and all the FDA enzymes in the and
decreased rapidly system were
all the FDAconsumed, leaving
enzymes in the system
the system enzyme-deficit
were consumed, leaving the
in the latter stages.
system enzyme-deficit in the latter stages. This phenomenon needs be
This phenomenon needs further investigation and can a potential
further investigation
objective in forthcoming
and can be astudies.
potential objective in forthcoming studies.

Figure 2.ofThe
Figure 2. The activities FDAactivities
(a), AMO of FDA (a), AMO
(b), HAO (b), HAO
(c), and (c), and component
the principal the principalanalysis
component analysis of
of FDA,
FDA, AMO, and HAO (d).
AMO, and HAO (d).

Few studies
Few studies have focusedhave focusedactivities
on AMO on AMO during
activitiesthe
during the composting
composting process, process,
while while
most studies have always evaluated AMO activities by the abundance of AOA or AOB
most studies have always evaluated AMO activities by the abundance of AOA or AOB
genes [8,9,33]. As shown in Figure 2b, the four treatments decreased sharply during days
genes [8,9,33]. As shown in Figure 2b, the four treatments decreased sharply during days
0–4, and the AMO activities reached a minimum on day 4, with the values of 1.20, 1.23,
0–4, and the AMO
1.39, activities reached−1a·min
and 1.32 nmol·mg minimum on dayThis
−1, respectively. 4, with the values
decrease may beofdue
1.20,
to1.23, 1.39,
the ammoxida-
·mg−
and 1.32 nmoltion 1 ·min−1 , respectively. This decrease may be due to the ammoxidation
of large quantities of NH4-N consuming a large amount of AMO enzyme. After that,
of large quantities of NH
the AMO 4 -N consuming
activities a large amount
gradually increased with theof AMO enzyme.
decrease in NH4-N After
(Figurethat, the aver-
1c). The
AMO activitiesaged
gradually increased
AMO activities withthe
during theentire
decrease in NH
process in the4 -N
CK,(Figure
GE, GE1c).
+ The
BC, and averaged
GE + PL treat-
AMO activitiesments
during the1.50,
were entire
16.0,process
1.70, and in1.67
the nmol·mg
CK, GE,−1GE·min +−1BC, and GE + PL treatments
, respectively.
The
were 1.50, 16.0, 1.70, HAO
and 1.67is the
nmolmain −1 ·minfor
·mgenzyme −1converting hydroxylamine into nitrite nitrogen, and
, respectively.
is directly related to the NO -N of the
The HAO is the main enzyme for converting hydroxylamine
3 end product compost [8]. Similar
into to AMO
nitrite activities,
nitrogen,
and is directly related to the NO3 -N of the end product compost [8]. Similar to AMO
activities, HAO activities in the four treatments reduced drastically during days 0–4,
and then gradually increased until the end of the composting period (Figure 2c). The
rapid decrease in HAO activity also corresponded to the rapid increase in NO3 -N content
(Figure 1d). The average values of the HAO in the CK, GE, GE + BC, and GE + PL treatments’
activities during the entire process were 0.41, 0.43, 0.43, and 0.45 µmol K3 Fe (CN)6 h−1 ,
respectively. These results showed that GE combined with BC and PL increased the
AMO and HAO activities and elevated the process of ammoxidation, thus improving the
subsequent nitrification process and augmenting the final NO3 -N content of the compost.
Furthermore, the PCA analysis showed that the three treatments (the GE, GE + BC,
and GE + PL treatments) affected the FDA, AMO, and HAO activities relative to the control
Agronomy 2024, 14, 1162 8 of 15

treatment (Figure 2d) during the whole composting period. Among the three treatments,
samples for the GE + BC treatments on days 4, 13, and 24 were very different to the samples
for the GE and GE + PL treatments, which indicated that BC connected GE could effectively
change enzymatic activity and has a greater impact on composting process compared to
the GE and GE + PL treatments.

