Ecotoxicology and Environmental Safety 247 (2022) 114251

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Ecotoxicology and Environmental Safety

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

Phytotoxicity of farm livestock manures in facultative heap composting

using the seed germination index as indicator
Yilin Kong a, 1, Guoying Wang a, 1, Wenjie Chen a, c, Yan Yang a, Ruonan Ma a, Danyang Li b,
Yujun Shen b, Guoxue Li a, c, Jing Yuan a, c, *
a
Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation, College of Resources and Environmental Science, China Agricultural University, Beijing,
China
b
Institute of Energy and Environmental Protection, Academy of Agricultural Planning & Engineering, Ministry of Agriculture and Rural Affairs, Beijing 100125, China
c
Organic Recycling Institute (Suzhou) of China Agricultural University, Wuzhong District, Suzhou 215128, China

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

Edited by Dr Muhammad Zia-ur-Rehman Static facultative heap composting of animal manure is widely used in China, but there is almost no systematic
research on the phytotoxicity of the produced compost. Here, we evaluated the phytotoxic variation in compost
Keywords: produced by facultative heap composting of four types of animal manure (chicken manure, pig manure, sheep
Livestock manure manure, and cattle manure) using different plant seeds (cucumber, radish, Chinese cabbage, and oilseed rape) to
Facultative heap composting
determine germination index (GI). The key factors that affected GI values were identified, including the dy­
Phytotoxicity
namics of the phytotoxicity and microbial community during heap composting. Sensitivity to toxicity differed
Germination index
Dissolved carbon and nitrogen depending on the type of plant seed used. Phytotoxicity during facultative heap composting, evaluated by the GI,
Bacterial community was in the order: chicken manure (0–6.6 %) < pig manure (14.4–90.5 %) < sheep manure (46.0–93.0 %) < cattle
manure (50.2–105.8 %). Network analysis showed that the volatile fatty acid (VFA) concentration was positively
correlated with Firmicutes abundance, and NH+ 4 -N was correlated with Actinobacteria, Proteobacteria, and
Bacteroidetes. More bacteria were stimulated to participate in conversions of dissolved organic carbon, dissolved
nitrogen, VFA, and ammonia-nitrogen (NH+ 4 -N) in sheep manure heap composting than that in other manure.
The GI was most affected by VFA in chicken manure and cattle manure heap composting, while NH+ 4 -N was the
main factor affecting the GI in pig manure and sheep manure compost. The dissolved carbon and nitrogen
content and composition, as well as the core and proprietary microbial communities, were the primary factors
that affected the succession of phytotoxic substances in facultative heap composting, which in turn affected GI
values. In this study, the key pathways of livestock manure composting that affected GI and phytotoxicity were
found and evaluated, which provided new insights and theoretical support for the safe use of organic fertilizer.

1. Introduction
The fast-growing livestock husbandry industry in China generates
3.8 billion tons of animal waste annually. Without effective and timely
treatment, these vast amounts of livestock manure constitute a threat to
the environment (Zhang et al., 2021a; Zhan et al., 2021). Composting is
a cost-effective and ecologically beneficial way to dispose of and recycle
organic solid waste. Static facultative heap composting, where the
manure is simply piled without further control and ventilation, is the
most common and frequently used way of treating animal waste by
farmers in China (Zhu et al., 2021). This technology is now extensively
used on small-scale farms because of its low cost and straightforward
operation, and in China more than 70 % of animal manure is directly
returned to farmland following static facultative heap composting at
relatively low temperatures (30–50 ◦ C) (Wang et al., 2022a,b).
Facultative heap composting differs from thermophilic composting
in that the oxygen diffusion depth is constrained and the pile provides a
facultative anaerobic environment, resulting in a lower, near-ambient
temperature (Wang et al., 2022b). Therefore, the organic matter is not
degraded uniformly, leading to a compost with varying quality (Liu

* Corresponding author at: Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation, College of Resources and Environmental Science, China
Agricultural University, Beijing, China.
E-mail address: jingyuan@cau.edu.cn (J. Yuan).
1
Authors contributed equally to this work.

https://doi.org/10.1016/j.ecoenv.2022.114251
Received 17 June 2022; Received in revised form 27 October 2022; Accepted 28 October 2022
Available online 31 October 2022
0147-6513/© 2022 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Kong et al.
et al., 2020a). The final compost is often used as an organic amendment
to improve soil fertility and plant (crop) growth. However, the maturity
and phytotoxicity of the compost are important concerns before use on
farmland (Luo et al., 2018). There have been several studies on the
maturity of thermophilic aerobic composting in recent years (Li et al.,
2018; Wang et al., 2022a), while only few reports have investigated the
maturity and phytotoxicity of compost produced by facultative heap
composting.
The germination index (GI) is a sensitive and authoritative biological
indicator to evaluate compost maturity and phytotoxicity (Zhan et al.,
2021), and was first proposed by Zucconi et al. in 1981, with a GI of
more than 80 % indicating phytotoxicity-free and mature compost
(Zucconi et al., 1981). Currently, the newest standard of organic fertil­
izer (NY525–2021) introduced GI as the compost maturity evaluation
index and added a requirement for GI ≥ 70 %. The accuracy of GI in
determining compost maturity is affected by the sensitivity of the
selected seed species to biotoxicity (Yang et al., 2021).
