Effects of different conditioners on humus composition and humic acid structural characteristics in Black Soil under the combined application of pig manure and straw

Purpose The objective of this work was to evaluate the variation in the amount and structure of humic acid (HA) after the application of different conditioners under the combined application of manure and straw. Methods This was measured by Fourier transform infrared (FTIR), �uorescence spectroscopies, and 13 C nuclear magnetic resonance ( 13 C CPMAS NMR). The experiment involved corn straw combined with pig manure (SZ), pig manure plus biochar (SCZ), pig manure plus boron slag (SBZ), pig manure plus biological agent (SJZ), and pig manure plus bio-organic fertilizer (SOZ), while corn straw only was used as control (SCK). Results The results demonstrated that pig manure combined with straw (PM-S) improved soil organic carbon (SOC), and the application of biochar and boron slag was the effect for improving the accumulation of SOC and humus C fractions. The elemental composition and 13 C CPMAS NMR results demonstrated that PM-S enhanced the ratio of H/C and the aliphatic C/aromatic C ratio of HA, reduced the O/C ratio, indicating enhanced aliphatic and conducive to simplifying HA molecular structure.


Introduction
Soil organic carbon is an important component of the soil carbon pool (Lehmann and Kleber, 2015), and its carbon content is three times greater than atmospheric carbon stocks (Zhao et al. 2018).Increasing soil organic carbon (SOC) has been shown to be an effective way of improving soil fertility and soil quality (Shu et al. 2021).Changes in soil organic carbon are in uenced by a number of factors, including soil type, environmental factors (temperature and moisture), biological characteristics (i.e., microbial community composition and abundance), and organic matter incorporation (Berhane et al. 2020; Wang et al. 2022).In particular, the application of straw, as well as biochar and organic fertilizers, plays an important role in improving SOC sequestration and stability (Dong et al. 2022;Yang et al. 2022;Zhang et al. 2019).
Humus is the main component of SOC and is a relatively stable organic carbon fraction in soils, accounting for 60%-90% of SOC, which has a regulatory function on soil fertility and plays an important role in soil nutrient cycling and carbon sequestration (Hernandez et al. 2019) (Shan et al. 2010).The stabilization potential of C in the soil is mainly regulated by HA structural characteristics (Stevenson, 1982), which are affected by the organic material (Chen et al. 2020;Cui et al. 2017;Zhang et al. 2020).The co-incorporation of organic manure and straw could enhance SOC (Gao S, 2018),improves soil humus composition, increase the content of alkyl carbon in HA (Chien et al. 2006), and simpli es HA molecular structure (Chen et al. 2019;Diacono and Montemurro, 2011).Therefore, the elucidation of HA structure is of great importance for carbon sequestration on farmland.However, the decomposition process of straw is slow when directly applied to the soil and as the quantity of straw diminishes, organic C inputs are decreased, which may eventually affect the soil humus composition and structural characteristics of humic acid.In recent years, domestic and foreign scholars have paid much attention to conditioners, while others focused on exogenous conditioners as an alternative practice to effectively sequester soil C and improve humus composition, given the shortfalls of direct soil application of straw in the soil.
Biochar application can promote humus formation during composting by modulating the speci c metabolic characteristics of the microbiome (Liu et al. 2023), accelerate soil humi cation (Jin et al. 2023), enhance the aliphatic characteristics of soil HA and reduce the proportion of aromatic structure of HA (Wang et al. 2023).Wang et al. (Wang et al. 2014) showed that the addition of 3% biochar improver to pig manure composting promoted the process of compost humi cation and the synthesis of humic acid.Li et al. (Li et al. 2021) studied that the addition of 10% coconut shell biochar stimulated humus formation and enhanced humi cation during green waste composting.Bai et al. (Bai et al. 2023) indicated that the addition of ash as a soil conditioner is expected to increase the soil humic substance pool and also alter its chemical properties and functions.Bioorganic fertilizers increase the humic acid carbon content of the soil and improve the soil humi cation rate and humi cation index (Sedlar et al. 2023).The addition of fungicides can effectively increase the decomposition rate of straw and the content of various forms of organic carbon in soil within a short period of time (Wang et al. 2021), promote the content of various components of soil humus (Wu et al. 2022), and improve the degree of soil humi cation (Lu et al. 2019).At present, although there have been more studies have investigated the effects of straw with different manure applications on soil nutrients and crop yields, few studies have compared the contribution of the differences in the changes of carbon composition and HA structure characteristics of humus in straw and organic manure under different conditioners.
The goal of this research is to evaluate the variation of humus composition and humic acid structural characteristics in black soil under different conditioners after the application of pig manure and straw (PM-S).
The results will help to identify the most suitable conditioners for improving the properties of soil and provide a theoretical basis for soil fertilization.

