Ecotoxicology and Environmental Safety 209 (2021) 111804

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Ecotoxicology and Environmental Safety

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

Effects of C/N ratio variation in swine biogas slurry on soil dissolved

organic matter: Content and fluorescence characteristics
Lilong Yan *, Cong Liu , Yudan Zhang , Shuang Liu , Ying Zhang *
School of Resource and Environment, Northeast Agricultural University, Harbin 150030, China

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

Edited by Dr Hyo-Bang Moon Nutrient-rich biogas slurry shows favorable prospects for application as an organic fertilizer in farmland. At the
same time, due to differing sources and treatment methods, the C/N ratio of biogas slurry varies greatly. The
Keywords: effect of differences in C/N of biogas slurry on soil organic matter properties remains unclear. In this experiment,
Biogas slurry pig farm biogas slurry differing in C/N (3, 6, 8.84 and 12) was applied instead of fertilizer. Fluorescence spec­
C/N
troscopy combined with parallel factor analysis and principal component analysis were used to determine dis­
DOM
solved organic carbon (DOC) and fluorescent dissolved organic matter (FDOM) in soil dissolved organic matter
Fluorescence spectra
Humification (DOM). The experimental results showed that the DOC and FDOM contents of soil could be significantly
increased at the initial stage of biogas slurry application. Compared with CK, on the 60th day, biogas slurry with
a C/N of 12 exhibited the greatest improvement in DOC, FDOM as well as for Component 1, Component 2 and
Component 3 contents in soil FDOM, 40.93%, 66.25%, 65.35%, 40.47%, and 78.42% respectively. However,
compared with the 0th day, by the 60th day, biogas slurry with a C/N of 8.84 exhibited the greatest decrease in
Component 4 content in soil FDOM, 74.68%. Biogas slurry with a C/N of 8.84 exerted the greatest promotion
effect on corn growth, the utilization and transformation of Component 4 by plants and microorganisms in the
soil. And it showed the strongest improvement in the degree of FDOM humification in the soil, the humification
index increased from 4.16 ± 0.17 to 4.92 ± 0.58, compared with CK. This study provided new insights for the
utilization of biogas slurry with respect to soil physical and chemical properties and maize plant growth.

1. Introduction
As one of China’s leading agricultural industries, the output value of
pig breeding in China is as high as 1.3 trillion yuan, approximately
56.6% of the total output value of livestock and poultry breeding in
China. The wastewater produced from pig breeding contains a high
concentration of organic matter (5000–30,000 mg/L) (Lu et al., 2020),
and is generally treated with an environmentally friendly anaerobic
digestion system to recover energy and materials and produce biogas
slurry (Chen and Liu, 2017). Biogas slurry, which contains a variety of
amino acids, such as leucine, glutamic acid, lysine and so on, is often
used to produce liquid fertilizer (Yang et al., 2019b). At the same time, it
is often used as a popular green agricultural fertilizer because its rich
nutrient content can improve soil quality and increase crop yield (Yan
et al., 2019). Biogas slurry reused in farmland not only can improve the
activity of enzymes influencing soil nitrogen and phosphorus contents
and enrich the soil for various nutrients but can also treat a large
quantity of agricultural wastewater to protect the environment (Chen
et al., 2015a). It can also reduce the pollution of the ecological envi­
ronment caused by fertilizer application and reduce the cost of agri­
cultural production (Chaoui et al., 2003).
Declining soil organic matter (SOM) leads to the deterioration of soil
structure and reduces soil productivity (Yilmaz et al., 2019). Biogas
slurry contains not only nutrients such as nitrogen and phosphorus but
also much organic matter. Biogas slurry reused in farmland can increase
the content of organic matter in soil, especially dissolved organic matter
(DOM), thus improving the soil structure (Yan et al., 2019). Therefore, it
is feasible to use biogas slurry in agricultural production to reduce
environmental degradation (Du et al., 2019). However, there are many
types of antibiotic residues and heavy metals in biogas slurry. When a
single application of biogas slurry is too large or when biogas slurry is
used in large quantities for a long time, potential risks include leaching
of nutrients such as nitrogen and phosphorus from soil, soil hardening
and accumulation of antibiotics and heavy metals is applied (Niyungeko
et al., 2019). Therefore, rational application is the premise of biogas
slurry application in farmland.

* Corresponding authors.
E-mail addresses: yanll98@163.com (L. Yan), zhangying_neau@163.com (Y. Zhang).