Many studies have shown that high salt concentrations, organic
acids, metals, and NH+ 4 in compost are phytotoxic substances that affect
the GI (Meng et al., 2019; Wang et al., 2020a; Chen et al., 2021a; Gong
et al., 2021). Facultative heap composting is a biological decomposition
process of organic matter accompanied by the dynamic activity of
various microbial populations (Wei et al., 2019). Solid macromolecular
organic matter was decomposed into micro-molecular water-soluble
substances through microbial degradation in facultative heap com­
posting. Only soluble organic materials may pass through microbial cell
membranes, so soluble components represent the most active compo­
nent and have been used to evaluate the humification and phytotoxicity
of compost (Jin et al., 2022). Small organic acids, phenolic acids (PA),
humic acids (HA), polysaccharides (PS), reducing sugars (RS) and amino
acids (AA) can all affect seed germination (Wang et al., 2022c). Dis­
solved organic carbon (DOC) is also an essential index of compost
maturity because organic acids can hinder seed germination (Yu et al.,
2019) and humus is inversely correlated with phytotoxicity (Wang et al.,
2022a). Furthermore, the microbial population is crucial to the trans­
formation and decomposition of dissolved organic waste during heap
composting (Wang et al., 2022c). However, there are currently few
studies on the key factors that affect GI during livestock manure facul­
tative heap composting, especially concerning variation in water-soluble
substances that directly influence the GI.
Traditionally, the composition and concentration of phytotoxic
substances in animal manure varied significantly depending on the an­
imal species, lifestyle, and diet. (Hussein et al., 2017; Chen et al., 2019).
For example, the manure of granivores (pigs and chicken) contains less
lignocellulose than that of herbivores (sheep and cattle); therefore,
herbivore manure has a higher C/N ratio than granivore manure (Curien
et al., 2021). Different manure characteristics determine the microbial
communities and organic matter degradation rates in the compost (Wei
et al., 2019). Although the thermophilic composting of livestock manure
has been extensively investigated, little research has been conducted on
the phytotoxicity of the facultative composting system; therefore,
identification of key phytotoxic substances affecting seed germination is
essential for soil and crop health.
The objectives of this study were (1) to systematically analyze the
relationship between dissolved carbon and nitrogen (DOC, volatile fatty
acids [VFAs], PA, HA, PS, and RS; dissolved nitrogen [DN], AA, NH+
and NO−3 ) and GI during facultative heap composting of manure from
different types of livestock (pig, chicken, sheep, and cattle); (2) to
investigate the dynamics of bacterial communities and their roles in
organic matter degradation; and (3) to identify key factors, bacterial
communities, and pathways that affect the GI in phytotoxicity tests
through structural equation model (SEM). This study investigated the
variation in phytotoxicity of livestock manure produced through facul­
tative heap composting, which provides alternative directions for the
precise regulation of compost maturity and the safe use of farmland.
Ecotoxicology and Environmental Safety 247 (2022) 114251
2. Materials and methods
2.1. Raw materials and experimental design
The feces of chicken, pigs, sheep, and cattle were collected from four
farms in Beijing, China. The physical and chemical properties of the raw
materials are shown in Table S1. Approximately 30 kg of each type of
manure was piled separately to create four treatments: CM (chicken
manure), PM (pig manure), SM (sheep manure), and CaM (cattle
manure). The composting piles were heaped for 49 d at the Shangzhuang
Research Station at the Chinese Agricultural University. The composting
system comprised a polyvinyl chloride cylindrical chamber (volume 60
L, height 0.62 m) with a temperature sensor for recording the temper­
ature automatically; a detailed description can be found in Wang et al.
(2021a). Duplicate samples of each composting pile were collected after
completely mixing the piles on days 0, 3, 7, 14, 21, 28, 35, and 49. One
sample was stored at 4 ◦ C for analysis of physicochemical indexes and
seed germination tests; the other sample was stored at − 20 ◦ C for
bacterial community analysis.
2.2. Sample collection and analyses
2.2.1. Physiochemical parameters
A 10-g aliquot of the fresh compost sample was mixed with 100 mL
distilled water (1:10 wt/volume ratio) and the mixture was shaken at
180 rpm and 25 ◦ C for 0.5 h. The supernatant was passed through a filter
and then analyzed. The pH and electrical conductivity (EC) were
measured using a pHS-3 C pH/EC meter (YK Scientific Instrument Co.,
Shanghai, China). The total carbon and total nitrogen contents of air-
dried samples were determined using a Vario MACRO Cube elemental
analyzer (Elementar Analysensysteme, Langenselbold, Germany). The
cucumber, radish, Chinese cabbage, and oilseed rape seeds were used for
a seed germination experiment, and the GI was determined using the
method described in the Chinese Standard for Organic Fertilizer (NY
525–2021) and calculated with the following equation:
number of germinated seeds in extract root length in extract
GI = × × 100%
number of germinated seeds in control root length in control
2.2.2. Dissolved substances
DOC and DN contents were determined using a TOC-L liquid chro­
matograph (Shimadzu, Kyoto, Japan). A Technicon Autoanalyzer (Bran
+ Luebbe, Hamburg, Germany) was used to analyze the content of NH+ 4-
N and NO−3 -N. VFA concentrations (formic, acetic, propionic, and
butyric acid) were determined using a high-performance liquid chro­
matograph (SPD–M20A, Shimadzu) with a diode array detector (Shi­
madzu). AAs were detected based on the ninhydrin color liquid reagent.
