Stabilization of microbial residues in soil organic matter after two years of decomposition

Journal Pre-proof Stabilization of microbial residues in soil organic matter after two years of decomposition Chao Wang, Xu Wang, Guangting Pei, Zongwei Xia, Bo Peng, Lifei Sun, Jian Wang, Decai Gao, Shidong Chen, Dongwei Liu, Weiwei Dai, Ping Jiang, Yunting Fang, Chao Liang, Nanping Wu, Edith Bai PII:

S0038-0717(19)30351-7

DOI:

https://doi.org/10.1016/j.soilbio.2019.107687

Reference:

SBB 107687

To appear in:

Soil Biology and Biochemistry

Received Date: 10 July 2019 Revised Date:

16 November 2019

Accepted Date: 23 November 2019

Please cite this article as: Wang, C., Wang, X., Pei, G., Xia, Z., Peng, B., Sun, L., Wang, J., Gao, D., Chen, S., Liu, D., Dai, W., Jiang, P., Fang, Y., Liang, C., Wu, N., Bai, E., Stabilization of microbial residues in soil organic matter after two years of decomposition, Soil Biology and Biochemistry (2019), doi: https://doi.org/10.1016/j.soilbio.2019.107687. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Stabilization of microbial residues in soil organic matter after two years of

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decomposition

3 4

Chao Wanga, Xu Wanga,b, Guangting Peia,b, Zongwei Xiaa, Bo Penga,b, Lifei Suna, Jian Wanga,

5

Decai Gaoa,c, Shidong Chend, Dongwei Liua, Weiwei Daia, Ping Jianga, Yunting Fanga, Chao

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Lianga, Nanping Wue & Edith Baia,c,*

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a

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Chinese Academy of Sciences, Shenyang, 110016, China

CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology,

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b

University of Chinese Academy of Sciences, Beijing, 100049, China

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c

School of Geographical Sciences, Northeast Normal University, Changchun, 130024, China

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d

College of Geographical Sciences, Fujian Normal University, Fuzhou, 350007, China

13

e

Department of Geology, University of Maryland College Park, Maryland, 20742, USA

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*Corresponding Author:

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Edith Bai

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Institute of Applied Ecology, Chinese Academy of Sciences,

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No. 72 Wenhua Road,

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Shenyang, Liaoning,

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110016, China

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Telephone: +86-24-83970570

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E-mail: [email protected] Page 1

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ABSTRACT

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Microbially-derived nitrogen (N) has been considered as one of important contributors to soil

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organic N, but few studies have quantified the rate of necromass N decomposition. Here, via

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an in situ incubation of

27

stabilized in the soil as non-living organic N after 803 days of incubation. Bacterial, fungal,

28

and actinobacterial necromass N showed similar decomposition pattern and mean residence

29

time. The decomposition of microbial necromass N was best simulated by a two-pool model

30

where a labile pool decomposed rapidly (0.4 years), and a more recalcitrant pool decomposed

31

at a much slower rate. This finding contrasted with the decomposition of plant litter N, which

32

was better simulated by a single-pool model. The stabilization of necromass N in soils after

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more than two years suggests the important contribution of microbial residues to soil organic

34

N, which is most likely due to mineral protection from decomposition.

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N-labeled necromass, we found that 33.1-39.5% of the initial

15

N

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Keywords: Soil microbial necromass; Microbial biomass; Soil organic matter; Soil nitrogen

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cycles; Stable nitrogen isotope; Analytical modeling

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1. Introduction

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Soil organic matter (SOM) is not only an important carbon (C) pool in the global C cycle, but

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also a reservoir of nutrients, such as nitrogen (N) and phosphorus (P), for plant growth and

41

soil microbial and animal activities (Elser et al., 2007; LeBauer and Treseder, 2008). Classical

42

views and models assume that SOM mainly is derived from recalcitrant plant residues, while

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soil microorganisms are considered as the primary agents of SOM decomposition

44

(Kögel-Knabner, 2002; Schmidt et al., 2011; Lehmann and Kleber, 2015). Yet, recent results

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show that microbial matter including their metabolic excretion and their senesced biomass

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(necromass) may be a significant part of SOM itself (Simpson et al., 2007; Miltner et al., 2012;

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Throckmorton et al., 2012; Schurig et al., 2013; Kögel-Knabner, 2017; Liang et al., 2017;

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Kästner and Miltner, 2018). Therefore, there is an emergent demand for a better

49

understanding of SOM due to paradigm shift for its formation and stabilization (Schmidt et al.,

50

2011; Lehmann and Kleber, 2015).

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The structure and chemical composition of plant litter are different from that of soil

52

microbes, which could result in different stability of SOM (Kögel-Knabner, 2002; Liang et al.,

53

2017). For example, the global average C/N ratio of plant litter (~53) (Yuan and Chen, 2009)

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is much higher than that of microbial biomass (~7) (Xu et al., 2013). First, the decoupling of

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C and N cycling may happen if microbes select more N-containing materials for their

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demands. Then, once the selected plant litter is processed by microbes for their growth and

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the formation of microbial biomass, the molecular structure and characteristics of

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microbially-derived SOM may also be different from those of plant-derived SOM

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(Kögel-Knabner, 2002; Liang et al., 2017). Ultimately, whether microbial necromass could Page 3

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contribute significantly to SOM formation over a long-term period depends on its pool size

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and its decomposition rate in situ, which could be affected by climatic factors, the mineralogy

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of the soil, as well as the ‘sorptive affinity’ of a particular necromass materials to the solid

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phase (Castellano et al., 2015; Sokol et al., 2019). Although the active microbial biomass

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carbon is measured to be less than 2% (Dalal, 1998), the rapid turnover of this biomass could

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leave behind a large amount of necromass, which could contribute to more than 50% of SOM

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(Simpson et al., 2007; Liang et al., 2011). Once the microbial necromass is physically

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protected by soil mineral particles and aggregates, its mean residence time (MRT) in soil may

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be much longer than previously thought and necromass could be a major contribution to

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stable SOM (Simpson et al., 2007; Kästner and Miltner, 2018). Experiments on the

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decomposition of microbial necromass considering the actual conditions in soil including

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physical protection and the variation of climatic factors are needed to better understand SOM

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formation and to incorporate microbially-derived SOM into biogeochemical models.

