INTRODUCTION

Microbial growth and activity in soil are regulated by substrate availability and environmental factors such as temperature,moisture,and pH.Thus,these factors are the main drivers of decomposition used in modeling soil processes(e.g.,Sierra et al.,2015).A less studied,but equally important,factor is which nutrient is limiting microbial activity in soil.The type and extent of nutrient limitation will not only affect decomposition rates in soil,but may also affect carbon(C)use efficiency(Schimel and Weintraub,2003).The most common way to infer nutrient limitation in soil has been to measure respiration after adding nutrients singly or in combinations.It is difficult,however,to differentiate the type of limitation using respiration measurements.This will,e.g.,be the case when differentiating between C and nitrogen(N)limitations for microbial growth in soil.In both cases adding easily available C(e.g.,glucose)will in the short term result in increased respiration(Cochran et al.,1988;Sørensen et al.,2006;Kamble and B˚a˚ath,2016).Adding N in N-limiting conditions may also result in problematic interpretations,since this can,at least theoretically,result in decreasing respiration due to changes in the partition of C into growth and respiration(Schimel and Weintraub,2003).Thus,as stated by Sistla et al.(2012),microbial N limitation is difficult to demonstrate because it is a challenge to measure microbial growth in soil and to separate biomass growth from changes in metabolism.However,using leucine(Leu)incorporation into bacteria as a proxy of bacterial growth and acetate incorporation into ergosterol(Acin-erg)as a proxy of fungal growth,changes in growth after alleviating nutrient limitation in soil could easily be determined(Ald´en et al.,2001;Demoling et al.,2007;Kamble et al.,2013).The primary limiting nutrient will then be the one that increases microbial growth,with no effects of adding other nutrients.Microbial growth in soils has usually been found to be limited by lack of C;however,phosphorus(P)or N limitation or limitation by two or more nutrients simultaneously has also been demonstrated(Ald´en et al.,2001;Sørensen et al.,2006;Demoling et al.,2007;Ri-nnan et al.,2007;G¨o ransson et al.,2011;Heuck et al.,2015;Schmidt et al.,2016).Clear examples of N limitation,however,are rare,except when studying fresh litter degradation(Cochran et al.,1988;Henriksen and Breland,1999).

In an earlier study,acidic soils from a temperate climate,which were originally C-limited for microbial growth,were altered into N limitation by amending with a C-rich substrate(Kamble and B˚a˚ath,2016).The effect of alleviating N limitation on fungal and bacterial growth and respiration was then studied.Alleviating C limitation by adding easily available C(glucose)in the unamended,C-limited soil resulted in increased respiration and growth,whereas N addition had no effect.Adding C to the substrate-amended,N-limited soil also resulted in increased respiration,but with no effect on growth,whereas N addition resulted in both increased growth and respiration.The growth increase was mostly of bacterial or fungal origin depending on the type of substrate used to induce N limitation.This study thus demonstrated differences between growth and respiration based on the evaluation of nutrient limitation.

Soil pH has been shown to be canonical in determining the balance between fungal and bacterial growth in soil(Rousk et al.,2009,2011),with bacteria being favored by high pH and fungi by low pH.Thus,the response of fungal and bacterial growth and activity after alleviating C and N limitations may not be the same in low-pH soils compared to high-pH soils.Furthermore,soils from tropical regions are not well studied.We thus repeated the experiment of Kamble and B˚a˚ath(2016)using soils from India with high pH(pH ＞ 8)to compare the effects of alleviating C and N limitations in these soils compared to the earlier studied low-pH soils.These soils were earlier shown to be C-limited for bacterial growth(Kamble et al.,2014).

MATERIALS AND METHODS

A nutrient-rich,clayish soil from Pravaranagar(India)was used.The soil is classified as a Vertisol,and the regional name is regur(black cotton soil).Soil organic matter content(as loss on ignition at 600°C)was 80 g kg−1,with a pH of 8.4 at a soil∶water ratio of(1∶5,weight∶volume).Soils from this area have earlier been studied by Kamble et al.(2014),showing bacterial growth being favored over fungal growth at this high soil pH.