Reducing sugars (RS) and polysaccharide (PS) concentrations were
determined using dinitro-salicylate reagent (Cao et al., 2013; Wu et al.,
2017). PA was measured according to the method described by Wang
et al. (2020b) with appropriate adjustments. HA was extracted and
quantified using the Chinese standard method to determine the HA
content of water-soluble fertilizers (NY/T 1971–2010).
2.2.3. High-throughput sequencing
4, Genomic DNA was extracted from compost samples from days 0, 14,
28, and 49 using the FastDNA SPIN Kit for Feces, following the manu­
facturer’s instructions (MP Biomedicals, Santa Ana, CA, USA). A Nano­
Drop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA) was used to determine the concentration and purity of DNA.
The V3–V4 hypervariable region of bacterial 16S rRNA genes was
amplified by polymerase chain reaction (PCR) using primers 341F (5′ -
CCTAYGGGRBGCASCAG-3′ ) and 806R (5′ -GGACTACNNGGGTATC­
TAAT-3′ ) (Zhu et al., 2019). High-throughput sequencing of PCR prod­
ucts was performed on the Illumina MiSeq PE250 platform at Shanghai
Oebiotechnology Co. Ltd. (Shanghai, China). Sequencing data were
2
Y. Kong et al.
analyzed as described by Wang et al. (2022a).
2.3. Data and statistical analysis
All physicochemical properties used for analyses were determined in
triplicate. Pearson correlation was used to determine the relationships
between environmental parameters and GI in SPSS 25.0 software (IBM
Corp., Armonk, NY, USA). The significance level for differences was set
at P < 0.05. Differences between means were analyzed using a linear
model for analysis of variance. Canoco (v5.0) was used to evaluate
changes in bacterial diversity by principal coordinate analysis (PCoA).
All networks were visualized using Gephi 0.9.2. SEMs were constructed
to assess the relationships among key environmental factors, bacterial
communities, and GI values. SEM is an a priori method for supporting
causal interpretations in a system by fitting data to models using AMOS
23.0 software (IBM Corporation Software Group, Somers, NY) with the
maximum-likelihood estimation method. The nonsignificant Chi-
squared (χ2 > 0.05) test, a high goodness-of-fit index (GFI > 0.90), a
low Akaike information criterion (AIC < 0.05), and a root-mean-square
error of approximation (RMSEA) < 0.05 ensured a good fit of the
models (Wang et al., 2021b).
3. Results and discussion
3.1. Seed germination index
GI is used to evaluate the phytotoxicity of compost because it in­
dicates the effects of toxicity on seed germination and seedling growth
(Wang et al., 2020a). The GI changes for the four kinds of seeds are
shown in Fig. 1. The GI values for the various seeds were all lower than
100 %, indicating that the raw livestock manure contained toxic sub­
stances (Barje et al., 2013). The GIs of radish, Chinese cabbage, and rape
seeds were always zero in the CM heap compost, although the GI of
Fig. 1. Germination indexes (GI) of different seeds during livestock manure heap composting. (a) Cucumber, (b) Radish, (c) Chinese cabbage, and (d) Oilseed rape.
CM: chicken manure, PM: pig manure, SM: sheep manure, CaM: cattle manure.
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Ecotoxicology and Environmental Safety 247 (2022) 114251
cucumber increased slightly after a period of composting (6.61 %). In
the other three types of manure compost, the GI values of the four seed
types were higher than in CM. The CM showed the highest phytotoxicity,
which was attributed to the higher NH+ 4 -N and VFAs in chicken manure
(Chen et al., 2019; Wan et al., 2021). The GI of cucumber seeds climbed
quickly during the first 7 days of composing, while the GI of the other
three seed types increased gradually with composting from days 1–28.
This shows that the sensitivity to toxicity of the individual seeds used in
the experiments varied significantly. The GI of cucumber seeds
increased rapidly to > 70 % through the composting process and was
higher than the GI values for the other seed types, as also reported by
Wei et al. (2022). The GI was higher in the CaM treatment
(50.16–105.75 %) than in the other manures (CM, 0–6.61 %; PM,
14.40–90.49 %; SM, 45.99–92.98 %) over the whole facultative heap
composting process. The degradation of phytotoxins in the raw manure
and the production of humus throughout the composting process
increased the GI for all treatments (Wang et al., 2022a). In general, the
phytotoxicity of the four kinds of feces was in the order: chicken feces
> pig feces > sheep feces > cattle feces (note that the sheep manure
used in this study was from sheep raised in the house, rather than by
grazing).
3.2. Maturity parameters
Temperature is an important indicator of facultative heap compost­
ing; it affects microorganism activity, organic matter degradation and
composting efficiency (Nakasaki and Hirai, 2017). The four treatments
had significant differences in temperature (P < 0.01) (Fig. 2a); the
temperatures of the treatments ranged from 15◦ to 45 ◦ C. SM had the
highest temperature, which was probably because the higher porosity
allowed more oxygen to enter the compost, resulting in higher aerobic
microbial activity and a higher organic matter degradation rate. The
temperature of the PM and CaM was slightly lower than that of the SM.
