Preferential Alternatives to Returning All Crop Residues as Biochar to

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Agricultural and Environmental Chemistry

Preferential Alternatives to Returning All Crop Residues as Biochar to the Crop Field? A Three-source 13C and 14C Partitioning Study Xiaowen Ji, Evgeny Vasilievich Abakumov, Xianchuan Xie, Dongyang Wei, Rong Tang, Jue Ding, Yu Cheng, and Jie Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03323 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Journal of Agricultural and Food Chemistry

Preferential Alternatives to Returning All Crop Residues as Biochar to the Crop Field? A Three-source 13C and 14C Partitioning Study Xiaowen Ji,†,‡ Evgeny Abakumov,‡ Xianchuan Xie,*,† Dongyang Wei,§ Rong Tang,‖ Jue Ding,┴ Yu Cheng,† and Jie Chen#

†State

Key Laboratory of Pollution Control and Resource Reuse, School of the

Environment, Nanjing University, 210093, Nanjing, P. R. China ‡Department

of Applied Ecology, Saint Petersburg State University, 199178, Saint

Petersburg, Russian Federation §South

China Institute of Environmental Sciences, Ministry of Environmental

Protection, 510655, Guangzhou, P. R. China ‖School

of Environment and Ecology, Jiangsu Open University, 210036, Nanjing, P. R.

China ┴Key Laboratory of Integrated Regulation and Resource Development on Shallow Lake

of Ministry of Education, College of Environment, Hohai University, 210098, Nanjing, P. R. China #School

of Geography and Ocean Science, Nanjing University, 210023, Nanjing, P. R.

China *

Corresponding Author: Dr. Xianchuan Xie ([email protected])

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ACS Paragon Plus Environment


ABSTRACT: The simultaneous effects of biochar on soil organic matter (SOM, C4) and sweet potato (SP) residue (Ipomoea batatas, C3) mineralization were studied over 180 days via 13C

and

14C

isotopic label partitioning. Upon concomitant SP residue addition, biochar

mineralization decreased by 11% of total added biochar-C. Compared with positive priming effects induced by biochar amendment alone on SOM (0.46 mg C g−1 soil) at 180 days, amendment solely with SP residues induced significantly larger effects (1.5 mg C g−1 soil). Combination biochar and SP residue addition reduced SOM mineralization by 20.5% and increased SP residue mineralization by 10.1%. Biochar addition caused preferential uptake of SP residues over SOM by microbes. Thus, the lower priming effects on SOM and CO2 emission induced by biochar amendment with or without SP residues compared to that from SP residue addition alone may result in crop residues being partly pyrolyzed to biochar in the cropland.

KEYWORDS: biochar, priming effect; sweet potato residue, three C sources, C isotope label

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INTRODUCTION In recent years, the conversion of plant biomass to biochar has attracted considerable attention because biochar as a C source has been widely utilized in agronomy owing to its dual benefits of improving soil fertility and C sequestration (negative priming effects) to prevent greenhouse gas emissions such as CO2, CH4, and N2O.1-2 Owing to the inert C of biochar, biochar amendment can be maintained in the soil for hundreds or thousands of years.3 Therefore, biochar derived from crop residues may serve as a long-term C sink for counteracting CO2 emissions. However, although biochar C may facilitate soil C storage, it is very important that it not activate native soil organic matter (SOM) stores or cause other negative environmental consequences in order to be adopted by policymakers or cropland owners as a climate change abatement strategy.4 Alternatively, returning crop residues to the soil can promote organic matter sources and increase soil water-holding capacity (WHC) along with the overall quality of soil moisture and fertility.5 Crop residues as main C sources in soil can alter microbial activation, consequently producing either positive or negative priming effects on SOM.6 Numerous studies have examined the interaction between biochar and both native SOM and crop/plant residues.6-9 Nevertheless, the effects of biochar amendment on native SOM decomposition are controversial.8-9 Short term soil incubation with biochar caused a positive priming effect on SOM; i.e., 15% SOM mineralization increase,10 but also negative priming effects with 8.6% decreased SOM mineralization over 6 months.11 The priming of C mineralization in soil over a short period was closely associated with the labile C in biochar.12 Additionally, SOM degradation by microbes benefitting from the labile C in biochar may produce a positive priming effect.13 Various factors underlie these discrepancies including SOM physicochemical properties, pyrolysis and feedback temperature, available soil nutrients, and soil microbial communities.10 To date, few studies have differentiated the sources of C pools during biochar-induced priming using 14C labeling and 13C natural abundance for differentiating heterogeneous C pools

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in biochar-enriched soils. For example, Luo et al.14 observed that 14C-labeled glucose (substrate) addition caused a positive priming effect in biochar-enriched soils over 28 days, which were 53% (German Luvisol) and 140% (Chinese Luvisol) higher than those in biochar free soils. However, Luo et al.14 pointed out that natural substrates such as root exudates and plant residues should be examined in long term field experiments for confirmation of these posited effects. Cui et al.9 revealed that the incorporation of biochar into soil consisting of maize litter sped up litter decomposition albeit decreased SOM decomposition as shown by dual 13C/14C isotopic labels. Notably, the process of organic matter decomposition is quite dynamic, which may be influenced by varying degrees of biochar and different chemical characteristics.15 Although interaction between the simultaneous decomposition of biochar, SOM, and litter has been demonstrated,9 few studies have shown that the negative priming of SOM mineralization by the plant rhizosphere might be due to the biochar addition into systems involving plants and soil.1617