https://doi.org/10.1016/j.ecoenv.2020.111804
Received 26 September 2020; Received in revised form 4 December 2020; Accepted 10 December 2020
Available online 22 December 2020
0147-6513/© 2020 The Author(s). 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/).
L. Yan et al.
In the process of anaerobic digestion, organic matter containing
carbon can be degraded and transformed to be removed, but the nitro­
gen removal ability of anaerobic digestion is limited, so that the biogas
slurry generated has a C/N imbalance (low carbon content and high
ammonia nitrogen content) (Roger, 2014). In addition, excessive biogas
slurry can exceed the carrying capacity of natural ecosystems and cause
secondary pollution of the environment (Tampio et al., 2016). There­
fore, secondary treatment of biogas slurry is needed to reduce the
environmental pollution of nitrogen and phosphorus, and a lot of re­
searches have been conducted on this aspect (Lin et al., 2017; Qu et al.,
2020). Magnesium ammonium phosphate (MAP) is often used for a
cost-effective, high-yield and easy-to-operate chemical method for sec­
ondary treatment of biogas slurry. It can remove nitrogen and phos­
phorus from biogas slurry while producing a concentrated and
marketable slow-release fertilizer (Lin et al., 2017). This method can
recover more than 80–95% nitrogen and phosphorus in biogas slurry
(Taddeo et al., 2016), but it also further changes the C/N of biogas
slurry. Previous results have shown that enhancing the C/N of biogas
slurry can prevent N leaching, thus increasing N fixation and soil deni­
trification (Cheng et al., 2017). Moreover, with the increase of C/N, soil
microbial communities will have higher diversity (Cheng et al., 2017)
and increase soil CO2 emissions while reducing soil N2O and CH4
emissions (Cheng et al., 2020). In recent years, a lot of researches have
carried out on the biological treatment of wastewater under different
C/N conditions (Sepehri and Sarrafzadeh, 2018, 2019; Sepehri et al.,
2020), but there have been few investigations of the influence of biogas
slurry with different C/N on DOM. Studying the influence of differences
in C/N of biogas slurry on soil DOM and exploring the changes in
components content and structure of soil DOM with biogas slurry
differing in C/N are helpful to fully understand the influence of biogas
slurry application on soil ecosystems, and provide certain theoretical
support for the migration and transformation of organic pollutants and
trace metals in soil after biogas slurry is applied.
The portion of organic matter that can filter through a 0.45 μm filter
membrane is called DOM. It is mainly composed of hydrophilic and
hydrophobic components and is the most mobile and active form of SOM
(Hu et al., 2016). DOM is considered to be the most active component of
SOM in agricultural ecosystems and plays an important role in the global
carbon cycle, pollutant and nutrient transport, and agricultural envi­
ronments (Bodmer et al., 2016). The types and contents of hydrophilic
components, as well as the types and quantities of functional groups,
affect the interactions between pollutants and DOM, causing pollutants
in soil to show different changes and characteristics (Yuan et al., 2016).
Carbon accounts for approximately 65% of DOM (DOM is usually rep­
resented by dissolved organic carbon (DOC) (Qualls et al., 2013)). At the
same time, due to the molecular complexity of each component in DOM,
there is limited information regarding protein-like and humus-like
substances in DOM. However, due to the molecular complexity of
each component in DOM, DOC has limited utility as an indicator of
protein-like and humic acid-like compounds in DOM. In view of the
large quantity of fluorescent substances in DOM, fluorescent dissolved
organic matter (FDOM) can be used to characterize protein-like and
humic acid-like compounds and distinguish them based on differences in
energy intensity released and absorbed by different substances at each
fluorescence wavelength (Rodriguez-Avella et al., 2020). The fluores­
cence signal strength of FDOM is positively correlated with DOM con­
centration (Cory et al., 2011); measurement of FDOM is another method
for characterizing DOM.
At present, as a fast and sensitive analytic method with small-sample
capacity, three-dimensional excitation-emission matrix (3D-EEM)
spectroscopy, is widely used to understand the fluorescence character­
istics of DOM components with different molecular weights, which is a
valuable technique for examining DOM (Wei et al., 2016). 3D-EEM and
synchronous fluorescence technique can be used to rapidly and sensi­
tively determine the fluorescent substances contained in DOM (Chen
et al., 2015b). Because DOM is a mixture, so that many fluorescence
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Ecotoxicology and Environmental Safety 209 (2021) 111804
peaks from different components move and overlap with each other,
spectral signals are difficult to recognize. Therefore, 3D-EEM alone
cannot accurately reflect the real information in DOM (Guo et al., 2019).
Parallel factor analysis (PARAFAC), which can decompose EEMs into a
single fluorescent component, features the functions of spectral correc­
tion, calibration, scattering elimination, identification and removal of
outliers, and model validation (Lu et al., 2019). In addition, it can
effectively identify and distinguish foreign or local FDOM components
in the environment (Henderson et al., 2009). As a fast in situ tool to
characterize DOM in soil, compost and fresh water, 3D-EEM combined
with PARAFAC can solve the problem of superposed peaks (Guo et al.,
2020). It can also quantitatively and qualitatively characterize the
fluorescent components in DOM, distinguish potential DOM sources,
and permit estimation of the relative concentrations of different organic
components. Principal component analysis (PCA) can use statistical
programs to reduce the number of variables, while retaining most of the
relevant information (Hu et al., 2017; Rodriguez-Avella et al., 2020; Yan
et al., 2019).
In view of these experiments, a pot experiment was designed in this
study. Biogas slurry was adjusted to different C/N ratios (3, 6, 8.84 and
12) by a chemical precipitation method and then added to soil in pots.
Fluorescence spectroscopy combined with PARAFAC and PAC was used
to determine DOC, FDOM and various components of soil DOM. The
research objectives of this article were to: (1) Clarify the influence of
applications of biogas slurry differing in C/N on soil DOM content and
fluorescence characteristics; (2) Clarify changes in DOM content and
composition during crop growth; and (3) Optimize the C/N of biogas
slurry from the perspective of influencing soil DOM properties and crop
growth, to provide reference for practical application of biogas slurry.
These results provided important theoretical support for exploring the
conditions for optimal application of biogas slurry.
2. Material and methods
2.1. Experimental design
The biogas slurry used in this experiment was obtained from a pig
farm in Heilongjiang Province. The organic matter content in biogas
slurry was 16.97 mg/mL, the total nitrogen and total phosphorus con­
tents were 1.92 mg/mL and 51.04 mg/L, respectively. The soil used was
black soil from Xiangyang Farm of Northeast Agricultural University.
The nitrate nitrogen, ammonium nitrogen and total phosphorus contents
of the black soil were 6.65, 32.35 mg/kg and 19.06 g/kg, respectively;
pH was 5. 81. 5 kg of black soil was put into each pot (24 cm in height
and 24 cm in diameter at the bottom) for a pot experiment. Six condi­
tions (CK, BS3, BS6, BS, BSNP and BS12) were set up in the experiment,
and each was repeated three times. In the experiment, a chemical pre­
cipitation method and exogenous ammonium chloride were used to
adjust the C/N of biogas slurry to avoid influencing organic components
of biogas slurry to the extent possible, and phosphate was added as
needed. No biogas slurry or chemical fertilizer was added to CK pots; BS
and BSNP were treated with untreated biogas slurry with C/N 8.84; and
biogas slurry with C/N of 3, 6 and 12 was applied to BS3, BS6 and BS12,
respectively. The total amount of nitrogen and phosphorus applied in
each treatment was the same, except for in CK. The amount of biogas
slurry applied in each treatment was converted according to the amount
of fertilizer applied by local residents (180 kg N/hm2), and the volume
of biogas slurry applied in each treatment was guaranteed to be the same
(an insufficient amount of biogas slurry was supplemented with distilled
water). At the beginning of the experiment, in order to investigate the
effect of soil microorganisms on the consumption of organic matter in
biogas slurry, corn seed was not planted in the BSNP treatment, and
three corn seeds were planted in each pot under other conditions. The
seedlings were thinned after the seedlings emerged. At last, one seedling
in good growth condition was kept in each pot.
L. Yan et al. Ecotoxicology and Environmental Safety 209 (2021) 111804

