Evolution of humus dynamics in composting of swine manure combined with wood vinegar

Aerobic composting was carried out in a greenhouse using home-made compost bins, and the changes in humus(HS) composition of composted manure with different proportions of wood vinegar(WV) added to pig manure and rice straw as raw materials were studied. Since the temperature, water content and pH were very different from those of the summer compost, they were used as basic evaluation indices of compost maturity. Rice straw(RS) and Swine manure (SM) were used as test materials, and the water-soluble carbon (CWSS), extractable humic carbon (CHE), humic carbon (CHA), humic carbon (CFA) and humic carbon (CHU) of the materials were investigated, and the humus decomposition coefficients were dynamically analyzed. The findings demonstrated that 1.25% and 1.75% wood vinegar affect humification. Meanwhile, the results of Ratio of absorbance of water-soluble organic substances at 465 and 665 under UV spectrophotometer (E4/E6) indicated that the aromatic ring of 1.75% WV solution was more stable after condensation, and the decomposition of hominin as a HS stabilizing component was more complete at the concentration of 1.75%. Consequently, this study revealed that WV at a concentration of 1.75% was more efficient in stimulating pile decomposition in cold conditions.


Introduction
(Zhou et al 2023)With the increase in livestock production in recent years, poultry manure has become an obstacle to environmental sustainability (Liu et al 2022, Feng et al 2023).Improper disposal of livestock manure results in a significant waste of resources and increases the potential for disease transmission.Composting is known to be a biological oxidation process of organic waste that involves mineralization and partial humification of organic material to produce a stable end product with specific humic properties and without phytotoxicity or infectivity (Zhao et al 2022, Chen et al 2023).Composting is a spontaneous biodegradation process of organic materials in an aerobic environment.This process of converting organic material into stable, usable organic matter is carried out by bacteria, fungi and other microorganisms, including microarthropods (Yang et al 2023).Composting also reduces waste production, eliminates weed seeds and removes pathogenic microorganisms (Qian et al 2023).It is one of the main solutions for livestock and agricultural wastes contaminated with many organic wastes and suppresses air pollution and soil contamination caused by organic wastes.
(Guo et al 2022, Wang et al (2022c)).HS is a complex polymeric compound that is the main nutrient produced during the composting process.Field experiments with targeted fertilization for institutional tomato production have shown that the quality of humus is the main indicator of composting performance.HS can be divided into two categories: humic acids (HE) and humans (HU).Humic acids are further subdivided into fulvic acid (FA) and humanin acid (HA) (Peng et al 2022, Wei et al 2023).In a previous study (Wang et al 2023) it was found that the addition of wood vinegar to compost accelerated humus production, induced the conversion of HU and HE to active humus fractions, accelerated lignin decay, increased the degree of decay, shortened the composting cycle and increased the rate of composting.
As biomass waste, SM and WV solution are rich in organic matter and can be used to improve soil quality, promote plant growth and control pests and diseases (Liu et al 2020, Wu et al 2022).The organic acids in the WV solution can accelerate the rate at which the organic matter in SM is broken down by microorganisms, improving the quality of the compost.And WV has more advantages for composting in the current composting process than chemical additives with secondary pollution(Wurzer and Mašek 2021, Yang et al 2021) and biological additives (Duan et al 2020, Wang et al 2021) that are difficult to use.In the previous research, the amount of WV added in the composting process was limited to 0%-2% based on the biotoxicity produced (Wang et al 2018(Wang et al , O'reilly et al (2021)), Mohammadi-Aragh et al 2021), but the nutrient yield of WV within the range of biotoxicity has not been studied, and this paper determines the amount of nutrients and the quality of compost in the composting process by the yield of HS as well as the dynamic evolution of the composting process.SM was used as the raw material and rice straw as the filler for the aerobic composting tests in this study, along with different concentrations of WV as additives.Using temperature, moisture content and pH as end-ofcomposting indicator indices and HS composition as the basis for decay assessment, studies were carried out to determine the ideal concentration of WV for use in composting under low temperature conditions.

