The impacts of oxytetracycline on humification during manure composting can be alleviated by adjusting initial moisture contents as illustrated by NMR

FENG Yao,WANG Gui-zhen,LIU Yuan-wang,CHENG Deng-miao,FAN Shuang-hu,ZHAO Quansheng,Jianming XUE,ZHANG Shu-qing,LI Zhao-jun

1 Key Laboratory of Plant Nutrition and Fertilizer,Ministry of Agriculture and Rural Affairs/China-New Zealand Joint Laboratory for Soil Molecular Ecology,Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences,Beijing 100081,P.R.China

2 College of Life Science,Langfang Normal University,Langfang 065000,P.R.China

3 College of Biology and the Environment,Nanjing Forestry University,Nanjing 210037,P.R.China

4 Scion,Christchurch 8440,New Zealand

Abstract Oxytetracycline (OTC) residues have been found in soil and water,and they may pose potential risks to agricultural ecological environments. One of the most impactful ways for OTC to enter the soil and water environments is through excrement used as organic fertilizer. Therefore,it is important to remove OTC during manure composting and to understand the transformation of the organic materials during composting in the presence of OTC. In the present paper,chicken manure and wheat sawdust spiked with OTC were composted under different initial moisture contents (MC) to evaluate the degradation of OTC and changes of organic matter during the composting process. The MC has a significant effect on OTC degradation during composting. A higher MC of 65% was more conducive to OTC degradation (77.4%) and compost maturity compared to the lower MC. However,the higher MC of 65% could increase the ammonia volatilization by promoting nitrification compared to the lower MC. An increase in the initial MC could improve the composting temperature. NMR results illustrated that the presence of OTC could affect the internal transformation of aliphatics,and OTC inhibited compost humification. Thus,an initial MC of 55-65% can alleviate the impacts of OTC on humification during manure composting.

Keywords:oxytetracycline degradation,humification,moisture content,composting,physico-chemical property

1.Introduction

The treatment and effective resource utilization of agricultural wastes is one of the hot topics related to current environmental protection. In recent years,however,antibiotic drugs such as oxytetracycline (OTC) have been widely applied during livestock and poultry breeding,and as a result,the abundance of antibiotic residues in manure seriously limits the further use of animal manure as fertilizer in agriculture (Kumaret al.2005a). The detection of tetracycline antibiotic residues on the typical large-scale farms of seven Chinese provincial regions indicated that the average concentration of OTC in chicken manure was 5.97 mg kg-1,and it could reach up to 23.43 mg kg-1(Zhanget al.2004). Another study showed that the concentration of antibiotic residues in chicken faeces could reach up to 200 mg kg-1(Kumaret al.2005b). Using faeces containing OTC residues may lead to the accumulation of antibiotics in the soil environment if the faeces are directly applied to farmland as an organic fertilizer without harmless disposal. The OTC residues that enter the soil may have impacts on the microorganismal community structure and its activity (Chenet al.2014). The presence of OTC in soil could also result in increased antibiotic resistance in soil microorganisms as well as reduced plant growth and development (Diehlet al.2010;Zhang Cet al.2019).

The aerobic composting of animal manure is one effective biooxidative process for reducing the amount of antibiotics in manure (Arikanet al.2007;Wuet al.2011;Selvamet al.2012). Wuet al.(2011) reported that chlortetracycline,tetracycline and OTC in swine manure could be degraded by 74,70,and 92%,respectively,after 52 days of pilot scale composting. Selvamet al.(2012) found that spiked chlortetracycline and sulfadiazine could be completely removed from the compost pile within 21 and 3 days,respectively;however,the removal rate of ciprofloxacin only ranged from 69 to 83% during a 56-day composting period. The levels of extractable OTC in the compost mixture could be decreased by 95% within the first six days of beef manure composting (Arikanet al.2007). The diverse degradation rates of antibiotics may be correlated with the antibiotic properties,manure types,composting parameters and microbial activities (Liuet al.2017).