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Due to the individual composition of compound-classes of different decomposability,

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necromass of different microbial groups might reflect a wide range of decomposition rates

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(Kögel-Knabner, 2002; Six et al., 2006). Chitin is the basic unit of cell walls of fungi

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(Bartnicki-Garcia, 1968; Kögel-Knabner, 2002). Additionally, cell walls of some fungi also

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contain relatively high proportions of proteins and melanin (Kögel-Knabner, 2002). Some

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studies have suggested that melanin is a recalcitrant polymer in fungal necromass and may

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decompose slowly in soils (Fernandez and Koide, 2013; Fernandez et al., 2019). Bacterial cell

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walls are mainly composed of carbohydrate, which is built largely from amino sugars

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(Kögel-Knabner, 2002). Despite the different decomposability of microbial cell wall Page 4

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components, Throckmorton et al. (2012) found no difference in the MRT of necromass C in

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soil among bacteria, actinobacteria and fungi. If there is a difference in the MRT of necromass

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N among diverse microbial groups is still unclear.

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Although previous researchers have conducted pioneering works to estimate microbial

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necromass C decomposition rate using

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spectroscopy in laboratory (Nelson et al., 1979; Jawson et al., 1989; Kindler et al., 2006;

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Miltner et al., 2009; Spence et al., 2011; Throckmorton et al., 2012), the in situ decomposition

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rate of microbial necromass N has never been studied. Previous studies have shown that the

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portion of microbially-derived soil organic N (SON) to total SON may be higher than the

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portion of microbially-derived SOC to total SOC (Simpson et al., 2007), pointing to the

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importance of microbially-derived N to soil N cycling. Although C and N are often coupled in

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different types of compounds of microbial detritus, selective preservation of either C or N

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during the decomposition of these necromass materials may decouple C and N decomposition,

95

resulting in different decomposition rate of microbial C and N in SOM (Knowles et al., 2010;

96

Veuger et al., 2012). However, at present, a quantitative assessment of the decomposition rate

97

of microbial necromass N is still lacking. In addition, previous studies on microbial

98

necromass C have mostly been done in the lab and at short time scales (Kindler et al., 2006;

99

Schweigert et al., 2015), which may not represent the real conditions in the field.

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Here we utilized a

15

13

C and

14

C isotope labeling techniques, and NMR

N labeling approach to explore the in situ turnover of bacterial,

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fungal, and actinobacterial necromass N in a temperate forest soil. Four members of each

102

microbial group were isolated from the soil, labeled with

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temperate Korean pine and broad-leaved mixed forest. We traced the labeled 15N in microbial Page 5

15

N, sterilized and incubated in a

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necromass into bulk total soil N (TN), soil microbial biomass N (MBN), soil inorganic N

105

(NO3--N and NH4+-N), and gaseous N (N2O) pools for 803 days after the addition of tracer. A

106

decay model was used to estimate MRT of microbial necromass 15N in soil to compare it with

107

the MRT of necromass 13C from previous studies on 13C-labeled necromass (Throckmorton et

108

al., 2012). Additionally, the necromass N turnover rate was estimated by introducing an

109

analytical model that included necromass N turnover, necromass N production, biomass N

110

turnover and microbial NH4+ immobilization processes. We further compared the estimated

111

necromass N decomposition rate with the decomposition rate of plant litter N in the same site.

112

We hypothesized that: (1) the MRT of necromass N in soil would be longer than 2 years (the

113

duration of a necromass C decomposition in the filed (Throckmorton et al., 2012)) and

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necromass N is an important contributor to soil organic N; and (2) necromass N of three

115

groups of microbes (bacteria, fungi, actinobacteria) would differ in their decomposition rate in

116

soil due to their different chemical materials of cell walls.

117

2. Methods

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2.1. Study site and experimental design

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The study was conducted at the Changbai Mountain Forest Ecosystem Research Station,

120

which is located in the east of Jilin Province in northern China (42.70o N,127.63o E). This

121

region is characterized by a typical temperate climate, with annual mean precipitation and

122

temperature at 745 mm and 3.6 oC, respectively. The growing season is from May to

123

September, with a mean temperature of 16.7 oC. The study site is in a natural Korean pine and

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broad-leaved mixed forest at 700-720 m above sea level, dominated by the species Pinus

125

koraiensis, Quercus mongolica, Tilia amurensis, and Fraxinus mandshurica. The soil is a dark Page 6

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brown soil developed from volcanic ash (Albic Luvisol) and its basic physicochemical

127

characteristics are shown in Table S1.

128

In May 2015, four experimental plots (20 m × 20 m) were randomly established with

129

a >10 m buffer zone between two adjacent plots. Then, twenty O-horizon soils from each plot

130

were randomly sampled using a corer (5 cm in diameter) after removing the litter layer. The

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soils were combined into one composite sample and immediately transported to the laboratory

132

in a box with ice bags for later microbial isolation.

133

To trace the fates of microbial necromass N in soil, we used a mesocosm approach to

134

incubate soil microbial necromass in situ. A PVC collar with an outer diameter of 11 cm (10

135

cm inter diameter) and a depth of 15 cm was randomly installed into the top 14 cm soil at

136

each plot mentioned above, leaving 1 cm above soil surface for gas sampling. On the PVC

137

wall, nine holes (2.5 cm in diameter) along three lines were drilled evenly and then covered

138

with a 450-µm mesh screen to allow fungal hyphae and fine roots to explore the mesocosm.