2.2. DOM extraction of soil samples Table 1

Plant height of each treatment on days 30, 45 and 60.
During the experiment, soil samples were collected after the 0th, 30 d 45 d 60 d
15th, 30th, 45th and 60th days respectively. Each soil sample was air-
ck 47.75 ± 6.01 62.25 ± 1.06 70.00 ± 1.41
dried, ground and added to 0.01 M CaCl2 solution (soil-water ratio: BS3 55.17 ± 6.90 77.10 ± 5.24 90.00 ± 7.00
1:10, W/V) prepared with ultra-pure water. Each sample solution was BS6 59.25 ± 6.72 83.15 ± 7.99 97.50 ± 6.36
extracted at a shock rate of 125 rpm/min for 24 h in darkness at 25 ◦ C BS 65.50 ± 3.53 90.25 ± 3.89 102.00 ± 4.24
and then centrifuged at 5000 rpm/min for 10 min. Finally, the super­ BS12 53.27 ± 3.85 73.87 ± 6.88 84.67 ± 8.38

natant of each obtained solution was filtered through a 0.45 μm filter

membrane to produce a soil DOM solution (Fan et al., 2019). A TOC
analyzer (Multi N/C 2100S, Germany) was used to determine the DOC
concentration of each sample.

2.3. Fluorescence spectral determination

After adjusting DOM concentrations (DOC = 5 mg/L, pH = 7.42 ±

0.05), samples were subjected to an F-7000 fluorescence photometer (F-
7000; Hitachi, Japan) measured 3D excitation-emission matrix inter­
ference (Yan et al., 2019). The fluorescence intensity of each undiluted
sample was obtained by multiplying EEMs with the dilution factor
(Shatrughan et al., 2013).
Each sample solution after adjusting DOM concentration was scan­
ned for synchronous fluorescence spectroscopy with an F-7000 fluo­
rescence spectrophotometer in a 1 cm quartz pond (Guo et al., 2013).

2.4. PARAFAC analysis

MATLAB 2014B and a free downloaded toolbox (DOMFluor) (www.

models.life.ku.dk) were used to conduct PARAFAC analysis of EEM
spectral data following Bro and Stedmon tutorials. The identified com­
ponents were verified by visual inspection, split analysis and residual
analysis, and the separated components were determined according to
the locations and shapes of the peaks (Song et al., 2017). In this study,
the maximum peak intensity (Fmax) in Raman units (R.U., nm-1) was
used to estimate the relative concentrations of components.

2.5. PCA

PCA was used to identify principal component (PCs) that could be

interpreted as type spectra and then identify compounds contained in
fluorescence spectra (Barker et al., 2010). In this study, SPSS 26 was
used for PCA analysis. Bartlett and KMO tests were used to evaluate the
partial correlations and dependence of the data, to eliminate the possible
influence of nonindependence of spectral data on PCA results.

2.6. Fn(355), fluorescence index (FI), and humification index (HIX)
analysis
Fluorescence index (FI) is the ratio of fluorescence intensity at Em =
450 nm to that at 500 nm, when Ex = 370 nm (Sepp et al., 2019). FI
value can reflect the origin of soil DOM. FI < 1.4 indicates that DOM is
mainly derived from land (the heterogenicity is obvious), and the
contribution of microbial activities to DOM is relatively low; FI > 1.5
signifies that DOM is dominated by endogenous input (the autogenous
source has obvious characteristics), and microbial activity is the main
source of DOM (Sepp et al., 2019). Fn(355) was used to represent the
relative concentration of FDOM, based on fluorescence intensities at Ex
= 355 nm and Em = 450 nm. When Ex = 254 nm, the ratio of the Em
∫
integral value in the region of 435–480 nm ( 435–480) to the Em in­
∫
tegral value in the region of 300–345 nm ( 300–345) was taken as the
humification index (HIX), which was used to characterize the degree of
DOM humification (Ohno et al., 2007).
Fig. 1. (a) DOC content on days 0, 15, 30, 45 and 60; significantly different
rates (p < 0.05) are indicated by the lowercase letters; (b) Correlation analysis
of each indicator (* significant correlation found at the 0.05 level (bilateral), **
significant correlation found at the 0.01 level (bilateral)).
3. Results and discussion
3.1. Effects of biogas slurry with different C/N on plant height of corn
As shown in Table 1, in the planting stage of corn, plant height was
higher in each biogas slurry treatment than in CK. As corn plants grew,
the gap in plant height between CK and biogas slurry treatments became
larger. The application of biogas slurry was able to promote the growth
of corn. This result is consistent with findings of the literature review by
Ghimire et al. (2015). On the 60th day, corn plant height first increased
and then decreased with increasing biogas slurry C/N, with BS yielding
the tallest corn plants and the most favorable corn growth. Therefore, it

3
L. Yan et al. Ecotoxicology and Environmental Safety 209 (2021) 111804

Fig. 2. (a) Fluorescence spectra of dilute biogas slurry and (b) soil DOM in different treatments.