Experimental site
A one-year eld experiment in 2021was conducted in Yushu County (44°26′59″N, 125°21′37″E), Jilin Province, Northeast China.The region is very cold during winter and hot during summer, having a temperate continental monsoon climate.The average annual precipitation is 331.9 mm, mainly from June to September.The soil type is typical black soil with the following basic properties: organic matter 22.92 g.kg − 1

Field experiment design
A eld experiment was conducted in May 2021.The eld was arranged in a randomized block design consisting of twelve plots (100 m 2 each) with six treatments in three replicates.The treatments comprised corn straw only (SCK), corn straw plus pig manure (SZ), corn straw and pig manure plus biochar (SCZ), corn straw and pig manure plus boron slag (SBZ), corn straw and pig manure plus biological agent (SJZ), corn straw and pig manure plus bio-organic fertilizer (SOZ), The straw strip composting method was used for the returning of straw.
The straw and/or pig manure were put into in the eld in May 2021.In this method, furrows were rstly created to about 20 cm deep using plow followed by putting the pig animal manures together with the straw into the furrows.The incorporated organic materials were covered with the surrounding soil.The application rates per community were 15 kg for biochar, 15 kg for boron slag, and 20 kg for pig manure,20 kg for bio-organic fertilizer,1 kg for biological agent.In the study, the cropping system was corn cropping.Corn crop was planted in May and harvested in October.Three random undisturbed soil samples were collected from each treatment in the 0-20 cm soil depths in July 2022.After visible animal and plant debris and stones were removed, soil samples were air-dried and passed through a 2-mm sieve

Analysis methods
Soil humus composition was analyzed following the International Humic Substances Society method (Kumada et al. 1967).Humic acid (HE) was extracted from the 30 mL of a mixture of 0.1 M alkali solution (NaOH + Na 4 P 2 O 7 ) as the extractant, then 0.5 M H 2 SO 4 was used to separate the humic acid (HE), which was left overnight to obtain humic acid (HA) and fulvic acid (FA), and the remaining solid was humin (HM).The C content was determined by the wet modi ed-oxidation method, by treating 0.2 g of soil with K 2 Cr 2 O 7 -H 2 SO 4 solution.
HA samples were extracted and puri ed by the IHSS method (Kuwatsuka et al. 1992), 50 g of soil samples over 2 mm were weighed and added with 1 M HCl at a soil-to-water ratio of 1:1, and after being placed for 1h, 0.1 M HCl was used to adjust to the nal soil water ratio of 1:10.Then 0.1 M NaOH solution was used to wash and precipitate and adjust to the nal soil water ratio of 1:10, and 1 M NaOH was used to adjust pH = 13-14.The resulting liquid HA was obtained after low-speed centrifugation, high-speed centrifugation, electrodialysis, rotary evaporation, freeze-dried and other processes.
Elemental analyzer (Vario-EL-III Hanau, Germany) was used to assess the C, H, N, and S contents of solid HA.HA infrared spectrum was measured by AVATAR 360 FTIR spectrometer, covering a frequency range of 4000 to 400 cm − 1 , under 4 cm -1 wave number resolution, 16 scans and pure KBr spectra as a background.Spectra were recorded using a Perkin Elmer FL 6500 uorescence spectroscope.Fluorescence spectra was obtained with the range of Em in 350-600 nm, and Ex in 300-550 nm.Em and Ex slits were xed at 5 nm.Scanning speed was 12 000 nm min -1 .HA solid 13 C-NMR spectra were measured using a Swiss Bruker AV400 NMR instrument.The spectra was recorded in the following conditions: spinning rate was 8 kHz, contact time was 2 ms, sampling time was 34 ms, relaxation delay time was 5s.The integral area is calculated using MestRe-Nova software, and the relative content of each carbon is expressed as a percentage of the integral area within a certain chemical shift interval to the total integral area

Data analysis
Analysis of variance was conducted to evaluate the effects of different straw returning modes on all measured soil parameters using SPSS software (IBM Statistics 21.0).Signi cance differences among straw returning mode means were evaluated using the least signi cant difference test at P < 0.05.All spectra graphs were compiled using Origin 8.5 software.

