Journal Pre-proofs

Quantification and Molecular Characterization of Organo-mineral Associations

as Influenced by Redox Oscillations

Mohammad Z. Afsar, Christopher Goodwin, Thomas P. Beebe, Deb P. Jaisi,

Yan Jin

PII: S0048-9697(19)35447-6
DOI: https://doi.org/10.1016/j.scitotenv.2019.135454
Reference: STOTEN 135454

To appear in: Science of the Total Environment

Received Date: 9 September 2019

Revised Date: 3 November 2019
Accepted Date: 7 November 2019

Please cite this article as: M.Z. Afsar, C. Goodwin, T.P. Beebe, D.P. Jaisi, Y. Jin, Quantification and Molecular
Characterization of Organo-mineral Associations as Influenced by Redox Oscillations, Science of the Total
Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135454

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1 Quantification and Molecular Characterization of Organo-mineral

2 Associations as Influenced by Redox Oscillations

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7 Mohammad Z. Afsar1, Christopher Goodwin2, Thomas P. Beebe Jr2, Deb P. Jaisi1, and Yan
8 Jin1*
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11 1Department of Plant and Soil Sciences, University of Delaware, Newark, DE-19716, USA

12 2Department of Chemistry and Biochemistry, University of Delaware, DE-19716, USA

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15 *Corresponding author: Tel, (302)831-6962; Email: yjin@udel.edu
16 473 Interdisciplinary Science and Engineering (ISE) Lab
17 221 Academy Street
18 Department of Plant and Soil Sciences
19 University of Delaware
20 Newark, DE 19716, USA
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28 Quantification and Molecular Characterization of Organo-mineral

29 Associations as Influenced by Redox Oscillations

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34 Mohammad Z Afsar1, Christopher Goodwin2, Thomas P. Beebe Jr2, Deb P. Jaisi1, and Yan
35 Jin1*
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38 1Department of Plant and Soil Sciences, University of Delaware, Newark, DE-19716, USA

39 2Department of Chemistry and Biochemistry, University of Delaware, DE-19716, USA

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42 *Corresponding author: Tel, (302)831-6962; Email: yjin@udel.edu
43 473 Interdisciplinary Science and Engineering (ISE) Lab
44 221 Academy Street
45 Department of Plant and Soil Sciences
46 University of Delaware
47 Newark, DE 19716, USA
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55 Graphical abstract

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73  Abstract
74 Organo-mineral association is one of the most important stabilization mechanisms of soil organic

75 matter. However, few studies have been conducted to assess the retention, transformation, and

76 transportation of colloids (1-1000 nm) and associated organic carbon (OC) in soil. Given the

77 particularly significant role that wetland soils play in carbon storage and cycling, we quantified

78 the dynamics of organo-mineral association within colloidal size range by conducting three

79 consecutive 35-day redox (reduction-oxidation) oscillation experiments using a wetland soil.

80 Molecular compositions of natural nanoparticle (NNP, 2.3-100 nm), fine colloid (100-450 nm),

81 and particulate (450-1000 nm) fractions were measured using isotope ratio mass spectrometry

82 (IRMS) and x-ray photoelectron spectroscopy (XPS). Results showed that NNP and fine colloids

83 constituted up to 8.94 ± 0.50% and 22.19 ± 7.52% of bulk C concentration (2.3-1000 nm),

84 respectively; indicating substantial contributions of these two fractions to the operationally

85 defined “dissolved” (<450 nm) fraction. There was significant enrichment in heavier δ13C

86 isotopes (p < 0.001) with size: NNP (-29.64 ± 0.32 ‰) < fine colloid (-28.81 ± 0.31 ‰) <

87 particulate (-28.34 ± 0.25 ‰) fractions. NNP had the highest percentages of carbonyl/carboxyl C

88 (C=O); while fine colloid and particulate fractions contained more reduced aromatic or aliphatic

89 C (C-C, C=C, C-H). OC became more δ13C-enriched (‰) in microbial-derived C with increasing

90 particle size as well as with repeated redox oscillations. Our findings clearly demonstrate

91 limitations of using the operationally defined “dissolved” fraction (<450 nm) to assess C cycling

92 in ecosystems such as wetlands. Increase in colloid and OC concentrations and presence of more

93 microbial-derived C in larger size fractions additionally imply that redox oscillations promote the

94 formation of molecularly diverse sub-colloid sized organo-mineral associations. Being a

95 composite unit of soil microaggregates, organic-mineral associations can thus influence the

96 overall stability of OC in wetland soils that undergo frequent redox oscillations.

97

98 Keywords: Wetland; Redox oscillation; Soil organic matter; Natural nanoparticle (NNP);

99 Colloid; organo-mineral association

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118 1. Introduction
119 Soil organic matter (SOM), an important and complex component of the ecosystem, exists as

120 a continuum from freshly produced plant residues to microbial-derived organic fractions
121 (Lehmann and Kleber, 2015). Soil comprises at least 3 times more carbon (C, 2500 Gt) than the

122 vegetative and atmospheric pools combined (Lal, 2004). SOM in wetlands is particularly

123 important as concerns for climate change increase (Schmidt et al., 2011). Although wetlands

124 cover only 8-10% of the world’s land surface (Sahagian and Melack, 1998), their geographical

125 location and high C content (20-30% of terrestrial C) make them highly vulnerable to climate

126 change (Mitsch, 2007). Any changes in the frequency and duration of oxic and anoxic events can

127 make wetlands biogeochemical “hotspots” for the (de)stabilization of SOM. The stability of

128 SOM is largely controlled by the formation of organo-mineral associations that accounts for up

129 to 90% of total soil C (Baldock and Skjemstad, 2000; Kleber et al., 2007; Wagai and Mayer,

130 2007). However, despite wetlands’ recognized significance, comprehensive understanding of

131 biogeochemical transformations of these associations resulting from redox (reduction-oxidation)

132 oscillations remains inadequate.