During the manure composting period,microorganisms decompose and transform organic matter under appropriate conditions through specific biochemical processes (Liuet al.2018). Bernalet al.(2009) summarized the main factors that affect microbial development during manure composting,which include the bulk density,porosity,particle size,nutrient contents,C/N ratio,temperature,pH,moisture,and oxygen contents. The moisture is also a key physical factor of the composting system that directly influences the physiological characteristics of microorganisms,the physical structure of solid matrices,and even the compost maturity and quality during the composting of animal manure (Lianget al.2003;Kimet al.2016). A high initial MC in composting would form an anaerobic environment and negatively affect cellulose degradation as a result of poor ventilation,even if other conditions such as the carbon and nitrogen (C/N)ratio and other factors are optimal. McCartney and Tingley (1998) indicated that low moisture during composting could cause early dehydration and inhibit the transport of the dissolved nutrients required for the metabolic and physiological activities of microorganisms. However,the compost pile temperature increases slowly and has a short high-temperature period,which is unable to satisfy the biological stabilization and harmlessness requirements of compost,when the MC was 40-50% (Jianget al.2015).A change in the moisture content has a marked effect on the concentrations of five fluoroquinolone antibiotics during broiler manure composting (Yanget al.2018). Shenet al.(2009) indicated that an initial moisture content of 60% is conducive to the degradation of tetracyclines during swine manure composting. However,there has been no research thus far on the degradation of OTC in chicken manure at different initial moisture contents. In the presence of OTC,microorganisms and their activity might be impacted,which could in turn affect the composting process,especially the transformation of organic materials during manure composting. In addition,the MC could also impact the interaction between OTC and microorganisms,and the manure surface (Zhang Met al.2019). Therefore,the objectives of the present paper are to investigate the effects of different initial moisture contents on OTC degradation and changes in the organic material structure and its transformation during the composting of chicken manure.

2.Materials and methods

2.1.Materials and reagents

Fresh and unfermented chicken manure was collected from a private ecological farm located in Beijing,China. Airdried wheat straw was collected from the experiment site (Langfang,Hebei) managed by the Chinese Academy of Agricultural Sciences (CAAS),and it was chopped into small 1.0-5.0 cm fragments and used to adjust the initial C/N ratios of the compost piles. As raw materials,both the chicken manure and wheat straw were divided into two parts:one part was used for the immediate determination of the OTC content,and the other part was used to measure selected physical and chemical properties,including the pH,moisture content,total organic carbon (TOC) and total nitrogen (TN) (Table 1). The OTC residue content in the chicken manure was 3.98 mg kg-1,probably due to incomplete uptake of OTC by the chickens. The wheat cultivar used for the germination index (GI) trials was Zhongmai 12,which was purchased from the Institute of Crop Sciences of CAAS.

Table 1 Selected physico-chemical properties of the composting materials1)

Oxytetracycline (OTC,98.9%) was purchased from Merck &Co.,Inc.(USA). The reagents for the OTC determination were chromatographically pure (Fisher Scientific,Waltham,MA,USA),and the others were analytically pure.

2.2.Experimental design and implementation

The optimum MC for composting varies with the composted materials,but generally,the mixture should should be at 50-60% (Bernalet al.2009). Regarding this percentage,the three moisture levels of 45% (LMC),55% (MMC),and 65% (HMC) were used in the present study. Each level was tested in triplicate. Chicken manure and shredded wheat straw were blended manually at the ratio,by dry weight,of 2 (chicken manure):1 (wheat straw) to give a final C/N ratio of approximately 20:1. The aqueous solution containing OTC at 1.0 g kg-1was then sprayed on the manure-straw mixtures and blended carefully to mix the OTC well with the mixtures and to ensure that the initial OTC concentration was 100 mg kg-1dry matter (DM) in each treatment (i.e.,each MC level of 45,55,and 65%),referring to the actual residuals of OTC in China. The purified water produced by a Milli-Q Plus Water System (Millipore,Billerica,MA,USA) was used to obtain the desired moisture content for all the composting mixtures. Then,the mixtures for each treatment were divided into three heaps weighing 20.0 kg each,which were transferred into homemade aerobic composting reactors for 42 days.

2.3.Sampling and analytic methods

The samples were collected on days 0,1,3,5,10,17,24,31,and 42 by completely mixing the upper,central and lower portions of the three sites uniformly distributed in the reactor to achieve representativeness and homogeneity as high as possible. The samples were stored at -20°C to analyse the OTC,temperature,pH,electrical conductivity (EC),TOC,TN,C/N radio,water-soluble organic carbon (WSOC),ammonium nitrogen (NH4+-N),nitrate nitrogen (NO3--N),seed GI and spectral characteristics during the different composting periods.