Materials and methods
2.1.Composting materials and procedure SM and WV were used as composting materials.SM was supplied by a farm in Alar, Xinjiang, China, and was subjected to wet-dry separation to retain solids and then air-dried to a moisture content (MC) of about 60%.WV was obtained from cotton straw by the Key Laboratory of Engineering, Tarim University, Xinjiang, China.RS was obtained from a farm in Alar, Xinjiang, China.The physics-chemical parameters of composting materials are shown in table 1.
A handmade compost bin 100 cm high, 50 cm wide and 60 cm long was used.According to the literature (O'reilly et al (2021)), the feedstock consisting of SM and RS (volume, 20:1) was manually homogenised to obtain mixtures with a low C:N=25 in order to process more livestock manure to reduce the environmental pressure on soil nutrients.Subsequently, WSS were mixed at 0.65%, 1.25% and 1.75% (V/W) (T1: treatment with 0.65% WV; T2: treatment with 1.25% WV; T3: treatment with 1.25% WV), while the treatment without WV served as control (CK).Each treatment had three replicates.The composting materials were manually turned each week in a 38-day composting process to ensure a supply of oxygen and homogeneity of feedstock.And maintain moisture content at about 50% humidity to provide a better habitat for the microorganisms(Zhao et al 2023).Temperature was measured daily at different position (top, middle, and bottom layers) inside composting windrows.Samples were taken from the upper, middle, and lower layers of the heaps on days 1, 3, 10, 17, 24, 31and38.The piles were stirred completely, freeze-dried, pulverized, and sieved through 100 mesh before being stored in a refrigerator at −3 degrees for testing.

Humic composition
HS and its components were extracted using the modified humus component extraction method described by (Zhang et al 2011).The procedure was as follows: 1.00 g of sample was weighed and mixed with 30 ml of deionised water in a 50 ml shaking tube.The sample was then extracted by shaking at 70 °C for 1 hour and then centrifuged twice at 4000 rpm for 10 minutes each time.The supernatant after centrifugation was collected in a 50 ml volumetric flask.The filter paper was rinsed three times with an appropriate volume of distilled water and the rinse water was combined with the supernatant from the first collection and fixed.The liquid collected at this point is the water-soluble(WSS) fraction.The remaining residue on the filter paper was rinsed again with a mixture of 0.1 mol/L NaOH and 0.1 mol/L Na 2 P 2 O 7 in a shaking tube and separated; the supernatant extracted in this step was HE.The residue on the filter paper was washed with distilled water into a centrifuge tube and mixed with the previous residue.The residue in the tube was washed several times until the pH of the paper was neutral, then placed in a test tube rack and oven baked at 55 °C until a constant weight was reached, at which point the solid residue was HU.
A total of 30 ml of HE solution was aspirated, the pH was adjusted to approximately 1.3 with 0.5 mol l −1 H 2 SO 4 and the sample was placed in a constant temperature water bath at 70 °C for 1.5 hours.The sample was then removed and left to stand for over 4 hours.At this point, the bottom of the tube contained a sediment and a brown flocculent material floated to the top.The sample was filtered into a 50 ml volumetric flask and the volume was fixed; the filtrate was FA.The brown residue was rinsed into a 50 ml volumetric flask with warm 0.05 mol/L NaOH and the volume was fixed; the liquid was brown without impurities and corresponded to HA. C WSS , C HE , C HA and C HU were determined by the potassium dichromate sulphonate method and the absorbance values of the HA base solutions at 465 nm and 665 nm (OD465, OD665) were determined using a TU-1810 UV-visible spectrophotometer and calculated from the formula E4/E6 = OD465/OD665.

Physico-chemical analysis
Three replicates were used in the analysis of all parameters, and the results were reported as mean value ± standard deviation.pH were analyzed using Remagnetics Analyzer detector (pHS-3 C).TC and TN were measured using a Elemental analyzer vario MICRO cube (Elementar, Germany).Moisture content was determined by drying at 105 °C for 24 h.

Statistical analysis
The experimental data were statistically analyses using Excel 2016 and Origin 2019b quantitative statistical analysis, and the LSD method was used to test the significance of differences.

Physico-chemical parameters
Temperature, the heat from the SM and RS degraded by microorganisms(Bui et al 2023), can directly reflect composting efficiency and microbial activity.According to the dynamics of composting temperature, the composting process can be divided into four phases: the thermophilic phase (d-3 to d-5, < 50 °C), the hyper thermophilic phase (d-5 to d-15, 50 °C-65 °C) and the cooling phase (d-15 to d-20, < 50 °C), and the aging phase (d-20 to d-40, < 30 °C).Temperature values of the remaining treatments, except for CK, the thermophilic phase in all treatments was maintained above 50 °C for 5 d, during the warming and high-temperature phases.Among them, T3 treatment appeared the most with the highest temperature reaching 54.9 °C (figure 1).It shows that the compost reached the standard sterilization effect (Chinese 'Sanitary requirements for the harmless treatment of night soil', GB7959-2012).
During the cycle of composting, the moisture content showed an overall decreasing trend, and the rise and fall at 24 days were not significantly affected by trace rainfall resulting in a decrease in moisture content, and there was no significant difference in moisture content changes among all treatment.During d1-17 of composting, pH values initially increased, then decreased and reached the lowest value on day 17, while EC values showed a non-significant upward trend for all treatments with added WV solution initially and began to decrease on d-3, except for T2 where EC values increased again on d-24 and EC values decreased overall (figure 1).Compared to the different treatments, the pH value of the CK treatment was slightly higher than the other treatments; the T3 treatment showed a more pronounced decrease in d-3 due to the higher temperature.During the composting period, TC values continued to decrease, while TN values, excluding the CK treatment, initially decreased and then increased in the remaining, reaching a peak on d-3 (figure 2).The addition of WV solution increased the TC content.The TC values were ranked as T3 > T1 > T1 > CK, and the TN values increased in the order of T1 < T2 < T3 < CK.During the composting process, the C: N ratio showed a decrease.Similar to TC values, C: N ratios in T1, T2, and T3 treatments were higher than in CK treatment and increased with the increase of biochar content.