Y. Kong et al.
Fig. 2. Variation of maturity indexes during livestock manure heap composting. (a) Temperature, (b) pH, (c) electrical conductivity (EC), and (d) carbon/nitrogen
ratio (C/N). CM: chicken manure, PM: pig manure, SM: sheep manure, CaM: cattle manure.
These composts increased slowly from 22 ◦ C to 33.31–38.94 ◦ C (the
peak value) over the first 14 days and then stabilized to ambient tem­
perature during days 28–49. The temperature of the CM, which had
higher moisture and density than the other composts, was significantly
positively correlated with the ambient temperature, resulting in an
anaerobic environment and limited activity of aerobic microorganisms.
The microbial metabolic activity varied with temperatures, resulting in
differences in the organic matter transformation in the four kinds of
animal manure during heap composting.
The pH values of the four treatments followed a similar and stable
trend (Fig. 2b). The initial pH values of the four manures were between
7.69 and 8.37. SM had the highest pH among the treatments. During the
first 21 days, the pH increased gradually (8.37–8.75), then stabilized at
8.76 during the next 28 days. In the first 3 days of CaM heap composting,
the pH increased because of VFA volatilization and the accumulation of
NH+ 4 in the feedstock (Li et al., 2018). The decreasing trend in pH in PM
during days 14–21 may have been caused by the incomplete degradation
of organic matter and ammonia volatilization. The pH of the CM treat­
ment did not change appreciably. The pH change during facultative
heap composting occurred much more slowly than in thermophilic
aerobic composting because of the limitation of oxygen supply, which
slowed organic matter transformation and breakdown (Kong et al.,
2020).
The EC reflects the degradation and mineralization of organic mat­
ter, and a higher EC inhibits the GI (Meng et al., 2019). The EC of the
PM, SM, and CaM treatments slightly increased during the early stage of
composting, but the overall variation was stable (Fig. 2c). The EC of the
CM treatment was significantly higher than that of the other treatments
(P < 0.01). This result is consistent with the lower GI for CM. The EC
values of the PM, SM, and CaM treatments decreased during the early
stage of heap composting, which might be explained by ammonia
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Ecotoxicology and Environmental Safety 247 (2022) 114251
volatilization (Guo et al., 2020). The increased EC after 14 days is
related to the degradation of organic matter, and EC values in the final
composts were all > 4 dS⋅m− 1, which is commonly regarded as
exceeding the limit for safely growing plants (Yuan et al., 2016).
The C/N ratio is an index that reflects available nutrition for mi­
croorganisms and the maturity of compost (Cui et al., 2017). The C/N
ratio trended downward during composting of the four treatments
(Fig. 2d), which could be caused by decomposition and stabilization
during heap composting (Wang et al., 2015a). A significantly higher C/N
ratio was observed in the SM treatment than in the other treatments
(P < 0.01) because of the high carbon content in sheep manure.
3.3. Water-soluble organic carbon and nitrogen substances
Facultative heap composting is not only a process of organic
component degradation, but also a process of HA formation (Xie et al.,
2017). Composting causes organic matter to be degraded into low mo­
lecular weight chemicals such PS, RS, and AA, which can serve as humus
precursors, and the phytotoxicity of compost can be reduced by forming
humus (Guo et al., 2019; Jiang et al., 2021). The concentrations of
water-soluble carbon and nitrogen compounds will be directly impacted
by the decomposition and transformation of organic materials. DOC,
DN, NH+ 4 , VFA, and PA have been shown to be phytotoxic and inhibit
seed germination (Chen et al., 2021a; Gong et al., 2021).
Fig. 3 shows the variation in the concentrations of water-soluble
organic carbon components in the composts. The PS concentration
changed during heap composting, and the PS contents in the CM, PM,
SM, and CaM treatments were 146.75, 150.63, 102.87, and
67.10 mg⋅L− 1, respectively. Increased PS concentrations in CM, PM, and
SM show that PS degradation was more active than PS use in heap
composting (Wu et al., 2017). However, the PS concentration decreased
Y. Kong et al.
Fig. 3. Variation of water-soluble organic carbon components during livestock manure heap composting. (a) Polysaccharide, (b) reducing sugar, (c) phenolic acid,
(d) humic acid, (e) total volatile acids, and (f) dissolved organic carbon (DOC). CM: chicken manure, PM: pig manure, SM: sheep manure, CaM: cattle manure.
in CaM composting because of higher utilization by microorganisms and
the formation of humic substances (HS) (Wu et al., 2017), and 42.85 %
of the PS was transformed into humus. This phenomenon was consistent
with the higher GI of the CaM compost than the other composts. RS, the
important precursor for HS synthesis, is the primary product of amylo­
hydrolysis and lignocellulose hydrolysis (Tan, 2014; Zhou et al., 2022).