The OTC contents in the compost samples were extracted and analysed according to the method described by Fenget al.(2016). Briefly,1.0 g samples (AdventurerTMOHAUS/Florham Park,NJ,USA) were extracted with 5 mL of 0.1 mol L-1Na2EDTA-McIlvaine buffer through vortexing (Berlin Wiggens,Berlin,Germany) for 1 min followed by sonication for 15 min in a 40 kHz/300 W ultrasonic bath (Ningbo Scientz,Ningbo,China). The extracts were then subjected to centrifugation (Sartorius Sigma,St.Louis,MO,USA) at 3 396×g for 10 min at 4°C. The supernatant was then decanted into a glass bottle,and the sediment was repeatedly extracted with 4 mL of 0.1 mol L-1Na2EDTA-McIlvaine buffer according to the above steps. Twice-extracted supernatants were merged and 1 mL of 20% trichloroacetic acid was added. After standing for 1 h at 4°C,the extraction was centrifuged for 10 min at 3 396×g. A total of 1.0 mL aliquot of extract liquid was collected for purification,and it was concentrated using a Speedisk Column H2O-Philic DVB (6 mL,500 mg) cartridge from JT Baker (Phillipsburg,NJ,USA),which had been preconditioned with 3 mL of methanol and 5 mL of DI water. The analyte was passed through the cartridge at a flow rate of 1 mL min-1,and the cartridge was rinsed with 5 mL of distilled water and dried under a vacuum for 5 min,followed by sample elution using 3 mL of 0.01 mol L-1methanolic oxalic acid. Following N2evaporation,the concentrated extracts were reconstituted in 1 mL of the mobile phase consisting of 0.01 mol L-1oxalic acid,acetonitrile and methanol (85:10:5,v/v) for OTC analysis using an Agilent 1100 Series High-Performance Liquid Chromatograph (Sunnyvale,CA,USA) on an Agilent ZORBAX SB-C18 column (3.5 μm,4.6 mm×150 mm). The flow rate was maintained at 1 mL min-1,the injection volume was 20 μL,and the detection wavelength was 355 nm. The calibration curves were typically generated using results from 20.0 μL injections of OTC standards ranging from 1.0 to 50.0 mg kg-1. The extraction recoveries of OTC were 76.4-92.6%.

The pH and EC of each sample were determined for a slurry consisting of 1:10 (w/v) compost:distilled water with an MP521 pH/EC meter (Shanghai Sanxing,China). The TOC was determined with a ‘Liqui TOC’ Total Organic Carbon Analyser (Elementar Analysensysteme GmbH,Germany). The TN was determined using a modified micro-Kjeldahl procedure with an KDY-9830 automatic Kjeldahl apparatus (Changzhou Henglong,China). The WSOC was determined with a WSOC automatic analyser. The ammonium and nitrate nitrogen were determined by indophenol blue colourimetry and the phenol disulfonic acid colourimetric method,respectively.

The GI was measured using the method described by Ivoneet al.(2008). Fresh compost samples were placed in distilled water at a ratio of 1:10 (w/v). The suspensions were shaken for 1 h,and the extracts were subjected to centrifugation (3 396×g,20 min) and then passed through medium-speed qualitative filter paper to remove the suspended particles. Each 10 mL of filtrate was added to a sterile culture dish (90 mm diameter) lined with two sheets of filter paper. Twenty wheat seeds were evenly distributed on the filter paper and incubated at 25°C in the dark for 72 h. The controls were treated in the same way,but with distilled water rather than compost filtrate. Filtrate or distilled water was added as needed to maintain the moisture. Each treatment was represented by three replicate dishes. The numbers of germinated seeds and the lengths of the root radicals were determined for each dish every 24 h. The GI was calculated for each treatment according to the following formula:

To investigate the changes in the organic matter during composting in the presence of OTC,the composting samples collected on days 1,17,and 42 were crushed into powder,dissolved in 0.1 mol L-1sodium hydroxide and then subjected to nuclear magnetic resonance (NMR) analysis with an AVANCE III 400 MHz Digital NMR Spectrometer (Bruker,Germany),with the use of 7-mm-diameter sample tubes. Data acquisition conditions were as follows:spectrometer frequency,100.46 MHz;spinning speed,7 kHz;pulse width,90°;acquisition time,69 ms;and pulse delay time,1 s. About 10 000 scans were collected for the organic matter of the compost samples.

2.4.Statistical analysis

The biodegradation of antibiotics in solid waste can be described by a pseudo first-order reaction equationCt=C0e-kt,whereCtis the residual concentration (mg kg-1) of OTC at sampling timet(d),C0is the initial concentration (mg kg-1) of OTC (t=0),kis the reaction constant (d-1),andtis the sampling time (d). The half-life (t1/2) was calculated according to the formula:t1/2(d)=0.693/k. All the statistical analyses were performed with Excel 2013 and SPSS 19.0,and the data were plotted using Origin 8.0.

