Lei Zhang ·Junqiang Zheng ·Xu Han ·Junhui Zhang·Chengxu Li·Shicong Geng ·Shijie Han

Abstract In addition to bacteria,the contribution of fungi to nitrous oxide (N2O) production has been recognized but the responses of these two broad and unrelated groups of microorganisms to global environmental changes,atmospheric nitrogen (N) deposition,and precipitation in terms of N2O production are unclear.We studied how these two microbial-mediated N2O production pathways responded to soil moisture conditions and to N addition in an N-limited temperate forest.Soils from a long-term N addition experiment in Changbai Mountain,northeastern China were incubated.Varied concentrations of cycloheximide and streptomycin,both inhibitors of fungal and bacterial activity,were used to determine the contributions of both to N2O production in 66%,98% and 130% water-f illed pore spaces(WFPS).The results showed that N2O production decreased significantly with increasing cycloheximide concentration whereas streptomycin was only inhibiting N2O emissions at 98% and 130% WFPS.The bacterial pathway of N2 O production in N-addition (Nadd) soil was significantly more dominant than that in untreated (Namb) soil.The difference in the fungal pathway of N2O production between the soil with nitrogen addition and the untreated soil was not significant.Net N2O emissions increased with increasing soil moisture,especially at 130% WFPS,a completely flooded condition.Bacteria dominated carbon dioxide (CO2) and N2O emissions in Nadd soil and at 130% WFPS regardless of N status,while fungi dominated CO2 and N2O emissions in soil without N addition at 66% and 98% WFPS.The results suggest that flooded soil is an important source of N2O emissions and that bacteria might be better adapted to compete in fertile soils under anoxic conditions.

Keywords Nitrous oxide·Fungi·Bacteria·Nitrogen addition·Soil moisture conditions

Introduction

Nitrogen oxide (N2O) is a greenhouse gas and ozone-depleting substance and its emission represents a loss of nitrogen (N),an important component of ecosystem N cycling(IPCC 2007;Butterbach-Bahl et al.2013).Forest soils are important sources of N2O emissions,and N2O is the byproduct and end product of nitrif ication and denitrif ication,respectively,in forest ecosystem nitrogen cycling.For over a century,bacterial pathways have been thought to be the only means of producing N2O,and that denitrif ication is carried out solely by bacteria,whereas the ability of fungi to perform denitrif ication and their contribution to N2O production have been unrecognized (Zumft 1997;Laughlin and Stevens 2002).However,a few studies have shown that isolated fungi have the ability to produce N2O (Bollag and Tung 1972;Kurakov et al.2000,2002;Lavrent’ev et al.2008),and soil N2O production through the fungal pathway may even exceed that of the bacterial pathway (Laughlin and Stevens 2002;Crenshaw et al.2008;Blagodatskaya et al.2010).Zhu et al.(2015) reported that fungi,not bacteria,dominated heterotrophic nitrif ication in incubation experiments from subtropical coniferous forest soil.Although a few studies have found that fungal rather than bacterial pathways contributed significantly more to soil N2O fluxes in subtropical forests,grasslands and farmlands (Laughlin and Stevens 2002;Chen et al.2014;Li et al.2014),this relationship in temperate forests is largely unknown.Elucidation of this relationship is essential for understanding the microbial mechanisms of N2O emissions in temperate forest ecosystems.

Under the backdrop of global climate changes,the response of the long-lived greenhouse gas,N2O,to atmospheric N deposition and variations in precipitation should be underestimated.A meta-analysis showed that the N2O emission response to increasing N input is exponential rather than linear (Iurii et al.2014).Variations in soil moisture also reportedly affect forest N2O emissions,but studies have focused on the effects of soil moisture saturation and lower moisture levels on N2O emissions,ignoring the effect of anaerobic flooded conditions just after rainfall (Chen et al.2015).According to in situ observations in our previous study,long-term N addition significantly increases soil N2O emissions compared to soils without the addition of nitrogen(Geng 2017).Furthermore,the response to precipitation of N2O emissions in N addition plots was different from that in plots without nitrogen addition (Geng et al.2017).Nitrogen addition and soil moisture have been hypothesized to play important roles in determining the dominant microbial pathway of N2O production (Blagodatskiy et al.2008;Laughlin et al.2009;Vries et al.2011;Maeda et al.2017).Previous studies have focused on the combined effect of N addition and soil moisture on N2O emissions,while the responses of the two main N2O production pathways to the interaction of the addition of nitrogen and soil moisture,especially under anaerobic flooded conditions,have rarely been explored(Geng et al.2017) .Research has shown that fungal:bacterial ratios vary in response to forest soil fertility,with relatively lower biomass ratios in N-rich plots (Boyle et al.2008).Compared to fungi,bacteria are more sensitive to N addition and bacterial activities predominate under high nutrient availability and anoxic conditions (Myers et al.2012;Lee et al.2015).Therefore,it is important to quantify how longterm N addition and soil moisture alter the dominant status of fungi and bacteria as a basis for predicting N2O emissions in forest ecosystems under future climate change.