The RS content increased from 49.13 to 93.53–317.27–352.97 mg⋅L− 1
in the CM and PM treatments, while the RS only increased slightly from
52.83 to 63.93–102.58–124.19 mg⋅L− 1 in SM and CaM treatments. Ac­
cording to these findings, humification rates were greater in the CM and
PM treatments than in the SM and CaM treatments. The RS concentra­
tion decreased in the early stages of PM and CM heap composting
because they were readily accessible for microorganisms as a carbon
source (Cao et al., 2013). Nevertheless, the RS concentrations of the CM,
PM, and CaM treatments were significantly increased at the end of
composting, which was attributable to the lower C/N ratio, and the use
of nitrogen-containing organic compounds by microorganisms led to the
accumulation of RS (Wu et al., 2017). HA was the main component of
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Ecotoxicology and Environmental Safety 247 (2022) 114251
the HS, which has a complex structure and can absorb phytotoxic sub­
stances (He et al., 2018). The significant difference in the variation of
HA content suggested that humification was associated with the com­
posting of raw materials (Zhu et al., 2021).
PA can inhibit seed germination by affecting enzyme activity in
plants (Yao et al., 2020). According to previous reports, PA in envi­
ronmental media could alter indole-3-acetic acid levels in plant tissues
and prevent the creation of ATP (Zenk and Muller, 1963). At the end of
facultative heap composting, large amounts of PA were accumulated in
the CM and PM treatments (18.89 and 19.81 mg⋅L− 1), with relatively
low amounts accumulated in the SM and CaM treatments (5.35 and
1.86 mg⋅L− 1). The reduced PA content in the SM and CaM treatments
indicated that the PA had been converted into humus and aromatic
compounds. This phenomenon was consistent with the higher GI values
of SM and CaM, and the lower GI values of CM and PM. The VFA present
in compost has been shown to cause phytotoxicity, and is produced by
incomplete degradation of organic matter (Liu et al., 2020b). The initial
total VFA contents were 190.44–1787.31 mg⋅L− 1; higher VFA
Y. Kong et al.
concentrations resulted in higher phytotoxicity to seed germination
(Shou et al., 2017; Wang et al., 2022a). The VFA contents were signif­
icantly different in the different treatments (P < 0.05). CM had the
highest VFA content because it accumulated in the anaerobic environ­
ment. VFA concentrations were significantly lower in the CaM treatment
than in the other treatments (P < 0.01), probably because the higher
porosity of this manure and its existing microorganisms promoted VFA
volatilization and degradation. This result was consistent with CM
having the lowest GI value and CaM having the highest. The DOC con­
centration reflects the stabilization of organic matter stabilization and
the maturity of compost (Wang et al., 2018; Yu et al., 2019). The DOC
concentration of each treatment decreased during facultative heap
composting; this was mainly because of the microbial degradation of
low molecular weight substances such as sugars, lipids, and peptides
(Badhwar et al., 2020; Chen et al., 2021b). The DOC content was
significantly different among the treatments (P < 0.01), and the
decrease in the DOC content was in the order: CaM treatment (56.72 %)
> SM treatment (56.68 %) > CM treatment (32.33 %) > PM treatment
(31.74 %). The CaM treatment had the highest degradation ratio of DOC
(56.72 %), indicating that the most toxicity in the compost had been
reduced.
The changes in AA concentration during livestock manure facultative
heap composting are shown in Fig. 4a. The AA concentrations in all
treatments increased gradually in the first 14 d. This can be attributed to
the degradation of proteinaceous substances by microbial metabolism
(Zhang et al., 2021b). Afterward, the AA concentrations decreased until
day 28 of heap composting. The AAs were mainly used as nutrition for
microbes or as precursors to synthesize HS during heap composting.
NH+4 -N can be produced from the decomposition of nitrogenous matter
during composting, and it has phytotoxic and inhibitory effects on seed
germination (Chang et al., 2019). The NH+ 4 -N concentrations in the raw
materials were 589.80, 214.75, 302.75, and 47.75 mg⋅L− 1 in the CM,
PM, SM, and CaM treatments, respectively (Fig. 4b). The NH+ 4 -N
Fig. 4. Variation of water-soluble nitrogen components during livestock manure composting. (a) Amino acid, (b) NH+
chicken manure, PM: pig manure, SM: sheep manure, CaM: cattle manure.
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Ecotoxicology and Environmental Safety 247 (2022) 114251
concentration generally decreased in PM (from 214.15 mg⋅L− 1 to
31.75 mg⋅L− 1), SM (from 302.75 mg⋅L− 1 to 47.20 mg⋅L− 1), and CaM
(from 47.75 mg⋅L− 1 to 9.22 mg⋅L− 1), which may be caused by ammox­
idation, nitrification, or conversion into NH3 (Zhang et al., 2016a).
During the whole process, the NH+ 4 -N content in the CM treatment
remained relatively high (531.15–589.80 mg⋅L− 1), this could explain
the reason that the CM treatment had the lowest GI value. The NO–3-N
concentration for each treatment was lower than the NH+ 4 -N concen­
tration (Fig. 4c). The NO−3 concentration increased in the CaM treat­
ment, probably because of the increased abundance of nitrite-oxidizing
and nitrifying bacteria during the later stage of composting. The DN
concentration decreased by 30.32–64.13 % in all treatments (Fig. 4d),
which was mainly a result of ammonia loss by volatilization. NH+4 -N was
the primary component of DN, and the GI has been shown to have a
significant negative correlation with NH+ 4 concentration (Wang et al.,
2022a). The DN concentration was significantly higher in the CM
treatment and lower in the CaM treatment (P < 0.01), which negatively
affected seed germination (Luo et al., 2018; Wang et al., 2022a).
3.4. Dynamics of bacterial community structures
Changes in the α-diversity index (the Chao1 and Shannon index) for
all treatments are shown in Fig. 5a and b, which indicated the changes of
richness and diversity of bacterial community (Zhang et al., 2021c). The
Chao1 and Shannon indexes decreased and then increased in the four
treatments, which was probably because of the presence of readily
degradable organic substances serving as nutrients for the rapid prolif­
eration of microorganisms. These phenomena have also been observed
in previous studies of livestock manure composting (Estrella-González
et al., 2020; Ma et al., 2018; Yan et al., 2021). Although the four ma­
nures had similar trends in facultative heap composting process, the
richness and diversity trends were opposite. The CM and PM treatments
had a higher Chao1 index and lower Shannon index, which indicated a
4 , (c) NO3 , and (d) dissolved nitrogen (DN). CM:
−
Y. Kong et al.
Fig. 5. Variations in the bacterial community during heap composting. Alpha diversity indices of bacterial community (indicated by (a) Chao1 and (b) Shannon-
Winner index), (c) principal coordinate analysis (PCoA) of bacterial community, (d) relative abundance (phylum level) of bacteria community, and (e) relative
abundance (genus level) of bacteria community. CM: chicken manure, PM: pig manure, SM: sheep manure, CaM: cattle manure.
higher richness and lower diversity of the bacterial community. These
results were consistent with previous studies showing that the gut
microbiomes of herbivores are more diverse than those of omnivores
(Ley et al., 2008). Temperature, nutrients, and the initial C/N ratio may
all impact the variety and richness of the bacterial community (Wei
et al., 2022).
PCoA using Bray–Curtis distance indicated that the β-diversity of the
bacterial communities clustered into four groups during facultative heap
composting of CM, PM, SM, and CaM; therefore, microbial diversity was
affected by the manure type, not the heap composting time (Fig. 5b).
The contribution of the first principal coordinate explained 44.52 % of
the bacterial community difference among all the composts. Distance
matrixes of the bacterial community structure in the CM and CaM
treatments were similar during heap composting, which implied that the
bacterial community compositions during different composting phases
were similar in the CM and CaM treatments. However, the results of the
PCoA showed that the bacterial community structure in the PM and SM
treatments changed significantly during the different composting pha­
ses; this was mainly caused by temperature changes (Wang et al.,
2015b). The succession in the bacterial community could accelerate
carbon and nitrogen transformation.
Fig. 5c and d show the bacterial community succession at the phylum
and genus levels during facultative heap composting. In this study,
Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the
dominant bacterial phyla for all four treatments, and the total of their
relative abundance was > 90 %. At the beginning of composting, their
abundances fluctuated depending on the different types of livestock
manure. However, the bacterial abundances in the three treatments
other than CM changed significantly. In the PM, SM, and CaM treat­
ments, the relative abundance of Firmicutes decreased from 28.98 % to
94.52 % to 10.76–30.98 % during heap composting, which indicated
that Firmicutes can survive in higher temperature conditions and play
7
Ecotoxicology and Environmental Safety 247 (2022) 114251
an important role in lignocellulosic degradation (Li et al., 2021). Some
studies found that the relative abundance of Firmicutes was higher in the
initial and thermophilic stages and lower in the cooling and mature
stages (Zhang et al., 2016b; Antunes et al., 2016). Actinobacteria (from
0.56 % to 3.77 % to 22.63–30.53 %), Proteobacteria (from 0.56 % to
26.00 % to 17.72–31.75 %), and Bacteroidetes (from 0.38 % to 14.92 %
to 24.85–29.15 %) increased during heap composting. Previous reports
also showed that the relative abundance of Actinobacteria gradually
increased during livestock manure composting (Wan et al., 2021). Spi­
rochaetae were abundant in the initial phase of SM and CaM treatments,
and these phyla play a significant role in cellulolysis and proteolysis
(Song et al., 2014).
Firmicutes was the main phylum throughout the heap composting of
CM (79.57–88.99 %), confirming that Firmicutes can adapt to nutrient-
poor environments (Tian et al., 2013). The dominant genera in CM
belonged to the Firmicutes, including Atopostipes (13.17–18.07 %),
Romboutsia (6.89–15.59 %), Gallicola (11.39–16.64 %), Lactobacillus
(3.30–5.62 %), Tissierella (2.16–3.31 %), W0503 (2.38–3.99 %), Fas­
tidiosipila (1.31–5.03 %), Facklamia (0.30–7.45 %), and Streptococcus
(0.53–0.84 %). Atopostipes, Lactobacillus, and Streptococcus all belong to
the Lactobacillales, which can produce VFA during heap composting
(Tran et al., 2019).
The abundances of Clostridium_sensu_stricto_1 (14.63–31.45 %), Ter­
risporobacter (6.08–17.73 %), Romboutsia (2.21–6.88 %), Turicibacter
(2.38–5.08 %), Atopostipes (0.58–5.09 %), Streptococcus (0.04–7.82 %),
and Lactobacillus (0.12–4.76 %), belonging to the Firmicutes, decreased
during PM heap composting. Streptococcus, Clostridium_sensu_stricto_1,
and Terrisporobacter include pathogenic bacteria and are widely present
in livestock manure (Kumar Awasthi et al., 2019).
Specifically, the dominant genera were within the Actinobacteria
(Pseudomonas, 0.25–4.39 %; Dietzia, 0.61–3.45 %; and Enteractinococcus,
0.60–1.26 %), Proteobacteria (Psychrobacter, 0.06–20.61 %),
Y. Kong et al.
Bacteroidetes (Proteiniphilum, 0.99–10.75 %), and Firmicutes (Proteini­
clasticum, 0.36–2.71 %; Atopostipes, 0.09–3.23 %; and Jeotgalicoccus,
0.05–0.64 %). The acid-producing bacteria Proteiniphilum, Proteiniclas­
ticum, Atopostipes, and Jeotgalicoccus decreased during CaM heap
composting.
In the SM treatment, the actinobacterial genera Corynebacterium_1
(3.70–11.32 %), Nocardiopsis (0–8.41 %), and Enteractinococcus
(0.36–5.30 %) were dominant; these genera are associated with the
biodegradation of organic matter (Xie et al., 2021). Actinobacteria are
usually considered to be an indicator of compost maturity (Xiao et al.,
2011); higher abundance of Actinobacteria at the end of heap com­
posting indicates a more stable compost product. Additionally, Actino­
bacteria play a crucial role in carbon and nitrogen conversion (Ma et al.,
2018), and help with the elimination of pathogenic microbes by
secreting many antibiotics (Franke-Whittle et al., 2009). These findings
indicate that facultative heap composting may reduce the phytotoxicity
of compost products, especially in the SM and CaM treatments.
3.5. Correlations and network analysis
The Pearson correlation heatmap for environmental factors, carbo­
naceous substances, nitrogen substances, and the GI values of different
seeds for all treatments are shown in Fig. S1. The DOC and DN are better
indicators of organic matter transformation than total nitrogen and total
carbon (Gong et al., 2021). A significant negative correlation was found
between DN, DOC, and GI (P < 0.05), demonstrating that the conversion
of dissolved carbon and nitrogen had a major influence on the GI during
facultative heap composting of livestock manure. For all treatments, the
NH+ 4 -N and VFA concentrations were significantly negatively correlated
with the GI values of all four seed types (P < 0.05), indicating that high
concentrations of NH+ 4 -N and VFA inhibited seed germination. This
Fig. 6. Network analysis of key factors and related bacteria at operational taxonomic unit level according to Pearson correlation analysis (P < 0.05) in heap
composting. (a) CM: chicken manure, (b) PM: pig manure, (c) SM: sheep manure, and (d) CaM: cattle manure. The Red line represents a positive correlation and blue
line represents a negative correlation. VFAs: total volatile acids, DOC: dissolved organic carbon, DN: dissolved nitrogen.
8
Ecotoxicology and Environmental Safety 247 (2022) 114251
result was consistent with the findings of a previous study (Yuan et al.,
2016). However, the key factors influencing seed germination in various
animal manures remained unclear; therefore, this study also investi­
gated the relationships between the main dissolved components and
microbial community structures.
The variation in the bacterial community inevitably affects carbon
and nitrogen transformations during composting (Qi et al., 2021).
Co-occurrence network analysis is a powerful tool for identifying core
bacteria involved in the formation of DOC, DN, VFA, and NH+ 4 -N based
on Pearson correlation at the genus level (P < 0.05). As a result, 27, 68,
109, and 45 nodes were identified for the CM, PM, SM, and CaM
treatments, respectively. Similarly, there were 27, 92, 227, and 46
network edges for the CM, PM, SM, and CaM treatments, respectively
(Fig. 6, Tables S2-S6). The correlation of core bacteria with DOC, DN,
VFA, and NH+ 4 -N varied significantly for the four treatments. There were
more nodes and edges for the SM treatment than for the other groups,
and fewer for the CM treatment. These results indicated that more
bacteria were stimulated to participate in DOC, DN, VFA, and NH+ 4 -N
conversion during SM heap composting. The PM treatment also had a
similar result to that of the SM treatment. Notably, nodes related to
bacteria with NH+ 4 -N and DN in PM and SM were higher than in the other
groups (CM: 18; PM: 33 and 35; SM: 22 and 69; CaM: 10 and 9). NH+ 4 -N
components and bacteria (Actinobacteria, Proteobacteria, and Bacter­
oidetes) were negatively correlated in the PM and SM treatments. These
results were consistent with the higher degradation rate of nitrogen
concentrations by bacterial activity in the PM and SM treatments. In SM
composting, the top 30 genera included those involved in the conversion
of NH+ 4 -N, including Atopostipes, Lactobacillus, Nocardiopsis, Pussillimo­
nas, Marinimicrobium, Psychrobacter, Galicola, and Halomonas. However,
the core genera involved in NH+ 4 -N and DN conversion during SM heap
composting (except Lactobacillus and Romboutsia) were not among the
Y. Kong et al.
top 30 genera. Considering the conversion of NH+ 4 -N, the top 30 genera
might have lower working efficiency, while the core genera with low
relative abundance in SM heap composting might be more efficient
because of interspecies competition (Gravuer et al., 2020). According to
the bacterial composition in the networks, the relationships between
core bacteria and carbon and nitrogen conversion were similar in the
four treatments. A positive link between VFA and Firmicutes was found
in all treatments; therefore, the change in VFA content was consistent
with the change in the abundance of Firmicutes. The lower GI for the CM
treatment could be explained by the higher abundance of Firmicutes
(Lactobacillus and Streptococcus) during facultative heap composting,
and the lower abundance of Firmicutes (Atopostipes and Jeotgalicoccus)
explained the higher GI in CaM facultative composting. Atopostipes and
Jeotgalicoccus are anaerobes or facultative anaerobes, which metabolize
glucose to produce small organic acids (Watanabe et al., 2008). Wang
et al. (2022c) also determined the main bacteria related to the GI and
humus content in composting piles, and lactic acid secreted by Lacto­
bacillus could inhibit seed germination.
3.6. Key factors affecting seed germination
SEMs were employed to reveal the possible relationship among the
key factors, bacterial communities, and GI values during facultative
heap composting. The key factors were selected according to Pearson
correlation analysis (Fig. 7). In the CM treatment (Fig. 7a), VFA was
positively related to DOC and the bacterial community. The bacterial
community structure and diversity changed with carbon transformation
because DOC could be used as a source of energy and nutrients for
bacteria. In CM, the GI was negatively affected by VFA and the bacterial
community diversity. The lower GI of CM could be explained by the
higher content of VFA and Firmicutes, especially Lactobacillus and
Streptococcus. For the PM treatment (Fig. 7b), NH+ 4 -N had a correlation
relationship with DN, and DN negatively affected the GI. GI was also
related to the bacterial community, and the community diversity could
be inhibited by DN. As a result, the NH+
4 -N had a negative impact on the
GI both directly and indirectly.
Fig. 7. Structural equation model (SEM) showing effects of key factors on seed germination indexes during heap composting. (a) CM: chicken manure, (b) PM: pig
manure, (c) SM: sheep manure, (d) CaM: cattle manure. Path coefficients are adjacent to arrows. Red and blue lines indicate positive and negative pathways,
respectively. Dotted lines indicate insignificant relationships. Asterisks indicate significant (*, P < 0.05), highly significant (**, P < 0.01), and very highly significant
(***, P < 0.001) relationships.
9
Ecotoxicology and Environmental Safety 247 (2022) 114251
In the SM treatment (Fig. 7c), the NH+ 4 -N showed a negative asso­
ciation with the GI and a positive association with DN and the bacterial
community, and the GI was negatively related to DN and the bacterial
community. The result was consistent with interspecies competition of
bacteria. In the CaM treatment (Fig. 7d), VFA was the main factor
affecting DOC. High DOC inhibited the bacterial community and was
positively associated with GI. The bacterial community also showed a
significant impact on the GI. The increased GI was related to the
degradation of organic matter and the higher diversity of the bacterial
community. Overall, analyses of all the parameters of different livestock
manures related to phytotoxicity using the SEMs showed that VFAs were
the main factor affecting the GI in the CM and CaM treatments. In the PM
and SM treatments, NH+ 4 -N affected the GI directly and/or indirectly.
4. Conclusions
In this study, seed GI was used to characterize the phytotoxicity of
livestock manure during facultative heap composting. The phytotoxicity
of different livestock manure was significantly different, and the order of
phytotoxicity with the type of manure was: chicken manure > pig
manure > sheep manure > cattle manure. VFA and NH+ 4 -N were the key
factors affecting GI. In CM and CaM heap composting, the GI was most
affected by VFA, while NH+ 4 -N was the main factor affecting GI in PM
and SM. Network analysis showed that VFA was negatively correlated
with Firmicutes abundance, and NH+ 4 -N was mostly related to the
Actinobacteria, Proteobacteria, and Bacteroidetes. The dissolved carbon
and nitrogen content and composition, as well as the core and pro­
prietary microbial communities, were the primary factors affecting the
succession of phytotoxic substances in facultative heap composting,
which in turn affected GI values. This study provides new perspectives
on the precise regulation of compost maturity and the safe utilization of
farmland in facultative heap composting.
CRediT authorship contribution statement
Yilin Kong: Performed the research, proposed the methodology,
Y. Kong et al.
analyzed data, and wrote the paper. Guoying Wang: Performed the
research, proposed the methodology, analyzed data, and wrote the
paper. Wenjie Chen: Data curation and analysis. Yan Yang: Data
curation. Ruonan Ma: Data curation. Danyang Li: Data collection and
analysis. Yujun Shen: Writing− review & editing. Guoxue Li:
Writing− review& editing. Jing Yuan: Conceptualization, Methodology,
Software, Supervision, Software, Validation, Writing− review & editing.
All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This research was supported by the National Natural Science Foun­
dation of China (42207380), the Major Science and Technology plan of
Hainan Province in China (ZDKJ2021009), and the Natural Science
Foundation of Shandong Province in China (ZR2021QE019).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2022.114251.
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