To induce N-limiting conditions,we used an experimental approach that was earlier used in a low-pH,forest soil(Kamble and B˚a˚ath,2014,2016),where C-rich substrates were added to the soil 1 month before the measurement of limiting nutrients.Straw(C/N ratio=75)and starch were used,since the former favors fungal growth and the latter usually favors bacterial growth.Straw was cut,milled,and sieved,and the fraction ＜ 0.25 mm size was used.Straw(80 mg g−1)and starch(40 mg g−1)were amended to the soils,with each soil considered as one replicate.A higher concentration of straw was added,since it is less available to the microorganisms than starch(Kamble and B˚a˚ath,2014).A treatment with no substrate addition was used as a control,resulting in three substrate treatments,each with four replicates.Twelve jars(100 mL)with lids,each containing 20 g soil,were then preincubated for 4 weeks at 22°C.The jars were aerated weekly,and the moisture content was maintained at 40%water-holding capacity by adding distilled water if necessary.

Direct effects of substrate(straw and starch)amendments 4 weeks after pre-incubation were compared using separate analysis of variances(ANOVAs)for each microbial variable at P＜0.05.Limiting factors were compared for each substrate amendment separately using a full factorial two-way ANOVA.

Bacterial growth was estimated using the Leu incorporation technique adapted for soil(B˚a˚ath et al.,2001).Briefly,bacteria were extracted from 1 g soil with 20 mL distilled water using a combination of homogenization(vortex for 3 min)and low speed centrifugation(1000×g for 10 min).The extracted bacterial suspension(the supernatant)was incubated for 2 h at 22°C with3H-labelled Leu(37 MBq mL−1and 5.74 TBq mmol−1;Perkin Elmer,Waltham,USA)and unlabelled Leu( final concentration of 275 nmoL−1).Growth was terminated by adding trichloroacetic acid,and subsequent washing steps and measurement of radioactivity were performed following the procedure described by B˚a˚ath et al.(2001).The amount of Leu incorporated into extracted bacteria per hour and per gram dry weight of soil was used as an estimate of bacterial growth.

Despite high soil pH(＞8),a condition known to favor bacterial growth(Rousk et al.,2009,2011),the main determinant of whether fungal or bacterial growth increased after alleviating N limitation was the type of substrate initially used to induce N limitation.Starch amendment and to some extent straw amendment induced bacterial growth,whereas straw amendment mainly induced fungal growth(Fig.1).These are similar results as those earlier found in a low-pH soil(Kamble and B˚a˚ath,2016),suggesting that the effect of substrate was more important than pH in determi-ning which microbial group responded to alleviating N limitation.

After a 4-week pre-incubation,nutrients limiting bacterial and fungal growth and respiration were measured,allowing for the comparison of the effect of alleviating nutrient limitation on bacterial and fungal activities.Our main definition of nutrient limitation was that adding the limiting nutrient should increase fungal or bacterial growth(Kamble and B˚a˚ath,2016),while respiration measurements were made as a comparison,since respiration has often been used to elucidate nutrient limitation.This also allowed us to crudely infer changes in C use efficiency,defined as growth/(growth+respiration)(Geyer et al.,2016).Since the earlier results(Kamble et al.,2014)showed that C was the primary limiting nutrient and N was the secondary limiting nutrient for bacterial growth in these soils,only these two nutrients were tested.Glucose(5 mg g−1equivalent to 2 mg glucose-C g−1)and NH4NO3(0.142 mg g−1equivalent to 0.05 mg NH4NO3-N g−1)were added to the soils in a full factorial design,resulting in four treatments∶CK(only water-added control),C(glucose-added),N(NH4NO3-added),and C+N(glucose and NH4NO3-added).Glucose was used as a general,easily available C source,since the extent of C limitation is dependent on the C source chosen.The methodology was essentially as described by Demoling et al.(2007)with modifications by Kamble et al.(2014).One g of soil was placed in 50-mL centrifuge tubes with lids,and the different combinations of C and N were added in 100µL distilled water.Bacterial growth(Leu content),fungal growth(Ac-in-erg incorporation),and respiration were then measured after 4 d at 22°C.The treatment with only water added(CK)was used to measure direct effects of substrate amendment on growth and respiration after 4 weeks of pre-incubation.To summarize the effect of C and N additions on the different microbial variables,a C/N limitation index was calculated as the log of the activity after adding C divided by that after adding N.Positive and negative values indicate C and N limitations,respectively(Kamble and B˚a˚ath,2016).Respiration was measured using 1 g soil weighed into 20-mL glass vials.The vials were aerated with pressurized air,sealed with a crimp cap,and incubated for 24 h at 22°C,after which the CO2concentration was determined using a gas chromatograph(YL6500 GC,YL Instruments,Gyeonggi-do,Korea),equipped with a methanizer and a flame ionization detector.