An incubation experiment was carried out using soil from a mixed birch-poplar forest on Changbai Mountain in northeast China.Our objective was to quantify the relative N2O production via fungal and bacterial pathways in response to soil moisture under long-term N addition.Soil N2O production was examined under three different moisture conditions:(1) aerobic;(2) saturated aerobic;and,(3) an anaerobic flooded condition.It was hypothesized that:(1) long-term N addition would significantly promote N2O production via regulation of bacteria;and,(2) the effect of soil moisture on N2O production would become dominant in the regulation of both bacteria and fungi in flooded soils.

Materials and methods

Soil sampling and processing

Soils were collected from a long-term N addition forest located in Changbai Mountain National Nature Reserve,northeastern China (41°42′N,127°38′E).The region has a temperate climate and winters are long and cold.The mean annual temperature is 3.2 °C,with the highest temperature in mid-August and the lowest in early February.Mean annual precipitation is 700 mm,mainly as rain from May to September.The current wet deposition rate is 23 kg N ha−1a−1.Betula platyphyllaSuk.is the dominant species.The soil type is a dark brown developed from volcanic ash (Albi-Boric Argosols) (Geng 2017).The soil properties are listed in Table 1.

Table 2 Two-way ANOVA examining the interaction of N addition and soil moisture on soil N2O and CO2 production without inhibitor

Nine plots (30 m × 30 m) were sampled in three replicates for two levels of N and one without N.Plots were sprayed withanammoniumnitrate(NH4N O3) solutionfromJune to October atarateof 5 and 10 kgNha−1permonthfrom 2007 until 2020,while control plots were sprayed with the same amount of water (Geng 2017).In this study,soils were collected from untreated (Namb) plots and from plots with the highest N addition (Nadd) .In June 2015,soil was collected from the upper 15 cm layer at five randomly selected points in each plot to form a composite soil sample after litter removal.The samples were placed in plastic bags,stored on ice for transport to the laboratory,sieved and mixed thoroughly and each was divided into two parts.One part was air-dried for soil property analysis,the other was stored at 4 °C for laboratory incubation experiments.

The cutting ring method was used to determine the soil water holding capacity (Xu 2018a).Three cutting rings of intact soils from each plot were sampled back.We placed the cutting rings on a flat-bottomed container filled with water.At the bottom,the inner side with holes was covered with filter paper,and the water surface was 1—2 mm lower thanthe upper edge of the ring.After 24 h,the hole cover was removed and the cutting rings placed tightly on dry soil for 8 h.The soil moisture of the intact soil was recorded as the water holding capacity.The target adjusted soil moisture (%water-f illed pore space) was obtained by dividing the natural soil moisture content by water holding capacity.

Laboratory incubation experiments

To determine the relative contribution of fungi and bacteria to N2O production,cycloheximide and streptomycin were used,respectively,to block fungal and bacterial protein synthesis and to inhibit the activities of fungi and bacteria(Anderson and Domsch 1973;Velvis 1997;Lin and Brookes 1999;Bailey et al.2003).Eight treatments were carried out for each soil sample:(1) controls,soil with the addition of distilledH2O ;(2) streptomycin 1,soilwith streptomycin at 0.5mgg−1soil;(3) streptomycin 2,soilwithstreptomycin at 1.0 mg g−1soil;(4) streptomycin 3,soil with streptomycin at 2.0 mg g−1soil;(5) cycloheximide 1,soil with cycloheximide at 1.0 mg g−1soil;(6) cycloheximide 2,soil with cycloheximide at 2.0 mg g−1soil;(7) cycloheximide 3,soil with cycloheximide at 3.0 mg g−1soil;(8) soil with the addition of streptomycin at 1.0 mg g−1and cycloheximide of 2.0 mg g−1soil.The concentrations of antibiotics were selected according to Chen et al.(2015) to determine the optimal concentration to identify contributions.Soils(equivalent to 15 g dry weight) were packed into 250 mLconical glass bottles to a 1 cm depth.Antibiotics at different concentrations were then dispersed evenly onto the soil surface,and the bottles were pre-incubated overnight to allow the antibiotic solutions to diff use into the soil pores.