4
L. Yan et al.
indicated that biogas slurry with a C/N of 8.84 exerted the strongest
promoting effect on corn growth.
3.2. Effects of biogas slurry with different C/N on soil DOC content
Compared with CK, on the 0th day, DOC content in each treatment
soil increased significantly, showing a gradually increasing trend with
increasing C/N in biogas slurry (Fig. 1a). DOC content in soil was
significantly positively correlated with biogas slurry C/N (p < 0.01)
(Fig. 1b). DOC content of BS, BSNP and BS12 increased significantly, by
163.93%, 164.54% and 165.56% compared with CK, respectively. With
the growth of maize plants, DOC content in soil samples of BS6, BS and
BS12 showed a gradually declining trend. The content in soil samples of
BSNP decreased in the early stage and gradually stabilized in the later
stage, perhaps due to the aging phenomenon of the external carbon
source (Liu et al., 2019a; Speratti et al., 2018).
At the same time, with differing C/N, DOC content in each treatment
varied greatly with time. Soil DOC content in CK and BS3 showed a trend
of first increasing and then decreasing with the extension of experi­
mental time (Fig. 1a). The content of soil DOC is related to the organic
quality of soil degraded by microorganisms and the growth of corn. In
the early stage of corn growth, the increase in soil DOC content in CK
resulted from the decomposition of SOM by microorganisms, which
increased DOC content in the soil (Shan et al., 2008). With the growth of
corn plants, corn consumption of DOC increased, exceeding the micro­
bial increase in DOC. As a result, soil DOC content in CK showed a
downward trend. Organic matter in biogas slurry partially exists in
dissolved state, and irrigation of biogas slurry can significantly increase
soil DOC content. Meanwhile, compared with soil DOC, DOC in biogas
slurry was more easily used by microorganisms, consistent with findings
of previous research (Qin et al., 2017). When the amount of organic
matter added externally was limited (C/N = 3), the amount of organic
matter consumed by microorganisms was lower than the amount of DOC
produced by microorganisms. As a result, DOC content in soil increased
in a short time. At the 30th and 45th days of the experiment, the soil
DOC content of each treatment showed a trend of first increasing, then
decreasing and then increasing with increasing C/N of biogas slurry.
During the growth of corn seedlings, the fulvic acids and hydrophilic
compounds with low molecular weight in the soil were transferred to the
plants as nutrients and thus utilized by the plants, resulting in lower soil
DOC content (Ren et al., 2015).
Compared with the 0th day, by the 60th day the soil DOC content of
each treatment had declined by 9.23%, 23.48%, 56.75%, 59.37% and
51.83% (Fig. 1a), respectively. Soil DOC content in BS showed the most
significant decrease with a decrement 15.28% greater than that for
BSNP, which was much higher than the decrement in CK. This may be
because the biogas slurry with C/N of 8.84 had the strongest promoting
effect on corn growth, so that corn consumed more DOC. These results
demonstrated that biogas slurry with a C/N value greater than 3 was
conducive to promoting the utilization of DOC in soil by corn. By the
60th day, the soil DOC contents in BS3, BS6, BS, BSNP and BS12 had
increased by 20.20%, 15.88%, 18.14%, 52.09% and 40.93%, respec­
tively (Fig. 1a). Compared with CK, BSNP exhibited the most significant
increase in soil DOC content indicating that biogas slurry can increase
soil DOC content. Soil DOC plays an important role in biogeochemical
processes of soil C, N, P and S. It is a matrix of microorganisms, affects
redox-related activity in soil, forms a major pathway for carbon transfer
from terrestrial ecosystems to aquatic ecosystems, and has a beneficial
effect on carbon balance of terrestrial ecosystems (He et al., 2020). As a
reactive intermediate and highly dynamic soil organic carbon pool, DOC
can provide and stabilize carbon in deep soil and exert a beneficial
impact on the migration and biological utilization of nutrients and
pollutants (such as nitrogen, phosphorus, sulfur, organic pollutants and
trace metals) (Luo et al., 2019). DOC is also able to provide the necessary
energy for microbial activity, thus reactivating the decomposition of
deep organic carbon (Sébastien et al., 2007). Therefore, biogas slurry of
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Ecotoxicology and Environmental Safety 209 (2021) 111804
different C/N can indirectly affect the above functions and processes by
changing soil DOC.
3.3. Effects of biogas slurry with different C/N on three-dimensional
fluorescence characteristics of soil DOM
Through the analysis of the fluorescence spectra of the samples
(Fig. 2), five types of fluorescence peaks were found for soil DOM of each
treatment, tyrosine-like (zone I, B peak), tryptophan-like (zone II, T
peak), fulvic acid (zone III, Peak A), soluble microbial byproducts (zone
IV, peak D), and humic acid (zone V, peak C) (Xi et al., 2017). The B peak
represents tyrosine-like, and the T peak represents tryptophan-like, both
of which are related to aromatic cyclic amino acids in proteins and
belong to protein-like fluorescence peaks. Peak A represents fulvic acid,
and peak C represents humic acid, both of which are produced by humic