133 The interactions between soil minerals and organic C (OC), are a major SOM stabilizing

134 mechanism, which depend on three major factors: (1) the structural properties of SOM (Kalbitz

135 et al., 2005; Mikutta et al., 2007), (2) type (Kaiser et al., 2002), and (3) surface area

136 (Guggenberger and Kaiser, 2003) of the minerals involved. Interactions between organic and

137 mineral particles lead to the formation of organo-mineral associations, which act as composite

138 building units of soil microaggregates (Kogel-Knabner et al., 2008; Lehmann et al., 2007). There

139 has been considerable evidence supporting the claim that the C incorporated onto mineral

140 surfaces resembles microbial-derived C rather than plant-derived C. For example, in a conceptual

141 model of molecular soil C dynamics, Grandy and Neff (2008) argued that mineral-associated OC

142 has distinct composition related more to microbial-derived C than to plant-derived C. In another

143 study, Sollins et al. (2006) observed an increase in microbial C signature with increasing degree
144 of mineral association, which can be stabilized via organo-mineral interactions (Baldock and

145 skjemstad, 2000; Kleber et al., 2015). Furthermore, Huygens et al. (2008) demonstrated

146 microbial processing as an important step towards SOM stabilization and observed high degree

147 of microbial biomarkers in soil micro-aggregates. Active contribution of microbial metabolites to

148 the formation of organo-mineral associations additionally supports the conceptual three-

149 dimensional functional view of C turnover dynamics as proposed by Kleber and Johnson (2010).

150 According to this view, organo-mineral microstructures are formed around colloid to sub-colloid

151 sized (discussed in the next paragraph) microbial cells rather than random attachment to the

152 mineral surfaces. However, in redox oscillating environments, much of the OC associated with

153 redox sensitive minerals, e.g., Fe and Al hydr(oxides), can be released to the dissolved pool

154 through reductive dissolution of those minerals (Liptzin and Silver, 2009). In addition to

155 regulating microbial/mineral driven biogeochemical processes, redox oscillations can promote

156 formation of new colloids (Thompson et al., 2006a) and thus the stability of associated OC.

157 In soil, nano (1-100 nm) and colloid sized (1-1000 nm) (Everett, 1972; Guo and Santschi,

158 2007) organo-mineral associations are found in both immobile solid phase and colloidally

159 dispersed mobile phase (Buettner et al., 2014; Fritzsche et al., 2015; Totsche et al., 2018).

160 Mobile colloids are inorganic or organic entities and are the identifiable solid phase in the

161 suspension (Sposito, 1989). Due to their large specific surface areas, high reactive site densities,

162 and high mobility, colloids and colloidal OC (COC), along with associated elements, can

163 transport across long distances. They can also actively participate in the biogeochemical

164 processes in both terrestrial and aquatic ecosystems (Kalbitz et al., 2000; Zsolnay, 2003). In

165 aquatic and marine ecosystems, size-specific concentration and molecular composition of COC

166 are widely used as indicators of environmental changes, however, those data are scarce for
167 terrestrial ecosystems. Apart from the difficulty and labor-intensive nature of performing size-

168 fractionated sample analysis, the scarcity is also due to the conventional technique to separate

169 dissolved OC (DOC) from particulate OC (POC) using the cutoff size of 450 nm, which lumps

170 COC with the dissolved fraction. DOC is a heterogeneous mixture of diverse organic matter

171 (OM) that significantly contributes to the formation of organo-mineral associations (Kleber et al.,

172 2015). The conventional practice also overestimates the contribution of actual dissolved phase to

173 global C cycling by including colloids of diameter <450 nm in the dissolved fraction. For

174 example, Yan et al. (2018) estimated that COC accounts for 8-19% of the operationally defined

175 DOC pool (<450 nm) regardless of sampling source and location. This estimation corresponds to

176 riverine flux of 13.6-32 TgC year-1 of global DOC pool in global C cycle. Moreover, different

177 cutoff size ranges are often used in different disciplines to define DOC fractions, e.g., <700 nm

178 (DeVilbiss et al., 2016; Osburn et al., 2012), <450 nm (Kellerman et al., 2014; Poulin et al.,

179 2014), or <200 nm (Chen et al., 2004). Such discrepancies make it more difficult to compare

180 results in C behavior and processes between different ecosystems and environments.

181 A better understanding of both simple records of C concentration and molecular composition

182 of sub-micron sized organo-mineral associations is thus necessary to make more accurate

183 assessment of SOM stability (Kaiser and Kalbitz, 2012), the processes and dynamics of OC

184 (Tfaily et al., 2015), and the interactions of OC with soil mineral matrices. During SOM

185 decomposition, microbes preferentially respire the lighter C isotopes while they tend to

186 incorporate the heavier δ13C isotope into their biomass (Dijkstra et al., 2006; Emmerton et al.,

187 2001). Thus, stable δ13C values are a good indicator and correlate well with SOM composition,

188 stability, and turnover processes during decomposition (Chen et al., 2005). Hence, we aimed to

189 understand to what extent the concentration and molecular composition of organo-mineral
190 associations vary (1) among different size fractions and (2) with redox oscillation cycles. We

191 tested the hypothesis that concentration and composition of size-fractionated OC would vary

192 widely within colloidal size range. We systematically induced redox oscillations in soil

193 suspension in microcosms and analyzed the concentration and molecular composition of both

194 colloids and associated OC in multiple size fractions including natural nanoparticle (NNP, 2.3-

195 100 nm), fine colloid (100-450 nm), and particulate (450-1000 nm) fractions. Our

196 comprehensive approach and the use of state-of-the-art spectroscopic techniques (isotope ratio

197 mass spectrometry, IRMS; and x-ray photoelectron spectroscopy, XPS) yielded results that show

198 size-specific molecular composition of organo-mineral associations and their response to redox

199 oscillations. The results provide insights that improve the understanding of OC stability in redox-

200 dynamic environments such as wetlands in response to climate change.

201 2. Materials and Methods

202 2.1. Study site and soil sampling

203 Soil samples were collected from the top 0-15 cm layer in 2016 from the Devil’s Hole

204 wetland, a freshwater wetland and a part of the White Clay Creek (WCC) watershed, Delaware,

205 USA. Mean annual precipitation and water flow rate, respectively, are 105.4 cm and 50.8 cm per

206 year (Corrozi et al., 2008). Over the last two decades, WCC watershed has experienced more

207 frequent and intense floods and more severe droughts. Devil’s Hole is defined as a steady state or

208 static wetland and the hydroperiod of the wetland is “permanently saturated” (NTCHS, 2015).