139

We set 40 PVC mesocosms at each plot and got a total of 160 mesocosms. After 1 year of

140

stabilization,

141

decomposition study.

142

2.2. Microbial growth and addition

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The isolation and enrichment of soil microbial strains were based on the cultivation of

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microorganisms in the agar and liquid media (Alef and Nannipieri, 1995). Briefly, hundreds

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of soil microbial isolates were screened by adding soil extract on the culture plates with

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trypticase soy agar (TSA). Then, bacterial (n = 60), actinobacteria (n = 40) and fungi (n = 55)

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isolates were grown in beef extract peptone medium (pH = 7.4 - 7.6), gauserime synthetic

15

N-labeled microbial necromass was added into the collars for the

Page 7

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agar medium (pH = 7.4 - 7.6), and potato dextrose agar (natural pH) in 25 ml test tubes,

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respectively. Finally, four isolates of each microbial group that grew well in liquid media were

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selected to produce sufficient labeled biomass for the field experiment. The identification of

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bacterial and actinobacterial isolates was based on 16S rRNA genes, which were amplified

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using universal primer pair 27F-1492R (Seán et al., 1999). And the identification of fungal

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isolates was based on ITS regions, which were amplified using primer pair ITS1F-ITS4

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(Manter and Vivanco, 2007). The sequences of these isolates were identified by sequence

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similarity searches (≥ 97%) against the 16Sr RNA dataset for bacteria and actinobacteria in

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NCBI Genbank and the ITS dataset for fungi in UNITE, respectively. Finally, four isolates in

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each of bacteria, fungi and actinobacteria were selected for the

158

experiment (Table 1).

15

N labeling and field

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In order to label the microbial biomass with 15N, 5 ml of each 12 microbial isolate from

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25 ml test tube were transferred to M9 liquid medium with 99.5 atom% 15N-NH4Cl. The detail

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composition and content of M9 medium can be found in Table S2. These isolates were

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harvested when they grew to late stationary phase in 250 ml M9 liquid media at 37 oC for the

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bacterial groups and 28 oC for the fungal and actinobacterial groups. All isolates were

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centrifuged and subsequently washed with 200 mL phosphate buffer solution (0.1 M, pH =

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7.0) for three times. Then the isolates were sterilized using UV light for 24 hours and then

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freeze-dried for 48 hours to ensure the complete death of all microbes. Finally, 2-3 g dry

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microbial necromass materials for each of the 12 isolates were produced for biochemical

168

analyses and field experiment (Table 1).

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On 13 May 2016, four microbial isolates within each microbial group were combined Page 8

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into one mixture based on their relative proportion in the soil microbial OTUs (Table 1). Then,

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the mixture (60 mg dry wt.) of each group (bacteria, fungi, and actinobacteria) was suspended

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in deionized water and injected into one PVC mesocosm to 5-10 cm depth using a syringe.

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The needle was inserted at 10 cm depth and was gradually pulled out during injection to

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ensure even distribution of necromass within the 5-10 cm depth. In addition, the control

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mesocosm was injected with an equal quantity of deionized water without

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total of 96 PVC collars out of the 160 PVC collars initially installed were used (three

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microbial groups and one control × 3 replicates × 8 sampling times = 96). The added cell

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necromass N corresponded to about 7% of the natural microbial biomass N in the soil.

15

N labeling. A

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After injection, three PVC collars of each microbial group were collected for soil and gas

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analysis at 0.5, 9, 30, 60, 132, 362, 490, and 803 days. On each sampling date, we sampled

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soil N2O from the PVC collar using the closed chamber method. A PVC-made chamber (110

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mm inside diameter and 200 mm in height) with a three-way stopcock for gas sampling was

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placed on the entire collar, resulting in 2 L volume on the top of soil (Fig. S1). The connector

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between PVC chamber and collar was sealed using tapes in order to avoid gas leakage. Then

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150 ml gas sample was taken from each chamber at 0 hour and 1.5 hours using a syringe and

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stored in air bags for N2O concentration and isotope measurement. The N2O concentration

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and stable N isotope ratios were analyzed by a gas chromatography (HP 5890-II) and an

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IRMS (IsoPrime100, IsoPrime limited, UK) with a 112-slot auto sampler (Gilson GX-271,

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IsoPrime limited, UK), respectively.

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Following gas sampling, the whole PVC mesocosm was excavated and the soil was

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divided into O layer (0-10 cm) and A layer (10-14 cm) and stored into plastic bags separately. Page 9

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The soils were stored at 4 oC prior to processing within one week. In order to gain information

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on the incorporation of the

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web and the non-living SOM, the recovery of

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pool, soil NO3--N pool, and soil NH4+-N pool were analyzed. Since we did not find

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enrichment in the A layer, we only used the data in O layer in the following analyses.

15

N-labeled necromass into the living fraction in microbial food 15

N in bulk soil total N, microbial biomass N 15

N

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Fresh soil samples were hand processed to remove visible roots and woody debris and

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sieved through a 2-mm sieve. Subsequently, ~ 20 g sieved soils were air-dried at room

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temperature for bulk soil total N content and its isotope analysis, while the other sieved soil

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was used for inorganic N (NO3--N and NH4+-N) and microbial biomass N analysis. For soil

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inorganic N, 30 g fresh soil was extracted with 150 mL KCl (2 M) and shaken on a rotary

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mechanical shaker for 1 hour at 160 rpm at room temperature. Then N isotope ratios of

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NO3--N and NH4+-N were measured using the ammonium diffusion method described by Sun

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et al. (2016). Soil microbial biomass N content and isotope ratios were measured using the

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chloroform fumigation-extraction method (Widmer et al., 1989). Briefly, 30 g fresh soil was

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extracted with 0.05 M K2SO4 at a soil-to-solution ratio of 1:4, while another 30 g fresh soil

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was fumigated with chloroform for 48 hours followed by 120 ml 0.05 M K2SO4 extraction.