3.4. Dynamic Changes in the Abundances of Archaea and Bacteria of the amoA Gene
AOA and AOB participate in the oxidation process of ammonia during the compost-
ing process [8]. Figure 3 shows the abundance of AOA and AOB genes ranging from
1.12 × 105 –4.71 × 105 to 3.28 × 104 –9.13 × 104 copies g−1 . During the whole composting
process, the gene copies of AOA were higher than those of AOB, which shows that AOA
has a greater contribution in accelerated nitrification in this study. This result was in line
Agronomy 2024, 14, x FOR PEER
with REVIEW
the results of Yan et al. [34] on the composting of cattle manure and those of Zeng 9 of

et al. [8] on the composting of agricultural waste.

5x105 105
a CK
b
GE
4x105 GE+BC 8x104

AOB gene (Copies g−1)

AOA gene (Copies g−1)

GE+PL

3x105 6x104

2x105 4x104

105 2x104

0 0
4 13 24 4 13 24
Time (days) Time (days)
12
c
10

8
AOA/AOB

0
4 13 24
Time (days)

Figure
Figure 3. The 3. The abundances
abundances of AOA
of AOA (a) and AOB(a)
(b)and AOB
genes (b)the
and genes
ratioand the ratio
of AOA of AOA
to AOB (c). to AOB (c).

As shown in AsFigure 3a, theofAOA

the results gene in
the AOA andtheAOB
CK, genes
GE, GEshown,
+ BC, and
the GE + PL treatments
addition of GE slowed dow
during thethethermophilic phase (4 d) had the highest values, with values
growth of AOA and AOB, while the addition of BC and PL could eliminate of 3.27 × 105 , this inh
5
2.68 × 10 ,bition 5
4.23 ×during
10 , and 5
× 10 copies − 1
the4.10
thermophilic g In
stage. , respectively.
addition, theThe abundance
gene copies ofofAOAthe AOA
and AOB in G
gene in GE+ was lower than that in the control treatment because of the
BC and GE + PL were also greater than in the GE and CK treatments during inhibition of GE the who
by microorganisms. Whereas
process. This showsthose
that in
BCGE and+ BC
PL and GE + PL
combined were
with GEhigher
could than the control
improve the growth env
mainly due ronment for AOA and AOB, and strengthen ammoxidation during (13
to BC and PL alleviating the inhibition of GE. At the cool stage the d), the
composting pr
abundancecess,of the AOA gene decreased, which can be attributed to probable relatively
which significantly reduces N2O emissions (Figure 1b) and increases NO3-N conte
higher temperature during
(Figure 1d). the thermophilic
Meanwhile, stage slowing
other nitrogen down
functional the growth
genes, such asof theAOA. After monoox
ammonia
this stage, the temperature dropped and the abundance of the AOA gene simultaneously
genase (amoA), nitrite reductase (nirK and nirS), and nitrous oxide reductase (nosZ) gene
increased until compost
also had closematurity (24 d).
relationships with NH3 and N2O emissions [33]. Thus, further research w
Relative to AOA, the AOB gene
be necessary to evaluate all hadnitrogen-functional
a similar change duringgenesthe thermophilic
after the additionphase
of additive
(Figure 3b). The abundance of AOB genes in the GE + BC and GE + PL treatments on day 4
which is likely to reveal the mechanism of the nitrogen loss reduction. In addition, th
increased by 73.9% and 38.7%, while in the GE treatment it reduced by 33.8% relative to
also has important guiding significance for selecting suitable additives to reduce nitrog
the control. Wu et al. [10] also reported that BC promoted the abundance of AOB during
loss.
the composting of agricultural waste. Lin et al. [35] reported that biochar significantly
3.5. Community Composition of AOA and AOB
Based on the 16S rDNA sequence data of AOA, a total of 171, 053 high-quality s
quences were obtained from 12 samples, and clustered into 51 OTUs based on a ≥3% d
similarity cutoff. Meanwhile, 252,871 sequences and only 14 OTUs were obtained for AO
Agronomy 2024, 14, 1162 9 of 15