Concentration of organic C and humus substances
Compared to SCK, the SOC in SZ, SCZ, and SBZ treatments increased by 10.64%, 15.69%, and 8.18%, respectively.However, the SOC of SJZ and SOZ exhibited a different trend, that is being signi cantly lower in SJZ and SOZ treatments compared with SCK treatment (Fig. 1).The contents of humus showed the order of: HM > HA > FA (Table 2).The SZ, SCZ and SBZ increased HA-C content by 9.73%, 13.63% and 6.81% compared with SCK respectively, the FA-C content increased by 4.49%, 7.05% and 2.56% in SZ, SCZ and SBZ treatments compared with SCK respectively.PQ value was the proportion of HA in extractable humic substances and an important indicator of the degree of soil organic matter humi cation.The PQ values of SZ, SCZ and SBZ treatments increased by 73.51%, 73.69% and 73.22% compared with SCK (Table 2).This indicated that the addition of biochar and boron slag conditioner were both bene cial for improving the degree of soil humi cation, with the addition of biochar mixed treatment being the most signi cant; while bio-organic fertilizer and biological agent conditioner reduced the soil humi cation coe cient, which was not conducive to soil carbon sequestration.11g.kg − 1 , and O content was 374.62-507.89g.kg − 1 (Table 3).Elemental C and N contents in soil HA were higher in SCZ treatment than in other treatments.The O content was lower than that of other treatments, which may be due to the formation of surface functional groups and the desorption of O onto the surface of black carbon (Cheng et al. 2006).The H/C ratio and O/C ratio indicated a degree of condensation and oxidation.Compared to SCK, the H/C ratio of HA increased by 8.65%, 13.51%, and 5.10% and the O/C ratio decreased by 9.72%, 12.09%, and 3.92% for SZ, SCZ, and SBZ treatments, respectively (Fig. 2).This result was more evident for the SCZ treatment, indicating that addition of biochar was decreased with the oxidation and condensation of HA structure and simpli ed HA molecular structure.The FTIR spectra of all treatments showed similar peak characteristics (Fig. 3), only the absorption intensities differed (Table 4).The peaks located at 2920 and 2850 cm -1 is related to the symmetric and asymmetric stretching vibrations of aliphatic C-H bonds in CH 3 and CH 2 groups respectively.The peak located at 1720 cm -1   was attributed to C = O stretching, caused by carboxyl, ketone and aldehydes.The peak at 1620 cm -1 was associated with C = C vibrations of aromatic structures.The relative intensities of the absorption peaks of the HA infrared spectra for each treatment were shown in Table 4.There was no obvious pattern in the variation of the peaks, which differed only in the relative absorption intensities.The ratio of I 2920 /I 1720 and I 2920 /I 1620 which re ects the strength of the ratio of aliphatic C to carboxyl C and aromatic C in the HA structure, respectively, revealed that SCZ and SZ treatments increased the aliphatic/carboxyl C and aliphatic/aromatic C, while SOZ and SJZ treatments decreased the ratio of aliphatic/aromatic C.
Among all treatments, the lowest uorescence intensities (FI) of peaks A-C were recorded under SCZ (Fig. 5), this may be due to the presence of polymeric compounds which contain linearly condensed aromatic ring structures (Rodríguez et al. 2014).Compared to SCK treatment, the uorescence peak position of SCZ treatment showed a greater degree of redshift, with an increase in Ex/Em ratio, indicating that the molecule contains electron donating groups (Fellman et al. 2008), a conjugated unsaturated system with high resonance (Hautala et al. 2000), and more hydroxyl, alkoxy, and methoxy groups (Hu et al. 2018).This indicated that after adding straw, pig manure, and biochar to the soil, the HA structure tends to be simpli ed.