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All K2SO4-extracts were dried to constant weight at 65 oC before analysis. The fractions of

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soil particulate organic matter (POM) and mineral associated organic matter (MAOM) were

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fractionated by density and size as described in Fulton-Smith and Cotrufo (2019). Finally, the

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N content and isotope ratio of bulk soil total N, inorganic N, microbial biomass N, POM and

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MAOM were analyzed using an Elementar Vario EL Cube (Elementar Analysis system GmbH,

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Hanau, Germany) interfaced to an isotope ratio mass spectrometer (IsoPrime100, IsoPrime Page 10

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Limited, Stockport, UK).

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2.3. Decomposition of litter N

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A field litter incubation experiment was conducted to explore the plant litter N decomposition

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in the same site (Pei et al., 2019). First, 20 independent plots (3 m × 3 m) were randomly

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established near the necromass N decomposition experiment site, and the buffer zones

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between two adjacent plots were 5 m at least. Second, 10 ten-year old specimens of Pinus

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koraiensis and Quercus mongolica were selected and transplanted into pots (35 cm diameter,

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30 cm height) at the beginning of April 2014. The trees were kept in an open-air greenhouse

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and fertilized with 12 g

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solution, applied five times during the growing season from May to September 2014. The

224

15

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growing season in October. Since root materials of Quercus mongolica were damaged during

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the processing, only the leaves and twigs of Quercus mongolica and leaves, twigs and roots of

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Pinus koraiensis were used for the incubation experiment in the field.

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N m-2 yr-1 using

15

N double-labeled NH4NO3 (99 atom%

15

N)

N-enriched leaves, twigs and roots of the two plants were harvested at the end of the

228

Then, the twigs were cut into < 2 cm pieces, and then leaves and twigs were separately

229

inserted into a polyethylene cylinder (11cm diameter, 10 cm height), which was covered with

230

1-mm mesh at the top and bottom to allow the passage of water, but to prevent the entry of

231

natural litter fall from above and the loss of small litter particles from the bottom. The wall of

232

each cylinder was covered by 5 mm lateral meshes to provide access to decomposer

233

communities (Fig. S2). The cylinders were installed randomly on the soil surface after

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removing the existing litter in the plots previously installed and were separated from each

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other by at least 50 cm. We randomly collected the cylinders at 90, 182, 365, 456, 548, 820 Page 11

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and 1186 days after addition, resulting in a total number of 112 litter cylinders (7 sampling

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times × 2 plant species × 2 litter tissues (leaf and twig) × 4 replicates).

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The 15N-labeled roots of Pinus koraiensis were incubated in a litter-bag, which was made

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of nylon net with a mesh size of about 1mm. The root bags were placed within the top 10 cm

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of soil by cutting into the soil with a shovel at a 45 degree angle, inserting the root bag into

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the cut, and firmly pressing the overlying soil onto the bag. The root samples were randomly

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collected at 37, 90, 182, 456 and 820 days after addition. The total number of root-bags were

243

20 (5 sampling times × 1 plant species × 1 litter tissues (root) × 4 replicates). The gross N

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release from plant litter was estimated as the method described by Pei et al. (2019).

245

2.4. 15N Recovery calculation

246

The mass percentage of

247

equation:

248

mass % N =

249

15

N (mass %

% %

×

15

N) for each N pool was calculated by the following

×

(

%

) ×

× 100

Eqn. 1

Then the recovery (15NRec, %) of microbial necromass

15

N tracer in each soil N pool

250

(15Nrec), including bulk soil total N (TN), microbial biomass N (MBN), soil NO3--N, soil

251

NH4+-N, and N2O-N, was calculated by multiplying each pool’s mean N stock (MpoolN, g N

252

per PVC mesocosm) by the measured difference in mean mass % 15N between treatment (15NT)

253

and control (15Nc, the soils without

254

mass of added tracer (M15N-added), and then by multiplying by 100:

255

N

(%) =

%



15

%

# '()**+*

N necromass addition) values, divided by the total

! " × #$%%&'

× 100

15

N

Eqn. 2

256

The cumulative loss of 15N in N2O pool was calculated by integrating the mean 15N-N2O

257

recovery over the sampling intervals. The 15N recovery in N loss pool, i.e. the 15N lost from Page 12

258

the mesocosm, was defined as the difference between initial tracer 15N input and the recovery

259

15

260

using Eqn. 1 and 2. Due to the incomplete 15N recovery in bulk soil at 0.5 days, the recovery

261

of each N pool at the following sampling days was standardized to its initial recovery at t =

262

0.5 days. The unstandardized data were also presented in the Fig. S3.

263

2.5. Mathematical models

264

First, the 15N recovery in bulk soil total N and plant litter 15N recovery were separately fitted

265

to a one-pool decay model as below (Kindler et al., 2006):

266

,(-) = . × exp

267

N in bulk soil total N. Additionally, the recovery of

15

N-labeled litter was also calculated

23

Eqn. 3

where f (t) is the recovery of necromass

15

N remaining in bulk soil total N or the

268

recovery of plant litter

269

exponential rate constant (yr-1). The MRT (years) for the necromass 15N or plant litter 15N was

270

calculated according to the following equation (Collins et al., 1999):

271

456 = 1/8

272 273

15

N at time t. a describes the initial size of each N pool, the k is the

Eqn. 4

Second, we developed an analytical model based on the following assumptions to estimate decomposition rate of necromass 15N (Fig. 1):

274

1) The decomposed necromass 15N tracer (Nnecro, g 15N g-1 soil) was either taken up by the

275

living microorganisms at a constant fraction (ε, %) or released excess N as ammonium (A,

276

g

277

efficiency with similar definition to microbial carbon use efficiency.