HS-fractions
Composting, a biochemical process in which microorganisms degrade SM and RS (Manga et al 2023), is the main contributor to the dynamics of the C/N ratio.Meanwhile, CWSS (figure 3) is the most active fraction of compost and a source of nutrients for microorganisms (Sun et al 2023).Compared with the piles without the addition of WV, the piles with the addition of WV initially showed a decrease in water-soluble substances with increasing concentration.As the composting time increased, each pile showed a decreasing trend, with the content decreasing by 64%, 69%, 65%, and 88%.C HE is a polymer compound produced by the decomposition of organic materials under the action of microorganisms and enzymes, and its complex structure is the key to the fertilization efficiency of organic fertilizer (Ge et al 2022).
The treatment with the addition of WV had significantly higher values than the treatment without the addition of WV, and the carbon content of C HE was higher than T1 in the T3 and T2.The treatment showed different performance patterns as the composting time progressed, with the CK showing an increasingdecreasing-increasing trend; the rest of the treatment showed an increasing trend followed by a decreasing trend.The 1.25% and 1.75% treatment showed a decrease of more than 20% at the end of composting.The 1.75% treatment showed the most significant depletion of extractable humic acid carbon content at the end of composting.
C HA is a high-molecular-weight polymer containing a large number of active functional (Li et al 2022).As shown in figure 3, on the 1st day of composting, T1, T2, and T3 was significantly less than that of the CK.As the composting time advanced, the carbon content in each treatment showed a trend of first decreasing and then increasing.At the end of composting, except for CK, the C HA increased by 6%, and the rest of the groups were depleted, with decreases of 8%, 9%, and 13%.
The E4/E6 of C HA has an inverse relationship to the degree of polymerization of its molecules when a smaller value of E4/E6 indicates a more complex molecular formula (Cui et al 2022).As the composting time progressed, all treatment showed an increasing and then decreasing trend, with the E4/E6 of C HA increasing by 30%, 32%, 72% and 92% in the CK, T1, T2, and T3, respectively, compared to the values on d-1.  Figure 4 C HU is one of the components of humus, (Bao et al 2023) studied the effect of Aspergillus niger on the maturity and cellulose degradation of cattle manure compost, mentioning that it is tightly bound to minerals and is one of the more stable substances in compost humus.
On day 1, the C HU values of compost without WV were higher than those of other composts.With the extension of composting time, the C HU values of all treatments showed a decreasing trend, and at the end of composting, the C HU values of CK, T1, T2 and T3 treatments decreased by 64%, 65%, 68% and 71%, respectively, of which the C HU decomposition degree of T3 treatment was higher.Therefore, the addition of WV solution promoted the differentiation of humus to plant-available nutrients and improved the nutrient quality of compost.
(Fang et al 2022) In their study on the effect of faecal matter on the composting process of edible mushroom residues during composting, Fang et al mentioned that C HA /C FA is the key to determine the degree of decomposition of the compost (Irawan 2022, Valverde-Orozco et al 2023), and the higher the ratio, the higher the degree of decomposition, as shown in figure 4. All treatment showed a trend of decreasing and then increasing throughout the composting process (Chang et al 2023).Compared with the first day, all treatment increased at the end of composting, except for the CK, which decreased; the observed increases were 13%, 19%, 45% and 50%, respectively, which showed that C HA was gradually converted from the original C FA during the composting process (Li et al 2023, Wan et al 2023).