After pre-incubation,the bottles were brought to a stable temperature (25 °C).To examine the effect of soil moisture on carbon dioxide (CO2) and N2O production,the soil moisture was adjusted by adding 5—15 mL of distilled H2O to create 66%,98% and 130% water-f illed pore spaces (WFPS).The 130% WFPS represented anaerobic flooded conditions just after rainfall.A 1 mL nutrient solution of KNO3and C6H12O6was then added to each bottle.Altogether,144 glass bottles were prepared,including two nitrogen levels,three replicates,three moisture levels and eight antibiotic treatments.

Soil gas fluxes were measured at 4,12,20 and 32 h after the addition of nutrients.At each sampling,the flasks were tightly capped with rubber septa,50 mL of gas was collected immediately,and then sampled again one hour later from the headspace of the flasks using a syringe with a needle.Thereafter,the flasks were opened until the next gas sampling.CO2and N2Oconcentrations in the gas samples were analysed using an Agilent 6890 gas chromatograph equipped with an electron capture detector (ECD) (Agilent Technologies Inc.,Santa Clara,CA,USA).Soil gas fluxes were calculated using the following equation:

where,Fgasis the net production of CO2and N2O gas;CsampleandCairare the CO2and N2O concentrations of the gas sample and the air (ppbv),respectively;V is the volume of headspace (L);r is molar volume at 25 °C and 1 atm(24.436 L mol−1),m is the dry weight of soil (g),and t is the incubation time (h).

Data analysis

Two-way analysis of variance (ANOVA) was performed to examine the interaction of two influential factors,N addition and soil moisture content,on total soil CO2and N2O emissions using antibiotic control treatment only (no inhibitor).Three-way ANOVA was carried out to determine the interaction of three influential factors,antibiotics,the addition of nitrogen,and soil moisture content,on bacterial and fungal CO2and N2O emissions.A one-way ANOVA and multiple comparisons were used to compare means (P<0.05) for CO2and N2O emissions among different antibiotics,N addition,and moisture contents,using SPSS v.22.0 (IBM SPSS Statistics for Windows,ver.22.0;IBM Corp.,Armonk,NY,USA).All statistics are expressed as mean (standard error),and all the data sets tested for normal distribution and homoscedasticity.If data were non-normally distributed or heteroscedastic,they were log-transformed prior to statistical analysis or a non-parametric test was applied.

Results

Effects of antibiotics on soil CO2 and N2O emissions

CO2emissions decreased significantly with increasing strep tomycinconcentrationsexcept for the0.5mgg−1streptomycin treatment which were statistically similar to those without strep tomycin (Fig.1 d—f).The addition of 1 and 2 mg g−1streptomycin reduced N2O emissions and decreased with increasing streptomycin concentrations at 98% and 130% WFPS (Fig.2 e,f).However,N2O emissions increased significantly with increasing streptomycin concentrations at 66% WFPS (P<0.01) (Fig.2 d).

Fungal activities also decreased with the addition of cycloheximide,but CO2emissions fromNaddsoilin the 1and3 mgg−1cycloheximidetreatmentsweresimilar to those without cycloheximide (Fig.1 a—c).N2O emissions were lower with higher cycloheximide concentrations(P<0.05) (Fig.2 a—c).

Fig.1 CO 2 flux rates in response to different antibiotic treatments at three soil moisture levels.Error bars represent standard errors for n=3 (three replicates for each treatment).Letters indicate significant differences among antibiotic concentrations at P=0.05 (a,d,and g represent 66% WFPS,b,e,and h 98% WFPS,and c,f,and i 130%WFPS)

Fig.2 N 2O flux rates in response to different antibiotic treatments at three soil moisture levels.Error bars represent standard errors for n=3 (three replicates for each treatment).Letters indicate significant differences among antibiotic concentrations at P=0.05 (a,d,and g represent 66% WFPS,b,e,and h 98% WFPS,and c,f,and i 130%WFPS)

Therefore,2 mg g−1cycloheximide and 1 mg g−1streptomycin were selected as the optimal concentrations to compare the contributions of the two microbial pathways to N2O production.