278

2) The necromass

279

that of necromass total N.

15

N g-1 soil) at a constant fraction (1-ε, %). The ε is equal to microbial nitrogen use

15

N had a constant decomposition rate (knecro, yr-1), which is equal to

Page 13

280

3) The necromass

281

decomposition constant (ks, yr-1) and a rapid fraction (1-θ, %) with a rapid decomposition

282

constant (kr, yr-1).

283

4) The necromass 15N that was taken up by living microorganisms (B, g 15N g-1 soil) had a

284

constant turnover rate (m, yr-1), which is equal to the microbial biomass turnover rate.

285

5) The 15N-NH4+ was either taken up by the living microorganisms at a constant rate (u,

286

yr-1) or lost from the soil system at a constant rate (l, yr-1).

287

The two-pool parallel model was then described by the following equations (Ekblad et

15

N can be divided into a slow fraction (θ, %) with a slow

288

al., 2016):

289

9:;<=>?

290

9H

291

9M

292

OP

293

8DEFG = 8Q × θ + 8G × (1 − θ)

93 93 93 O

= @ × A − CDEFG × 8DEFG

Eqn. 5a

= CDEFG × 8DEFG × I + K × L − @ × A

Eqn. 5b

= CDEFG × 8DEFG × (1 − I) − K × L − K × N

Eqn. 5c

= K × N

Eqn. 5d Eqn. 5e

294

Based on the pattern of 15N recovery in bulk soil total N pool in this study, we divided

295

the data into two phases with t less than 0.4 years (rapid decomposition phase) and t more

296

than 0.4 years (slow decomposition phase). We set a range 0-20 for m, 0-20 for k, 0-0.5 for u

297

and 0-1 for ε for model simulation. Then by applying Eqn. 5a to 5e simultaneously, the

298

estimation of these unknown parameters at each phase was performed using Matlab 2014a

299

(MathWorks, Inc. US) ode45 algorithm 10000 times to minimize the sum of squared

300

residuals.

Page 14

301

2.6. Statistical analysis

302

The differences in the estimated parameters (knecro, kr and ks) among three microbial groups

303

were analyzed using one-way analysis of variance (ANOVA) and a Tukey HSD test. The

304

level of significance (α) was set at 0.05. The repeated measures ANOVA model (MANOVA)

305

considering microbial groups, time and their interactions as independent factors were used to

306

compare the mean difference in the recovery of necromass

307

NO3--N, NH4+-N, and loss N among microbial groups. All statistical analyses were conducted

308

using SPSS 19.0 (SPSS Inc., Chicago, IL, USA).

309

3. Results

310

3.1.

311

Four isolates within each microbial group were selected from more than one hundred cultured

312

isolates (Table 1). The average initial N content of fungal necromass was 5.3%, which was

313

lower than that of bacterial (9.2%) and actinobacterial (10.3%) necromass. Additionally, the

314

initial C/N ratio of fungal necromass was slightly higher than that of bacterial and

315

actinobacterial groups. The atom % 15N was similar among the three groups, with an average

316

atom% 15N at 65.0 ± 0.9%.

317

3.2.

318

The labeled necromass N decomposed rapidly during the first 132 days following application

319

to soil, and decreased by 55.5% for bacteria, 56.3% for fungi and 51.1% for actinobacteria

320

necromass, respectively (Fig. 2 and Fig. S3). Then the rate of labeled necromass N

321

decomposition declined and became nearly constant. At the end of the incubation (803 days),

322

about 37.7-44.3% of 15N was recovered in the bulk soil total N pool for the three groups (Fig.

15

N in bulk soil total N, MBN,

Initial necromass characteristics

Decomposition of necromass 15N and litter 15N

Page 15

323

2). There were significant differences in the recovery of microbial necromass

324

‘microbial groups’ and ‘time’, but their interactions (microbial groups × time) was not

325

significant (Table S3). The estimated MRT was 2.75 ± 0.04, 2.90 ± 0.26 and 2.52 ± 0.21 years

326

for bacteria, fungi and actinobacteria necromass N, respectively (Fig. 2). Additionally, the

327

decomposition constant (kr) of the rapid necromass N pool estimated by the analytical model

328

was 6.08, 5.31 and 6.88 yr-1 for bacterial, fungal, and actinobacterial groups, respectively,

329

while the decomposition constant (ks) of the slow pool was 0.95, 0.87 and 0.79 yr-1 for

330

bacterial, fungal, and actinobacterial groups, respectively (Table 2 and Fig. S4). The overall

331

necromass N decomposition rate (knecro) was 3.82, 3.62 and 4.08 yr-1 for bacterial, fungal, and

332

actinobacterial groups, respectively (Table 2). The overall decomposition rate of plant N in

333

the same site ranged from 0.18 to 0.60 yr-1, which were significantly lower than the

334

necromass N decomposition rate (Fig. S5).

335

3.3.

336

The atom%

337

necromass

338

persisted throughout the entire experiment (Fig. S6). The maximum

339

was at 30 days of experiment which reached 12.3% for bacteria, 11.0% for fungi, and 11.1%

340

for actinobacteria necromass, respectively (Fig. 3). By the 803 days, 4.3-4.9% of necromass

341

15

15

N among

Fate of necromass 15N 15

15

N in microbial biomass N showed immediate enrichment following the

N addition and the higher

15

N enrichment compared to the control treatment 15

N recovery in MBN

N was still recovered in the MBN pool (Fig. 3).