13 C CPMAS NMR of HA
The solid-state 13 C CPMAS NMR spectrum of HA was shown in Fig. 6.The spectrum was identi ed as alkyl C The spectral shapes of HA in different treatments were similar, but there were signi cant differences in the intensity of absorption peaks (Fig. 6).Each treatment showed characteristic peaks of COOH > C = O at 170ppm, aromatic ring characteristic peaks at 130ppm, and lipid alkyl peaks at low shifts (0-30ppm).Table 5 had the carbon content of different functional groups of HA for regional integration of the spectral peaks in Fig. 7. HA was mainly a mixture of alkyl C, alkoxy C, aromatic C and carboxy C. It contains higher contents of aromatic C and carboxy C (Table 5).Compared with SCK, SCZ and SZ treatments increased the content of aliphatic C, aromatic C and the ratio of hydrophobic C/hydrophilic C, indicating that the hydrophobicity of HA molecules was enhanced by the addition of pig manure and biochar.However, SOZ and SBZ treatments decreased the aliphatic C/aromatic C ratio, indicating that the aromaticity of HA was enhanced after the addition of biological agent and bioorganic fertilizers.

Effects of different treatments on SOC content and humus composition
After one-year, mixed application of pig manure and straw (PM-S) into soil was bene cial to the accumulation of organic carbon content in soil and humus components, and the addition of biochar signi cantly increased the carbon content of soil organic carbon fractions, while the addition of biological agent and bioorganic fertilizers were not conducive to soil organic carbon xation and reduced the humi cation coe cient.Gogoi et al. (2018) reported that application of pig manure signi cantly enhanced SOC content and our results agree with this (Gogoi et al. 2021).This might be because pig manure has low C/N and high soluble matter, which make it easy to be decomposed by microorganisms and can release soluble matter into the soil more quickly after application, which improves SOC accumulation (Thangarajan et al. 2013).The application of straw combined with pig manure can reduce SOC mineralization in native soil, strengthen the interaction between organic matter and minerals, facilitate the formation and stability of aggregates, and increase the content of soil organic matter (Li et al. 2020b).The addition of PM-S was found to be conducive to increasing SOC, HA-C and FA-C contents (Table 2), and the PQ value slightly increased, but the change was not signi cant.This indicated that the degree of maturation and fertility of the soil is changing an appropriate direction after returning to the eld, which is possible that the application of organic fertilizer can promote the process of soil humi cation and improve the quality of organic matter, thus contributing to the increase of soil humus, HA and FA content (Gregorich, 1996).
In the present study, we found that the application of biochar signi cantly increased the SOC and HA contents compared to other treatments (Fig. 1), and was consistent with previous studies (Novak et al. 2009).This was because that biochar has high C/N ratio, hard-to-degrade stable organic carbon and large porosity (Khare and Goyal, 2013), which provides microbial activity and nutrient sources (Lal, 2004), accelerates the decomposition of straw and conversion to humus and nutrients, which is conducive to the accumulation of SOC (Li et al. promoting the conversion of biochar to humus, increasing the content of organic carbon in humus components and affecting soil PQ value.However, we found that the SOC content in the SJZ treatment was lower than that in SCK treatment, which was inconsistent with the results of Li (Li et al. 2017).This may be due to the long winter time and low temperature in the north, which lead to slow decomposition of straw return, resulting in a slower rate of cellulose degradation.The decomposition of straw mainly relied on the microorganisms contained in the straw and soil itself, and there are differences in the population and quantity of soil microorganisms in different regions and straw.Therefore, it was di cult for the agent microbial community to have a synergistic effect with the soil microbial community, resulting in a less signi cant promoting effect on straw decay (Wen et al. 2013).
The lower C/N ratio of bio-organic fertilizers in the SOZ treatment was more likely to be decomposed by microorganisms and released as CO2 after mixing, which is not conducive to soil carbon xation.