Effects of N addition and soil moisture on soil microbe-mediated CO2 and N2O emissions

The addition of nitrogen without antibiotics significantly promoted total CO2and N2O emissions (Table 2).According to soil CO2emissions,N addition significantly promoted total fungal and bacterial activities (P<0.01)(Table 2).The increase in total N2O emissions from Naddsoil was mainly attributed to bacterial pathways because the response of fungal N2O emissions to nitrogen addition was insignificant (Table 3).

Soil respiration at 130% WFPS was significantly lower than that at 66% WFPS and 98% WFPS (P<0.01) (Fig.1).N2O emissions increased with increasing soil moisture and were significantly higher at 130% WFPS regardless of the type or concentration of antibiotic or whether the soil was from Nambor Naddplots (P<0.01) (Fig.2) (Table 2),exhibiting an opposite trend to CO2production.

Table 3 ANOVA F values for soil N2O and CO2 production with different nitrogen levels,soil moisture contents and concentrations of antibiotics

At 130% WFPS,microbial activities were dominated by bacteria (Fig.1 i),with the bacterial pathway dominant pathway for N2O emissions regardless of N status(Fig.2 i).However,at 66% and 98% WFPS,soils with added N showed reduced CO2and N2O emissions which were more pronounced with the addition of streptomycin than with the addition of cycloheximide.The decrease in CO2and N2O emissions in soils without added nitrogen was more pronounced with the addition of cycloheximide(Figs.1 and 2 g,h).

Discussion

Effects of antibiotics on N2O emissions

Cycloheximide and streptomycin have been widely used to explore the relative contributions of fungal and bacterial pathways to N2O emissions (Anderson and Domsch 1 973;Velvis 1997;Lin and Brookes 1999;Bailey et al.2003;Boyle et al.2008;Chen et al.2014,2015).In this study,increasing N2O emissions with streptomycin and lower negative N2O emissions at higher concentrations of cycloheximide at 66% WFPS indicate that bacteria consumed N2O,as reported by Blagodatskaya et al.(2010).Bacteria not only produce N2O through nitrif ication and denitrif ication but also metabolize N2O by producing N2O reductase (Kolb and Horn 2012),which further reduces N2O to N2(Blackmer and Bremner 1979).The application of streptomycin indirectly reduces the production of N2O reductase by inhibiting bacterial activities,resulting in the accumulation of N2O (Blagodatskiy et al.2008).Additionally,streptomycin can provide more available substrates for fungi or archaea by reducing bacterial utilization or by providing antibiotickilled cells for surviving microorganisms to increase fungal activity to promote N2O emissions (Parkinson et al.1971;Land et al.1993).Dendooven and Anderson (1994)and Dendooven et al.(1994) reported that the addition of chloramphenicol,another inhibitor of protein synthesis by prokaryotes,increased N2O and CO2production.They considered that chloramphenicol served as a carbon substrate for N2O production.If this hypothesis is valid,streptomycin,with a lower C/N relative to soil,could also be regarded as an available substrate to promote N2O production by regulating the carbon and N balance of the decomposing substrate.McLain and Martens (2006) also considered that antibiotics may serve as an additional,easily available substrate for microbial metabolism and N2O production.The lower N2O emissions with the addition of higher cycloheximide concentrations and the appearance of negative N2O emissions show that fungi produced N2O .In this study,the inhibitory effect on N2O production was intensified by higher concentrations of cycloheximide.Unlike streptomycin,higher C/N ratios of cycloheximide strengthen the N-limited conditions to restrict N2O emissions.Both cycloheximide and streptomycin inhibited soil N2O production,although bacteria played a consuming role in net N2O production at 66% WFPS.

Effects of N addition on soil microbe-mediated CO2 and N2O pathways

Soil nitrogen acts as a substrate for decomposition,and its addition to soil affects N2O emissions.In this study,the addition of nitrogen significantly promoted CO2and N2O emissions,which is consistent with studies by Hall and Matson(1999),Jenssen et al.(2002),and Bai et al.(2014).Nitrogen applied to an N-limited temperate forest can relieve the soil N-limited status and increase substrate availability,thus increasing microbial activity and promote N2O emissions(Basiliko et al.2006;Bragazza et al.2012).Additionally,at 66% and 98% water-f illed pore space,the bacterial pathway dominated N2O and C O2emissions in Naddsoil,while fungal pathways were more important in Nambsoils.These results support our hypothesis and can be explained by the increasing competitiveness of bacteria under high nutrient availability conditions (Myers et al.2012).Although the addition of nitrogen promotes both fungal and bacterial activities,bacteria prefer nutrient-rich soils and are more sensitive to the application of nutrients.Bacteria,therefore,predominate in soils with added nitrogen (Basiliko et al.2006;Killham and Prosser 2007;Bragazza et al.2012;Lee et al.2015).Xu (2018b) reported that bacterial biomass increased after the addition of nitrogen and recorded greater abundance ofnirK,nirSandnosZgenes in Naddsoils compared to soils without nitrogen added at our study site.This is another reason for promoting bacterial N2O production.In contrast,fungi have lower biomass N and other nutrient requirements,which allows them to better endure harsh conditions such as poor nutrient availability (Moore and Basiliko 2006;Horwath 2007).In general,changes in soil nutrient status not only influenced CO2and N2O emissions but also altered the dominant microbial-mediated pathways.