Discussion
In the first stage of conventional composting, simple organic carbon compounds are readily mineralized and metabolized by microorganisms, producing CO 2 , NH 3 , H 2 O, organic acids and heat, and this heat accumulation raises the temperature of the pile (Wang et al 2022a).Monitoring of compost temperatures showed shorter compost temperature retention times and slower compost warming compared to the results of (Zhang et al 2022), but current conditions meet the criteria for compost decomposition.
Throughout the fermentation cycle, the pH tends to rise, then fall, then gradually stabilise.This trend is due to a rapid increase in pH during the early stages of fermentation when easily degradable organic acids are biodegraded (Xi et al 2021), volatilised and nitrogen ammoniated, followed by a high decomposition of more difficult to degrade organic matter during the high temperature phase, producing low molecular weight acids and low molecular weight organic acids.In CK, aromatic hydrocarbons did not condense during the cooling phase and many nitrification reactions occurred (Afzal et al 2019).
This C WSS phenomenon can be attributed to the rapid heating after the addition of the WV solution, which led to the rapid multiplication of many microorganisms, the decomposition of organic matter and the consumption of WSS (Wang et al 2022b); this result suggests that WV has a certain promoting effect on microbial activities.The reason for the large fluctuation of T3 in C HE may be that a higher WV content induced the combined action of microorganisms and enzymes to accelerate the decomposition of macromolecules to form nutrients that are more easily absorbed by the plant (Bouket et al 2022).The T3 treatment of C HE resulted in relatively good nutrient quality of the piles, followed by the T2 treatment with good nutrient quality.The trend of C HA (E4/E6) shows that CK contains an enhanced degree of condensation and aromatization than the other treated molecules, while the other treated HAs have an enhanced degree of aliphaticization and a simpler structure.As shown in table 2, the correlation coefficient between C HA / C FA and C HA content was as high as 0.852, indicating that all treatments of compost HA showed conversion to C FA , which eventually formed C FA higher than C HA , and was ultimately beneficial to humus quality, C WSS and C HU reached 0.794 correlation, C WSS was rapidly decomposed by microorganisms after the more difficult to decompose part of the form of aromatic rings to become The correlation between C HE and C HA reached 0.784, which was mainly due to the component of C HE , and, the C HA content of T3 treatment was much higher than the other treatments.According to the change of the curve, it can be shown that the addition of WV can promote the quality of humus (Zhu et al 2021).This is consistent with the results study by Zhu et al

Discussion and conclusions
By analyzing the evolution of HS composition in the samples from the beginning of composting to complete decomposition at low temperatures, the following conclusions were drawn.
(1) Compared to ordinary compost, the preparation with added wood vinegar solution contains higher content of C WSS and C HA and lower content of C HU , which indicates that the WV solution promotes compost decomposition.
(2) C HE behaved unstably during composting and was easily decomposed.Some of the products condensed and formed humus substances with more stable structure and properties under the action of microorganisms.The addition of WV solution is more favorable to the ripening process and promotes the formation of HS.
(3) Changes in the molecular structure of C HA are often used to characterize compost maturation.Acetic acid solution is more conducive to the aromatization of compost C HA .The E4/E6 of C HA solution shows the degree of aromatization of the molecular structure, the smaller the ratio is, the higher the degree of molecular condensation and aromatization, the higher the molecular weight is, the degree of aromatization of the newly formed C HA molecules in the CK test group has not yet been condensed to the original complexity of C HA , C HA /C FA is the humification index (Jiang et al 2022), which is the main index for evaluating the quality of HS, the higher the ratio is, the higher the quality is, and it is obvious that in the WV treatment group, the C HA /C FA is the main index of humus quality.higher than the CK treatment group.
(4) C HU is the inert component of HS, with high molecular weight and stable nature, and it mostly combines with minerals and exists in the form of a complex under natural conditions.It decreased in all treatment groups, among which T3 decomposed C HU the most and transformed it into active HS components.
The main role of wood vinegar solution throughout the composting stage is mainly when added within a week, the pile internal rapid warming, a large number of microorganisms are provided with a reproductive environment, violent activity of microorganisms consume a large number of carbohydrates (Yu et al 2020, Guo et al 2021), carbohydrates are decomposed and metabolised by microorganisms into proteins, peptides, amino acids, ammonia, polyphenols and quinones, usually these substances will be condensed, the amino group of one amino acid and another amino acid The carboxyl group of one amino acid can be condensed to form a peptide, the amide group formed is called a peptide bond in protein chemistry (Yu et al 2020, Wu et al 2022).The amino group of one amino acid condenses with the carboxyl group of another amino acid to form a peptide.Proteins are sometimes referred to as 'polypeptides'.Some amino acids are converted into ammonia and polyphenols into quinones which then polymerise and condense to form humic substances (Zhong et al 2023).When carbohydrates are consumed to a certain level, microorganisms begin to break down organic matter for energy, breaking down the lignin in the substrate into polyphenols, which are converted to quinones for polymerisation and condensation to form humus.

Figure 1 .
Figure 1.Dynamic changes in temperature, pH and EC within composting process.

Figure 2 .
Figure 2. Dynamic changes in TC, TN and C/N within composting process.

Figure 3 .
Figure 3. Dynamics of C WSS , C HE and C HA during composting.
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pH, MC: moisture content; OM: organic matter; EC: electrical conductivity; TOC: total organic carbon; TN: total nitrogen.The experimental results are reported as mean ± standard deviation.

Table 2 .
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