increased the abundance of the AOA gene, and found that N2 O emissions had an extreme
relationship with the abundance of the AOA gene. As the composting process proceeded,
the inhibition of GE weakened or was eliminated after the thermophilic phase. This change
gradually supported the growth of AOA resulting in an increased abundance of AOA
genes during cooling stage. During the cooling and maturity stages, the abundance of the
AOA genes in the three additive treatments raged from 5 to 7 × 105 copies g−1 , while this
value was 3–4 × 105 copies g−1 in the control.
The ratio of AOA to AOB copies initially decreased during days 4–13, and then
increased during days 13–24 (Figure 3c). The results also showed that the AOA copies
increased at the cooling and maturity stages relative to AOB, which indicated that AOA
could grow well at lower temperatures during the composting process. Sims et al. [5] also
reported the better adaptation of AOA than AOB to the low-temperature environment of
natural wetlands. Meanwhile, AOB increased during the thermophilic phase, meaning that
the high temperature suited the growth of AOB, which also corresponded with the high
NH4 -N content (Figure 1c), providing further optimal conditions for AOB [34].
As the results of the AOA and AOB genes shown, the addition of GE slowed down the
growth of AOA and AOB, while the addition of BC and PL could eliminate this inhibition
during the thermophilic stage. In addition, the gene copies of AOA and AOB in GE + BC
and GE + PL were also greater than in the GE and CK treatments during the whole process.
This shows that BC and PL combined with GE could improve the growth environment
for AOA and AOB, and strengthen ammoxidation during the composting process, which
significantly reduces N2 O emissions (Figure 1b) and increases NO3 -N content (Figure 1d).
Meanwhile, other nitrogen functional genes, such as the ammonia monooxygenase (amoA),
nitrite reductase (nirK and nirS), and nitrous oxide reductase (nosZ) genes, also had close
relationships with NH3 and N2 O emissions [33]. Thus, further research will be necessary
to evaluate all nitrogen-functional genes after the addition of additives, which is likely to
reveal the mechanism of the nitrogen loss reduction. In addition, this also has important
guiding significance for selecting suitable additives to reduce nitrogen loss.

3.5. Community Composition of AOA and AOB

Based on the 16S rDNA sequence data of AOA, a total of 171, 053 high-quality se-
quences were obtained from 12 samples, and clustered into 51 OTUs based on a ≥3%
dissimilarity cutoff. Meanwhile, 252,871 sequences and only 14 OTUs were obtained for
AOB from the same samples. The results were in line with the relative abundances (Ras)
of AOA and AOB genes, and once more affirmed that AOA has a greater contribution to
nitrification in this study, relative to AOB.
Principal coordinates analyses (PCoAs), based on unweighted UniFrac distances and
the OUT level, were also conducted to explore the difference in species composition among
the different treatments. The results showed that the grouping ellipse sizes in GE + BC and
GE + BC were bigger than those in the CK and GE treatments, which indicated that BC and
PL correlated with GE significantly, which altered the community composition of the AOA
(Figure S1).
As shown in Figure 4a, only Crenarchaeota and Thaumarchaeota could be identified
as AOA during the composting processes, which is also in line with our previous study
when a urease inhibitor and a nitrification inhibitor were added during SS composting [36].
The RAs of theses four phyla were 27.55–64.21%, 34.59–63.33%, 0–24.33%, and 0–1.24%,
respectively. Significant differences in AOA between treatments (Figure S2) showed that
the RA of an unclassified phylum for the GE treatments was lower than for CK, while the
RAs for the GE + BC, and GE + PL treatments were greater than the control treatment
during thermophilic phase. In the cooling and maturity phases, the RAs of the unclassified
phylum in the GE, GE + BC, and GE + PL treatments were all higher than that in the control.
The RAs of Crenarchaeota in the GE + BC and GE + PL treatments were lower than in
CK during the entire composting process. However, the RA in the GE treatment during
thermophilic stage was higher than in the control, while the RA in the GE treatment during
Agronomy 2024, 14, 1162 10 of 15

the cooling stage was lower than in the CK treatment. Crenarchaeota widely exist in the
ocean, hot springs, and soil, and are the most abundant and most widely distributed AOA
in nature [37]. These results not only showed that BC and PL together with GE resulted
in the transition from Crenarchaeota to an unclassified phylum, but also indicated the
complexity of the community structure of AOA. Thaumarchaeota, as the newly discovered
AOA phylum, are highly diverse and widely distributed in various environments, but their
physiological metabolism, genetic characteristics, and ecological functions need further
study [38]. Thaumarchaeota were only found in the samples of GE + BC_13, CK_24,
Agronomy 2024, 14, x FOR PEER REVIEW 11 and
of 15
GE_24. At the genus level, unclassified_Archaea, norank_Crenarchaeota, Nitrososphera, and
norank_p_environmental_samples were the dominant genera of AOA (Figure 4b). These three
genera, unclassified_Archaea, norank_Crenarchaeota, and norank_p_environmental_samples,
detected in the samples GE + PL_4, GE_13, and GE + PL_13 with RAs of 20%, 10%, and
accounted for 75.7–100% of the total sequencing reads. Lin et al. [39] also reported that
20%, respectively.
all of the detected genera belonged to no rank or unclassified genus when treating saline
wastewater in biofilm reactors.
100 100
a b
80 unclassified 80
unclassified_Archaea
Relative abundance (%)
Relative abundance (%)