3.Results and discussion

3.1.Fates of oxytetracycline during composting

The OTC concentrations decreased quickly over the composting process (Fig.1). The degradation of OTC within the initial 10 days of composting was far more rapid than that at the curing stage,which was consistent with previous reports (Wuet al.2011;Wanget al.2015). The OTC concentrations in the treatments containing 45,55,and 65% MC decreased sharply,by 42.3,73.0,and 64.2%,respectively,after 10 days of composting. Then the OTC degradation became slower from day 10 until the end of the composting trial. The OTC degradation rates reached 68.8% (LMC),76.0% (MMC),and 77.4% (HMC) after 42 days of composting. The OTC here showed lower removal rates than the tetracyclines in previous studies,e.g.,Winckler and Grafe (2001) (＞99% for chlortetracycline,40% initial MC),Wuet al.(2011) (92% for oxytetracycline,56.9% initial MC),and Huet al.(2011) (97.2% for oxytetracycline,65% initial MC). Zhang Met al.(2019) reported that 79.6% of the OTC with an initial concentration of 22 mg kg-1was dissipated within 38 days of composting with an initial MC of 65%,which is consistent with our study. The degradation of antibiotics is simultaneously affected by biotic and abiotic factors during composting. In the present study,it is interesting that the OTC in the MMC and HMC treatments displayed significantly higher degradation rates than that of the LMC treatment (45% MC) during the entire composting period. This finding may be because the higher MC is conducive to the growth and activities of functional microbes,which in turn enhances their ability to biodegrade organic matter,including OTC,during composting (Lianget al.2003;Eunjonget al.2016).In addition,it was also found that the OTC degradation rates in the MMC treatment were higher than those of the HMC in samples taken before 24 days of composting;however,the corresponding rates were lower than those in the HMC treatment after 24 days of composting. Zhang Met al.(2019) presented lower removal rates for tetracyclines in the composting process,which are thought to be caused by the reduced moisture levels and low frequency turnings. An insufficient oxygen supply could lead to an anaerobic environment when the MC exceeds 60%,which inhibits microbial activity and hampers biological processes (Bernalet al.2009). The MC gradually diminished during the composting period,and the HMC treatment had a more suitable MC than the MMC treatment for the microbial activity in the later composting period.

Fig.1 Effect of the moisture content on oxytetracycline (OTC) degradation during composting. LMC,low moisture content (45%);MMC,middle moisture content (55%);HMC,high moisture content (65%). The results are the means of three replicates,and the bars indicate the standard error of the three replicates.

The changes in OTC contents during the entire composting process could be clearly described by the first order kinetics equation (Table 2),with correlation coefficients (R) between 0.9363 and 0.9709. In this study,all of the treatments showed a relatively fast dissipation during composting,with the half-life (t1/2) ranging from 1.9 to 5.6 days (Table 2). In general,the degradation rate constant (kvalues) of OTC obtained from the treatment with a high moisture content was greater than those from the lower levels (Table 2). Arikanet al.(2009) noted that thet1/2of OTC is 3.2-30 days during the composting of sludge and manure. Therefore,OTC degradation primarily occurred during the early stage of the thermophilic phase.

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Table 2 The fitted equation and half-life period of oxytetracycline (OTC) degradation during composting1)

3.2.Temperature profile

The temperature is one of the most critical factors,and it affects the microbial activity and even the maturity and quality of composting (Chenget al.2019). The changes that occured in the pile temperature and ambient temperature during composting are shown in Fig.2. During composting,the ambient temperature ranged from 15 to 23°C. The pile temperatures of the different MC treatments went through four typical phases,mesophilic,thermophilic,cooling and maturation. The pile temperature in the HMC treatment increased more rapidly than those in the other treatments. For example,the pile temperature in the HMC treatment reached a peak at day 5 of composting with a value of 61.76°C;however,the corresponding peak of the pile temperatures for the LMC and MMC temperatures appeared on day 8 of composting with values of 58.2 and 63.4°C,respectively. In contrast to the LMC and MMC treatments,the pile temperature in the HMC treatment had a longer thermophilic (＞50°C) period. This observation indicates that the HMC treatment could have more benefits than the other treatments,such as LMC and MMC,in terms of killing the pathogenic microorganisms in the pile body (Bernalet al.2009). During the maturation stage,the temperatures of the three treatments had gently declining trends towards the ambient temperature. Because of the active microorganisms,higher MCs (55 and 65%) were beneficial for reaching a higher maximum temperature and maintaining a longer thermophilic phase,allowing thermophilic microorganisms to degrade OTC.

Fig.2 Effect of the moisture content on the pile temperature during composting. LMC,low moisture content (45%);MMC,middle moisture content (55%);HMC,high moisture content (65%). The results are the means of three replicates at 9 a.m.and 4 p.m.every day.