RESULTS

Respiration was significantly higher(P＜0.001)4 weeks after straw and starch amendment compared to the non-amendment control(Table I).Straw amendment resulted in almost four times higher respiration and higher bacterial growth,fungal growth,and fungal biomass compared to the non-amended control(P＜0.001),whereas starch amendment had no significant effect at this time point.

(1)不浮选时煤泥的销售收入。选煤厂入洗能力120万t/a，根据生产实际情况，煤泥产率在25%左右，按年入洗原煤100万t计，煤泥产量约25万t，不浮选时煤泥价格245元/t，煤泥销售收入6 125万元。

To compare the effects of C and N additions on the different microbial variables,a C/N limitation index was calculated(Fig.1d).For the non-amended control soil,all microbial variables had positive C/N limitation indices,indicating a C-limiting condition.For bacterial and fungal growth,the amendment with starch or straw clearly transformed the soils from C limitation to N limitation,with only bacterial growth responding in the starch-amended soil and fungal growth responding mostly in the straw-amended soil.The transition from C to N limitation could not be elucidated by the C/N limitation index for respiration,being close to zero in both substrate-amended soils.

TABLE I Microbial activities and biomassa)in a nutrient-rich,clayish soil from Pravaranagar,India,without(control)or with amendments of starch(40 mg g−1)or straw(80 mg g−1)after 4-week incubation at 22 °C

a)Leucine(Leu)incorporation,acetate incorporation into ergosterol(Ac-in-erg),andergosterol(erg)contents were measured as estimates of bacterial growth,fungal growth,and fungal biomass,respectively. b)Means±standard errors(n=4). c)Means in the same column followed by different letters are significantly different at P ＜ 0.05.

Treatment Respiration Bacterial growth Fungal growth Fungal biomassµg CO2h−1g−1 pmol Leu h−1g−1 pmol Ac-in-erg h−1g−1 µg erg g−1 Control 1.1±0.11b)ac) 18.9±2.5a 4.0±0.4a 0.27±0.05a Starch 3.1±0.4b 21.4±2.2a 4.1±1.0a 1.40±0.82a Straw 8.7±0.8c 108.0±16.1b 15.5±1.9b 4.80±0.67b

Starch amendment transformed the soil into N limitation for microbial activity.This was most evident for bacterial growth(Fig.1b),with significantly increased growth 4 d after adding N but not C.Respiration,conversely,increased to the same extent with the addition of either C or N(Fig.1a),making conclusions less clear.Fungal growth was not significantly affected by C or N addition in the starch-amended soil.However,in the straw-amended soil,fungal growth was significantly in-creased by N,but not C addition Fig.1c),with similar,but less evident effect on bacterial growth(Fig.1b),showing N limitation for microbial growth.Similar to the starch-amended soil,respiration increased to the same extent by adding C or N in the straw-amended soil(Fig.1a),making conclusions on the limiting nutrient less straight forward.

DISCUSSION

民事审前程序最早起源于英美法系国家，由于其在促进案件集中审理及解决纠纷等方面发挥了重要作用，因此，目前无论是在英美法系国家还是大陆法系国家，审前程序都备受重视，多数国家都在民事诉讼程序中设置了该内容。与之形成鲜明对比的是，我国的民事审前程序长期以来被民事立法、司法和理论界所忽视，至今尚未建立严格意义上的审前程序。

In the non-amended control,microbial growth and activity were limited by the lack of C.This was seen as a significant increase in respiration(Fig.1a)and bacterial growth(Fig.1b)after adding glucose C,with no effect of N addition on either of these variables.The significant N addition effect on bacterial growth was due to higher growth when adding both C and N(P＜0.01),compared to only adding C.Fungal growth was not affected by C addition,whereas adding N decreased fungal growth(Fig.1c).

Fungal growth was estimated by Ac-in-erg(B˚a˚ath,2001).Soil samples were mixed with distilled water,[1-14C]-acetic acid sodium salt(7.4 MBq mL−1and 2.04 GBq mmol−1;Perkin Elmer,Waltham,USA),and unlabelled sodium acetate( final acetate concentration of 220 µmol L−1),and then incubated for 4.5 h at 22 °C in the dark.Formalin was added to terminate growth.Ergosterol was then extracted and quantified as described by Rousk et al.(2009).The amount of acetate incorporated into fungal ergosterol per hour and per gram dry weight of soil was used as a measure of fungal growth.The amount of ergosterol was also used as a proxy of fungal biomass in soil.