Effects of different treatments on HA structural characteristics
In the present study, we found that the mixed application of PM-S reduced the condensation and oxidation degree of HA, and the addition of biochar signi cantly enhanced the lipophilicity and hydrophobicity of HA molecules, rendering HA molecular structure simpler; while the addition of biological agent and bio-organic fertilizer conditioner enhanced the aromaticity of HA molecule and complicated the structure of HA.Chien et al.
(2006) reported that application of pig manure signi cantly enhanced the proportion of alkyl carbon in soil humic acid and our results agree with this (Chien et al. 2006).This might be because pig manure has more C that is di cult to decompose, which can increase the number of polysaccharides, aromatic compounds and aliphatic compound in the soil (Li et al. 2020c), and enhance the soil with aliphatic compounds.
The present study demonstrated that biochar to the soil increased H/C (Fig. 2), which implied that the addition of biochar reduced the degree of condensation and oxidation of soil HA molecules, enriched the soil with aliphatic compounds and rendered HA molecular structure simpler.This was attributed to the developed porosity Among all treatments, SCZ showed a signi cant increase in uorescence intensity in the A, B, and C peaks (Fig. 5), indicating that the structure of soil HA was simpli ed by the application of biochar.It was possible that the HA molecular structure contains electron-donating substituents such as hydroxyl, methoxy and amino groups, which increase the uorescence intensity of the HA molecule by increasing transition probability between the singlet and ground states (Zech et al. 1997).FT-IR spectroscopy provided evidence of structural differences in HA samples, which may be that the input of organic exogenous materials promoted the decarboxylation of peripheral functional groups of HA (Dou et al. 2008) and the decomposition of unstable structures in HA (Giovanela et al. 2010).
However, considering the complexity of HA peaks in uorescence spectroscopy, the structural characteristics of HA were further determined using solid-state 13 C-CPMAS-NMR techniques.In the present study, different organic materials had markedly different effects on the functional groups of HA structures, and the changes in HA structure were due to alkyl C, O-alkyl C, aromatic carbon, and carboxyl carbon (Table 5).
Alkyl C was a stable organic carbon component that is di cult to degrade (Ussiri and Johnson, 2003); O-alkyl C was the most readily decomposable organic carbon functional group in plant residues, and its decomposition was dominated by the cellulose and hemicellulose contained in plant residues (Kubar.2018), During the rapid phase of decomposition of plant litter, the alkoxy C from plant residues will rapidly lose into the soil (Mathers and Xu. 2003).Therefore, the ratio of alkyl C/ O-alkyl C can re ect the alkylation degree of humus and can be used as an indicator of the decomposition degree of the original plant materials or the humi cation degree of organic carbon.Higher alkyl C/ O-alkyl C content, was indicative of greater decomposition degree in organic materials, which plays a vital role in the stabilization of C (Zhao.2012).Aromatic C was one of the more di cult organic carbon fractions to decompose in the soil, with a higher proportion, possibly due to the selective retention of lignin from plant residues during the early stages of decomposition (Baldock et al. 1992), which increased the proportion of aromatic C. The carboxy carbon was mostly derived from the absorption of aliphatic acid, amino acids, amides, esters, ketones, and aldehydes (Mao et al. 2008), and its content re ected the degree of oxidative decomposition of soil organic matter (Quideau et al. 2000), In this study, the application of bioorganic fertilizers in the soil increased the relative content of carbonyl carbon (Table 5), possibly due to a large number of organic acids brought in by bio-organic fertilizers.The hydrophobicity of HA was determined by the ratio of hydrophobic C (aromatic C and O-alkyl C) to hydrophilic C (alkoxy C and carboxyl C).The ratio was higher, which implied that the hydrophobicity of HA was stronger.The aromaticity of HA was enhanced after adding biological agents and bio-organic fertilizer conditioners.In contrast, the addition of biochar signi cantly enhanced the aliphatic and hydrophobicity of HA, resulting in simplifying HA molecular structure, probably because the input of biochar promoted the decarboxylation of peripheral functional groups of HA (Dou et al. 2008) and the decomposition of unstable structures in HA (Giovanela et al. 2010).There was a size exclusion phenomenon in the micropores of biochar, resulting in smaller aliphatic molecules being more easily adsorbed than larger aromatic molecules (Smebye et al. 2016).

Conclusions
Pig manure with straw (PM-S) was more conducive in increasing SOC in the 0-20 cm soil depth, and increasing the proportion of humus.The application of biochar conditioners had a signi cant effect on the accumulation of soil organic carbon composition, the addition of biological agent and bio-organic fertilizers on the other hand The EEM uorescence spectrum of HA after the application of different treatments.