Crenarchaeota
norank_Crenarchaeota
60 60
Thaumarchaeota
Nitrososphera
environmental_samples
40 norank_p_environmental_samples
40
others
others

20 20

0 0

_4 _4 _4 L_4 _13 _13 _13 _13 _24 _24 _24 _24 _4 _ 4 _4 _ 4 _1 3 _1 3 _ 13 _1 3 _24 _2 4 _24 _2 4
CK GE +BC +P CK GE BC PL CK GE BC PL CK GE +BC +PL CK GE BC PL CK GE BC PL
E + E+ + E+
GE G
E + E+ + E+ GE G GE G GE G
GE G GE G
100 100
c d Nitrosospira
80 80
Relative abundance (%)
Relative abundance (%)

norank_f_environmental_samples
Proteobacteria norank_p_ammonia_oxidsing_
60 60 bacteria_ensemble
ammonia_oxidsing_
bacteria_ensemble norank_f_environmental_samples
40 40 _c_Betaproteobacteria
unclassified_k_norank
_d_Bacteria Nitrosomonas

20 20 unclassified_o_Nitrosomonadales

unclassiified_k_norank_d_Bacteria
0 0
_4 _4 _4 L_4 _13 _13 _13 _13 _24 _24 _24 _24 _4 _4 _4 L_4 _13 _13 _13 _13 _24 _24 _24 _24
CK GE +BC +P CK GE BC PL CK GE BC PL CK GE +BC +P CK GE BC PL CK GE BC PL
+ E+ + E+ E + E+ + E+
GE G
E
GE G GE G GE G GE G GE G

Figure 4. The community composition AOA at the phylum level (a) and at the genus level (b); the
Figure 4. The community composition AOA at the phylum level (a) and at the genus level (b); the
community
communitycomposition
compositionAOB
AOBat
atthe
thephylum
phylumlevel
level(c)
(c)and
andat
atthe
thegenus
genuslevel
level(d).
(d).

As shown in Figure 4c, only three phyla of AOB, Proteobacteria, ammonia_oxidsing_

3.6. Relationships between Physicochemical Indexes, Enzymatic Activity, and
bacteria_ensemble, and unclassified_k_norank_d_Bacteria, were detected in all of the sam-
Ammonia-Oxidizing Microorganisms
ples during the composting process. The Proteobacteria was the dominant phylum in
The RDA
all samples, shows
while that the selected environmental variables
ammonia_oxidsing_bacteria_ensemble was (EC,
onlyMC,foundNHin 4-N,
theNOGE_43-N,

and GE_13
and C/N) and AMO enzymatic
samples, with the RAs activities
of 30% could
andexplain 70.8% and 60.1%
50%, respectively. of the
At the variations
genus level,
in thewere
there AOA and AOBdifferences
significant community, in respectively (Figure 5a,b).
the AOA composition Among
between the seven
different selected
periods and
tactors, FDA
treatments and AMO
(Figure activities, NH
4d). Nitrosospira 4-N, NO3norank_f_environmental_samples
(0–100%), -N, and C/N significantly affect(0–100%),the AOA
and AOB community structures. FDA represented
norank_p_ammonia_oxidsing_bacteria_ensemble (0–50%),thenorank_f_environmental_samples_c_
overall level of all enzymes and
was closely related to the N transformation during the composting
Betaproteobacteria (0–40%), Nitrosomonas (0–20%), unclassified_o_Nitrosomonadales process [23]. The AMO
(0–20%),
enzyme
and is secreted directly by ammonia-oxidizing
unclassiified_k_norank_d_Bacteria (0–10%) were themicroorganisms,
main genera during consisting of AOA
the composting
and AOB.
process. TheWu et al. [10]
no-rank and also reportedgenera
unclassified that AMO activity
accounted forhad markedly
75.7–100% positive
of the relation-
total sequenc-
ships
ing withRelated
reads. AOA and AOB
to the numbers.
no-rank andAs the substrate
unclassified of ammonia-oxidizing
genera for AOA, these results microorgan-
indicate
isms,
the NH4-N content
complexity directly affected
of the community their of
structure structure
AOA and and abundance.
AOB NO3-N, as an
during composting, indi-
which
rect product
urgently of ammonia
requires advanced oxidation,
isolationcould
and reflect the evolution
identification of ammonia-oxidizing
techniques and the updatingmi- of
croorganisms
gene databases. toStudies
a certainhave
extent. Shi that
found et al.Nitrosospira
[6] found thatand NO 3-N solely explained
Nitrosomonas were the 27.3%
domi- of
nant genus forinnitrification
the variation in many
the AOB species ecosystems,
during such as
food waste sea [30], soil
composting. The[36], wastewater
initial C/N ratio [39].
of
composting substrates affects the reproduction of all microorganisms and plays a vital
role during the composting process. Lin et al. [35] reported that AOB were positively cor-
related with NH4-N and C/N, while having an opposite correlation with NO3-N.
Agronomy 2024, 14, 1162 11 of 15

On day 4, the RAs of Nitrosospira in the GE and GE + PL treatments were significantly

greater than in the CK and GE + BC treatments. This can be mainly attributed to the high
temperature and NH4 -N content in the GE + BC treatment, which ultimately inhibited the
Nitrosospira genus [40]. Going through the high temperature of the thermophilic stage, the
Nitrosospira in GE + PL and GE + BC could not be detected on day 13, meaning that the
composting conditions were accrual for this specific bacterial genus. With the decrease in
temperature during the maturity stages, Nitrosospira became the dominant genus with
RAs of 40%, 70%, 100%, and 40% in the CK, GE, GE + BC, and GE + PL treatments, and its
RAs in the GE + BC treatment reached up to 100%. The high NO3 -N content could support
this inference. Nitrosomonas was the dominant species among the AOB, and Lu et al. [31]
found that the RAs of Nitrosomonas were up to 79.8% in an anammox-inoculated wastewa-
ter treatment system. However, Nitrosomonas was only detected in the samples GE + PL_4,
GE_13, and GE + PL_13 with RAs of 20%, 10%, and 20%, respectively.

3.6. Relationships between Physicochemical Indexes, Enzymatic Activity, and

Ammonia-Oxidizing Microorganisms
The RDA shows that the selected environmental variables (EC, MC, NH4 -N, NO3 -N,
and C/N) and AMO enzymatic activities could explain 70.8% and 60.1% of the variations in
the AOA and AOB community, respectively (Figure 5a,b). Among the seven selected tactors,
FDA and AMO activities, NH4 -N, NO3 -N, and C/N significantly affect the AOA and AOB
community structures. FDA represented the overall level of all enzymes and was closely
related to the N transformation during the composting process [23]. The AMO enzyme
is secreted directly by ammonia-oxidizing microorganisms, consisting of AOA and AOB.
Wu et al. [10] also reported that AMO activity had markedly positive relationships with
AOA and AOB numbers. As the substrate of ammonia-oxidizing microorganisms, NH4 -N
content directly affected their structure and abundance. NO3 -N, as an indirect product of
ammonia oxidation, could reflect the evolution of ammonia-oxidizing microorganisms to a
certain extent. Shi et al. [6] found that NO3 -N solely explained 27.3% of the variation in the
AOB species during food waste composting. The initial C/N ratio of composting substrates
affects the reproduction of all microorganisms and plays a vital role during the composting
Agronomy 2024, 14, x FOR PEER REVIEW 12 of 15
process. Lin et al. [35] reported that AOB were positively correlated with NH4 -N and C/N,
while having an opposite correlation with NO3 -N.

70 4
a CK_24 CK b
60 CK
GE
3 GE
GEBC
50 AM O GE_BC
GEPL GE+BC_24
BG_24 GE_PL
40 2 GE_24
C/N
CN AM O EC
30 GE+PL_24
PG_24
GE+BC_13
BG_13 1 CK_13 CN
C/N
GE+PL_4
RDA2(20.27%)

RDA2(10.74%)

20 PG_4
CK_4 GE+BC_13
BG_13
GE_24 CK_24
10 0
NH4-N GE+PL_24
PG_24 GE+BC_4
BG_4 MC
NH4N NH4-N
NH4N
0
GE_13 -1
EC BG_4
GE+BC_4 NO3N
NO3-N
-10 GE+PL_4
PG_4 GE_4
MC PG_13
CK_13
PG_13 BG_24
GE+PL_13 GE+BC_24
-20 -2
CK_4 FDA
-30 GE_4
FDA
NO3N -3
NO3-N
-40 GE_13

-50 -4

-50 -40 -30 -20 -10 0 10 20 30 40 50 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3
RDA1(50.53%) RDA1(49.36%)

Figure
Figure 5.
5. The
TheRDA
RDAanalysis
analysisof
ofenzyme
enzymeactivities,
activities,physicochemical
physicochemical indexes,
indexes, and
and ammonia-oxidizing
ammonia-oxidizing
microorganisms at the genus level: the RDA of AOA (a) and AOB
microorganisms at the genus level: the RDA of AOA (a) and AOB (b). (b).

In
In order
order to analyze the relationships
relationshipsbetween
betweenthe
thethree
threeenzymes,
enzymes,eight
eightphysiochemical
physiochemi-
cal factors,
factors, andand
thethe detected
detected OTUs
OTUs (OTU
(OTU 51 for
51 for AOAAOA
andand
OUTOUT14 14
forfor AOB),
AOB), network
network anal-
analysis
ysis was conducted. The yielded nodes for the AOB community in the CK, GE, GE + BC,
and GE + PL treatments were only 13, 4, 3, and 3 (Figure S3), while the corresponding
nodes for the AOA community were 20, 25, 26, and 22, respectively (Figure 6). Therefore,
only the network analysis related to AOA is presented in the current study.
 -50 -40 -30 -20 -10 0 10 20 30 40 50 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3
RDA1(50.53%) RDA1(49.36%)

Figure 5. The RDA analysis of enzyme activities, physicochemical indexes, and ammonia-oxidizing
microorganisms at the genus level: the RDA of AOA (a) and AOB (b).
Agronomy 2024, 14, 1162 12 of 15
In order to analyze the relationships between the three enzymes, eight physiochemi-
cal factors, and the detected OTUs (OTU 51 for AOA and OUT 14 for AOB), network anal-
was conducted.
ysis was The The
conducted. yielded nodes
yielded for the
nodes forAOB
the AOBcommunity
communityin the
inCK,
the GE,
CK, GE
GE,+GEBC,+ and
BC,
GE +GE
and PL +treatments were only
PL treatments were13, 4, 3,13,
only and4, 33,(Figure
and 3 S3), while
(Figure thewhile
S3), corresponding nodes for
the corresponding
the AOA
nodes for community were 20, 25,
the AOA community 26,20,
were and 25,22,
26,respectively (Figure 6).
and 22, respectively Therefore,
(Figure only the
6). Therefore,
network analysis related to AOA is presented in the current study.
only the network analysis related to AOA is presented in the current study.