3.3.Changes in the composting physico-chemical properties

pH and EC valueThe differences in effects of the initial MC of the pile on the pH and EC are shown in Appendix A. The pH is one of the most key parameters in the composting process of antibiotics degradation (Fenget al.2020). The pH of the compost was slightly alkaline,and the values in the MMC and HMC treatments increased rapidly at the early stage of the composting and then decreased gradually at the later stage. It is interesting that the pH values of the LMC treatment kept increasing during the entire composting period. The changes of pH values in different treatments could result in the changes of MC and decomposition of organic matter (Atchley and Clark 1979). During the first 3 days of composting,the pH values of all the treatments increased dramatically,which could have resulted from the fact that the variation in ammonia volatilization occurred because of proteolysis and ammonification (Wonget al.2009). The subsequent decrease in the pH could be explained by the H+released from microbial nitrification,the decomposition of organic matter and the production of organic and inorganic acids,and CO2accumulation caused by a lack of aeration during composting (Cacereset al.2006). It was also found that the pH value of the LMC treatment increased more slowly than in the other two treatments. This result might be because organic acids are generated slowly in the pile when the MC is low (Shanet al.2007). In addition,the mineralization of nitrogen in which organic nitrogen compounds are decomposed gradually and then converted to more ammonia nitrogen could also lead to a higher pH value in the LMC than in the other two treatments at the later stage of composting (Tiquia and Tam 2000). It is interesting that the rising pH values in the different treatments were significantly different from one another. For example,during the first 10 days of composting,the rates of rising pH in the LMC,MMC and HMC treatments were 0.11,0.17,and 0.14 pH d-1,respectively,and were different from each other (P＜0.05).

The EC value reflects the degree of salinity in the composting,indicating possible phytotoxic/phyto-inhibitory effects on the growth of the plants (Huanget al.2004). The change in EC values of the piles among the three treatments during the composting period are shown in Appendix A. The initial EC values of the three composting materials were 5.3 mS cm-1(LMC),4.3 mS cm-1(MMC),and 3.9 mS cm-1(HMC),respectively. During the entire composting trial,the EC values of the LMC were significantly higher than those of the other two treatments. The EC values of piles followed the order LMC＞MMC＞HMC. It could be illustrated that the high moisture content facilitated a dilution effect,resulting in low EC values. At the earlier stage of composting,the EC values in MMC and HMC had similar increasing trends due to the release of mineral ions such as phosphates,ammonium and potassium through the degradation of organic materials.When the compost arrived at the curing stage (after 30 days of composting),the EC values gradually declined in the LMC and MMC treatments due to the volatilization of ammonia and the precipitation of mineral salts (Huanget al.2004),but they still increased in the HMC treatment,which might be caused by the reduced humic acid substances and cation exchange capacity of the pile (Huanget al.2006). At the end of composting,the EC values of the piles ranged from 4.3 to 5.3 mS cm-1,and significant differences were also found among the three treatments.

TOC,TN and C/N radiosThe TOC values of the three treatments presented a decreasing trend with the composting time (Fig.3-A). The TOC of the three treatments decreased dramatically within the first 17 days of composting,and the TOC values declined by 12.9% (LMC),20.8% (MMC),and 17.3% (HMC). After 17 days of composting,the TOC values decreased gradually. At the end,the TOC value of the piles in the LMC,MMC and HMC treatments were 35.7,34.0,and 32.0%,respectively. The results were consistent with those of Selvamet al.(2012). Overall,the TOC content of the LMC was the highest and that of HMC was the lowest,which was related to the microorganism activity. The slight activity of the microbes due to the limited moisture content led to weaker carbon usability.

At the earlier stage of composting,the TN values of all the treatments showed a sharp decline (Fig.3-B),which might be due to the volatilization of NH3(Jarviset al.2009;Shanet al.2019). However,significant differences in the TN changes were also found among the three treatments. The TN values in the LMC and HMC treatments decreased to 17.1 and 19.9 g kg-1DM during the first 17 days of composting,respectively,and then they increased after 17 days. This finding is consistent with Konget al.(2018) on the changes in nitrogenous components during the windrow composting of chicken manure with rice chaff. The TN content of the MMC treatment declined dramatically during the first 10 days of composting,followed by a continuous but more gradual decrease. The pile TN contents at the end of composting were 19.8 g kg-1(LMC),17.8 g kg-1(MMC),and 21.4 g kg-1(HMC) DM. This trend might be due to the inhibition of N-mineralization in the presence of OTC (Wonget al.1997),which in turn led to the increases in the TN content at the end of composting.

As shown in Fig.3-C,the pile C/N ratio had a decreasing tendency during the entire composting period. At the end,the pile C/N ratios in the LMC,MMC and HMC treatments were 18.0,19.2,and 18.0,respectively. Compared to LMC and MMC treatments,the HMC treatment showed a slightly smaller change in the C/N ratio due to its lower nitrogen loss.