substances with complex molecular structure and belong to the humic
fluorescence peak. The D peak can reflect soluble microbial byproducts
in soil DOM. Among the 5 peaks, peak B, peak D and peak T are closely
related to the life activities of microorganisms, including both micro­
organisms in the soil itself and microorganisms carried by exogenous
sources (such as biogas slurry) (Wu et al., 2015). To better understand
the influence of biogas slurry on the fluorescence characteristics of soil
DOM, the fluorescence characteristics of biogas slurry were analyzed
(Fig. 2a). The results showed that tyrosine-like, tryptophan-like, fulvic
acid, humic acid and soluble microbial byproducts accounted for
30.70%, 30.37%, 16.62%, 14.78% and 7.53% of the total fluorescent
substances in biogas slurry, respectively. The fluorescent components in
biogas slurry were mainly protein substances (such as tryptophan and
tyrosine), followed by humus. Humus in biogas slurry plays a dynamic
role in plant growth, plant nutrition and the improvement of soil fertility
(Selim and Mosa, 2012). In the early stage of the experiment, compared
with the control group, the fluorescence intensity of each treatment with
biogas slurry increased significantly, among which the fluorescence in­
tensities of zone I and zone II (protein substances) increased more
(Fig. 2b). Biogas slurry with C/N of 6, 8.84 and 12 can significantly
improve the fluorescence intensity of each component in soil DOM. With
the growth of corn, the fluorescence intensity of protein substances in
CK samples showed a trend of first decreasing and then increasing, and
the overall trend was upward, while the fluorescence intensities in zone
III and zone V (humic substances) showed a trend of first increasing and
then decreasing (Fig. 2b). The fluorescence intensity of each component
in each treatment treated with biogas slurry showed a downward trend,
which indicated that fluorescent substances in biogas slurry were easily
decomposed and utilized.
Compared with the 0th day, on the 60th day, the fluorescence in­
tensity of all components in CK had increased, and the fluorescence
intensity of protein substances were significantly higher (Fig. 2b).
Therefore, it demonstrated that planting corn would increase the con­
tent of fluorescent components (especially protein substances) in soil
DOM within a period of time. This result was consistent with the results
of Liu et al. (2019b). The proportion of proteins in each treatment
treated with biogas slurry was significantly reduced in the soil DOM,
while the proportion of humic substances was elevated. Therefore, it
demonstrated that biogas slurry was able to transform easily decom­
posed and biodegradable proteins in soil DOM into humic substances,
consistent with the experimental results of Yan et al. (2019). Compared
with CK, on the 60th day, the fluorescence intensities of peak A and peak
C in each biogas slurry treatment were higher, while values for peaks B
and T in each biogas slurry treatment were lower (Fig. 2b). The fluo­
rescence intensities of peaks A and C in BS12 were highest, followed by
BSNP. The fluorescence intensities of peaks B and T decreased the most
in BSNP, followed by BS. The fluorescence intensities of peak A and peak
C in BSNP were higher than that of BS, while values for peak B and peak
T in BSNP were lower than that of BS. It indicated that planting corn
reduced the degree of protein-to-humus conversion over time. The
content and composition of soil DOM can affect soil microbial activity
L. Yan et al.
Fig. 3. Total fluorescence intensity on days 0, 15, 30, 45 and 60; significantly
different rates (p < 0.05) are indicated by the lowercase letters.
and trace metal immobilization (Yang et al., 2019c). Increasing the
amount of humus in the soil can also stabilize soil pH, improve soil
structure, reduce leaching of fertilizers, and promote plant growth, and
increase plant drought resistance and nutrient uptake (Suh et al., 2015).
Biogas slurry with different C/N ratios can have a beneficial impact on
soil by influencing DOM content and structure.
3.4. Effects of biogas slurry with different C/N on the total fluorescence
values in soil DOM
The results obtained for organic compounds by fluorescence region
integration (FRI) (Fig. 3) can reflect the relative concentration of FDOM
to a certain extent, to quantitatively reveal the changes in FDOM for
each treatment (Xi et al., 2017). Compared with CK, on the 0th day, the
total fluorescence response value of DOM in all treatments treated with
biogas slurry was significantly higher, consistent with the results of Guo
et al. (2020). The total fluorescence response value of DOM was
significantly positively correlated with biogas slurry C/N at alpha levels
of 0.01 and 0.05 (Fig. 1b), respectively. It can be seen from CK that
during the growth process of corn, the concentration of FDOM in the soil
first increased and then decreased as the corn grew (Fig. 3). At the end of
the experiment, soil FDOM content had increased by 19.74% compared
with the initial value (the 0th day), because plant root exudates were
able to change soil DOM concentration during corn growth (Wang et al.,
2020). The FDOM of each biogas slurry treatment decreased gradually