Effects of moisture on potential CO2 and N2O production pathways

Soil moisture is the main controlling factor for O2contents and for the mobility of soluble organic matter,the respiration substrate and energy sources for soil respiration (Wang et al.2003;Rochette et al.2010).In this study,the lowest CO2emissions at 130% WFPS indicated the poorest soil aeration.The over-saturated moisture levels blocked oxygen diff usion from the atmosphere into the soil and reduced microbial abundance,decreasing the rate of microbial respiration (Madigan et al.2000;Lavigne et al.2004;Scott-Denton et al.2010).

N2O is produced mainly by nitrif ication and denitrif ication,depending on soil moisture conditions (Rogers and Whitman 1991;Skiba et al.1993;Schönwiese et al.1997;Wang et al.2017).In our study at 66% WFPS,there was the coexistence of both nitrif ication and denitrif ication,as determined by a preliminary experiment.This approximates the critical moisture level of 60—80%,which varies with soil type and O2content (Stevens et al.1998;Rudaz et al.1999;Bateman and Baggs 2005;Ruser et al.2006).Denitrif ication was the main process of N2O production at 130% WFPS.N2O emissions at 130% WFPS were much higher than at 66% and 98% WFPS.Chen et al.(2015) found that N2O emissions increased with increasing moisture when the soil was under the soil saturated threshold because the anaerobic environment was more conducive to denitrif ication (Schindlbacher et al.2004).Furthermore,the over-saturated condition was not only more suitable for the growth of anaerobic microbes,including fungi and bacteria,but was also beneficial for the diff usion of available nutrients to promote denitrif ication (Smith et al.1998;Dobbie and Smith 2010b;Xing 2017).Dobbie and Smith (2010a) showed that N2O emissions increased exponentially with increasing soil moisture up to an over-saturated level.Rapid flooding of soils results in high N2O production and consumption and increases net N2O emissions to the atmosphere (Jørgensen and Elberling 2012).Flooded soils substantially promote N2O production by providing more anaerobic microsites for denitrif ication compared to aerobic conditions.

In our study,the role of soil moisture became dominant in anaerobic flooded soils and bacteria predominated.Bacteria do not tolerate acidic conditions as do fungi but rather prefer neutral conditions for N2O production.The 130% WFPS provided a more neutral microenvironment for bacteria with the addition of more neutral deionized water to achieve the targeted moisture content.Additionally,fungal denitrif ication required a small amount of O2because nicotinamide adenine dinucleotide phosphate (NADPH) provided fungal nitric oxide reductase (i.e.,P450) with electrons and requires a small amount of O2to be generated (Zhou et al.2001).However,bacteria tend to replace simple organic molecules and oxygen with alternative electron acceptors for greater adaptation (Killham and Prosser 2007).Anaerobic conditions allowed the anaerobic bacterial pathway to dominate in N2O production.

Conclusion

By collecting long-term nitrogen added forest soils,and laboratory experimentation with different antibiotics and simulated water treatment,the complex association among N2O effl ux,soil fungi,soil bacteria,N addition and soil moisture has been elucidated.Although both microbial pathways of N2O production were promoted by the long-term addition of nitrogen,the bacterial pathway was more sensitive.Soil N2O emissions were higher at 130% WFPS than at 66% and 98% WFPS and the role of soil moisture became dominant in anaerobic flooded conditions,allowing bacteria to predominate,regardless of nitrogen status.The bacterial pathway of N2O production was more sensitive to the addition of nitrogen and adapted better to flooded conditions.Bacteria play important roles under over-saturated moisture conditions,which must be considered when analysing microbial mechanisms of N2O production in temperate forests.

AcknowledgementsWe appreciate AJE for language modification of the manuscript.