Fig.3 Effects of the moisture content on the pile total organic carbon (TOC;A),total Kjeldahl nitrogen (TKN;B),C/N ratio (C),water-soluble organic carbon (WSOC;D),NH4+-N (E) and NO3--N (F) during composting. LMC,low moisture content (45%);MMC,middle moisture content (55%);HMC,high moisture content (65%). Bars show standard deviations (n=3).

Water-soluble organic carbon contentsThe watersoluble organic carbon (WSOC) is an effective carbon source that can be used by microorganisms (Francouet al.2008). The WSOC contents of pile in 65% MC treatments were significantly higher than those of the 45 and 55% MC treatments during the entire composting process (Fig.3-D). The WSOC contents of the pile were 3.8% (LMC),3.5% (MMC),and 5.1% (HMC) on day 0 of the composting. The WSOC contents of the pile in the three treatments presented an increasing trend on days 1-5 and then a decreasing trend on days 5-24. The increases in WSOC contents on days 1-5 of composting might be because the microorganisms rapidly degraded the labile organic materials,such as the carbohydrates and fats,with the increasing temperature (Iannottiet al.1994). The deceased WSOC contents of piles on composting days 5-24 could be ascribed to microbial decomposition and the production of carbon dioxide,methane and volatile organic compounds containing carbon,which was basically identical to the research results of Iannottiet al.(1994). At the curing period (24-42 days) of compost,the WSOC contents in all the treatments appeared to show an increasing trend once again,which was related to the decomposition of refractory organics and the formation of small functional substances.

NH4+-N and NO3--N contentsAs shown in Fig.3-E,the changes in water-soluble NH4+-N contents of piles increased first and then decreased during the composting process. The NH4+-N contents of piles in the LMC and HMC treatments reached the maximum values of 1 179.5 and 1 513.0 mg kg-1DM on day 5 of composting,respectively,and the NH4+-N contents of the pile in the MMC treatment increased to a maximum of 1 429.0 mg kg-1DM on day 10 of composting. This trend could be caused by the conversion of organic nitrogen into ammonia through ammonification. The NH4+-N contents then decreased because of NH3volatilization at high temperature and high pH (Guoet al.2012),and the conversion of NH4+-N to NO3--N. The results also showed that the water-soluble NH4+-N content in the LMC treatment was obviously lower than those of the MMC and HMC,which was probably because the low moisture contents negatively affected the activity of microbes and the NH4+-N to NO3--N transformation capacity.Compared with the water-soluble NH4+-N contents for the initial composting,the NH4+-N contents declined to 469.2 mg kg-1DM (LMC),689.1 mg kg-1DM (MMC),and 578.8 mg kg-1DM (HMC) at the end of composting period for percentage decreases of 57.6,36.1,and 42.9%,respectively. This result indicated that the high MC of the initial pile benefitted the reservation of the nitrogen in the pile,which is consistent with the results of El Kaderet al.(2007).

The initial NO3--N contents were 606.9 mg kg-1DM (LMC),481.1 mg kg-1DM (MMC),and 582.7 mg kg-1DM (HMC),respectively,on day 0 of composting (Fig.3-F).The NO3--N content fluctuated within the first 10 days of composting,which was probably correlative with the reproduction of nitrobacteria at various moisture contents,high temperatures and high concentrations of OTC. After 10 days of composting,with the gradually reduced moisture content and OTC concentration,the NO3--N contents in all treatments increased due to the conversion of NH4+-N to NO3--N. At the end of the composting trial,compared with those on day 1 of composting,the NO3--N contents in LMC,MMC,and HMC treatments increased to 782.5,858.8,and 767.2 mg kg-1DM,respectively,for corresponding percentage increases of 28.9,78.5,and 31.7%,respectively.

The decreasing amounts of NH4+-N in all the treatments followed the order LMC＞HMC＞MMC,which was the opposite from the increasing amounts of NO3--N contents. These findings indicated that lower water (45% of MC) could accelerate ammonia volatilization due to high porosity. This result may be because a higher water content (65% of MC) might have a negative effect on the oxygen supply,and it further affected the ammonification and nitrification during composting (Guardiaet al.2008).