as the corn grew. Therefore, it indicated that within a period of time
after biogas slurry was applied, the FDOM in the soil was gradually
transformed and utilized by the microorganisms in the soil, and the
FDOM entering the soil with biogas slurry had higher bioavailability.
This is consistent with the findings of Singh et al. (2014). Compared with
the 0th day, by the 60th day, the total fluorescence response value of CK
had increased, while the total fluorescence response value of the other
treatments had declined by 48.32%, 50.66%, 51.02%, 52.83% and
48.16%, respectively (Fig. 3). The biogas slurry with a C/N of 8.84
exhibited the highest promotion effect on soil FDOM conversion and
utilization. Fluorescence response value of CK increased at the 60th day,
indicating that planting corn was able to increase FDOM concentration
in soil within a certain period of time. Meanwhile, the total fluorescence
response value of BSNP decreased the most and was higher than that of
BS, which further indicated that planting corn could increase FDOM
concentration in the soil within a certain period of time.
Further analysis found that the difference between the degree of
reduction in total fluorescence response values of BS and BSNP was far
less than the increase in total fluorescence response values of CK, which
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Ecotoxicology and Environmental Safety 209 (2021) 111804
Table 2
FI, HIX and Fn (335) statistics on day 60.
FI HIX Fn (355)
ck 1.66 ± 0.06 4.16 ± 0.17 365.27 ± 31.68
BS3 1.79 ± 0.02 4.20 ± 0.12 391.33 ± 61.20
BS6 1.76 ± 0.02 4.55 ± 0.52 457.77 ± 0.28
BS 1.86 ± 0.05 4.92 ± 0.58 486.82 ± 6.72
BSNP 1.81 ± 0.03 5.55 ± 0.89 460.62 ± 18.45
BS12 1.78 ± 0.09 4.55 ± 0.06 516.67 ± 35.21
indicated to be caused by the application of biogas slurry to improve the
bioavailability of soil FDOM (Fig. 3). Increasing soil DOM content is
conducive to the improvement of soil structure, increasing soil fertility
and promoting plant growth (Chen et al., 2014). Compared with CK, by
the end of the experiment, the total fluorescence response value of each
biogas slurry treatment was higher by 5.41%, 27.85%, 2.09%, 40.48%
and 66.25%, respectively. Application of biogas slurry can improve the
concentration of FDOM in soil, thus playing a role in soil improvement.
Biogas slurry with C/N of 12 showed the clearest soil improvement,
followed by biogas slurry with C/N of 8.84.
3.5. Effects of biogas slurry on FI, HIX, and Fn(355) of soil FDOM
Biogas slurry of different C/N and plants had different effects on FI,
HIX and Fn(355) of soil DOM (Table 2). The FI value of soil DOM in each
treatment exceeded 1.6, indicating that DOM in the soil was mainly
derived from microbial activity in the soil (Sepp et al., 2019). Compared
with CK, the FI index of each biogas slurry treatment was significantly
higher. The FI index of BS improved most significantly. Therefore, it
indicated that the addition of biogas slurry promoted the microbial ac­
tivity associated with DOM in the soil, and the biogas slurry with a C/N
of 8.84 had the greatest promotion effect. The FI index for BS was higher
than that of BSNP. Thus, it can be concluded that corn planting also
promoted the microbial activity associated with DOM in the soil, which
also provided an explanation for the change in soil FDOM concentration
in CK. Biogas slurry was able to increase the HIX index of soil DOM; that
is, biogas slurry could improve the degree of humification of DOM in
soil, thus making the structure of soil DOM more complex (Ohno et al.,
2007). By comparing the HIX index of each treatment, the HIX index of
BSNP was found to be highest, followed by that of BS. Therefore, it
demonstrated that biogas slurry with a C/N of 8.84 showed clearest
improvement in the degree of DOM humification in the soil. Although
biogas slurry with C/N of 12 exhibited the greatest increases in the
contents of DOC and FDOM in soil, its promotion of plant growth and
soil humus were weaker than that of biogas slurry with C/N of 8.84. It is
also likely to produce a large amount of greenhouse gases (El Hassani
et al., 2020; Zhao et al., 2017). Planting corn for a period of time could
reduce the degree of humification of corn by biogas slurry, which may be
caused by the absorption of some organic matter by the growth of corn.
Soil mineralization and humification can reduce the content of soil DOC,
explaining the experimental results regarding the influence of biogas
slurry on soil DOC. This result is consistent with findings in the literature
(Yemadje et al., 2016). The application of biogas slurry can also
significantly increase the Fn(355) value of DOM in soil; that is, appli­
cation of biogas slurry can improve the concentration of DOM in soil
(Ohno et al., 2007). Results from the current study indicated that biogas
slurry with a C/N of 12 increased DOM concentration the most in the
soil, followed by biogas slurry with a C/N of 8.84. The Fn(355) index for
BS was higher than that of BSNP, indicating that the concentration of
DOM in soil was able to be increased in a certain period of time when
planting corn. This further affirmed the previous result.
3.6. PARAFAC analysis
To eliminate the influence of fluorescence peak overlap (Guo et al.,
L. Yan et al. Ecotoxicology and Environmental Safety 209 (2021) 111804