Bruegel：美国关税政策旨在遏制中国技术崛起。4月10日，欧智库布鲁盖尔研究所（Bruegel）发文称，美国对华关税政策旨在遏制中国技术崛起，中国可将此视为加强与欧盟和亚洲邻国经贸关系的有利时机。美国此举或影响中国技术赶超速度，但效果取决于其他发达国家是否效仿美国的贸易保护主义措施。

Fig.1 Effects of limiting nutrient(C as glucose and N as NH4NO3)addition in a full factorial design after 4 d on respiration(a),bacterial growth(b),fungal growth(c),and C/N limitation index(log of the activity after adding C divided by that after adding N)(d)in a nutrient-rich,clayish soil from Pravaranagar,India,pre-incubated without(control)or with amendment of starch(40 mg g−1)or straw(80 mg g−1)for 4 weeks at 22 °C.Values are the means with standard errors shown by the vertical bars(n=4).*,**,and***indicate significant effects at P ＜ 0.05,P ＜ 0.01,and P ＜ 0.001,respectively,and ns indicates no significant effects of C,N or their interactions.Data in a–c were plotted as delta values,with the mean of no addition of C and N in the limiting nutrient assay subtracted,thus indicating extra respiration and growth after adding C and N.Leucine(Leu)incorporation and acetate incorporation into ergosterol(Ac-in-erg)were measured as estimates of bacterial and fungal growth,respectively.Positive and negative values of the C/N limitation index indicate C and N limitations,respectively.CK=only water-added control.

Using the respiration response in the simplistic way we used here,not following respiration closely over the first days(Nordgren,1992;Scheu,1993;Sørensen et al.,2006),did not result in the same clear results of the limiting factor as the growth-based evaluation methods,since adding glucose C always increased respiration(Fig.1a).Still,by comparing with the effect of adding N,respiration can also be used to elucidate nutrient limitation in soil.With C limitation,respiration increases after adding C,but not after adding N,whereas an increased respiration after N addition in the N-limiting situation always clearly indicated that N was the primary limiting nutrient,even if respiration also increased after adding C.However,to use the C/N limitation index as a rapid measure of nutrient limitation only worked when using the growth-based methods,since for respiration the C/N limitation index was close to zero in the N-limiting situation(Fig.1d),and thus no conclusive conclusions on the main limiting nutrient could be drawn.

The primary limiting nutrient was easily detected using the growth-based assays,both for C and N limitations.Earlier studies on limiting nutrients in soil mostly used only bacterial growth(e.g.,Demoling et al.,2007).When clear results on the primary limiting substance were found using bacterial growth,such as C limitation for the non-amended control soil(Fig.1b),measuring fungal growth will be less important.However,in situations with less clear indications of the limiting nutrient using bacterial growth,as in the case of N limitation induced by straw amendment,it might be necessary to study both fungal and bacterial growth to clearly demonstrate the limiting nutrient.

In the non-amended soil with C limitation,adding C increased both respiration and microbial growth(mainly bacterial growth),which is expected if respiration reflects microbial growth.Since there were no major effects of adding N on microbial growth and respiration,our results are in accordance with the theoretical model suggested by Schimel and Weintraub(2003).This was not the case,however,after alleviating N limitation,since their model suggests a decreased respiration concomitant with increased microbial growth due to a shift in the use of C for respiration and growth.This would result in a large increase in C use efficiency after adding N.Instead,alleviating N limitation increased both respiration and growth,which is in accordance with earlier results for a low-pH soil(Kamble and B˚a˚ath,2016),suggesting no drastic changes in C use efficiency.Sistla et al.(2012)also found a correlation between increased respiration and biomass after adding N to an extremely N-limiting tussock Arctic soil.They suggested that in a situation of severe N limitation,where the lack of N impairs enzyme production,alleviating N limitation would increase both respiration and microbial growth,similar to the present study.Since tussock soils,to a large extent,consist of decaying roots,i.e.,dead plant material similar to the substrates used here to induce N limitation,the extremely N-limiting situation in the tussock soils studied by Sistla et al.(2012)may thus be similar to the present study.On the other hand,if microbial activity is only moderately N-limited,there might be situations where alleviating N limitation could result in decreased respiration and increased microbial growth in soil,as previously suggested by Sistla et al.(2012).It remains to identify soils not rich in fresh plant material that are moderately N-limited to test this suggestion.

无线传感器网络在节点数据融合的应用中，时间同步对整个网络有着重要的影响[10-11]，如何实现节点之间的时间同步和获取节点准确的时钟信息是系统必须解决的关键问题。

ACKNOWLEDGEMENT

This study was supported by an Erasmus Mundi grant to the first author,Dr.Pramod N.Kamble.

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