( 0 -
50 ppm), alkoxy C (50-110 ppm), aromatic C (110-160 ppm) and carboxyl C (160-200 ppm).The maximum absorption peak of alkyl C was at 30 ppm for methylene C; the signal at 55-56 ppm for alkoxy C was methoxy C, 70-73 ppm and 104-105 ppm for carbohydrate C and polysaccharide dioxygen C, respectively; the maximum absorption peak of aromatic C was at 128-130 ppm; the small peak at 152-153 ppm was for phenolic hydroxyl C; the signal of carboxyl C was mainly at 171-173 ppm.The signal of carboxyl C was mainly concentrated at 171-173 ppm.
2020a), abating direct mineralization of carbon content(Yu et al. 2020), thereby increasing soil carbon storage(Qualls, 2004);The unstable organic matter released from biomass charcoal can promote the formation of soil aggregates(Zimmerman et al. 2011), inhibit the rapid decomposition of soil particulate organic carbon by microorganisms, and reduce the mineralization rate of soil organic carbon (Blanco-Canqui, 2017);The addition of biochar changed the physical properties of soil(Zhou et al. 2021), increased the contact area between soil microorganisms and straw, accelerated the transformation of straw to humus carbon, and led to the change of soil humus components.Additionally, its application will be partially degraded naturally and providing new carbon sources for microorganisms, thereby increasing microbial activity and activity(Pokharel et al. 2021), and huge speci c surface area of biochar (Jga et al. 2019), which adsorbs more aliphatic molecules and enhances the HA molecular structure more aliphatic (Jimenez-Gonzalez et al. 2019);The application of biochar provided carbon or nutrient sources for microorganisms(Liu et al. 2015), increased the abundance and activity of microorganisms(Pokharel et al. 2021) and promotes microbial decomposition(Zimmerman et al. 2011).The microbial activity of the microorganisms promoted the decomposition of part of the biochar into the soil, which was reconstituted by microbial action to form the chain structure in HA, and part of the C = C double bond was retained, thereby increasing the stability of the HA structure.The increase in peak area ratios of HA infrared spectra at 2920/1720 and 2920/1620 as shown in Table4also indicated that biochar can promote the formation of aliphatic carbon in the HA structure, and that the aliphatic carbon structure of biochar can be easily transformed into HA in soil through mineralization and decomposition(Cheng and Lehmann, 2008).Pospíšilová (2020) also found that biochar application altered the structure of soil humus(Pospíšilová et al. 2020), and reduced the condensation of soil HA(Zhang et al. 2016).

Figure 1 Figure 2 3 Page 18/ 20 FTIR
Figures , alkaline nitrogen 117.71 g.kg− 1, available phosphorous 28.56 g.kg − 1 , available potassium 126.33 g.kg − 1 .Corn straw was produced in situ in the experimental site; Waste biochar was commercially produced by Jilin Mingtai Renewable Energy Company; biological organic fertilizer was produced by Liaoyuan Xinrong Biological Fertilizer Company; the liquid biological agent was produced by Jilin Haoyu Biotechnology Company; the pig manure was produced by the Gain Agricultural Cooperative of Huancheng Township, and is directly returned to the eld after harmless treatment, meeting the requirements of the "Technical Speci cation for Returning Livestock and Poultry Manure to the Field" (GB/T 25246 − 2010); the boron slag comes was produced by Dandong East Magnesium Fertilizer Industry Company.The basic properties of other organic materials are shown in Table1.

Table 2
Soil organic carbon content of soil humus after the application of PM-S and different conditioners Note: Different letters indicate signi cant differences between treatments(P < 0.05).

Table 3
showed the elemental composition of soil HA across all treatments, and the range of each attribute was as follows: C content was 443.06 to 551g.kg − 1 , N content was 25.55 to 39. 41g.kg − 1 , H content was 23.49 to 35.

Table 3
Effects of the combined application of PM-S and different conditioners on the elemental composition of soil HA samples 3.3 FTIR spectra of HA

Table 4
Effects of the combined application of PM-S and different conditioners on the relative intensity of the main absorption peaks in FTIR spectra of HA in soil

Table 5
Effects of the combined application of PM-S and different conditioners on the relative intensity of the main absorption peaks in13C CPMAS NMR spectra of HA in soil