342

The atom% 15N in NH4+-N and NO3--N was also higher in treatments than in control soil

343

(Fig. S6). The overall 15N recovery in soil NH4+-N was low (< 0.7%) and only 0.02-0.04% of

344

necromass

15

N were recovered in NH4+-N at 803 days after tracer addition (Fig. 3 and Fig. Page 16

345

S7-S8). The recovery of

346

highest recovery at the first sampling time (9.0-13.1%) and lowest recovery at the end of the

347

experiment (0.1-0.2%). The cumulative 15N recovery in N2O was extremely low and was less

348

than 0.05% at 803 days (Fig. 3 and Fig. S9). In addition, 33.1-39.5% of necromass

349

recovered in the necromass pool and 56.0-62.3% was lost from the incubator at the end of the

350

incubation (Fig. 3).

351

4. Discussion

352

The analytical model results suggest that the overall decomposition rate of necromass N

353

ranged from 3.62 to 4.08 yr-1 in our study site, with an average value of 3.84 yr-1. The

354

two-pool parallel model showed significantly different decomposition rate constants between

355

the labile pool and the resistant pool (Table 2). While the labile pool had a very fast

356

decomposition rate, the resistant pool turned to be relatively slowly decomposed.

357

Nevertheless, 51.1% to 56.3% of necromass

358

132 days of incubation, suggesting that microbial necromass could be a significant source of

359

stable SOM and may contribute to the replenishment of the soil N reserve.

15

N in soil NO3--N showed a decreasing pattern with time, with

15

15

N was

N was recovered in the bulk soil total N after

360

Our results also indicated that the decomposition rate (knecro) of bacterial, fungal, and

361

actinobacterial necromass in soil was not statistically different among groups (Table 2 and

362

Table S4), which is inconsistent with our hypothesis. However, we found the decomposition

363

rate of both rapid (kr) and slow pool (ks) of fungal necromass N were lower than those of

364

bacteria and actinobacteria necromass N, and the living fungal biomass had a lower turnover

365

rate (m, Table 2). Additionally, fungi had a higher proportion of slow decomposition pool than

366

others and the recovery of necromass 15N in bulk soil total N was generally statistically higher Page 17

367

for the fungal group than the bacterial and actinobacterial groups during the entire experiment

368

(Fig. 2). These findings together indicated that although the biochemical composition of

369

microbial cells is one controller of long-term decomposition rates of necromass (Nelson et al.,

370

1979; Six et al., 2006; Lehmann and Kleber, 2015), the three groups showed a similar pattern

371

of decomposition and stabilization.

372

The mechanisms of the stabilization of necromass-derived N in soils may be the same

373

for the three groups. Recent studies have found that the intrinsic lability of organic matter is

374

not important in controlling the microbial necromass decomposition processes, especially

375

during slow decomposition phases (Amelung et al., 2008; Schmidt et al., 2011; Kästner and

376

Miltner, 2018). We propose that the necromass may be stabilized by interaction with oxides or

377

minerals of Fe and Al, which have very large specific surface areas, and a whole suite of

378

microbial biomass components has a high affinity to mineral oxides, resulting in necromass

379

stabilization against microbial decay (von Lützow et al., 2008; Kästner and Miltner, 2018).

380

Our soil fractionation results showed that more than 75% of necromass

381

associated with the mineral phase (Fig. 4), which also supported this statement.

15

N recovered was

382

Overall, we found the decomposition pattern of microbial necromass N was different

383

from that of plant litter N at the same site. Plant litter N was best modeled by the one-pool

384

first order decomposition model and the averaged litter N decomposition rates were 0.37 yr-1

385

and 0.26 yr-1 for aboveground plant litter and root, respectively (Fig. S5). These results were

386

comparable with a beech foliar litter N release rate in Vosges mountains, France (0.34 yr-1)

387

(Zeller et al., 2000), but were about 10 times lower than the decomposition rate of necromass

388

N (Fig. S5). The possible reason for the lower N decomposition rate of plant tissues may be Page 18

389

because N-containing components in plant litter, most likely proteins (Kögel-Knabner, 1997,

390

2002), are more resistant to decomposition and less accessible for soil microorganisms than

391

microbial necromass N. This finding is also consistent with the viewpoint that microbial

392

necromass is a source of C and nutrients for free-living microorganisms and plant growth

393

(Drigo et al., 2012; Miltner et al., 2012; Morrissey et al., 2015). However, more importantly, it

394

should be noted that the necromass N was best modeled by the two-pool first order

395

decomposition model and although the overall decomposition rate of necromass N was faster

396

than that of plant litter N, the decomposition rate of the resistant necromass N pool (0.79 -

397

0.95 yr-1, Table 2) was comparable to the decomposition rate of plant litter N. Besides, the

398

different experimental methods to assess plant (litter boxes or bags) and necromass (direct

399

injection) decomposition may cause uncertainty in the comparisons of their decomposition

400

rates. Therefore, the long term retention of these two kinds of N sources cannot be inferred

401

from these decomposition rates and still need to be studied.

402

The

15

N recovery in MBN increased first and then declined (Fig. 3), which may be

403

because that necromass contained substantial amounts of

404

could be immediately taken up by microorganisms. After the first 132 days,

405

the pools of MBN, NH4+, and NO3- kept declining (Fig. 3), indicating that the rate of

406

decomposition of the labeled necromass (input flux to these pools) become slower than the

407

rate of output fluxes of these pools after 132 days (i.e. microbial death for MBN, nitrification

408

and plant uptake for NH4+, and denitrification and plant uptake for NO3-). In addition, the new

409

microbes grown which took up the

410

after death may be more stable than the initially labeled necromass. After 803 days, the

15

15

N-NO3- and

15

N -NH4+, which 15

N recovery in

N tracer were likely attached to mineral particles, and

Page 19

411

remaining 15N in soil was mainly recovered in the non-living non-extractable N (33.1-39.5%

412

of initial 15N) and MBN (4.3-4.9% of initial 15N) pools and from 485 to 803 days the decline

413

in the recovery rate in non-living non-extractable N was only 1.2% to 1.8% (Fig. S6). These

414

results suggest that after the initial rapid decomposition of

415

recovered necromass 15N could be an important contributor to soil organic N.