Generally, these reports only included plant rhizosphere exudate rather than recalcitrant

vegetation residues and most did not differentiate whether the C fluxes affected by biochar originated from SOM or plants. Currently, although biochar has been widely used for agricultural amelioration as well as remediation and passivation of heavy metals in soils, the priming effects for short-term use with crop residues remain unclear. Here, in order to retain maximum soil fertility from an agricultural perspective and prevent SOM decomposition in the cropland, we hypothesized that a portion of crop residues after harvesting should be converted to biochar with the remainder retained in in soil. To understand the benefits of this strategy, the effects of biochar amendment in soil mixed with sweet potato (SP) stems and leaves on SOM versus SP residue mineralization over 6 month laboratory incubation were assessed. The three-source partitioning approach including 13C and 14C isotopic labels was used to partition SP residue, SOM, and biochar decomposition. Our aims were to (1) explore the SOM decomposition primed separately by SP residues versus biochar, (2) distinguish the priming effects induced by SP residue addition on SOM versus biochar in

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mixtures of soil and SP residues, and (3) offer a concept for the likely consequences to soil C pools caused by biochar amendment.

MATERIALS AND METHODS Soils, Biochar, and SP Residues. Soils were sampled (0–15 cm) using a stainlesssteel shovel from a plowing (Ap) horizon from an experiment plot (located in the north part of Vasilyevsky Island, in a field managed by the Department of Applied Ecology, Saint Petersburg State University) growing C3 perennial ryegrass (Lolium perenne) and soybean (Glycine max) without other land uses since 2013. The soils were classified as Plaggic Anthrosols according to the World Reference Base system soil classification scheme. The soil samples were handpicked to remove visible plant debris, fully homogenized through a 2 mm sieve, and adjusted to 50% WHC for incubation. Soil pH was potentiometrically determined using a pH meter (Multiparameter Bench Top Table Top pH Meter PHS-550, Hangzhou, China) in a supernatant suspension of a 1:2.5 soil liquid mixture consisting of a 1 M KCl solution (unbuffered). An elemental analyzer (Vario MAX CNS Element Analyzer, Elementar, Langenselbold, Germany) was used to determine total C by dry combustion. Roughly 25 mg of air-dried soil (milled < 200 μm) was placed in a small vial for measurement. Inside the apparatus, soil samples were heated to 975 C with a combination of helium carrier stream and oxygen for oxidation promotion. The generated CO2 was quantified using a thermal conductivity detector after being differentiated among other gases in the chromatography column. The output signal from detector was converted into C concentrations according to the mass of each soil sample. The Kjeldahl semimicro protocol for total nitrogen (N) analysis was used based on acid digestion followed by steam distillation of the resulting NH3 following alkali addition. Dry combustion was conducted simultaneously with analysis of C in the same elemental analyzer. Soil property characteristics are shown in Table 1.

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Biochar was generated from SP (Ipomoea batatas) stems and leaves evenly labeled with 14C.

Labeling procedures were performed according to Bromand et al.18 and were conducted in

a chamber (3.18 mm thick transparent polycarbonate walls and a 5.32 mm thick polycarbonate ceiling) wherein young SP seedlings were grown for approximately 1.5 months. The labeling chamber was checked for air leakage by filling with a high concentration of CO2 (800 ppm) and checking the concentration of CO2 retained overnight prior to the labeling experiment. Plastic beakers containing Na214CO3 (1.06 × 104 μg mL−1 and 15.9 × 103 Bq mL−1) and HCl (1M) were placed near the SP seedlings for reaction with the contained 14CO2. Atmospheric CO2 was maintained at about 300 ppm with consecutive resupply of Na214CO3 every 3 days. Following labeling, the stems and leaves were cut off, air-dried at 75 C, and stored in 1 C in a refrigerated chamber. To generate 14C-enriched biochar, 14C-labeled SP stems and leaves were milled with ZrO2, screened using a 2 mm sieve, then placed into air-tight platinum crucibles (20 mm diameter, 15 mm height, 0.22 mm wall thickness). The crucibles were placed in a duo-muffle furnace at an initial temperature of 25 C and slowly increased to 350 C at a rate of 5 C min−1, maintained at 350 C for 15 h, then naturally cooled to room temperature. Following carbonization, the biochar product mass was 28.7 ± 0.9% of the initial SP stem and leaf mass. The biochar was ground and screened using a 0.5 mm sieve prior to soil incubation experiments. Biochar

14C-specific

radioactivity was measured as previously described.19 Briefly,

approximately 5 g weighed biochar was digested for 10 min at 70 C in potassium dichromate (0.2 M, 20 mL) mixed with 50 mL acid mixture (H2SO4:H3PO4, 2:1). The evolved 14CO2 during digestion was trapped by NaOH (0.4 M, 50 mL). To determine trapped 14CO2 14C radioactivity, adsorbed

14CO

2

in NaOH was titrated electrically an automatic titrator (TitroLine® 5000,

Thomas Scientific, Waltham, MA) by the difference of conductivity between the carbonated NaOH and a pure reference solution. The separation of

14C

was analyzed according to its

radioactivity, as described in Isotopic and Chemical Analyses, below.

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Biochar pH was determined using a pH electrode at a ratio of 1:20 biochar:water. To measure the water-exchange fractions of components, the biochar was extracted thrice using distilled water (1:10, v:v) and the extracts were combined. The NH4+ concentrations in the water extracts were measured using the phenol-hypochlorite reaction for determination, and the NO3− was measured via steam distillation using Devarda's alloy. Dissolved organic C in biochar extracts was determined using an elemental analyzer (multi EA® 4000 for Macro-Elemental Analysis, Analytik Jena, Germany), and the total contents of C and N in the solid biochar were assessed following the LECO Combustion Analysis methods.20 Biochar δ13C value was determined using a stable isotope ratio mass spectrometer (253 Ultra, Thermo Scientific) with ± 0.30‰ accuracy, and calculated from:

δ13C (‰) =

[(

) ― 1] × 1000

𝑅𝑠

𝑅𝑉 ― 𝑃𝐷𝐵

(1)

where Rs and RV-PDB represent the biochar ratio of 13C/12C and the Vinna Pee Dee Belemnite (V-PDB) standard,21 respectively. SP plants were grown in a controlled chamber for 160 days. To ensure even labeling, SPs were pulse-labeled with approximately 99%

13CO

2

with regular intervals during the whole

growth period. The homogenous labeling of SP leaves and stems was confirmed using measurement of 13C values at residue degradation stages.6 Upon maturation, the upper part of the SP (leaves and stems) was harvested and gently washed for soil particle removal.

Experimental Design and Layout. To explore the priming effects on SP residues and SOM after biochar addition, a 180 day incubation experiment was carried out for four treatments with three replicates of each treatment: (1) soil without any addition (control), (2) soil+SP residues (leaves and stems), (3) soil+biochar, and (4) soil+SP residues+biochar. The biochar and SP residues were added to soils at a rate of 50 mg C g−1 soil as previously reported.14 SP residues and biochar were thoroughly milled before mixing with dry soils (50 g, freezedried weight). The weight of soil (50 g) in the various treatments was kept the same. The WHC of the mixtures was maintained at 40% ± 5% by deionized water addition on a gravimetric

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basis. To evaluate maximum WHC, all mixtures were treated similarly: the mixtures were moved to a funnel containing 0.45 μm filter paper to restrict soil or SP residues/biochar particle loss during the whole incubation. Mixtures permeated by distilled water were allowed to gravimetrically drain for 12 h prior to freeze-drying for WHC measurement. The incubation processes were as described by Shahbaz et al.22 with some modifications. All samples were placed into a 50 mL glass bottle and incubated in a 100 mL glass jar including a vessel containing 30 mL of 1.0 M NaOH solution (including three null control bottles, such as without soils) to trap the evolved CO2 and 3 mL of deionized water at the bottom to maintain soil mixture moisture. The glass bottles containing soil/mixture samples were covered with parafilm. Once the glass bottles were placed into the glass jars, the parafilm was removed and the glass jars were sealed with a foam liner and incubated at 20 C ± 0.5 C. Three blank jars were established without soil but with only deionized water and NaOH to provide atmospheric CO2 in the headspace of the incubation jar. The NaOH in the vessel was exchanged at 0, 3, 8, 10, 30, 50, 90, 120, 150, 160, and 180 days. These dates were chosen to prevent the traps being saturated through the use of more Na2CO3 than 60% of their capacity.

Isotopic and Chemical Analyses. To quantify the evolved CO2, 1 M BaCl2 was added for precipitation of the NaOH solution. The total quantity of trapped CO2 was determined by titration of excess NaOH with 0.05 M HCl and an indicator of phenolphthalein. Owing to the dual labeling method (13C-labeled SP residues and 14C-labeled biochar), the CO2 trapped by NaOH was specifically prepared. To measure 13C, 15 mL of trapped CO2 as Na2CO3 in NaOH was precipitated with the same amount of 1 M SrCl2 solution. The NaOH solution containing SrCO3 was then centrifuged for 20 min at 4000 rpm. The process was repeated with deionized water to eliminate excess NaOH and adjust the pH to 7. SrCO3 pellets were dehydrated and freeze-dried prior to 13C analysis. Pellet 13C values were measured using an isotope ratio mass spectrometer coupled with an elemental analyzer (multi EA® 4000 for Macro-Elemental Analysis).

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14C

activity determination was carried out immediately upon CO2-trapped NaOH vessel

replacement with new vessels during the incubation. The 14C activity of CO2 trapped in NaOH was determined by the decay of chemiluminescence in Rotiszint Eco Plus LSC-Universal Scintillation Cocktail (Carl Roth, Germany) using a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer, Waltham, MA). Soil microbial biomass C was measured at the end of the incubation using the chloroform fumigation-extraction method. Briefly, soils were destructively sampled, mixed completely, and 10 g of moist soil extracted with 35 mL 0.05 M K2SO4 at 25 C for 3 h. Another 10 g of moist soil was fumigated with ethanol-free CHCl3 for 48 h and extracted as described above. The obtained extracts were store at 3 C and 5 mL aliquots were then analyzed for C concentration using a TOC/TNb analyzer (Vario TOC cube, Elementar). The total amount of extractable microbial biomass C was calculated via the surplus between K2SO4-extracted C in fumigated and non-fumigated soils with a conversion factor of 0.45.23 Another 10 mL K2SO4 extract aliquot was freeze-dried at −60 C and later analyzed for 14C activity using a liquid scintillation counter (Tri-Carb 2100TR, PerkinElmer). The remaining 20 mL of K2SO4 extracts was freeze-dried for measuring the 13C composition of soil microbial biomass C with an isotope ratio mass spectrometer (253 Ultra). The contents of soil NH4+ and NO3− in water extracts were measured similar to biochar. The total C and N in soil was measured using an elemental analyzer (Multi EA® 4000 for Macro-Elemental Analysis, Analytik Jena, Jena, Germany).

Calculation and Statistics. CO2 efflux and K2SO4-extractable dissolved organic content (DOC) were differentiated in the soil mixtures for three sources: SOM, biochar, and SP residues as previously described.24 We partitioned C derived from biochar or SP residues/SOM according to 14C signal, then distinguished between SP residues and SOM-derived C according to their δ13C signatures. Firstly, the quantity of SOM-derived C in the corresponding pool was calculated as 14C activity in the pool as follows:

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CB ― drived =

(CS ― CBL) × 30 14CB/CB

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(2)

where CB-derived (mg C) represents the quantity of biochar-derived C; CS and CBL (decay per min, DPM) represent the 14C activity of the samples and the blank samples (in 1 mL NaOH solution), respectively. 14CB (DPM g−1 biochar) is the 14C radioactivity ratio of biochar; and CB (mg C g−1 biochar) is the biochar C concentration; 30 is the total NaOH amount (mL) applied to trap released CO2. δ13C values (‰) derived from SP residues and SOM (non-biochar sources) were calculated according to the mass balance model of stable isotopes:

𝛿13Cnon ― B =

𝛿13Ctotal × Ctotal ― 𝛿13CB × CB ― derived

(3)

Ctotal ― CB ― derived

Cnon ― B = Ctotal ― CB ― derived

(4)

where δ13Cnon-B and Cnon-B are the C originating from the non-biochar for δ13C values and C contents, respectively; and δ13Ctotal and Ctotal are the total C pool for δ13C values and C contents, respectively. Finally, C deriving from SP residues and SOM was calculated following:

CSP ― derived = Cnon ― B ×

𝛿13Cnon ― B ― 𝛿13CSOM

(5)

𝛿13CSP ― 𝛿13CSOM

CSOM ― derived = Cnon ― C ― CSP ― derived

(6)

where CSP-derived and CSOM-derived (mg C) represent SP- and SOM-derived C, respectively. δ13CSP and δ13CSOM represent the values of δ13C for SP residues and SOM, respectively. The control soils at each sampling day were used for mean δ13C values to assess δ13CSOM. For separate SP residue/biochar addition treatment, no apparent

14C

activity signals in

K2SO4-extractable DOC could be measured; thus, the relevant parameters were not included during the course of calculation. Soil microbial biomass C derived from SP residues during incubation can be calculated as:

Microbial biomass CSP ― derived = CSP ― derivedf ― CSP ― derivednf

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(7)

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Microbial biomass CSOM ― derived = Microbial biomass Ctotal ― Microbial biomass CSP ― derived

(8)

where microbial biomass Ctotal/SP-derived/SOM-derived refers to the total amount (μg C g−1 soil) of microbial biomass C, and those deriving from SP residues and SOM, respectively. CSP-derivedf and CSP-derivednf are the content (μg C g−1 soil) of K2SO4-extractable DOC originating from SP residues of the fumigated and non-fumigated soils respectively, which were calculated based on Eqs. (2)–(6). The priming effects/relative priming effects of separate biochar/SP residue addition on decomposition of SOM was calculated based on:

Priming effects = CSOM ― derivedamended ― CSOM ― derivednon ― amended Priming effects (%) =

CSOM ― derivedamended ― CSOM ― derivednon ― amended CSOM ― derivednon ― amended

(9) (10)

× 100

where CSOM-derivedamended and CSOM-derivednon-amended refer to the amount of SOM-deriving CO2-C (mg C g−1 soil) in soils amended with only biochar or SP residues and in control soils without amendment, respectively. Priming effects induced by addition of SP residues on biochar mineralization were calculated based on:

Priming effects = CB ― derivedSP + B ― CB ― derivedB

(11)

where CB-derivedSP+B and CB-derivedB represent the amount of biochar-derived CO2-C (%, in total input of biochar C) in the soil+biochar+SP residues and soil+biochar amendments, respectively. To compare the priming effects induced by biochar addition on SP residue/SOM mineralization simultaneously, the calculations were based on soil+SP residues and soil+SP residues+biochar treatments:

Priming effects on SP residues = CSP ― derivedSP + B ― CSP ― derivedSP Priming effects on SOM = CSOM ― derivedSP + B ― CSOMSP

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(12) (13)


where CSP-derivedSP+B/CSOM-derivedSP+B and CSP-derivedSP/CSOM-derivedSP represent the amount of SP residue-/SOM-originated CO2-C (mg C g−1 soil) from soil+SP residues+biochar and soil+SP residues, respectively. One-way ANOVA was applied to evaluate the significant different in C effluxes among treatments using Duncan’s post-hoc test. The significance of SP residue and biochar addition effects was evaluated at p < 0.05 (two-way ANOVA).

RESULTS Total Amount of CO2 Efflux. During the process of 180 day incubation, the total amount of cumulative CO2 evolved for control soil was 1.91 ± 0.02 mg C g−1; biochar-only amended soil released 2.12 ± 0.03 mg C g−1 CO2. The order of the evolved cumulative CO2 amount was: soil+SP residues = soil+SP residues+biochar > soil+biochar > soil only (Figure 1). The cumulative CO2 evolved from biochar-alone amendment increased significantly during 50–180 days (1.60 mg C g−1) (p < 0.05), significantly higher than that during 0–50 days (0.69 mg C g−1) (p 0.05). However, both soil+SP residues and soil+SP residues+biochar exhibited significantly higher CO2 effluxes than those of soil with biochar amendment and control soil.

Decomposition of Biochar With and Without SP Residue Addition. Biochar mineralization was calculated according to CO2 efflux. Over 180 day incubation, biochar decomposition occupied only 5.01–5.34% (average decomposition rate: 0.042% day−1) (Figure 2a). Highest biochar decomposition rate (5.23–7.62% day–1) during the first two days, especially for the soil+biochar treatments, followed by one order of magnitude declining trend during 3–140 days (0.08–0.14% day−1) then maintenance at 0.0003–0.0016% day−1 after 140 days (Figure 2b). Approximately 66% of total biochar-induced CO2 was produced in the first

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70 days (Figure 2a). The SP residues+biochar soil samples showed a lesser extent of biochar decomposition (Figure 2a). However, biochar mineralization rate for SP residue treatment was higher than that for treatments without SP residues at the last incubation day (Figure 2b).

Decomposition of SP Residues With and Without Biochar Addition. SP residues provided the highest contribution of CO2 emission from soil, representing 61 ± 2.3% of total produced CO2. During the whole incubation period, cumulative SP-derived CO2 was 8.45 ± 0.62 and 9.12 ± 0.46 mg C g−1 soil for soil+SP residues and soil+SP residues+biochar treatments, respectively (Figure 3a). This suggested a priming effect of approximately 7.3% on SP residue mineralization by biochar addition, with the cumulative positive priming effects occurring mostly in the first 50 days (Figure 3a). SP residue decomposition rates were highest during the first 50 days (mean = 0.24 mg C g−1 soil day−1); the rate decreased during 3–8 days, then increased to 0.31 mg C g−1 soil day−1 at 50 days for soil+SP residue treatments. However, the soil+SP residues+biochar rates increased from 3–8 days and decreased during 8–10 days, then increased during 10–50 days. Subsequently, the SP residue mineralization rates decreased until the end of incubation (approximately 0.0125 mg C g−1 soil day−1) (Figure 3b).

Release of CO2 from Protogenetic SOM. SOM decomposition decreased during 180 day incubation for all amendments, following the order (mg C g−1 soil): soil+SP residues (3.37 ± 0.41) > soil+SP residues+biochar (2.41 ± 0.28) = soil+biochar (2.36 ± 0.17) > soil only (2.1 ± 0.14) (Figure 4a). All amendments caused net positive albeit different priming effects on SOM decomposition over the entire incubation period compared with that of control soil, with the order (mg C g−1 soil): soil+SP residues (1.40 ± 0.21) > soil+SP residues+biochar

(0.70 ±

0.11) > soil+biochar (0.42 ± 0.06). Notably, these positive effects were observed at end of incubation rather than throughout the entire incubation period (Figure 4b). Soil + biochar cumulative priming effects were initially negative, then maximally negative at 8–10 days, gradually becoming positive after 90 days. Soil+SP residue effects were positive with steady increases throughout (Figure 4b). Soil+SP residues+biochar priming effects could be divided

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into three stages: (1) gradually increasing priming effects within the first 8 days; (2) decreasing to relatively small negative effect during 8–90 days; and (3) increasing to positive after 90 days.

Priming Effects Induced by Biochar on Decomposition of SOM and SP Residues in Soils Consisting of SP Residues. To evaluate the effects induced by biochar on SP residues and native SOM in mixtures of soil and SP residues, the CO2 emission originating from soil+SP residues+biochar amendment was compared with that from soil+SP residue amendment. Biochar appeared to have an opposing priming effect on SOM mineralization and SP residues (Figure 5). Biochar addition afforded a slightly positive effect on SOM only in the first 8 days, declined slightly during 8–50 days, then dramatically decreased to −4.2 ± −0.27 mg C g−1 soil at the end of incubation (relative priming effect = −20.5%). Conversely, during the first 8 days, the slightly negative priming effects on SP decreased, becoming positive after 30 days then increasing to 5.22 ± 0.26 mg C g−1 soil (relative priming effects = 10.1%) at 180 days.

Microbial Biomass. During the 180 day incubation period, the cumulative total amount of microbial biomass C in soils decreased, following the order (μg C g−1 soil): soil+SP residues+biochar (201 ± 20) > soil+SP residues (158 ± 16) > soil+biochar (163 ± 20) > control soil (143 ± 21) (Figure 6). The different sources of microbial biomass C indicated that biocharderived C could not be detected in soil+biochar treatments and only slightly in soil+SP residues+biochar treatments. Apparently, SOM-derived microbial biomass C constitutes the dominant outcome for all treatments, with the order (μg C g−1 soil): soil+biochar (143 ± 23) = control soil (141 ± 19) > soil+SP residues (132 ± 8) > soil+SP residues+biochar (112 ± 11). An obvious amount of microbial mass C (12%–32%) derived from SP residues, particularly for treatments of soil+SP residues and soil+SP residues+biochar (46 ± 5 and 64 ± 12 μg C g−1 soil, respectively), indicating that biochar facilitated SP residue conversion to SOM microbial biomass.

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DISCUSSION As 14C allows greater analyses sensitivity than that for stable isotopes, labeled 14C biochar can be effectively quantified for slow decomposition rates.8-9, 14 In the present study, following biochar addition, the mineralization of biochar only accounted for 5–10% at the last day of incubation with a decomposition rate (0.0016% day−1) approximately 2–4 times higher than those obtained using 14C-labeled biochar; i.e., made from rice (Oryza sativa) in soils with added maize (Zea mays)9 or from perennial ryegrass (L. perenne) in soils with added glucose.25 The average biochar decomposition rate (0.011% day−1) approached that of another 183 day 14Clabeled biochar incubation study (0.015–0.021% day−1)15 and a synthesis study for < 6 months (0.023% day−1).10 The higher mean decomposition rate may result from the high water content (40%) of our soils, which favored biochar decomposition.9 Moreover, the higher molar H/C ratio (0.63) in this study may have also favored biochar decomposition. The biochar mineralization rates during the first 100 days were 1–2 orders of magnitude faster than those at the end of incubation (140–180 days) in our study. Biochar C decomposition can be greatly influenced by SOM quality, with soil DOC increasing following biochar addition through soil microbes preferentially utilizing the new C input.3, 26 Therefore, the higher DOC content of our soils may facilitate initial labile biochar C decomposition. Our results showed the occurrence of negative priming effects induced by SP residues on biochar mineralization, comparable to the decreased 14C-labeled biochar observed upon straw addition.9 The SP residues used herein are more recalcitrant and afford a less simulative effect on soil microorganisms. Natural substrates such as SP residues generally utilize k-strategists with slow growth.27 Furthermore, SP residue-decomposed organic compounds may obstruct biochar, causing its slow mineralization.9 Consequently, different plant residues, and soil and biochar properties may yield different priming effects on biochar decomposition, which requires further study. CO2 effluxes increased following biochar addition, indicating positive priming effects on the decomposition of SOM. The degree of priming effects (0.42 mg C g−1 soil, 6 months) was

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similar to previous reports.9, 28 Biochar priming effects on soil SOM over a short period may be explained by labile organic substances in biochar via two mechanisms. First, soil microbes were “triggered” by biochar labile C to decompose SOM (co-metabolism).28 Significantly enhanced labile C mineralization of biochar may occur for several days following biochar amendment,28 causing the greatest priming effects in the first month.10 Second, compared to recalcitrant SOM in soil, biochar C is more available for microbes, leading to negative priming effects on SOM mineralization.11 Moreover, biochar containing carbonates can also produce CO2 effluxes in the initial period following amendment,29 potentially resulting in overestimation of biochar decomposition.30 This portion of CO2 emission should therefore be removed when calculating biochar-induced priming effects on SOM mineralization. However, the pH was neutral in the current biochar, indicating that this does not likely constitute an important pathway of CO2 emission in the current biochar. The highest biochar decomposition rate occurred over 50 day incubation with significantly negative priming effects. Specifically, microbes likely preferentially consumed the labile C rather than supporting co-metabolism; thus, the biochar was largely mineralized. Preferential microbial utilization of exogenous C sources is also consistent with a reported biochar-induced negative priming effect on SOM in the initial 10-20 days.31 During 50–180 day incubation, biochar mediated successively positive priming effects on SOM. The decomposition rate was as slow as 0.00043 mg biochar-induced C g−1 soil day−1, which was clearly lower than the magnitudes of SOM-induced CO2 emission (0.045 mg C soil day−1) and priming effects (0.062 mg C soil day−1). This cannot all be due to co-metabolism. Biochar amendment can alter soil conditions; e.g., improving nutrient and soil water availability;32 improving drainage and aeration;33 and influencing potential enzyme activities.34 In the last incubation day, microbial biomass C increased in soil+biochar treatment along with mineral N content (by 143%) (Table 2). This mineral N may originate from two sources: (1) biochar itself and (2) in part when SOM primed by the biochar labile C was mined by soil microbes for nutrients or C.35 Notably, this

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mechanism is specific to the SP residues utilized for biochar production as biochar made of lignocellulosic biomass appears as opposing N mineralization in soil.36 Microbes and nutrient substances in the soil changed by biochar addition can significantly influence SOM mineralization.37 Thus, the SOM decomposition induced by biochar positive priming effects observed in this study cannot compare to those upon the addition of simple organic substances such as glucose, but rather corresponds to long term changes in soil microbiology and fertility. Therefore, the priming would maintain stable even after exhaustion of labile substance from biochar.37 Generally, soils amended by biochar can enhance C sequestration, reflecting the long-term influence on soil nutrients and microbial activity. SP residue decomposition was increased after biochar addition to soil and SP residue mixtures along with decreased SOM, which was consistent with reported increased maize and switchgrass litter decomposition upon biochar addition,9, 38 albeit conflicted with other reports of decreased plant residue mineralization.3,

37

This discrepancy may derive from different

experiment conditions and materials used for biochar. During the entire incubation, biochar addition accelerated SP residue decomposition and reduced SOM (Figure 5), indicating that soil microbes could exploit SP residues rendered more readily available through biochar. Additionally, comparison between soil+biochar and soil+SP resides clearly showed more microbial biomass C originating from SP residues (Figure 6). Similarly, biochar addition to maize litter in soil and maize litter mixtures promoted maize litter decomposition compared with addition to soil only.9 The mechanisms of biochar effects on SP residues and SOM decomposition can be associated with SOM mining by microbes for their nutrient demands as microbes tend to utilize exogenous C substrates to increase SOM mineralization.8, 24 Thus, SP residues with high C:N ratio could evoke long term positive priming effects on SOM decomposition (Figure 4) consequent to mining of N in SOM by microbes.39 Additionally, C-rich SP residues with labile N may facilitate preferential microbial use of SP residues, decreasing their N acquisition and thereby reducing decomposition of SOM.39 The NH4+ from biochar itself increased NH4+ in soil

17



by about 100% (Table 1), which may partially meet the microbial N requirements to deplete SP residues. Furthermore, the content of NH4+ and NO3− in soil+biochar+SP residue mixtures was much lower than that in soil+SP residues, indicating that mineral N was immobilized upon SP residue addition whereas microorganism N utilization and retention in soil+SP residue mixtures occurred consequent to biochar addition. Thus, available N for microbial activities is markedly influenced by added exogenous C sources, highlighting that the process of biochar effects on N should be considered, rather than only the labile C in biochar, with regard to priming effects. However, a limitation of this study is that synchronous N-labeling was not utilized to illustrate N mineralization in different treatments. Biomass, such as litter and plant residues, is recommended for biochar production to segregate C that is readily decomposed in soils, thereby reducing CO2 emission as a greenhouse gas.40 Alternatively, full pyrolysis of crop residues would deprive soil of C sources from crop residue incorporation, thus requiring the management of crop residues in the agroecosystem to provide more beneficial effects; e.g., promoting soil fertility, crop yields, and microbial activities.40 However, the process of SOM/N induction by biochar addition remain unclear for agroecosystems, especially regarding crop yield at a large-scale. The present study presented an example of SP residues that alone induced the positive priming effects on SOM decomposition (> 50%), whereas this priming effect was significantly reduced (20%) following biochar addition to SP residue+soil mixtures. Alternatively, a previous study showed that the net soil emission of greenhouse gas (CO2, N2O, and CH4) was reduced by 37% annually over 2 years in a biochar-treated Miscanthus bioenergy crop.40 Cardinael, et al.41 suggested that straw did not induce a net loss of SOM; moreover, some reports demonstrated that plant straw residues can replenish SOM reserves.42-43 In practical perspective, plant residues can be used as a widely available feedstock for famers and can be regarded as a better soil conditioning agent than biochar with regard to certain characteristics, especially in the short term (i.e., for promoting microbial activity, cycling of nutrients, improving aggregation and structural stability, and being less susceptible to water/wind

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erosion). In comparison, biochar can promote some features in the long term.44-45 Therefore, considering that legislative and economic barriers may stifle large-scale adoption of biochar in crop production areas,46 we propose that crop residues might be partly pyrolyzed to biochar for beneficial agronomic effects along with preventing greenhouse gas emission. Additionally, retaining a portion of crop residues in this format allows more ready availability for soil microbes.

AUTHOR INFORMATION Dr. Xiaowen Ji ([email protected]) School of the Environment, Nanjing University, Nanjing, P. R. China Dr. Evgeny Abakumov ([email protected]) Department of Applied Ecology, Saint Petersburg State University, Saint Petersburg, Russian Federation Dr. Dongyang Wei ([email protected]) South China Institute of Environmental Sciences, Ministry of Environmental Protection, Guangzhou, P. R. China Dr. Rong Tang ([email protected]) School of Environment and Ecology, Jiangsu Open University, Nanjing, P. R. China Dr. Jue Ding ([email protected]) College of Environment, Hohai University, 210098, Nanjing, P. R. China Dr. Yu Cheng ([email protected]) School of the Environment, Nanjing University, Nanjing, P. R. China Dr. Jie Cheng ([email protected]) School of Geography and Ocean Science, Nanjing University, Nanjing, P. R. China Corresponding Author 19



*E-mail: [email protected]. Tel: +86-025-83594492 ACKNOWLEDGEMENTS We are very grateful to Dr. Christian Knoblauch from Institute of Soil Science, Faculty of Mathematics, Informatics and Natural Sciences, Universität Hamburg for good suggestions to our manuscript, and Miss Yu Su from School of Visual Art, BFA Computer Art for helping to draw supplementary journal cover. We also would like to thank three anonymous reviewers for good comments and suggestions.

FUNDING This work was supported by Water Pollution Control and Treatment of China (2015ZX07204 and 2017ZX07602), the National Natural Science Foundation of China (41203062 and 51438008), the Jiangsu Nature Science Fund (BK20151378 and BE2015708), Fundamental Research Funds for the Central Universities (090514380001), the Natural Science Fund for Colleges and Universities in Jiangsu Province (16KJB610004), the Natural Science Foundation of Guangdong Province (No. 2016A030313021), and a Guangzhou Science and Technology Plan Scientific Research Project (No. 201607010259).

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36. Rodríguez-Salgado, I.; Pérez-Rodríguez, P.; Campillo-Cora, C.; Gómez-Armesto, A.; Arias-Estévez, M.; Díaz-Raviña, M.; Nóvoa-Muñoz, J. C.; Fernández-Calviño, D., Nitrogen mineralization dynamics in acid vineyard soils amended with bentonite winery waste. Arch Agron Soil Sci 2018, 64 (6), 805-818. 37. Bruun, S.; el-zehery, T., Biochar effect on the mineralization of soil organic matter. Pesq Agropec Bras 2012, 47 (5), 665-671. 38. Novak, J. M.; Busscher, W. J.; Watts, D. W.; Laird, D. A.; Ahmedna, M. A.; Niandou, M. A. S., Short-term CO2 mineralization after additions of biochar and switchgrass to a Typic Kandiudult. Geoderma 2010, 154 (3), 281-288. 39. Chen, R.; Senbayram, M.; Blagodatsky, S.; Myachina, O.; Dittert, K.; Lin, X.; Blagodatskaya, E.; Kuzyakov, Y., Soil C and N availability determine the priming effect: microbial N mining and stoichiometric decomposition theories. Global Change Biol 2014, 20 (7), 2356-67. 40. Case, S. D. C.; McNamara, N. P.; Reay, D. S.; Whitaker, J., Can biochar reduce soil greenhouse gas emissions from a Miscanthus bioenergy crop? GCB Bioenergy 2014, 6 (1), 7689. 41. Cardinael, R.; Eglin, T.; Guenet, B.; Neill, C.; Houot, S.; Chenu, C., Is priming effect a significant process for long-term SOC dynamics? Analysis of a 52-years old experiment. Biogeochemistry 2015, 123 (1-2), 203-219. 42. Liu, X. B.; Han, X. Z.; Song, C. Y.; Herbert, S. J.; Xing, B. S., Soil organic carbon dynamics in black soils of China under different agricultural management systems. Commun

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Soil Sci Plan 2003, 34 (7-8), 973-984. 43. Malhi, S. S.; Lemke, R.; Wang, Z. H.; Chhabra, B. S., Tillage, nitrogen and crop residue effects on crop yield, nutrient uptake, soil quality, and greenhouse gas emissions. Soil Till Res 2006, 90 (1-2), 171-183. 44. Borchard, N.; Ladd, B.; Eschemann, S.; Hegenberg, D.; Moeseler, B. M.; Amelung, W., Black carbon and soil properties at historical charcoal production sites in Germany. Geoderma 2014, 232, 236-242. 45. Hernandez-Soriano, M. C.; Kerre, B.; Goos, P.; Hardy, B.; Dufey, J.; Smolders, E., Longterm effect of biochar on the stabilization of recent carbon: soils with historical inputs of charcoal. Global Change Biology Bioenergy 2016, 8 (2), 371-381. 46. Rittl, T. F.; Arts, B.; Kuyper, T. W., Biochar: An emerging policy arrangement in Brazil? Environ Sci Policy 2015, 51, 45-55.

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Figure legends Figure 1. Cumulative CO2 released during the while period of incubation (0–180 days). Error bars represent the mean standard error (n = 3). Figure 2. Cumulative CO2 produced from biochar (a) and biochar-induced C mineralization rate (b). CO2 appears as the percent of total added biochar-C. A larger scale of the y-axis for biochar mineralization rates during 150–180 day incubation is shown in an inset (b) for clarity. Error bars represent the mean standard error (n = 3). Figure 3. Cumulative CO2 derived from (sweet potato) SP residues (a) and SP residue-induced C mineralization rate (b). Error bars represent mean standard errors (n = 3). Figure 4. Amount of cumulative CO2 derived from soil organic matter (SOM) (a) and the cumulative priming effects induced by different treatments on SOM decomposition during the whole incubation period (b). The priming effects (b) were calculated according to comparison to the control soil (soil only). Numbers in (b) represent the significant differences among treatments (NS: no significance, p > 0.05). Error bars represent the mean standard error (n = 3). Figure 5. Priming effects induced by biochar on soil organic matter (SOM) and sweet potato (SP) residue mineralization in mixtures of soil and SP residues. The priming effects were assessed by the differences of SOM mineralization between the treatments of soil+SP residues+biochar and soil+SP residues. Error bars represent the mean standard error (n = 3). Figure 6. Microbial biomass C deriving from different sources during incubation. Numbers refer to significant differences of total microbial biomass C; letters refer to significant

27



differences (p < 0.05) of microbial biomass C-derived from soil organic matter (SOM) for different treatments. Error bars represent the mean standard error (n = 3). SP, sweet potato.

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Table 1. Characteristics of soil, biochar, and sweet potato residues Soil*

Biochar**

Sweet potato residue

pH

8.13 ± 0.12

7.12 ± 0.11

ND

NH4+ (mg N g−1)

0.021 ± 0.00005

0.0481 ± 0.001

ND

NO3− (mg N g−1)

0.032 ± 0.002

0.0012 ± 0.0003

ND

Total C (%)

2.17 ± 0.04

46.7 ± 0.70

52.1 ± 0.78

Total N (%)

0.213 ± 0.003

4.89 ± 0.007

1.17 ± 0.01

C: N

10.18 ± 0.03

9.56 ± 0.01

44.5 ± 0.03

DOC (mg C g−1)

0.036 ± 0.0005

3.15 ± 0.047

ND

ND

7.43 ± 0.11

ND

−15.3 ± 0.22

−12.4 ± 0.18

−10.1 ± 0.15

14C

activity (Bq mg−1) δ13C (‰)

* NH4+ and NO3− were extracted by 1 M KCl; dissolved organic content (DOC) in soil was extracted by 0.5 M K2SO4. pH was measured using a ratio of 1:2.5 soil: 1 M KCl. ** The determinations of NH4+, NO3−, and DOC were in water extracts for biochar. pH was measured using a ratio of 1:20 biochar:water. ND: not determined.

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Table 2. Characteristics of soil at the end of the incubation period. Treatment

pH

NH4+

NO3-

Total C

Total N

(mg N g−1)

(mg N g−1)

(%)

(%)

DOC * C: N ratio (mg C g−1)

Soil only

6.98±0.01 a

21±0.03 a

32±0.4 a

2.32±0.01 a

0.24±0.004 a

9.67±0.1 a

5.5±0.09 a

Soil+SP residues

6.78±0.1 a

19±0.02 b

135±2.0 b

2.46±0.01 b

0.25±0.003 b

9.84±0.1 a

10.4±0.02 b

Soil+biochar

6.64±0.09 b

11±0.01 c

112±1.8 c

2.67±0.01 c

0.27±0.001 c

9.89±0.1 a

12.3±0.18 c

Soil+SP residues+ biochar

6.69±0.01 c

7±0.01 d

143±2.1 d

3.36±0.01 d

0.28±0.001 d

12±0.2 b

14.6±0.22 d

*K2SO4 extracts (0.05 M) were measured. The letters in the table represent significant differences for different treatments (p < 0.05). Duncan’ test was applied for comparing differences. SP, sweet potato.

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Figure 1.

Figure 2.

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Figure 3.

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Figure 4.

33



Figure 5.

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Figure 6.

35



Graphical abstract

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Preferential Alternatives to Returning All Crop Residues as Biochar to

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