209 Water table fluctuates +/- 5 cm of the soil surface throughout the year and the wetland usually

210 has no inundation during periods of drought. The soil is classified as fine-silty, active, mesic,

211 Thapto-Histic Fluvaquents according to the U.S. Department of Agriculture (USDA) soil

212 taxonomy (Soil Survey Staff, 2015). Soil samples were immediately transported to the lab and
213 stored at 40C until they were used for the incubation study. A sub-set of the collected soil sample

214 was air dried and passed through a 2-mm sieve to study the basic characteristics of the soil. A

215 summary of the soil physical and chemical composition is listed in Table S1 in Supplemental

216 Information (SI).

217 2.2. Redox oscillation experiment

218 Moist soil samples were transferred from 4C storage to a 95%:5% N2:H2 glovebox (Coy,

219 Grass Lake, MI) and stored for 24 h to thoroughly equilibrate the samples with the gases to

220 ensure anoxic conditions. Any visible plant debris and rocks were removed manually. Initially,

221 soil was suspended in deoxygenated 2.0 mM NaCl solution at a soil: solution ratio of ~1:11 in

222 the absence of light and without any supplemental C sources. Approximately 85 g (oven dry

223 weight) of soil was weighed into a 1 L microcosm; a total of 21 microcosms were prepared. The

224 suspension was shaken for 5 min and then passed through a 1-mm sieve for several times

225 (Thompson et al., 2006a). The precise weight of the soil was determined by oven-drying (110

226 0C) 10 mL of the suspension. Microcosms were always capped with polypropylene screws fitted

227 with tygon tubing extended at the bottom of the jars. A complete redox oscillation was

228 designated by 30-day of reducing half-cycle (RHC) followed by 5-day of oxidizing half-cycle

229 (OHC). Microcosms were divided into 7 subsets to individually run (1) control (Initial soil, 0-

230 day), (2) 1st RHC (30-day), (3) 1st RHC + OHC (1st redox oscillation, 35-day), (4) 2nd RHC (65-

231 day), (5) 2nd RHC + OHC (2nd redox oscillation, 70-day), (6) 3rd RHC (100-day), and (7) 3rd

232 RHC + OHC (3rd redox oscillation, 105-day), with 3 replicates for each treatment. The RHC was

233 initiated by injecting nitrogen (N2) gas intermittently for 2-h through tygon tubing at the bottom

234 of the soil suspension. Later, no N2 gas was injected for the rest of the RHC period. During the

235 OHC, microcosms were removed from the globe box, and intermittently purged with O2 gas
236 (grade 4.7) at the bottom of the suspension. Microcosms were shaken for 5 min in every day to

237 homogenize the soil suspension. Complete procedure for the redox oscillation study is outlined

238 in Fig. S1.

239 2.3. Sample collection

240 Soil suspension was destructively collected from triplicate microcosms after each treatment.

241 Initially, microcosms were shaken for 5 min and allowed to settle for 2 h before supernatants

242 were collected for size fractionation. The supernatants were transferred into four 250 ml

243 centrifuge tubes and were fractionated into <1000 nm (6-min, 220 rcf), <450 nm (8-min, 810 rcf)

244 and <100 nm (6-min, 22040 rcf) sized fractions using Sorvall centrifuge (Thermo Scientific) with

245 a F14-6*250Y rotor. The centrifugation speed and time to achieve desired particle sizes were

246 estimated from the Stokes’ law (Gimbert et al., 2005) assuming spherical particles and particle

247 density of 2.65 g cm-3 (Section S1), which is a typical value used for mineral soils (Murphy et al.,

248 1993; Sainz Rozas et al., 2001; Taylor et al., 1992). Although the two assumptions do not

249 consider heterogeneity in particle size and composition, we chose centrifugation over filtration

250 for size fractionation (except at 2.3 nm) as it avoids the inaccuracies associated with

251 filter/membrane filtration due to pore clogging. Conventional filtration using 450 nm or 200 nm

252 membrane filters can remove more soil particles than the corresponding centrifugation

253 procedures particularly due to the influence of clogging of pores, particle shape (e.g., platey

254 structure) and interactions between colloid particles and membrane surface (Gimbert et al.,

255 2005). We acknowledge that organic materials having density <2.65 g cm-3 would likely remain

256 in supernatants when mineral particles with the same size would settle at a given centrifugation

257 speed, thus be underestimated. We passed the <100 nm fraction through an ultrafiltration system

258 (Amicon® stirred cell) equipped with Ultracel® 10 kDa membrane ultrafiltration discs (EMD
259 Millipore Corporation) to separate the dissolved, <10 kDa (~2.3 nm) fraction (Buettner et al.,

260 2014; Guo and Santschi, 2007). The particulate, fine colloid and NNP fractions were collected

261 from the precipitates at each centrifugation steps and freeze-dried. During the RHC events,

262 Microcosms were kept and opened inside the glove box for all subsequent procedures. pH and

263 Eh of the supernatants were measured using corresponding probes (Fisher Scientific, Hampton,

264 NH).

265 2.4. IRMS analyses

266 Stable δ13C and δ15N isotope values and total C and N concentrations of the freeze-dried, size-

267 fractionated samples were determined using an IRMS (DELTA V plus, Thermo Fisher

268 Scientific) equipped with an elemental analyzer (EA). Stable δ13C or δ15N values were calculated

269 as (Rsample/ Rstandard -1) x 1000, where R is the ratio of heavier to lighter isotope (13C/12C, or

270 15N/14N), and expressed in parts per thousand (‰) relative to their international standards

271 Vienna-Pee Dee Belemnite (PDB) limestone and atmospheric N2 standards. USGS-40 (L-

272 glutamic acid, 40.8% C and 9.52% N) and USGS-41 (L-glutamic acid enriched in 13C and 15N,

273 41.9% C and 9.76% N) were used as reference materials. Because there was insufficient amount

274 of samples at 2.3-100 nm after 1st and 2nd complete oscillations, replicate samples were

275 combined to run the IRMS analyses.