Germination indexThe GI based on the seed germination rate and root elongation is determined in a plate assay,and it is a sensitive indicator of composting maturity and phytotoxicity (Tiquiaet al.1996). The changes of GI in all treatments are shown in Appendix B. The leaching solution of the composting material showed strong biological toxicity during early composting. The GIs of all the treatments had minimum values of 10.0% (LMC),1.3% (MMC),and 8.6% (HMC) on day 1 of composting. This observation could be explained by the generation of low molecular weight and short-chain volatile fatty acids (primarily acetic acid) and ammonia. The GIs in all the treatments gradually increased with the degradation of the OTC and the decomposition of the toxic materials. The GI values were 18.1% (LMC),45.1% (MMC),and 77.4% (HMC) on day 17. Significant differences were found in the GI values among the treatments. A GI value greater than 50% indicates that the compost has almost no toxicity,and a GI value of greater than 80% indicates that the composting products are not phytotoxic and mature (Zucconiet al.1981). At the end of composting,the GIs in the three treatments were 65.3% (LMC),70.3% (MMC),and 89.8% (HMC),which showed that the process for the manure-straw compost containing OTC had been basically finished and the biological toxicity displayed a clear decrease. Among the treatments,the GI in the treatment with 65% MC was significantly higher than those of the 45 and 55% MC treatments. The increased GI was related to the degradation of OTC,the volatilization of ammonia and the decomposition of small molecule organic acids. Overall,composting at an initial moisture content from 55-65% is more conducive to OTC degradation and the decline of biological toxicity.

13C-NMR spectral characteristics during compostingHumic acid and fulvic acid are formed during the aerobic composting of chicken faeces,especially at the later stage of composting (Shiet al.2018). The humic acid content and the complicated nature of its structure are the important decisive factors in assessing the quality and maturity of compost products because humic acid has a very important influence on the soil physical and chemical properties and its biological characteristics,which can prevent nutrients from escaping soils and can also promote crop growth and development (Tahiret al.2011). As a non-destructive method,nuclear magnetic resonance (NMR) technology is a very effective means for illustrating the structure of humic substances (Lambert and Buddrus 2015). It can provide information on the humic functional groups and the transformation of organic ingredients during composting (Caricasoleet al.2010). Because of the various distribution ranges of carbon chemical shifts with different hybrid modes,the specific structural unit can be derived by one or more of the resonance absorption peaks. The13C-NMR spectra were divided into the five functional groups shown in Fig.4,namely,alkyl C (0-50 ppm),alkoxy C (50-112 ppm) such as sugars,alcohols,esters and amines composed of C-O and C-N,aromatic C (112-163 ppm) including fat carbon and phenol class carbon,carboxyl and amide C (163-190 ppm) and carbonyl C (190-215 ppm). The intensity distributions of the chemical shift groups in the respective chicken faeces composting processing are summarized in Appendix C,which shows the changes in the spectral characteristics of the LMC,MMC,and HMC treatments.

Fig.4 13C-NMR spectra of the samples at day 1 (A),day 17 (B),and day 42 (C) of composting. HMC,high moisture content (65%);MMC,middle moisture content (55%);LMC,low moisture content (45%). The spectra show the five functional groups over different chemical shifts (ppm),including alkyl C (0-50 ppm),alkoxy C (50-112 ppm),aromatic C (112-163 ppm),carboxyl and amide C (163-190 ppm) and carbonyl C (190-215 ppm).

The intensity distributions of C-containing functional groups were largely consistent in the three treatments during the different composting stages. Among them,the highest content was of alkoxy C (65.0-72.1%) followed by alkyl C (12.3-21.9%),aromatic C (6.3-10.1%),carboxyl and amide C (4.8-6.9%) and carbonyl C (0.9-1.9%).During composting,the relatively higher contents of alkyl C and alkoxy C,which are primary the aliphatic and carbohydrate structures (aliphatic C),indicated that organic carbon was present in the forms of fat,carbohydrates and polysaccharides. Compared with the intensity distributions at the beginning of the composting process,the contents of the alkyl C in the three treatments at the end of composting were decreased by 40.9% (LMC),16.7% (MMC),and 19.28% (HMC). The alkoxy C contents in the three treatments were increased by 6.8% (LMC),8.6% (MMC),and 2.3% (HMC). However,no significant differences were found in the aliphatic C contents among the three treatments.Fuenteset al.(2007) showed the opposite results,in which the alkyl C increased and the alkoxy C decreased during composting without OTC,and aliphatic materials were transformed through intense microbial activities into more stable humic compounds. Our previous results showed the microbial degradation pathway of OTC,which indicated that the OTC (m/z=461,C22H24N2O9) was transformed into its product (m/z=463,C22H26N2O9) by hydrogenase (Qiet al.2019). A portion of the hydrogen ions might be derived from paraffin. Therefore,it is deduced that OTC could affect the internal transformation of aliphatic substances and weaken the reduction reaction of the aliphatics,thus inhibiting the decomposition of aliphatic substances to stable humic compounds (Amiret al.2004). With regard to the aromatic C,the LMC and HMC contents increased by 53.0 and 30.2%,respectively,and the aromaticity also increased;while in the MMC,the content decreased by 6.0% and the aromaticity also decreased at the end of the composting process. Bernalet al.(2009) noted that the humification of the organic matter during composting is related to the increasing aromatic characteristics. Castaldiet al.(2005) reported that the aliphatic C was reduced by the transformation of the polysaccharides to other oxygenated compounds,particularly carboxylic and ester groups,with an increase of aromatic structures during composting.The decline of aromatic C in MMC may be due to the increase of the microbes that could degrade OTC under moderate MC and the decrease of microbes facilitating the formation of aromatic compounds. Additionally,combined with the GI results for the three treatments,it is concluded that the aromatic substances cannot be used as the sole criterion for maturity. The carboxyl and amide C contents (163-190 ppm) in all the treatments decreased during the high-temperature stage and then increased at the curing stage,which indicated that the organic matter such as lipids containing carboxyl and amide C in the initial materials was decomposed into small molecule substances under the high temperature (Almendroset al.2000),and the conversion of organic-N to inorganic-N caused the volatilization of ammonia gas. At the later composting,the carboxyl and amide C contents in HMC displayed a larger increase than in the LMC and MMC treatments. These results could indicate that a higher water content contributes to increased amides and is conducive to reducing nitrogen loss due to the fixation of nitrogen in emerging organic matter during composting. Overall,the presence and microbial degradation of OTC is not conducive to the humification of compost.