Fig. 4. (a), (b), (c), and (d) four separate components in the DOM of soil samples; (e) Fmax distributions of the four components on days 0, 15, 30, 45 and 60.

7
L. Yan et al.
2020), PARAFAC analysis was performed on soil FDOM, to further
characterize the influence of biogas slurry differing in C/N on the
changes in content of different components in soil FDOM. After PAR­
AFAC, it was determined that there were four main independent com­
ponents in the sample (Fig. 4a–d). Component 1 (C1) showed a peak at
the excitation/emission (Ex/Em) of 230 nm/420 nm and a secondary
peak at the Ex/Em of 335 nm/420 nm, consistent with humic-like and
fulvic-like substances. It was mainly a folic acid-like substance, which is
generally related to terrestrial humus (Guo et al., 2020). Component 2
(C2) also showed two fluorescence peaks at Ex/Em of 275 nm/475 nm
and 355 nm/475 nm, respectively. It was thought to be a humus-like
compound caused by microorganisms, usually composed of phenols
and other aromatic structures (Gui et al., 2020). C2 was closely related
to many refractory organic materials (Guo et al., 2020). C1 and C2 were
composed of humus fluorophores of anthropogenic origin, similar to the
combination of traditional humus peak A and peak C. They are
humus-like substances and usually occur in agriculture-oriented areas
(Huang et al., 2017). Component 3 (C3) had maximum excitation value
at 280 nm and maximum emission value at 350 nm, respectively. C3
was similar to biologically unstable substances, proteins or amino acids
in aquatic environments, related to tryptophan-like substances, and
closely related to microbial decomposition (Guo et al., 2020). Compo­
nent 4 (C4) contained two types of peaks (protein-like peak and folic
acid-like peak), which could not be separated by the PARAFAC model.
Among them, the fluorescence peak at Ex/Em (225 nm/300 nm) was a
protein peak, which was mainly related to tyrosine-like substances,
while the fluorescence peak at 275 nm/410 nm was a furetic peak,
which was related to highly aromatic humus-like components (Hu et al.,
2017; Guo et al., 2020). C4 was related to hydrophobic compounds
according to the location of the folic acid peak of C4 (Chen et al., 2015c).
Generally, C3 and C4, which were closely related to proteins, were
bioavailable organic substances that could predict bacterial activity
(Hudson et al., 2008). Compared with C3, C1 and C2 featured richer
aromatic structure, higher molecular weight and more complex molec­
ular structure, because amino acids, carboxylic acids, reducing sugars,
polysaccharides and polyphenols can promote the formation of humus
(Wu et al., 2017). Thus, tryptophan in C3 and tyrosine in C4 could both
be energy sources for microorganisms and precursors to humus
polymers.
PARAFAC analysis was used to further analyze the relative contents
of each component in FDOM (Ma et al., 2018). The results showed that
(Fig. 4e), the FDOM in the soil used in the experiment was mainly
dominated by C1 and C2 (see CK). On the 0th day, the relative contents
of all components increased with the increasing C/N of biogas slurry.
Among them, the relative contents of protein-related C3 and C4 were
significantly elevated, indicating that biogas slurry had an obvious in­
crease in the relative contents of protein-like proteins in soil DOM,
consistent with our previous research results (Yan et al., 2019). With the
passage of time, the four components in each treatment showed different
trends, among which the biogas slurry with C/N of 3 had little influence
on each component. The contents of C1 and C2 in CK and BS3 showed a
trend of first increasing and then decreasing, while the above group of
other treatments showed a gradually declining trend. C3 of CK and BS3
showed a trend of first rising and then decreasing, while other treat­
ments showed a trend of first declining, then increasing and then
declining again, with an overall downward trend. C4 of CK showed an
upward trend. Surprisingly, C4 of BSNP almost disappeared after the
15th day. C4 of other biogas slurry treatments showed a decreasing
trend and then an increasing trend. It can be concluded that planting
corn was able to increase C4 content for a period of time. After the 15th
day, C4 content was negatively correlated with biogas slurry C/N
(Fig. 1b), which indicated that increasing organic matter in biogas slurry
was able to promote the decomposition, transformation and utilization
of C4 by plants and soil microorganisms.
Compared with the 0th day, by the 60th day, the content of all
components in CK showed increasing trends, while the contents of all
8
Ecotoxicology and Environmental Safety 209 (2021) 111804
components in biogas slurry treatments had decreased to varying de­
grees (Fig. 4e). C1, C2, C3 and C4 of CK rose by 11.93%, 28.48%,
26.22% and 43.61%, respectively. Thus, it demonstrated that planting
corn for a period of time increased the concentration of each component
of soil FDOM and greatly improved the C4 concentration. For C1 and C2,
the decrement of the two components in each biogas slurry treatment
increased first and then decreased with increasing biogas slurry C/N. For
C3, except for in CK and BS3, the degree of reduction in C3 in the other
biogas slurry treatments showed a pattern of first increasing and then
decreasing with increasing biogas slurry C/N, declining most clearly in
BSNP by 74.85%. BS showed the second greatest decline, with a
decrease of 66.52%. For C4, in addition to the increasing trend in CK, the
decrements of C4 components in other biogas slurry treatments showed
a pattern of first increasing and then decreasing with increasing biogas
slurry C/N. C4 in BSNP and BS decreased by 100% and 74.68%,
respectively. C4 is related to hydrophobic compounds, which are easily
absorbed by plants and biodegraded (Ren et al., 2015). This was also an
important reason for the decrease in soil DOM concentration. The
reduction of C1 and C2 in BS was greater than that in BSNP, and the
reduction of C3 and C4 in BS was less than that in BSNP, which further
demonstrated that planting corn for 60 days was able to increase C4 in
soil and reduce the transformation of protein-like substances in soil
DOM into humus-like substances. On the 60th day, compared with CK,
C3 and C4 of BSNP were lower by 8.71% and 100%, while C1 and C2 of
BSNP were higher by 39.98% and 30.19% respectively. This result
further verified that application of biogas slurry promoted the trans­
formation of protein-like substances in soil DOM into humic substances.
On the 60th day, compared with CK, BS12 exhibited the greatest in­
crease C1, C2 and C3 contents, 65.35%, 40.47% and 78.42%, respec­
tively, followed by BS.
Meanwhile, corn planted for 60 days weakened or delayed the
transformation of protein-like substances into humic substances in soil
DOM, because the increases in C1 and C2 in BSNP were greater than
those for BS, and the C3 content of BSNP decreased, while the C3 content
of BS increased (Fig. 4e). As the main component of soil organic matter,
humic substances account for more than 75% of soil organic matter,
which is a perfect indicator of environmental conditions (Labaz et al.,
2014). Increasing soil humus can promote plant growth, increase crop
yield, and restore used land. Humic components constitute an important
indicator to measure the maturity and stability of soil organic matter.
Soil humification is conducive to improving the maturity and stability of
soil organic matter, thus stabilizing soil structure and reducing
groundwater pollution, air pollution and greenhouse gas production (El
Hassani et al., 2020; Zhao et al., 2017). However, under natural con­
ditions, humus takes a long time to form (Yang et al., 2019a; Lwin et al.,
2018).
Further analysis of the proportions of each component in DOM
showed that, compared with the 0th day, by the 60th day, the pro­
portions of C1 and C2 in each biogas slurry treatment first increased and
then decreased with increasing biogas slurry C/N, increasing most
clearly in BSNP, followed by BS (Fig. 4e). However, proportion of C3 in
each biogas slurry treatment had decreased, and the decrement first
increased and then decreased with increasing biogas slurry C/N. C3
proportion declined most clearly in BSNP, followed by BS. It has been
predicted that biogas slurry can promote microorganisms in the soil to
transform protein-like substances with simple molecular structure in soil
DOM into humic substances with more complex molecular structure and
higher aromatic properties, thus stabilizing soil structure and improving
plant yield and nutritional quality (Yang et al., 2019a; Lwin et al., 2018;
Selim and Mosa, 2012). It indicated that biogas slurry with a value of
C/N 12 had a stronger effect on soil fertility increase, while biogas slurry
with a value of C/N 8.84 more strongly promoted soil structure
improvement and crop growth.
L. Yan et al. Ecotoxicology and Environmental Safety 209 (2021) 111804

Fig. 5. (a), (b), (c), (d) and (e) synchronous fluorescence spectra of soil DOM samples on days 0, 15, 30, 45 and 60, respectively; (f) PCA factor scores and syn­
chronous spectra loading on PC2 relative to PC1; (g) synchronous spectra loading on PC2 relative to PC1 on days 0, 15, 30, 45 and 60.

9
L. Yan et al.
3.7. Synchronous fluorescence spectra and PCA
The results of PARAFAC were verified by synchronous fluorescence
spectra to further enhance the reliability of the experimental results
(Fig. 5a–e). There are mainly three fluorescence peaks of different in­
tensities for soil DOM, and the fluorescence peaks with excitation
wavelengths between 270 and 280 nm are related to protein-like sub­
stances. There were fluorescence peaks related to flavonoid acid and
humic acid in the vicinity of 350–370 nm. At the 15th day, BSNP and
BS12 showed a tryptophan-related acromion at 300 nm (Determann
et al., 1998). This peak appeared in the remaining treatments on the
30th day, and two new protein-related fluorescence peaks appeared at
265 nm and 290 nm, disappearing after the 45th day. In the early stage
of biogas slurry application, compared with CK, the fluorescence in­
tensities of protein-like substances and humic substances in the biogas
slurry treatments were greatly enhanced. With the extension of experi­
mental time, the fluorescence intensities of protein-like substances and
humic substances in the biogas slurry treatments decreased overall, and
the fluorescence intensity of protein-like substances decreased greatly,
because protein substances are more biodegradable than humic sub­
stances (Liu et al., 2019b). Compared with the 0th day, the fluorescence
intensities of protein-like substances and humic substances in CK had
increased by the 60th day. The fluorescence intensities of protein-like
substances and humic substances in the biogas slurry treatments had
decreased. On the 60th day, compared with CK, fluorescence intensity of
protein-like substances in the biogas slurry treatments was lower in all
treatments except for BS12. For humic substances, compared with CK,
the fluorescence intensity of humic substances in the biogas slurry
treatments was greater on the 60th day. The fluorescence intensities of
protein-like and humic substance varied the most in BSNP, followed by
BS. Thus, results again verified that biogas slurry promoted the trans­
formation of protein-like substances in soil DOM into humic substances,
with the biogas slurry with C/N of 8.84 showing the strongest promoting
effect on this process, and indicating that planting corn can weaken the
transformation of protein-like substances into humic substances in soil
DOM for a period of time.
The experimental results for multipeaked synchronous fluorescence
spectra exhibited complex characteristics, which made it difficult for us
to use it to analyze a large number of complex spectral changes. At this
point, a PCA technology was needed that could decompose complex
multivariable signals into linearly independent components for analysis
(Yan et al., 2019). PCA correlation test results (Fig. 5f) showed that the
partial correlations between variables were small (KMO = 0.922), and
the correlations between variables was significant (P < 0.001). There­
fore, these experimental samples were suitable for PCA. The character­
istic roots of PC1 and PC2 were 27.07 and 2.59, respectively, and their
combined variance contribution was 98.87%, this covered most of the
information. PCA mainly identified six major fluorescence peaks at 265,
275, 290, 302, 345 and 362 nm. PC1 had a main fluorescence peak
associated with aromatic amino acids at 275 nm, mainly reflecting
tyrosine. At 340 nm, there was a secondary fluorescence peak, which
was related to humus and maybe fulvic acid (Yan et al., 2019). The main
fluorescence peak associated with PC2 appeared at 362 nm, which was
the fluorescence peak representing humic acid. This mainly reflected
fulvic acid (Yan et al., 2019). There was a secondary fluorescence peak
associated with protein in PC2 at 265 nm. PC2 also showed two peaks at
290 nm and 302 nm, associated with protein in DOM (Barker et al.,
2010). These two peaks appeared successively in the synchronous
fluorescence spectra of soil samples in each treatment on the 15th day
and the 30th day and then disappeared after the 45th day.
PC1 and PC2 of each treatment were affected by biogas slurry C/N
(Fig. 5g). At the 0th day, the dominant fluorophore of DOM of soil
samples in the biogas slurry treatments was inclined to migrate towards
PC2 related to humic substances. The dominant fluorophore of DOM of
soil samples in BS6 shifted to PC2 the most, followed by BSNP. When the
planting time of corn was extended from the 0th day to the 30th day, the
10
Ecotoxicology and Environmental Safety 209 (2021) 111804
dominant fluorophore of DOM gradually shifted from PC1 to PC2. This is
due to the complex, highly aromatic substances and a large number of
polymers of apparent molecular weight greater than 10,000 Da pro­
duced by microorganisms from simple organic compounds (Hunt and
Ohno, 2007; Sheng and Yu, 2006). Similarly, the results of Laurent et al.
(2020) also showed that the application of organic fertilizer was able to
increase the concentration of DOM in soil and improve the aroma of
DOM in soil. From the 45th day to the 60th day, the dominant fluo­
rophore of DOM of soil samples was transformed from PC2 to PC1.
Among them, the dominant fluorophore of DOM of soil samples from
CK, BS3 and BS6 was still PC1. The PC1 load of BS, BSNP and BS12 was
not significantly different from that of PC2; that is, soil DOM treated
with high C/N biogas slurry had no obvious dominant fluorophore.
Compared with the 0th day, by the 60th day, the dominant fluorophore
of soil samples from biogas slurry treatments had shifted to PC2. BSNP
was the most offset, followed by BS. This result indicated that planting
corn for a period of time could prevent the dominant fluorophore in the
DOM of the soil sample from migrating towards PC2 related to humus.
Biogas slurry, especially biogas slurry with a C/N of 8.84 could promote
this process.
4. Conclusion
Planting corn for a period of time could reduce the concentration of
DOC in soil and weaken the humification of soil but increase the con­
centration of FDOM and its components in soil. Applying biogas slurry
with different C/N could promote the growth of corn, improve the
content of soil DOM (DOC and FDOM), promote soil humification, in­
crease the bioavailability of FDOM, increase the content of humus and
protein in FDOM, and promote the decomposition, transformation and
utilization of C4 by plants and soil microorganisms. The optimum C/N
ratio for biogas slurry application in farmland was 8.84. The biogas
slurry with C/N of 12 exhibited the greatest increase in the content of
DOC and FDOM in soil, 40.93% and 66.25%, respectively. However,
biogas slurry with C/N of 8.84 exerted the strongest promotion effect on
corn growth, soil humification, bioavailability of FDOM, and the
transformation and utilization of C4.
CRediT authorship contribution statement
Lilong Yan: Conceptualization, Investigation, Formal analysis,
Validation, Writing - review & editing. Cong Liu: Data curation,
Methodology, Visualization, Supervision, Writing - original draft.
Yudan Zhang: Supervision, Methodology. Shuang Liu: Supervision.
Ying Zhang: Writing - review & editing.
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.
Acknowledgments
This work was supported by Key Technologies Research and Devel­
opment Program of China (2018YFD0800105).
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