276 2.5. XPS analyses

277 The photoelectron spectra of the size-fractionated, freeze-dried samples were taken at the

278 University of Delaware’s Surface Analysis Facility (SAF) using a Thermo Scientific K-alpha+

279 XPS (East Grinstead, United Kingdom). Samples were dispersed uniformly on double-sided

280 conducting tapes and affixed to a stainless-steel plug. The samples were excited with a
281 monochromated aluminum Kα radiation (Eexc = 1486.6 eV) at an incident angle of 57.2 with a

282 spot size of 100 μm wide ellipse; charge neutralization was carried out with a low energy

283 electron flood gun. Elemental composition of soil minerals and their associated OC was

284 measured by a survey scanning from 0 to 1200 eV using a 1 eV step size and 100 eV pass

285 energy. The high-resolution scans for every element in the samples were obtained using a step

286 size and pass energy of 0.1 eV and 20 eV, respectively. During operation, vacuum was

287 maintained at <3 x 10-9 mbar. The chemical composition was revealed by deconvoluting C 1s, N

288 1s, Si 2p, Fe 2p3/2, Al 2p, Mn 2p, Mn 3s, O 1s, Mg 1s, Mg 2p, Ca 2p, Cl 2p, Na 1s peaks (Fig.

289 S2) into sub-peaks by fitting Gaussian-Lorentzian functions and Shirley background correction

290 of the XPS spectra using the CasaXPS version 2.3.16 (Teignmouth, UK). Molar fractions of the

291 various elements were calculated from peak areas based on acquisition parameters and atomic

292 sensitivity factors calculated from the electronic cross-section of each element and orbital

293 (Scofield, 1976).

294 A typical C spectrum obtained by XPS (Fig. S3) is deconvoluted into three components

295 representing aliphatic and aromatic C[0] (C-C, C=C, C-H; at 284.6 eV), ether, alcohol, and amine

296 C such as those in polysaccharides or amino acids, C[+1] (C-O, C-N; at 286.5±0.2 eV), and

297 carbonyl and carboxyl C, C[+2] (C=O, at 288.6±0.2 eV), depending on the specific bonding

298 environments or oxidation states (Gerin et al., 2003; Mikutta et al., 2009; Proctor and Sherwood,

299 1982). Though carbonyl/carboxyl C peaks are generally easily discerned, the effect of different

300 counter ions (H, Na, K) and low concentration might shift the peak position. Therefore, a

301 combined peak shape was used for the fitting. Weight % of surface C (Csurface) was calculated by

302 multiplying the molecular weight of all elements with their measured atom % assuming that

303 hydrogen has negligible contribution (Arnarson and Keil, 2001, 2007).
304

305 2.6. Statistics

306 Statistical analyses were performed using R 3.3.1 (R Core Team, 2016). Two-way analysis of

307 variance (ANOVA) was performed to test for redox oscillations and size effects on measured

308 parameters using the Tukey’s Honestly Significant Difference (HSD) test. Pearson’s correlation

309 coefficients between C and N concentration measured in IRMS and XPS analyses, and stable

310 δ13C and C concentrations were obtained using Origin (OriginLab, Northampton, MA).

311 Regression plots of XPS-based surface atom % of Al, Fe, Si, Mg, and N with C atom % and

312 between OC and colloid mass were obtained using the Origin. Differences of means were

313 significant at p < 0.05 unless otherwise noted.

314 3. Results and Discussion

315 3.1. Changes in soil solution chemistry

316 Changes in solution pH and pE as a function of redox oscillations are summarized in Fig. S4.

317 As expected, RHCs were characterized by an increase in pH and decrease in pE, whereas OHCs

318 exhibited opposite trends. These changes in solution chemistry are consistent with results from

319 Thompson et al. (2006a), who studied colloid mobilization in wet tropical Hawaiian soils. Final

320 pH of the suspension was decreased from 6.18 ± 0.40 after 1st RHC to 5.28 ± 0.31 after 3rd RHC.

321 However, we did not observe wide variations in pE after 2nd and 3rd RHCs, which might be due

322 to nitrate (NO3-) accumulation through ammonia oxidation during intermediate OHC periods.

323 Being a terminal electron acceptor, NO3- produced under aerobic condition through ammonia

324 oxidation tends to stabilize redox potential during successive reducing events (Reddy and

325 Patrick, 1975).

326

327

328 3.2. Distribution of C and N in size-fractionated particles

329 C and N concentrations ([C]particle and [N]particle) of the size-fractionated particles based on

330 their freeze-dry weight are shown in Fig. 1. Both [C]particle and [N]particle values at NNP fractions

331 dropped sharply after 1st RHC and fluctuated narrowly throughout the remaining redox

332 oscillation events. Sharp drop in the concentrations after 1st RHC indicates that most of the labile

333 OC was mineralized during that period. We also observed an overall decrease in redox-

334 dependent fluctuation in [C]particle and [N]particle with increasing particle size, following the order

335 of NNP > fine colloid > particulate fractions (Fig. 1a-b). The smallest concentration changes in

336 particulate size fraction indicates that OC in the larger size fractions is less redox dependent. The

337 higher [C]particle and [N]particle in the small size fractions might be due to (1) greater specific

338 surface area, and/or (2) detachment/transformation of particulate OM (Guo and Santschi, 1997)

339 with decreasing size of the particles. In fact, Schulten and Leinweber (2000) also reported that

340 OC and total N contents increased exponentially with decreasing particle sizes in soils with

341 different clay and silt contents. Strong positive correlation between [C]particle and [N]particle at 2.3-

342 1000 nm (Fig. 1c; r2 = 0.97, p < 0.001) additionally supports that they are mostly from soil

343 organic matrices rather than from inorganic sources.