Correlations among OTC,substance transformationand physical-chemical conditionsAccording to the RDA analysis shown in Fig.5,variations in the occurrence of OTC and physical-chemical conditions could explain 96.59% of the variance in the transformation of organic materials. The positive and negative correlations among OTC residues,organic material transformation and the physical-chemical conditions of the composting process were determined based on the Pearson correlation statistics (Appendix D),which were supported by the acute or obtuse angles among them as shown in Fig.5. The OTC concentration was positively correlated with TOC,TN and NH4+-N,indicating that microorganismal activities play a major role in OTC degradation. However,the OTC was negatively correlated with the pH and NO3--N (P≤0.01). OTC has a series of functional groups (e.g.,amines,carboxylics and phenolics) that exhibit different species. There are four species (H3OTC+,H2OTC±,HOTC-and OTC2-) of OTC under different pH values (Chenget al.2018). Adriana and Michael (2000) stated that neutral and high pH values were significantly adverse to OTC stability. Due to the changes in the pH during the entire composting process (7.39-9.16),the OTC was predominantly present as the anionic forms rather than the cationic species (Liet al.2019). In addition,both OTC and pH showed significant correlations with the transformations of alkyl C and alkoxy C,e.g.,OTC with alkyl C (r=0.787,P≤0.05),OTC with alkoxy C (r=-0.784,P≤0.05),pH with alkyl C (r=-0.856,P≤0.01) and pH with alkoxy C (r=-0.834,P≤0.01). It was deduced that the composting pile pH affected the distribution of the functional groups and thus influenced the OTC,alkyl C and alkoxy C levels. The TOC,NH4+-N and NO3--N had significant correlations with the alkyl C,but not with the alkoxy C. In general,the occurrence of OTC affected the transformation of alkyl C and alkoxy C due to the change in pH. The TOC and inorganic N (NH4+-N and NO3--N) were the most important physical-chemical factors during the composting process.

Fig.5 Redundancy analysis (RDA) of the relationships between physico-chemical conditions,i.e.,the pH,EC,total organic carbon (TOC),total Kjeldahl nitrogen (TKN),NH4+-N,NO3--N,and oxytetracycline (OTC),and the organic material transformation during chicken manure composting spiked with the OTC. LMC,low moisture content (45%);MMC,middle moisture content (55%);HMC,high moisture content (65%).The triangle,star and circle symbols denote data obtained from adding OTC at low,middle and high moisture contents,respectively. Orange,green and purple denote data collected on days 1,17,and 42,respectively.

4.Conclusion

Having a moderate moisture content (55-65%) is more beneficial to OTC degradation. The moisture content of the composting materials affected the pile temperature,pH and EC during the composting process. The high moisture content accelerated the increases of temperature and pH,and led to a lower EC. Having an initial moisture content between 55-65% favours the reduction of extractable biological toxicity,the degradation of the OTC and the maturity of the compost containing OTC during manurestraw composting. The13C-NMR spectra showed that the presence and microbial degradation of OTC is not conducive to the humification of compost.

Acknowledgements

This study was jointly supported by the National Key R&D Program of China (2018YFD0500206),the National Natural Science Foundation of China (31772395),the Fundamental Research Funds for Central Non-profit Scientific Institutions,China (1610132019046),and the Science and Technology Support Project of Langfang,Hebei,China (2019013125).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm