Effect of lignite amendment on carbon and nitrogen mineralization from raw and composted manure during incubation with soil

Robert IMPRAIM,Anthony WEATHERLEY,Deli CHEN and Helen SUTER

Schoolof Agriculture andFood,Facultyof VeterinaryandAgriculturalSciences.The Universityof Melbourne,Parkville,Victoria 3010(Australia)

ABSTRACT The application of animal manure as a source of plant nutrients requires the determination of the amount and pattern of nutrient mineralization from manure.A laboratory incubation study was conducted to investigate the influence of lignite amendment and lignite type on carbon(C)and nitrogen(N)mineralization in raw (feedstock) and composted cattle manure following application to soil at 30 and 60 t ha-1. The mineralization of C and N was determined by measuring changes in CO2 evolution and mineral N(NH+4 -N+NO-3 -N)over 40 d.The results showed that lignite amendment suppressed the amount of manure C mineralized in both feedstock and compost,with the effect being more pronounced in the compost.Over the 40-d incubation,the percentage of applied C mineralized was 26.4%–27.8%and 16.3%–21.4%in unamended and lignite-amended feedstocks,respectively.The corresponding C mineralized in the composts was 12.4%–14.1%and 3.5%–6.5%.Lignite had no significant effect on the net N mineralized in compost(4.8%–6.7%and 2.5%–7.8%in unamended and lignite-amended composts,respectively).Lignite had either no effect or increased the net N mineralized in feedstock(from 3.2%–8.7%without lignite to 10.4%–13.5%)depending on the type of lignite used.This study suggests that using lignite-amended manure,especially when composted,has the potential to build up soil organic C without limiting the availability of mineral N.

KeyWords: CO2 evolution,manure mineralization,mineral N,nitrification,soil incubation

INTRODUCTION

Animal manure,raw or composted,is a valuable source of organic matter to soil and nutrients to plants;therefore,its application to improve soil quality and crop yield remains a common agricultural practice(Bernalet al.,1998;Risseet al.,2006;Abbasiet al.,2007).To meet the nutrient requirements of a crop through manure application and minimize the associated environmental risk(e.g.,leaching of excess nitrate(NO-3)),the rate and timing of manure application should be based on the expected nutrient mineralization from the manure(Hartzet al.,2000;Eghballet al.,2002;Qian and Schoenau,2002).

Following the application of manure to soil, the mineralization of manure nutrients(e.g.,nitrogen(N))occurs through microbially mediated processes,which are affected by temperature,soil properties,and manure characteristics(Eghballet al., 2002; Qiuet al., 2008; Azeez and Van Averbeke, 2010). Aulakhet al. (2000) reported that the amount of manure N mineralized is inversely related to the carbon(C):N ratio and directly related to the N content of the manure.According to Chadwicket al.(2000),the C:organic N ratio of manure accounted for 40%of the variation in N mineralized in contrasting manure types.The C:ammonium(NH+4)ratio of manure has also been reported to correlate with the rate of nitrification and the amount of NO-3accumulated when applied to soil (Griffinet al., 2005). In addition,the composting of manure alters its characteristics and influences the pattern and rate of nutrient mineralization(Eghballet al.,2002).Usually,composted manure has lower C and N mineralization rates than raw manure because of the conversion of labile N to inorganic N, as well as the loss of labile forms of C as carbon dioxide(CO2)and the stabilization of organic matter during the composting process(Hartzet al.,2000;Eghballet al.,2002;Bernalet al.,2009).

Lignite (brown coal) is a product of the early stages of coalification and intermediate between peat and subbituminous coal(Závodská and Lesný,2006).Owing to its unique properties, such as high cation exchange capacity(CEC),high C content,high pH buffering capacity,and low pH, the use of lignite to suppress N loss during manure management is considered a viable technique.In fresh cattle manure,lignite was able to suppress ammonia(NH3)loss by 30%–66% when applied to pen surfaces at a rate of 3–6 kg dry lignite m-2(Chenet al., 2015; Sunet al.,2016).Danaet al.(2020)reported a 47%–91%reduction in NH3loss from broiler litter following the application of 10%–30%lignite.During composting,lignite was able to reduce NH3emissions from cattle manure by 54%(Baiet al., 2020). In a recent study (Impraimet al., 2020),we observed that manure amended with 20%lignite had a lower microbial activity(indicated by lower CO2evolution),compared with unamended manure,during composting.In addition,Caoet al.(2020)reported that composted ligniteamended manure(lignite rate of 5%–15%)contained 25%more total N and 65%more mineral N(NH+4-N+NO-3-N)than unamended manure compost.

As lignite,at application rates mentioned above,alters the properties of manure,it is expected that mineralization of C and N in lignite-amended manure will differ from that in unamended manure when applied to soil.Therefore,it is important to assess the influence of lignite amendment on manure mineralization to understand how lignite-amended manure is likely to meet the N requirements of a crop.Further,there is a need to establish manure-specific fertilizer recommendation rates that are adequate for crop nutrient requirements without adverse environmental impacts(Azeez and Van Averbeke,2010).Very few studies have reported on the effect of lignite on C and N mineralization rates when applied to soil.Schefeet al.(2008)and Tranet al.(2015)both reported an inhibition of C mineralization(measured as CO2emission from microbial respiration)following lignite application to soil.Lignite inhibition of microbial respiration has been suggested to occur through the release of substances that are toxic to microbes,physical protection of substrates,or substrate adsorption (Whiteley and Pettit, 1994; Tranet al.,2015).For N mineralization in soil,Tranet al.(2015)observed that the effect of lignite was minor and inconsistent.The authors observed that lignite had no effect on NH+4-N and NO-3-N contents,although lower levels of extractable NH+4-N in the lignite-treated soil were observed at the initial stage of incubation,whereas a decrease in the availability of NO-3-N was observed at the later stage of incubation.Although some studies have reported on the effect of lignite on C and N transformations in soils, they only examined the direct application of lignite to soil.It remains unknown how the amendment of manure with lignite affects C and N mineralization from manure when applied to soil. In addition, it is unknown whether the effect of lignite on C and N mineralization from raw manure differs from that of composted manure. As lignite is increasingly being used(e.g., up to 60 t dry lignite ha-1in cattle feedlot pens)to mitigate N loss from both raw and composted manures(Chenet al.,2015;Sunet al.,2016;Baiet al.,2020),it is necessary to examine the effect of lignite amendment on the C and N mineralization patterns of manure that is often applied to agricultural lands.

The objective of this study was to assess the impact of lignite amendment on the C and N mineralization dynamics of raw and composted cattle manure under incubation with soil in the laboratory. We hypothesized that lignite amendment of manure would suppress the mineralization of manure C and N due to the lignite’s ability to inhibit microbial activity.Two application rates,30 and 60 t ha-1,within the range of organic amendment application rates generally used by farmers,were assessed.Depending on the soil and crop,manure and compost application rates generally range between up to 30 t ha-1for broadacre agriculture and up to 145 t ha-1(with 56 t ha-1being the recommended rate)for horticultural systems(Paulin and O’Malley,2008;Quilty and Cattle,2011).

MATERIALS AND METHODS

Soil,lignite,feedstock,andcompost used

The soil used for the incubation was a clay loam(Mottled-Mesonatric Red Sodosol),collected from the top 20 cm of a pasture field in Victoria,Australia.The soil was air-dried and sifted with a 2-mm sieve prior to use in the study.The manure(M)was collected from a commercial cattle feedlot pen located in Victoria,Australia.Two types of lignites,Bacchus Marsh(BM)and Loy Yang(LY),were sourced from different open-cut mines in Victoria.A previous characterization study (Impraim, 2020) involving five different Victorian lignites showed that the BM and LY lignites differed in their capacity for N retention, especially at their inherent pH.In addition,these lignites differed in their ability to inhibit microbial activity during composting(Impraimet al.,2020).Lignite-amended feedstocks(raw material for composting)were prepared by mixing separate samples of manure with BM lignite(manure+BM)and LY lignite(manure+LY)so that the proportion of lignite in the manure-lignite mix was 20%(equivalent to 45 t dry lignite ha-1),which was identified by Chenet al.(2015)and Sunet al.(2016)to be a lignite rate suitable for N retention in feedlot cattle manure.A third feedstock,comprising manure alone,served as the control.These previous studies involved the application of lignite to the surfaces of pens prior to the introduction of cattle.This enabled the lignite to retain the N excreted predominantly in the form of urinary urea by the cattle.As the manure collected for this incubation study was not freshly excreted,a large proportion of the excreted N would have already been lost as NH3.Thus,to simulate the generation of lignite-amended manure from a feedlot,as well as allowing for N retention by the lignite, urea solution (ca. 60 g urea kg-1dry manure)was added to all feedstocks at 2-d intervals for a period of 10 d. The quantity of urea added was based on the difference between the expected N content(50 g kg-1)in freshly excreted manure(Wattset al.,2011)and the present N content(21.3 g kg-1)of the manure.The feedstock was mixed after each addition of urea to ensure uniform distribution and was collected 4 d after the final urea addition.Afterwards,a portion of each feedstock was composted,while the rest remained non-composted.In brief,the composting process, which lasted for 21 d, involved adjusting the initial moisture content of the feedstock to 550 g kg-1and then loading it into the composting vessels,at 7 kg dry matter per vessel,with a forced aeration system.Composting was accomplished without adjusting the initial C:N ratio of the feedstock,similar to typical manure composting in the cattle feedlot where the manure was collected.Details of the composting methodology have been reported by Impraimet al. (2020). The chemical properties of the soil,raw materials used in feedstock preparation,feedstocks,and composts are presented in Table I.

Incubation experiment

Twenty grams of dry soil were weighed into 250-mL plastic vials and the water-filled pore space (WFPS) was brought to 60%with MilliQ water.The volume of water(Vw,cm3)required to attain 60%WFPS was calculated according to Adviento-Borbeet al.(2006):

where WFPS is in the targeted value of 60%,BDtotis the bulk density of soil or soil plus amendment(g cm-3),2.65 is the specific density of the soil(g cm-3),andmtotis the mass of soil or soil plus amendment to be incubated(g).

The vials were then pre-incubated in the dark in an incubator at 25°C for 7 d to overcome the Birch effect(Planteet al.,2011).This was necessary to avoid an increase in microbial activity and nutrient transformations following soil wetting, which may override the treatment effect. At the end of the pre-incubation period,the soil(20 g)in each vial was completely mixed with the feedstocks or composts(M alone and incombination with BM/LY)at two rates(258 and 516 mg feedstock or compost per vial),equivalent to 30 and 60 t ha-1, respectively, as the treatments M30, M60,MBM30,MBM60,MLY30,and MLY60,and soil with no added feedstock or compost(unamended soil)served as a control (Table II). The vials were then capped (lids were placed over the vials without tightening)to restrict moisture loss while allowing for aeration and returned to the incubator.

TABLE II Treatments set up in the laboratory soil incubation study

Throughout the pre-incubation and incubation period,60% WFPS in the soil was maintained by replenishingwith MilliQ water as required, based on the mass loss measurements of the incubated sample,twice per week.A jar of MilliQ water was kept in the incubator to maintain a humid environment to further minimize moisture loss from the incubated soils.The vials were uncapped and fanned twice a week to maintain aerobic condition.To allow for destructive sampling,there were a total of 448 incubated vials(i.e.,32 per treatment,including control,with four replicates)for the eight sampling intervals.On days 0(within 30 min of treatment application),1,3,6,10,20,30,and 40 of incubation,four replicates of each treatment were destructively sampled and analyzed for mineral N as a measure of N mineralization.Carbon mineralization was determined by measuring CO2evolution from the samples(Bernalet al.,1998)at the same sampling intervals as those for mineral N.

TABLE I Chemical properties of the soil,raw materials,feedstocks(after urea addition),and composts used in the laboratory soil incubation study

Sample analysis andmeasurement of C evolution

Soil,feedstock,and compost samples were analyzed for pH and electrical conductivity(EC)using a pH/EC meter(smartCHEM-LAB,Australia)at a sample-to-water ratio of 1:5(weight/volume).Total C and total N were analyzed using a LECO TruMac CN Elemental Analyzer (LECO, USA).Organic matter was determined after drying the samples at 105°C for 24 h in an oven and ashing at 550°C for a further 24 h in a muffle furnace (Awasthiet al., 2015).Mineral N was analyzed using a Skalar SAN++segmented flow analyzer(Skalar,Netherlands)following extraction of the samples with 2 mol L-1KCl at a sample-to-extract ratio of 1:5(weight/volume)(Rayment and Lyons,2011).

For CO2evolution measurement, gas samples were collected from the headspace of three sets of incubated vials(per treatment)using a polypropylene syringe into a 12-mL exetainer vial (Gagnonet al., 2016). The lids of this set of incubated vials were fitted with a gas sampling system comprising a metal port,Teflon tubing,a three-way valve,and a needle.Prior to gas sampling,the lids of the incubated vials were removed and fanned for 30 s to remove any gas that might have accumulated in the headspace of the vial.During gas sampling,with the lids of the vials tightly fitted,20 mL of headspace was drawn with a 25-mL syringe connected to the gas fittings into the pre-evacuated 12-mL exetainer vial. The headspace gas was homogenized by performing five pumping movements with the syringe before drawing the gas.On each day of sampling,a background gas sample was collected (time zero) and three samples were taken from the vials after capping them at 1-h intervals to allow for flux calculations. After gas sampling, the lids of the vials were removed and the vial headspace was replaced with background air before the lids were placed over the vials(without tightening)and the vials were returned to the incubator.The gas samples were analyzed for CO2using an Agilent 7890 A gas chromatograph(Agilent,USA)with a thermal conductivity detector.

Calculations

whereFt1andFt2are theFtat timest1 andt2,respectively.

Statisticalanalysis

A one-way analysis of variance(ANOVA)test was performed on the initial properties of the feedstocks and composts. To compare the main effects (lignite, composting,application rate, and time) and their interactions with the measured parameters during the incubation study,a four-way ANOVA was performed.Where necessary,analysis was performed on log-transformed data,i.e.,log10(x+0.5),to meet the ANOVA assumptions.If the ANOVA showed a significantPvalue(＜0.05),treatment means were separated using Tukey’s honestly significant difference test. To assess the effect of lignite on manure mineralization,statistical analysis and multiple comparisons were limited to soils amended with feedstock and compost,with and without lignite.All analyses were performed using GenStat 16th edition(VSN International,UK).RESULTS

Carbon mineralization during incubation

The C mineralization rate, measured as CO2flux, remained relatively low (＜38 mg C kg-1d-1) in the unamended soil (control) throughout the incubation period(Fig.1).In soils amended with feedstock or compost,there was a decrease in CO2flux between days 0 and 1. Afterwards,the CO2flux increased until day 3 and then decreased gradually over the remainder of the incubation period.In all treatments,the highest C mineralization occurred during the first 10 d of the incubation period(64%–79%of the mineralized C in the feedstocks and 46%–72%in the composts),with peak mineralization occurring on day 3 of incubation.The presence of lignite did not affect the pattern of change in CO2flux in either the feedstocks or composts.

Fig. 1 Rate (CO2 flux) and cumulative amount of C mineralized in soil amended with feedstock or compost (the corresponding feedstock after 21-d composting)during the 40-d incubation experiment.Error bars represent standard errors of means(n =3).CK=control with no amendment;M30=manure applied at 30 t ha-1 (i.e.,12.9 g kg-1 soil);MBM30=manure-Bacchus Marsh lignite mixture applied at 30 t ha-1;MLY30=manure-Loy Yang lignite mixture applied at 30 t ha-1;M60=manure applied at 60 t ha-1 (i.e.,25.8 g kg-1 soil);MBM60=manure-Bacchus Marsh lignite mixture applied at 60 t ha-1;MLY60=manure-Loy Yang lignite mixture applied at 60 t ha-1.For the manure-lignite mixture,the proportion of lignite(Bacchus Marsh or Loy Yang)was 20%by weight(equivalent to 45 t dry lignite ha-1).

The cumulative amount of mineralized C was higher in the feedstocks than in the composts(Fig.1).In both feedstocks and composts, the presence of lignite significantly(degree of freedom(df)=2,P=0.04)inhibited the mineralization of applied C,with the lignite inhibitory effect being more pronounced in the composted manure.Over the 40-d incubation period,the percentages of C mineralized from the M30,MBM30,and MLY30 feedstocks were 26.4%,21.4%,and 17.3%of the applied total C,whereas the percentages of C mineralized from the corresponding composts were 14.1%,6.5%,and 3.5%,respectively.For the M60,MBM60,and MLY60 feedstocks,the percentages of C mineralized were 27.8%,20.4%,and 16.3%of the applied total C,whereas the percentages of C mineralized from the corresponding composts were 12.4%,5.9%,and 4.9%,respectively.For a same feedstock,the rate of application did not significantly influence the percentage of applied C mineralized.A similar observation was made in the composts.For both the feedstocks and composts,at 30 or 60 t ha-1,the magnitude of cumulative percentage of the applied C mineralized was in the order of M＞MBM＞MLY.

Nitrogen mineralization during incubation

(df=2,P ＜0.01), rate(df=1,P ＜0.01), composting

There was negligible NO-3-N in all the feedstocks and composts used for the incubation(Table I).Nitrification of NH+4in both the feedstock-and compost-amended soils led to NO-3accumulation over the incubation period(Fig.2).The ANOVA test showed that net nitrification was significantly(df=6,P ＜0.01)affected by incubation time.Similar to the reduction in NH+4-N content,the net nitrification in all treatments increased rapidly for the first 20 d of incubation compared with the rest of the incubation period.For each application rate, feedstock and compost with lignite had higher net nitrification,especially after 10 d of incubation,though non-significantly(df=6,P=0.077).At the higher rate of application of the lignite-amended composts (i.e.,MBM60 and MLY60),there was net negative nitrification for the first 3 d of incubation.

Fig.2 NH+4 -N content and net nitrification in soil amended with feedstock or compost(the corresponding feedstock after 21-d composting)during the 40-d incubation experiment.Error bars represent standard errors of means(n=4).CK=control with no amendment;M30=manure applied at 30 t ha-1 (i.e.,12.9 g kg-1 soil);MBM30=manure-Bacchus Marsh lignite mixture applied at 30 t ha-1;MLY30=manure-Loy Yang lignite mixture applied at 30 t ha-1;M60=manure applied at 60 t ha-1 (i.e.,25.8 g kg-1 soil);MBM60=manure-Bacchus Marsh lignite mixture applied at 60 t ha-1;MLY60=manure-Loy Yang lignite mixture applied at 60 t ha-1.For the manure-lignite mixture,the proportion of lignite(Bacchus Marsh or Loy Yang)was 20%by weight(equivalent to 45 t dry lignite ha-1).

Net N mineralization.In both feedstock-and compostamended soils,there was an initial decrease in the net organic N mineralized before a general increase for the rest of the incubation period(Fig.3).Relative to the feedstocks,the period of decline was shorter for the composts.The decrease in organic N mineralized in the MBM60 and MLY60 feedstocks continued until day 10 and 20,respectively,compared with day 6 in the M60 treatment.The M30 and M60 feedstocks had negative net organic N mineralized values between days 3 and 10.This coincided with high CO2evolution during the same period(Fig.1).In the composts,the net organic N mineralized decreased until day 3 in all treatments except in M60 where the decrease continued until day 6 and in MLY60 where the net N mineralized increased throughout the incubation period.Similar to the feedstocks,the decrease in net organic N mineralized in the composts corresponded with an increase in CO2evolution on day 3(Fig.1),with the subsequent drop in CO2evolution coinciding with increased net organic N mineralized.Compared with the CO2evolution,which consistently decreased from day 3 until the end of the incubation period,the changes in net organic N mineralized fluctuated with incubation time.

Fig. 4 Percentage of net organic N mineralized in soil amended with feedstock or compost(the corresponding feedstock after 21-d composting)during the 40-d incubation experiment.Error bars represent standard errors of means(n=4).Columns with the same letter(s)(across all treatments)are not significantly different(P ＞0.05)according to Tukey’s test.M30=manure applied at 30 t ha-1 (i.e.,12.9 g kg-1 soil);MBM30=manure-Bacchus Marsh lignite mixture applied at 30 t ha-1;MLY30=manure-Loy Yang lignite mixture applied at 30 t ha-1;M60=manure applied at 60 t ha-1 (i.e.,25.8 g kg-1 soil);MBM60=manure-Bacchus Marsh lignite mixture applied at 60 t ha-1;MLY60=manure-Loy Yang lignite mixture applied at 60 t ha-1. For the manure-lignite mixture, the proportion of lignite(Bacchus Marsh or Loy Yang)was 20%by weight(equivalent to 45 t dry lignite ha-1).

Over the 40-d incubation,there was a net mineralization of N(gross mineralization exceeding gross immobilization,leading to net release of N)in all treatments(Fig.4).The percentages of applied organic N mineralized were higher in the feedstocks than in the corresponding composts,and higher at lower application rates than at higher rates.Lignite had a mixed effect on N mineralization from manure.For MLY feedstock,at 30 and 60 t ha-1,the N mineralized was significantly higher(10.4%–13.5%)than that in the MBM(4.1%–9.8%)and M(3.2%–8.7%)feedstocks.However,this LY lignite effect diminished in the composts,especially at the lower rate of application.The presence of lignite in the compost did not significantly affect the percentage of applied N mineralized over the 40-d incubation period.The initial organic N contents in the manure and lignite treatments(feedstock or compost)were similar:20.8±0.2,19.8±0.2,and 18.9±0.4 g kg-1in the M,MBM,and MLY feedstocks,with corresponding contents in the composts being 22.6±0.1,22.3±1.0,and 22.8±0.4 g kg-1,respectively.

Fig.3 Net organic N mineralized in soil amended with feedstock or compost(the corresponding feedstock after 21-d composting)during the 40-d incubation experiment.Error bars represent standard errors of means(n=4).M30=manure applied at 30 t ha-1 (i.e.,12.9 g kg-1 soil);MBM30=manure-Bacchus Marsh lignite mixture applied at 30 t ha-1;MLY30=manure-Loy Yang lignite mixture applied at 30 t ha-1;M60=manure applied at 60 t ha-1 (i.e.,25.8 g kg-1 soil);MBM60=manure-Bacchus Marsh lignite mixture applied at 60 t ha-1;MLY60=manure-Loy Yang lignite mixture applied at 60 t ha-1.For the manure-lignite mixture,the proportion of lignite(Bacchus Marsh or Loy Yang)was 20%by weight(equivalent to 45 t dry lignite ha-1).

DISCUSSION

The high C mineralization(CO2evolution)at the initial stage of incubation was likely a result of the onset of increased microbial growth and activity owing to the readily degradable C in the feedstocks and composts(Limet al.,2012).The increased microbial activity following the application of organic matter to soil may stimulate the decomposition of indigenous soil organic matter(priming effect)(Bernalet al., 1998), therefore contributing to the observed CO2evolved by the amended soil.However,the amendment of soil with feedstock or compost was assumed to have a negligible priming effect on soil organic matter decomposition(Marstorp and Kirchmann,1991;Van Kessel and Reeves,2002).The high C mineralization at the onset of incubation coincided with a decline in mineralized N in both feedstockand compost-amended soils, most likely due to microbial immobilization of the mineral N.The subsequent decrease in CO2evolution with incubation time can be attributed to the depletion of readily degradable C(Bernalet al.,1998).The lower C mineralization in the compost-amended soils,compared with the feedstocks,could be attributed to a more stabilized C fraction in the compost (Hartzet al., 2000;Cambardellaet al.,2003).During the composting process,the labile C pool is lost as CO2and the remaining C is transformed into a more stable form,which is less amenable to microbial mineralization(Eghballet al.,2002).Although the presence of lignite in both feedstocks and composts did not alter the pattern of change in CO2evolution,all treatments with lignite had low amounts of CO2evolved over the incubation period.The C mineralized(expressed as a percentage of the applied C)in the feedstocks with no lignite was 6.4–11.5 percentage points higher than that of feedstocks with lignite.In addition,C mineralization in the composts with no lignite was 7.6–10.6 percentage points higher than that in the composts with lignite.The bioavailability of lignite C is relatively low(Chabbiet al.,2006;Yoonet al.,2016).Therefore,most of the CO2evolved from the lignite-amended feedstocks and composts was expected to come from manure C. Hence,the inhibitory effect of lignite on manure C mineralization may be less than that observed,as lignite constituted 20%of the manure-lignite mix.In the feedstocks,there was only a minimal reduction in C mineralized in the MLY treatments(0.4%less than that in unamended feedstock),whereas in the MBM treatments,there was an increase(4.5%–5.7%)after standardizing for the amount of lignite present (i.e.,expressing the C mineralized as a percentage of the applied manure C).Standardizing the amount of lignite present in the compost,however,still showed that lignite significantly inhibited the mineralization of manure C(2.7–8.1 percentage points lower in composts with lignite).Lignite’s capacity to reduce CO2evolution in amended soils is consistent with the findings of Schefeet al.(2008)and Tranet al.(2015).The findings of this study show that lignite has the capacity to inhibit microbial activity either when applied directly(as reported in previous studies)or when mixed with manure(raw or composted)before being applied to the soil.

The percentages of applied C mineralized(16.3%–27.8%for all feedstocks and 3.5%–14.1%for all composts)were within the ranges of values reported in the literature.Antilet al.(2011)reported 11%–16%mineralization of applied composted cattle manure C over a 168-d incubation period.Limet al.(2012)reported 1.6%–11.4%of C mineralized in composted poultry,swine,and cattle manures over a period of 100 d. A range of 12%–40% of the applied organic C from poultry manure was mineralized over a period of 60 d(Martínet al.,2012).In a 24-week incubation,Hartzet al.(2000) reported that 35% of manure C was mineralized,whereas only 14%of composted manure C was mineralized.In a study by Hébertet al. (1991), 1%–8.2% of the total applied C from composted sheep,pig,and cattle manures was mineralized after 28 d of incubation.

Owing to the relatively stabilized organic matter of composts,their application is preferred to raw feedstocks with regard to building up soil organic matter (Bernalet al.,1998).This study suggests that,as a soil amendment,ligniteamended manure(especially when composted)has the potential to build up soil organic matter reserves, and thus improve long-term soil health owing to lignite’s ability to suppress C mineralization.

Relative to the unamended feedstock and compost,the high initial NH+4-N content in the lignite-amended feedstocks and composts can be attributed to the ability of lignite to retain NH+4-N in both raw manure and during manure composting(Chenet al.,2015;Sunet al.,2016;Caoet al.,2020).As incubation progresses,the reduction in NH+4-N has been attributed to processes such as nitrification,biological immobilization,NH3volatilization under alkaline pH condition,and denitrification under predominantly anaerobic condition (Bernal and Kirchmann, 1992; Azeez and Van Averbeke,2010;Moreno-Cornejoet al.,2014).In this study,the loss of NH+4through NH3volatilization was expected to be insignificant given the acidic pH of the soil(pHH2O=6.4,pHCaCl2=5.4),the low amount of amendment(pHH2O=7.6–8.2) used relative to the soil (0.26–0.52 g in 20 g of soil), and the static nature of the incubation experiment.The incubation vials were expected to remain predominantly aerobic over the incubation period,as the vials were aerated twice a week and kept at 60% WFPS, so the NH+4loss through denitrification(as gaseous NO and N2O)from NO-3produced from nitrification was expected to be insignificant as well.The production of N2O increases significantly with the depletion of soil oxygen, and a moisture content in the range of 70%–80% WFPS is considered optimum for N2O emission (Butterbach-Bahlet al., 2013). Hence, the observed reduction in NH+4content over the incubation period was mainly due to the nitrification of NH+4,leading to the accumulation of NO-3over the same period.Because of the higher initial NH+4content in the lignite treatments,providing more substrate for nitrification,the accumulated NO-3over the incubation period was also higher in the lignite treatments than in the control. In all treatments,especially from day 10 onwards,the decrease in NH+4content was less than the corresponding increase in NO-3content.This suggests that mineralized N was continuously nitrified without changes in NH+4content(Bernal and Kirchmann,1992).The net nitrification remained positive throughout the incubation period,except in the lignite-amended composts at 60 t ha-1, where there were negative values for days 1 and 3.Although increased net nitrification(accumulation of NO-3)is important for plant growth,NO-3can also leach into groundwater or denitrify to gaseous N2and N2O,leading to environmental pollution and health risk. The negative NO-3-N values observed at the start of incubation could be indicative of net immobilization of the initial NO-3-N inherently present or produced in the soil(as there was negligible NO-3-N in both the initial feedstocks and composts used for incubation)(Moreno-Cornejoet al.,2014).However,according to Calderónet al.(2004,2005),a negative NO-3-N value is not necessarily equivalent to immobilization,as it could also be due to denitrification occurring in anaerobic microsites in aerobic microcosms.

Composting of the manures,although necessary for destroying pathogens and improving the handling of the initial material (e.g., haulage and off-site application), reduces manure N mineralization owing to the conversion of labile organic matter into a more stable form(Hartzet al.,2000;Bernalet al.,2017).During this process,easily mineralizable N is transformed into inorganic forms with the remaining organic N less subject to mineralization (Eghball, 2000;Eghballet al., 2002). In this study, the percentage of applied organic N mineralized in the feedstock-amended soils(3.2%–13.5%)was higher than that in the compost-amended soils (2.5%–7.8%). The net N mineralization in all treatments could be attributed to the low C:N ratio(11:1–13:1)of the feedstocks and composts. A C:N ratio of less than 20 usually leads to net mineralization(Ralebitso-Senior and Orr,2016).Although lignite inhibited the mineralization of C from both feedstock and composts,the effect of lignite on the net organic N mineralized over 40 d was inconsistent.While lignite had no significant effect on the net organic N mineralized in the compost,the feedstocks containing LY(i.e.,both MLY30 and MLY60)had significantly higher net organic N mineralization.The net N mineralized was also higher,though non-significantly,in the MBM30 feedstock relative to the unamended feedstock.It is unclear why LY resulted in higher net N mineralization than BM. Further studies comparing BM and LY lignites for their effects on net N mineralization are recommended.Lignite’s ability to inhibit microbial activity most likely minimized the microbial immobilization of mineralized N,thus causing the higher net mineralized N,particularly in the MLY feedstock,despite its lower cumulative CO2evolution(Schimel,1986;Songet al., 2011). The application of lignite-amended manure is therefore likely to minimize the initial microbial immobilization of mineral N (which then becomes available to the crop) often associated with the application of organic amendments to soil. Overall, the percentages of applied organic N mineralized(2.5%–13.5%)across all treatments were similar to values reported in the literature.Hartzet al.(2000)reported that the mineralization of organic N from non-composted manure and composted manure was 7%and 1%, respectively, after 12 weeks of incubation with soil.Eghball(2000)found that over a period of 8 weeks,21%of applied organic N was mineralized in manure and 11%in composted manure.Other N mineralization values reported in the literature are as follows:4%–5%in composted cattle manure over 168 d(Antilet al.,2011),-29%(net immobilization)to 55%in dairy manure over 56 d(Van Kessel and Reeves,2002),and 21%,19%,and 13%in chicken,pig,and cattle manure, respectively, over 161 d(Li and Li, 2014).The wide variation in the mineralization of organic N has been attributed to factors such as soil properties, manure pre-treatment and characteristics,and incubation conditions(Hartzet al.,2000;Abbasiet al.,2007).

The net mineralization of 2.5%–13.5%of applied organic N observed in this study may not be adequate to sustain the productivity of certain crops;therefore,fortification of the feedstock or compost application with inorganic N fertilizers may become necessary(Abbasiet al.,2007;Azeez and Van Averbeke, 2010). Although more manure may be applied to compensate for the lower N mineralization, repeated over-application could have negative effects on soil and the environment,e.g., increased EC, excessive P, and loss of NO-3to water bodies(Mukhtaret al.,2003;Cabreraet al.,2009).

CONCLUSIONS

Lignite amendment inhibited the mineralization of C in both raw and composted cattle manure during laboratory soil incubation,with the inhibitory effect being more pronounced in the compost(even after standardizing the amount of lignite present in the manure).Although there was no significant effect of lignite amendment on the net organic N mineralized in the composted manure at the end of the 40-d incubation period,the feedstock containing LY lignite had significantly higher net organic N mineralization. Lignite addition to manure, especially when composted, has the potential to increase soil organic matter reserve and improve long-term soil health owing to the inhibitory effect of lignite on manure C mineralization.Hence,the application of lignite to mitigate N loss from manure can improve the subsequent effectiveness of manure as a soil amendment.Further studies(including field studies) involving the use of different soil types and extended incubation periods are recommended to elucidate the effect of lignite on C and N mineralization in manure.In addition,the long-term effects of the continuous application of lignite-amended manure on soil health and plant yield should be assessed.

ACKNOWLEDGEMENTS

This research was supported by the Meat and Livestock Australia (No. B.FLT.0148). We would like to thank the Trace Analysis for Chemical, Earth, and Environmental Sciences (TrACEES) platform of The University of Melbourne,Australia for analytical and technical support.We also acknowledge the support of the Statistical Consulting Centre of The University of Melbourne for helping with data analysis.