344 An opposite trend was observed in total mass and concentration when the size-fractionated

345 particles and associated OC were expressed in per kg oven dry soil (Fig. 2a-d, Table S2). Among

346 the different size fractions, least amount of C was partitioned at smaller size fractions; NNP (3 ±

347 1 to 54 ± 8 mg kg-1), followed by fine colloid (17 ± 5 to 120 ± 50 mg kg-1), and then particulate
348 (49 ± 10 to 414 ± 32 mg kg-1) (Fig. 2c-d). The average percentages of NNP- and fine colloid-

349 bound C were between 2.10 ± 1.18% to 8.94 ± 0.50% and 17.88 ± 1.40% to 22.19 ± 7.52% of

350 the bulk C concentration (2.3-1000 nm), respectively, indicating substantial contributions of

351 these two fractions to the operationally defined dissolved fraction (<450 nm). Consistent with

352 other studies (Grybos et al., 2009; Hagedorn et al., 2000; Olivie-Lauquet et al., 2001), RHC

353 samples contained more C than the samples from complete oscillations (RHC + OHC). Under

354 reduced condition, dissolution of Fe-oxide coatings and/or concomitant changes in pH can

355 mobilize OC associated with the minerals (Ryan and Gschwend, 1994; Thompson et al., 2006a).

356 Until 2nd complete oscillation, RHC treated samples had 3-10 times more C than OHC. However,

357 following the 3rd RHC, size-fractionated particles had only 1-2 times more C than the 3rd OHC.

358 The changes in OC concentration along the redox oscillations might be due to the variation in

359 redox potentials and possible de novo synthesis colloid particles with different surface charge

360 properties. Microcosms had the lowest pE values after the 2nd RHC (-0.13 ± 0.8) compared to the

361 1st (2.62 ± 0.53) and the 3rd (0.24 ± 0.58) RHCs (Fig. S4), which would favor Fe reduction and

362 concomitant release of more colloids and OC during the 2nd RHC. In a closed system, Thompson

363 et al. (2006b) repeated that redox oscillations could increase Fe crystallinity in soil by converting

364 short-range ordered Fe minerals into less soluble, more crystalline Fe-oxides (e.g., goethite and

365 hematite). Further, colloid and OC concentrations after each complete oscillation also increased,

366 which might be due to the increase in colloid stability resulting from redox oscillations, as

367 proposed by Thompson et al. (2006a). Overall, after the 3rd complete redox oscillation, C

368 concentration in the respective NNP, fine colloid, and particulate fractions increased by

369 349±101%, 159±38%, and 281±33% relative to the concentration measured for the initial

370 (control) soil (Table S2).

371 3.3. Stable isotopic (δ13C, δ15N) signatures

372 Stable isotopes of C (δ13C) and N (δ15N) values show the molecular heterogeneity among

373 size-fractionated particles (Fig. 3 and Table S3). The δ13C values enriched significantly (p <

374 0.001, Table S4) with increasing size: NNP (-29.64 ± 0.32 ‰) < fine colloid (-28.81 ± 0.31 ‰) <

375 particulate (-28.34 ± 0.25 ‰). Similar trends were also observed in the δ15N isotope signatures,

376 ranging from 1.72 ± 0.56 ‰, 1.95 ± 0.69 ‰ and 2.81 ± 0.81 ‰ for NNP, fine colloid, and

377 particulate size fractions, respectively. Higher δ13C and δ15N values indicate enrichment of

378 heavier isotopes (13C and 15N; Fry, 2006). Enrichment of heavier stable isotopes in larger size

379 fractions is also consistent with results from Guo and Macdonald (2006), in which a consistent

380 increase in δ13C and δ15N values was reported from colloid (1 Kda-450 nm) to particulate (>450

381 nm) OM fractions in samples from the upper Yukon river.

382 Enrichment of heavier isotopes is often speculated to be due to greater microbial processed

383 OC (Dijkstra et al., 2006; Ehleringer et al., 2000; Sollins et al., 2009). Microbes preferentially

384 utilize 13C-depleted molecules for respiration, while 13C-enriched molecules tend to be used for

385 the synthesis of microbial biomass and metabolites (Dijkstra et al., 2006; Emmerton et al., 2001).

386 As a result, decomposition of SOM leads to an enrichment of δ13C in microbial biomass and

387 residual C pool. Gradual δ13C enrichment (and analogously δ15N) from NNP to particulate

388 fraction thus indicates presence of more microbial-derived C than plant-derived C in the larger

389 size fractions. Previous studies have reported that plant-derived lignin compounds are usually

390 depleted in heavier δ13C isotopes than other types of SOM (Benner et al., 1987; Schulten and

391 Gleixner, 1999). Size-dependent heterogeneity in isotope signatures became more visible when

392 δ13C and δ15N values were plotted in “isotope space” (Fig. S5a).
393 With respect to the effects of redox oscillations, all size fractions became enriched in δ13C

394 values compared to the initial soil except the 2nd OHC, indicating an abundance of more

395 microbial metabolites upon redox reactions. After 3rd redox oscillation, NNP, fine colloid, and

396 particulate fractions became enriched in heaver δ13C values by 0.77 ± 0.19 ‰, 0.53 ± 0.55 ‰,

397 and 0.27 ± 0.11 ‰, respectively. However, anomalous trends were observed in δ15N values along

398 the redox oscillations, that is, δ15N values decreased by ~1 ‰ during the 1st redox oscillation.

399 Smaller particles responded more quickly to redox changes as shown by their early drop in δ15N

400 values. However, size fractionated particles became enriched during the 2nd redox oscillation

401 followed by a continuous drop in the heavier δ15N isotopes. The changes in stable δ15N values

402 might be related to the biogeochemical cycling of organic N, however, we could not make any

403 definite conclusion due to the lack of mass balance of total N. Further research is needed to

404 investigate the size specific biogeochemical cycling of organic-N under redox oscillation

405 processes. Here, we posit that changes in δ15N might reflect a shift from N assimilation to N

406 dissimilation process, driven by respective higher C availability at the beginning and lower C

407 availability at the later stages of our incubation study (Dijkstra et al., 2006). The observed

408 depletion of δ15N is consistent with the reported isotope fractionation range (-1.6 to +1 ‰)

409 during N assimilation by microorganisms (Hübner, 1986). Other N transformation processes,

410 e.g., NH3 volatilization, nitrification and denitrification cause higher isotope fractionation than

411 those associated with N assimilation and dissimilation (kendall, 1998). It should also be noted

412 that the C and N pool sizes can also influence the measured isotope values. If the C pool is small,

413 isotope fractionation would trigger a bigger change in the δ13C values due to the OC recycling. A

414 proportional recycling is possible but rare. Among the three size fractions, the least amount of C
415 was partitioned at NNP, which had gradual δ13C enrichment indicating the influence of OC pool

416 sizes on final δ13C values after cumulative redox oscillations.