Figure 6. The network analysis of the enzymes, physicochemical characteristics, and the ammonia-
Figure 6. The network analysis of the enzymes, physicochemical characteristics, and the ammonia-
oxidizing archaea (AOA) at the OTU level for CK (a), GE (b), GE + BC (c), and GE + PL (d) treatments,
oxidizing archaea (AOA) at the OTU level for CK (a), GE (b), GE + BC (c), and GE + PL (d) treatments,
respectively. A connection represents a significant correlation (p < 0.05) according to Spearman’s
respectively.
rank analysisA(rho
connection
> 0.6). represents a significant correlation (p < 0.05) according to Spearman’s rank
analysis (rho > 0.6).
For the AOA, the analysis revealed 20 nodes and 102 edges, with 11 physicochemical
For the AOA, the analysis revealed 20 nodes and 102 edges, with 11 physicochemical
indexes and 9 OTUs in CK. The AMO and HAO activities had negative relationships with
indexes and 9 OTUs in CK. The AMO and HAO activities had negative relationships with
OTU 9, OTU 6, OTU 20, OTU 29, OTU 27, and OTU 21 (Figure 6). In the GE treatment,
OTU 9, OTU 6, OTU 20, OTU 29, OTU 27, and OTU 21 (Figure 6). In the GE treatment,
there were 25 nodes and 144 edges, with 11 physicochemical indexes and 14 OTUs. The
AMO and HAO activities had positive relationships with OTU 32, OTU 35, and OTU
50, while they negatively correlated with OTU 38, and OTU 41 OTU. For the GE + BC
treatment, the analysis revealed 26 nodes and 166 edges, with 11 physicochemical indexes
and 15 OTUs. The AMO and HAO activities had positive relationships with OTU 6, OTU
9, OTU 12, OTU 23, OTU 39, and OTU 46, while they had negative relationships with
OTU 20 and OUT 50. For the GE + PL treatments, there were 22 nodes and 86 edges, with
11 physicochemical indexes and 11 OTUs. The AMO and HAO activities had positive
relationships with OTU 29 and OTU 37, while they had negative relationship with OTU 4.
The OTUs had positive relationships with all AMO and HAO activities belonging to
the unclassified_Archaea and norank_Crenarchaeota genera, indicating that these two genera
most probable secreted ammoxidation-related enzymes. Among the three treatments, the
close relationships between environmental factors, enzymatic activities, and AOA, were
significantly promoted as revealed by the increased numbers of nodes and edges, and the
OTUs that had a positive relationship with AMO and HAO activities were also increased
in the GE + BC treatment, which showed that the combined usage of GE and BC has
the greatest potential to enhance the relationship between enzymes and microorganisms
related to ammonia oxidation processes compared to other treatments. Accordingly, this
treatment increased the rate of nitrification and reduced NH3 emissions and N loss during
the composting process. Similar results were also reported by Jiang et al. [17] during
SS composting.
Agronomy 2024, 14, 1162 13 of 15

4. Conclusions
The GE + BC and GE + PL treatments clearly promoted the activities of AMO and
HAO and the gene copy numbers of AOA and AOB, thus improving NH4 -N oxidation
and NO3 -N production. Meanwhile, the GE + BC and GE + PL treatments decreased NH3
emissions by 23.8% and 8.3%, respectively. Network analysis showed that the co-application
of garbage enzyme with biochar enhanced the positive connections between environmental
factors, enzymatic activities, and the dominant genus within AOA. In addition, RDA
indicated that FDA and AMO activities, NH4 -N, NO3 -N, and C/N significantly affect the
community structures of AOA and AOB. Therefore, GE combined with BC might be a useful
strategy to reduce NH3 emissions and accelerate ammoxidation during SS composting. Due
to the complexity of the community structures of AOA and AOB during composting, further
research will be necessary to exploit advanced isolation and identification techniques and
the updating of the gene database.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/agronomy14061162/s1, Table S1: The analyze of variance inflation
factor; Figure S1: The principal co-ordinates analysis (PCoA) analysis based on unweighted UniFrac
distances for the ammonia-oxidizing microorganisms on OUT level: (a) the PCoA nanlysis of AOA,
(b) the PCoA nanlysis of AOB; Figure S2: Significant differences in ammonia-oxidizing archaea (AOA)
at the phylum level between treatments; Figure S3: The network analysis among the enzymatic
activities, physicochemical characteristics, and the ammonia oxidizing bacteria (AOB) at the OTU
level. A connection represents a significant correlation (p < 0.05) according to Spearman’s rank
analysis (rho > 0.6).
Author Contributions: Funding acquisition, Supervision, Writing—review &editing, J.J. Writing—
original draft, Investigation, H.C. Data curation, Formal analysis, Investigation, P.B. Writing—review
& editing, C.C.C.C. Funding acquisition, Supervision, F.Y. Methodology, Resources, Writing—review
& editing, Supervision, Validation, D.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (41805123),
the Natural Science Foundation of Henan Province (232300421247), and the Yunnan Revitalization
Talent Support Program ‘Young Talent’ Project (YNQR-QNRC-2019-025).
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding author.
Acknowledgments: The bacterial community analyses were conducted on the online platform of
Majorbio Cloud Platform.
Conflicts of Interest: The authors declare no conflicts of interest.

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