15

N-labeled necromass N, the

416

We compared our estimated MRT of necromass N in soil with a previous estimation on

417

the MRT of necromass C (Throckmorton et al., 2012) and found the averaged MRT of

418

necromass N in this study (2.72 years) was lower than the MRT of necromass C in a

419

temperate forest soil (5.19 years), but was comparable to the MRT of necromass C in the

420

tropical forest soil (2.25 years). This is not consistent with the previous view that necromass

421

N had longer turnover time than necromass C (Simpson et al., 2007; Kindler et al., 2009;

422

Miltner et al., 2009). The possible reason may be that N-containing materials in necromass

423

may be utilized by soil microbes at a higher rate than the C-containing materials due to the

424

labile nature of high N-containing materials such as protein and amino sugars (Kögel-Knabner,

425

2002; Knowles et al., 2010). However, it should be noted that the soil and climate conditions

426

of necromass C decomposition experiment in Throckmorton et al. (2012) were different from

427

this study, and a real assessment of the difference between MRT for necromass C and N

428

should be based on the decomposition experiment of 15N and 13C double-labeled necromass.

429

We found that 56.0-62.3% of necromass

15

N was lost from the mesocosm system after

430

803 days of incubation (Fig. 3). Since we found the recovery of

431

the entire experiment (< 0.05%, Fig. S9), other gaseous N losses such as NOx and N2 might

432

also have been low due to their connections among each other via nitrification and Page 20

15

N in N2O was low during

433

denitrification processes. In addition, the

434

very low during the entire experiment (data not shown) due to the low downward leaching in

435

the study area, which was consistent with the previous result of a 15N tracer experiment in the

436

same site (Liu et al., 2017). Therefore, we speculated that plant uptake N was an important

437

pathway for the loss of necromass N from the mesocosm.

438

Although we found significant

15

15

N content in the deep soils (10-14 cm) was also

N enrichment in roots in 15N treated plots than in the

439

control plots, we can not estimate the recovery rate of necromass

440

because root N is translocated to shoots continuously. In a different lab incubation study

441

without plants, we found accumulation of NH4+ in the soil (unpublished data), which suggests

442

that the loss of NH4+ from the mesocosm was by plant uptake when plants were present.

15

N in plant biomass

443

While our study provides the experimental evidence for the necromass N decomposition

444

and stabilization in field in a temperate forest soil, some limitations still exist. First, the added

445

necromass was not physically protected during the initial stage of our study, while under

446

natural conditions microbial necromass may be already physically attached to soil particles as

447

it derives from formerly living microbial cells that developed in biofilms. This limitation

448

could cause an over-estimation of the decomposition rate of necromass N. Second, the

449

chloroform fumigation extraction method only targets the microbial cytoplasmic components,

450

which can not represent whole microbial cells (Gunina et al., 2017), resulting in an

451

under-estimation of necromass

452

bacterial and fungal necromass might be selectively used by microorganisms and incorporated

453

into cellular components with different turnover rates, so that the chloroform fumigation

454

method might not catch the real variations of necromass

15

N recovery in microbial biomass pool. Additionally, the

Page 21

15

N in microbial biomass pool over

455

time. Thus, our results of necromass

456

Third, the parameters (m and ε) in the analytical model could affect the value of

457

decomposition rate of necromass N. Although our results of m value were in the ranges of

458

previous studies (Wieder et al., 2013; Xu et al., 2017), site-level observations of m and ε in

459

future studies may reduce the uncertainties in predicting necromass N decomposition rate.

460

More future studies on microbial nitrogen use efficiency is also urgently needed to better

461

estimate the contribution of microbial residues to soil organic nitrogen.

15

N recovery in microbial biomass were conservative.

462

In summary, we found that a portion of necromass decomposed rapidly and could be an

463

important nutrient source for microbial growth. Experiment results showed that 33.1-39.5% of

464

microbial necromass 15N still remained in soil as non-living non-extractable N after more than

465

2 years of incubation, suggesting the resistant characteristics of microbial residues. Modeled

466

overall decomposition rate of necromass N was much higher than the decomposition rate of

467

plant litter, and the decomposition pattern and model structure were different between

468

necromass N and plant N, suggesting the necessity of separating these two pools in

469

process-based models. Nevertheless, the contribution of microbial residues to stable organic

470

matter should not be neglected and one important controlling factor was the in situ

471

decomposition rate of microbial biomass, which should be studied more in the future.

Page 22

472

Acknowledgements

473

We appreciate the many helpful comments from two anonymous reviewers that greatly

474

improved the manuscript. We thank Sun, Hao and Li, Xu for the assistance on isolation and

475

enrichment of soil microbial strains, Zhenzhen Fan, Ying Tu, Linlin Song, Ziping Liu and Qu,

476

Lingrui for laboratory analyses, Xianlei Fan for model running and Dr. Ember Morrissey for

477

the comments on the preliminary version of the manuscript. This work was financially

478

supported by the Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC006),

479

National Key R&D Program of China (2019YFA0607300), the National Natural Science

480

Foundation of China (31830015 and 41601255), the Youth Innovation Promotion Association

481

CAS to Chao Wang (2018231) and the National Program for Support of Top-notch Young

482

Professionals (to Edith Bai). The data that support the findings of this study and the code used

483

for model estimation are available from the corresponding author upon reasonable request.

Page 23

484

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Yuan, Z., Chen, H.Y.H., 2009. Global trends in senesced-leaf nitrogen and phosphorus. Global Ecology and Biogeography 18: 532-542. Zeller, B., Colin-Belgrand, M., Dambrine, E., Martin, F., Bottner, P., 2000. Decomposition of

616

15

617

Oecologia 123: 550-559.

N-labelled beech litter and fate of nitrogen derived from litter in a beech forest.