417 To further examine dynamics of C under redox oscillations, we plotted the δ13C values of

418 size-fractionated OC against the logarithm of its concentration (Fig. S5b). The regression plot

419 was highly significant (p < 0.001) and the δ13C values increased with decreasing C

420 concentrations along a size continuum from NNP to particulate. Relatively large variations in

421 δ13C and δ15N values thus confirm our hypothesis that NNP, fine colloid, and particulate

422 fractions have widely diverse molecular compositions. Therefore, quantification and molecular

423 characterization of size-specific organo-mineral associations are crucial for better understanding

424 of the dynamics of OC in wetlands.

425 3.4. Linking surficial elemental composition of soil minerals to associated organic C and N

426 Complement to the IRMS, XPS analyses provide information on surface composition of

427 mineral matrices and functional groups of associated OC. Normalized atomic percentages of Al,

428 Si, Fe, C, and N in size-fractionated particles are presented in Fig. S6. Additionally, minor

429 amounts of K, Mg, Mn, and Ca were also detected. Among the different size fractions, NNP had

430 significantly (p < 0.001) the lowest % of Al, Si, and Fe and the highest % of C and N than the

431 fine colloid and particulate fractions. The highest surface atomic % of C and N in NNP is also

432 consistent to their bulk concentrations as determined from IRMS analyses. The strong negative

433 correlations between C and other elements e.g., Al (r2 = 0.95, p < 0.01), Fe (r2 = 0.32, p < 0.05),

434 Si (r2 = 0.93, p < 0.01), and Mg (r2 = 0.58, p < 0.01) indicate an overall increase in surface

435 coating of OC over the underlying mineral phases with increasing C concentration (Fig. S7).

436 Woche et al. (2017) found similar strong negative relationship between C and other elements

437 during biogeochemical interface (BGI) formation. Additionally, strong positive relationship
438 between C and N (r2 = 0.79, p < 0.01) suggests that both C and N originated from organic

439 fractions and agrees well with the values obtained from IRMS analyses (r2 = 0.97, p < 0.001).

440 The location of the deconvoluted N spectra confirms that SOM is the main source of the surface

441 N within 2.3-1000 nm size range. N spectrum detected at a binding energy of 399-400 eV

442 usually corresponds to organic-N, while mineral-N provides peaks at ~401.2 eV (NH4+) and 406-

443 407 eV (NO3-) (Gerin et al., 2003). In our study, mineral-N was observed mostly at <2.3 nm size

444 fraction (Fig. S8).Size-dependent heterogeneity in the composition and reactivity of minerals is

445 likely to play an important role in organo-mineral interactions. Strong linear regression between

446 size-fractionated colloid particles and associated OC (Fig. S9) points to the significance of

447 organo-mineral associations to the sequestration of OC in soil that have been receiving

448 increasing attention over the past three decades (Baldock and Skjemstad, 2000; Sollins et al.,

449 1996). Due to sample scarcity, we were unable to run x-ray diffraction (XRD) analysis to

450 investigate the mineralogical composition of different size fractions. However, XPS could

451 compensate to some extent as it provides quantitative information of all surface elements (except

452 H and He). For example, Mg/Al ratio (indicative of smectites) was the highest in NNP and

453 decreased with increasing particle sizes (Fig. S10), which suggests presence of less smectite

454 minerals in larger size fractions. In soil, phyllosilicates, e.g., smectites are typically smaller than

455 kaolinite and illite and are mostly concentrated in <100 nm size particles (Grim, 1953; Jepson,

456 1984; Muir et al., 1957; Tsao et al., 2013). Consistent with previous studies (Arnarson and Keil,

457 2001; Barr et al., 1999) and based on measured Mg/Al ratios, it is likely that the smaller fractions

458 are more abundant in Mg-smectites. Having a large specific surface area enables these minerals

459 to have more OC attached to the NNPs (Bock and Mayer, 2000; Ransom et al., 1997).

460 3.5. Speciation of organic C and selective sorption on mineral surfaces

461 The C 1s XPS spectrum was de-convoluted into 3 main peaks representing the most reduced

462 (C-C, C-H) aliphatic or aromatic C (44.58%), the C-O/C-N peak that is indicative of amines

463 and/or alcohols like those in polysaccharides and amino acids (28-34%) and the most oxidized

464 (C=O) carbonyl/carboxyl C (12-33%) (Fig. S11). Selective adsorption of reduced aliphatic and

465 aromatic C over oxidized carbonyl/carboxyl C agrees well with Kothawala et al. (2012).

466 However, considerable evidence suggested that the concentration and composition of dissolved

467 organic matter (DOM) can influence the selectivity of OC functional groups to mineral surfaces.

468 For example, Avneri-Katz et al. (2017) studied adsorption of biosolid DOM on soil mineral

469 surfaces and observed preferential sorption of hydrophobic DOM components on mineral

470 surfaces relative to hydrophilic components at high DOM concentration. Hydrophobic DOM

471 components include aromatic units associated with aliphatic moieties (Chefetz et al., 1998).

472 Young et al. (2018) demonstrated that the non-selective sorption of nitrogen rich, biosolid DOM

473 on Fe(III)-montmorillonite surfaces contributed substantially to the formation of organo-mineral

474 association. The NNP fraction had a greater proportion of most oxidized C species than other

475 larger size fractions, while the particulate fraction had higher proportion of aliphatic/aromatic C

476 groups. Such size dependent heterogeneity in C functional groups clearly demonstrates the

477 selective sorption of more degraded and highly oxidized C species in smaller size fractions. The

478 ratios of aromatic or aliphatic C (most reduced) to alcohol/amine C and to carbonyl/carboxyl C

479 (most oxidized) increased from NNP to particulate fractions (Fig. 4). Together with the δ13C and

480 δ15N stable isotope data, an increase in the reduced to oxidized form of C species suggest an

481 increasing abundance of more stable, microbial-derived C in the larger size fractions. Based on

482 XPS and solid state C13-nuclear magnetic resonance (NMR) spectroscopy analyses, Gentsch et

483 al. (2015) also linked an increase in reduced to oxidized C species to the presence of more
484 decomposed, microbial-derived C. Microbial-derived C has been recognized to form stable SOM

485 (Gleixner et al., 1999; Huang et al., 1998; Knicker, 2011; Kogel-Knabner et al., 2008). Microbes

486 preferentially utilize more labile plant constituents, produce metabolites that serve as the main

487 precursors of decomposition-resistant, stable SOM, and promote aggregation with soil matrix

488 (Cotrufo et al., 2013). Stabilization of SOM is further favored by thermodynamic limitations of

489 oxidation of highly reduced C compounds under anoxic conditions (Boye et al., 2017). Our

490 findings collectively suggest the significance of microbial-derived C to form organo-mineral and

491 organo-metal oxide associations, which are considered as one of the important stabilization

492 mechanisms of SOM (Baldock and Skjemstad, 2000; Kleber et al., 2015).

493 3.6. Size dependent mineral-OC interactions

494 The assemblages of SOM to mineral surfaces may depend on the heterogeneity in the C and

495 N bonding environment. The selective attachment and release of C to/from different mineral

496 surfaces are controlled by their associated bonding mechanisms. For example, enrichment of

497 oxidized C species (e.g., C-O, C=O) supports the significance of ligand exchange reaction and/or

498 H-bonding with the mineral matrices of NNPs. Phyllosilicate (e.g., smectite) dominated fraction

499 was found to have the highest proportion of oxidized C species in protonated amide forms (Jones

500 and Singh, 2014). Here, the basal oxygen planes of smectite minerals can act as weak electron

501 donors and form H-bonds with oxidized C species (Jones and Singh, 2014; Mortland, 1986).

502 Additionally, Fe oxide minerals have higher affinity for highly oxidized, lignin-derived organic

503 molecules (Kaiser et al., 1997; Mikutta et al., 2007) because ligand exchange reaction is the

504 dominant bonding mechanism between SOM and iron oxides (Jones and Singh, 2014). Presence

505 of more oxidized C species and higher Mg/Al ratios at NNP indicate greater degree of oxidative

506 alteration and/or a stronger retention of OM components to mineral surfaces. Different from the
507 NNP fraction, fine colloid and particulate fractions were enriched in aliphatic and aromatic C

508 species. For soils with higher aliphatic C species, non-ionic lipids and ligand bonds are proposed

509 to dominate the interactions between SOM and mineral surfaces (Gillabel et al., 2010; Jones and

510 Singh, 2014; Kleber et al., 2015). Thus, organo-mineral associations might dominantly have

511 ligand bonds at fine colloid and particulate size fractions that contain mostly microbial-derived C

512 based on IRMS and XPS analyses.

513 Increase in soil particle and C concentrations (Fig. 2) and presence of more microbial-derived

514 C in NNP to particulate size fractions following redox oscillations are in good agreement with

515 the three-dimensional conceptual view of C turnover dynamics as proposed by Kleber and

516 Johnson (2010). Previously, the two-dimensional concept considered soil as a system composed

517 of adsorbent (mineral surface) and adsorbate (SOM). This concept is now extended to the three-

518 dimensional view assuming that colloid to sub-colloid sized organo-mineral associations are

519 considered as an important composite building units of soil microaggregates (Kogel-Knabner et

520 al., 2008; Lehmann et al., 2007; Totsche et al., 2018). Based on the surface properties, SOM and

521 minerals can adhere together to form the association and in contrast to the plant-derived C,

522 microbial-derived C actively contribute to the formation of such stable nano and colloid sized

523 organo-mineral associations (Asano and Wagai, 2015). Formation of organo-mineral

524 associations are common in soil both in the mobile and immobile phases (Buettner et al., 2014;

525 Totsche et al., 2018). Here we posit that microbial-derived C enhanced the formation of more

526 nano and colloid sized organo-mineral associations as indicated from the increase in soil particle

527 and C concentrations following the redox oscillations processes.

528 3.7. Layer thickness of surficial OM

529 Although XPS measurements do not provide information about the spatial distribution of

530 adsorbed materials on mineral surfaces, layer thickness (t, nm) of surface SOM (Y) coating is

531 often determined following a simplified model (Pantano and Wittberg, 1990; Woche et al.,

532 2017). This approach ideally assumes a homogeneous and continuous coating of OM on mineral

533 surfaces, although direct analyses by transmission electron microscopy and nano-scale secondary

534 ion mass spectrometry (nanoSIMS) have shown that OM exists in patches on mineral surfaces

535 (Heister et al., 2012; Vogel et al., 2013). Briefly, the thickness is calculated from the potential

536 energy (PE) mean free path λ (nm) of an element X in the underlying minerals. The content of X

537 is determined for both coated and non-coated surfaces. For a non-coated surface, the value of X

538 is calculated from the regression analysis using a PE with similar λ of an element Y occurring in

539 the coating (Section S2). Here, we assumed that the roughness of all samples was the same on

540 the scale of the x-ray spot size and calculated mean layer thicknesses for all particle size

541 fractions (n= 17 or 21). For simplicity, an additional assumption was made that there were only

542 two layers to the material. The bottom layer was mainly Si (Xi; Si2p, λ=3.2 nm; λSi) and Al (X2;

543 Al2p; λ= 3 nm; λAl) and the surface layer was mostly OC (Y; C1s; λ=3.0 nm) and the two layers

544 did not overlap. The assumption is based on the highest atom % of Si and Al as revealed from

545 XPS analyses (Fig. S6). This model, of course, is an oversimplification for the non-ideal,

546 heterogeneous soil particles. The rough soil surface can also emit electrons at many angles, and

547 therefore may vary in the thickness of adsorbed components. Nevertheless, this model has been

548 previously used (Gerin et al., 2003; Woche et al., 2017) to estimate an average thickness of OC

549 attached to the mineral surfaces. The calculation revealed that the NNP had the highest mean

550 layer thickness of surface OC coating (3.91 ± 1.25 nm), followed by fine colloid (1.55 ± 0.46

551 nm) and particulate (1.27 ± 0.36 nm) fractions. The significant relationships between mean layer
552 thickness and atom C% in NNP (r2 = 0.74), fine colloid (r2 = 0.80), and particulate fractions (r2 =

553 0.93) indicate SOM as the main coating component on mineral surfaces (Fig. 5a). The variations

554 in the thickness of OM coating on mineral surfaces may be an indication that the associations

555 between mineral and SOM surfaces vary at sub-micron size fractions, which is discussed further

556 in the following section.