618

Page 30

619

Figure legends:

620

Fig. 1. The conceptual model to analyze necromass

621

includes four dynamic N pools, which are necromass N (Nnecro), biomass N (B), NH4+-N (A)

622

and loss N (mainly plants uptake N and leaching N). Decomposition consists of five processes:

623

necromass N uptake by microbial biomass, necromass N mineralized to NH4+-N, NH4+-N

624

absorbed by microbial biomass, necromass N production via death of microorganisms and

625

NH4+-N loss from soil. The m (yr-1) and k (yr-1) describe the turnover constant of biomass N

626

and necromass N, respectively. The u (yr-1) and l (yr-1) describe the NH4+-N uptake rate

627

constant by microbial biomass and loss rate from soil, respectively. The ε (%) is proportion of

628

the decomposed necromass

629

microbial nitrogen use efficiency.

630

Fig. 2. Recovery of microbial necromass

631

incomplete 15N recovery in bulk soil at 0.5 days, the recovery of each N pool at the following

632

sampling days was standardized to its initial recovery at t = 0.5 days. The unstandardized data

633

were also presented in the Fig. S3. Data were shown as mean values and standard errors. The

634

remaining 15N in soils was well fitted by the one-pool decay model (Eqn. 3), with a MRT of

635

2.75 ± 0.04, 2.90 ± 0.26, and 2.52 ± 0.21 years for bacterial, fungal and actinobacterial group,

636

respectively.

637

Fig. 3. The fates of necromass

638

soil N pools included microbial biomass N (MBN), soil NO3--N, soil NH4--N, N2O-N, loss N

639

(mainly plants uptake N and leaching N) and necromass (includes the undecomposed N and

15

15

N transformations in soil. This model

N uptake by living microorganisms, which is the same as

15

15

N in bulk soil total N pool (0-10 cm). Due to the

N with time. Results show the recovery of

Page 31

15

N in different

640

new formed necromass N) at depth 0-10 cm after tracer addition.

641

Fig. 4. The relative recovery of necromass

642

mineral associated organic matter (MAOM) pool at depth 0-10 cm. The relative recovery of

643

necromass

644

incubation by the recovery of non-living non-extractable necromass 15N.

15

15

N in particulate organic matter (POM) and

N in POM and MAOM was calculated by dividing their recovery at the end of

Page 32

645

Table 1 Soil microbial isolates used in this study. Percentage of a given species in OUT for

646

a given cell type, the mass proportion of treatment mixture for a given cell type, and the

647

microbial necromass N (%), C/N ratio and atom %

648

estimated deviation is given in parentheses.

Groups Bacteria

Fungi

Actinobacteria

Taxonomic classification Bacillaceae Bacillaceae Paenibacillaceae Enterobacteriaceae Zygomycota Mortierellaceae Penicillium Coniochaetaceae Streptomycetaceae Streptomycetaceae Microbacteriaceae Nocardiopsaceae

15

N for the mixture of each group. The

Genus / species

Percentage in OTU (%)

Mass proportion (%)

Bacillus simplex Bacillus sp. Paenibacillus sp. Serratia liquefacien Umbelopsis isabellina Mortierella amoeboidea Penicillium spinulosum Lecythophora hoffmannii Streptomyces avidinii Streptomyces atratus Microbacterium sp. Nocardiopsis sp.

0.10 0.10 0.05 0.001 0.78 4.70 0.13 0.02 0.42 0.42 0.02 0.01

39.2 39.2 21.2 0.4 13.9 83.3 2.3 0.5 48.3 48.3 2.3 1.1

649

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N (%)

C/N ratio

Atom% 15 N

9.2 (0.7)

4.1 (0.1)

65.0 (1.5)

5.3 (0.1)

5.9 (0.3)

64.9 (0.1)

10.3 (0.2)

4.0 (0.1)

65.1 (0.1)

650

Table 2 Modeled microbial necromass N decomposition rate and biomass N turnover rate. Results from an analytical model

Groups Bacteria Fungi Actinobacteria 651 652 653 654

knecro (yr-1)

kr (yr-1)

ks (yr-1)

m (yr-1)

u (yr-1)

R2

3.82 (2.81-4.83) 3.62 (2.12-4.55) 4.08 (3.19-5.21)

6.08 (4.31-7.85) 5.31 (3.22-7.40) 6.88 (5.12-8.64)

0.95 (0.91-0.99) 0.87 (0.82-0.92) 0.79 (0.73-0.85)

6.26 (5.42-7.10) 5.35 (4.57-5.35) 7.27 (4.67-9.87)

0.001 (0.0003-0.0013) 0.001 (0.0001-0.0020) 0.005 (0.0036-0.0076)

0.97 0.98 0.97

knecro is soil microbial necromass N decomposition rate constant. kr is the decomposition rate constant of the rapid decomposition pool. ks is the decomposition rate constant of the slow decomposition pool. m is the turnover rate constant of microbial biomass N. u is the constant of N uptake rate by living microbial biomass. The estimated deviations are given in parentheses.

Page 34

Fig. 1.

Page 1

Fig. 2.

Page 2

100

100

(b) Fungi Recovery of necromass 15N (%)

Recovery of necromass 15N (%)

(a) Bacteria 80

60

40

20

0

80

60

40

20

0 0

0.5

9

30

60

132

362

490

803

0

0.5

9

Time (days) Loss N Necromass N N2O-N

Recovery of necromass 15N (%)

(c) Actinobacteria 80

NH4+-N NO3--N MBN

60

40

20

0 0.5

9

30

60

132

60

132

Time (days)

100

0

30

362

490

803

Time (days)

Fig. 3.

Page 3

362

490

803

Fig. 4.

Page 4

Highlights

We showed the microbial necromass N decomposition rate in a temperate forest soil. Approximately 40% of microbial necromass 15N was recovered in soil after more than two years incubation in situ. We found no difference in necromass N decomposition rate among microbial groups.