557 3.8. Relationship between Surface and Bulk C

558 Fig. 5(b) shows the surface C concentration (Csurface, measured by XPS) as a function of its

559 bulk concentration (Cbulk, measured by IRMS) for different size fractions. The linear relationship

560 between Csurface vs Cbulk of NNPs deviated more from the 1:1 line than other size fractions,

561 indicating that the surfaces of NNP are more enriched in C (Gerin et al., 2003). The C

562 enrichment factors (Csurface/Cbulk) under alternating redox oscillation conditions for different size

563 fractions show a significant increase in the surface C concentration relative to the bulk with

564 increasing particle size and the values ranged from 1.92 ± 0.22, 2.47 ± 0.50, and 3.36 ± 0.66 for

565 NNP, fine colloid, and particulate fractions, respectively (Fig. 5c, Table S5). Repeated redox

566 oscillations decreased the enrichment factor in the particulate size fraction while increased in

567 other two size fractions. These observations indicate that OM primarily exists as coatings on

568 minerals surfaces rather than being dispersed or incorporated into soil particle structures on

569 larger size fractions while the smaller fractions are mostly organic materials with some minerals

570 glued to them. Previous studies (Arnarson and Keil, 2001; Jones and Singh, 2014) also observed

571 an increase in C enrichment factor from lighter to heavier fractions and suggested that the

572 heavier fractions are mostly mineral aggregates with increasing C concentration on the mineral

573 surfaces relative to the bulk C concentration.

574 4. Conclusions
575 Being a coastal state in the Northeast region of the United States, Delaware is already facing

576 several challenges due to climate change, such as extreme precipitation, inundation, and sea level

577 rise (Melillo, 2014). Any changes in the frequency, duration and degree of reduced conditions in

578 wetlands will have profound influence on soil biogeochemistry that can modify organo-mineral

579 associations and hence the stability of SOM. Moreover, soluble C formed during anoxic events

580 in wetlands has significant influence on biogeochemical cycles in terrestrial and downstream

581 aquatic ecosystems. We designed our batch incubation study to evaluate how anoxic-oxic events

582 would influence the organo-mineral associations within colloid size range. From this study, we

583 observed that size-fractionated COC varied widely both in concentration and molecular

584 composition. Our results clearly showed that NNP and fine-colloid fractions constituted up to

585 8.94 ± 0.50% and 22.19 ± 7.52% of bulk C concentration (2.3-1000 nm), respectively, that

586 would be otherwise lumped as “dissolved fraction” (<450 nm) in the operational definition of

587 DOM. The cutoff size range of DOM at 450 nm thus can significantly overestimate the dissolved

588 phase OC concentration, which can lead to inaccurate assessment of OC fate and transport

589 behavior in the environment. Therefore, understanding of the size-specific molecular

590 composition of COC could improve the accuracy in assessing the mobility, reactivity and

591 environmental functions of OC in redox sensitive wetlands. Stable δ13C and δ15N isotope

592 signatures and XPS analyses demonstrated that microbial metabolites are the pervasive SOM

593 components in fine colloid and particulate size fractions, while NNPs were enriched in plant-

594 derived C. Size-specific, selective attachment and/or release of C to/from different mineral

595 surfaces additionally give weight to the three-dimensional conceptual view of soil

596 microaggregates by incorporating the microbe-mineral interactions.

597 Acknowledgements
598 This study was supported by the USDA-NIFA (grant no. 2013-67019-21361) and USDA

599 hatch funds. We would like to thank Dr. Bruce Vasilas, for providing access to his wetland

600 research site; Gerald Poirier, Director of the Advanced Materials Characterization Lab (AMCL)

601 at the University of Delaware for his assistance and support in the laboratory.

602 Supporting Information

603

604

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850

851
852

853
854

Reducing half-cycle (RHC) Oxidizing half-cycle (OHC)

300 NNP Fine colloid Particulate
[C]particle (g kg-1)

(a)
200

100

0
40 (b)
[N]particle (g kg-1)

30

20

10

0
Initial 1st RHC 1st OHC 2nd RHC 2nd OHC 3rd RHC 3rd OHC
soil Treatment

40 (c)
2
[N] = 0.12 x [C] - 1.18, r = 0.97
[N]particle (g kg-1)

30

20

10

0
100 200 300
[C]particle (g kg-1)
855

856 Fig. 1. Changes in bulk (a) C ([C]particle, g kg-1 size-fractionated particles) and (b) N concentrations

857 ([N]particle, g kg-1 size-fractionated particles) as a function of redox oscillations measured by IRMS

858 equipped with EA. (c) Regression plot between C and N concentrations within 2.3-1000 nm size range.

859 The error bars show standard deviations among replicates.

860

861
862

Reducing half-cycle (RHC) Oxidizing half-cycle (OHC)

NNP (a)
Mcolloid (mg)

600 Fine colloid

400 Particulate

200

0
8 NNP Fine colloid Particulate
(b)
Colloid conc.
(g kg soil)

6
-1

4
2
0
40
Total C (mg)

30 (c)

20
10
0
500
C conc. (mg kg-1 soil)

400
300 (d)

200
100
0
Initial 1st RHC 1st OHC 2nd RHC 2nd OHC 3rd RHC 3rd OHC
soil Treatment
863
864 Fig. 2. (a) Total mass of size-fractionated particles ([M]particle, mg); (b) particle concentrations

865 ([particle]soil, g kg-1 soil); (c) total mass of C (MC, mg); and (d) C concentrations ([C]soil, mg kg-1 soil).

866 Concentrations are calculated based on the oven-dry weight of soil suspension. The error bars indicate

867 standard deviations among replicates.

868

869

870
871

Reducing half-cycle (RHC) Oxidizing half-cycle (OHC)

-27 NNP Fine colloid Particulate
(a)
13C (‰)
-28

-29

-30

4
(b)

3
15N (‰)

0
Initial 1st RHC 1st OHC 2nd RHC 2nd OHC 3rd RHC 3rd OHC
872 soil Treatment

873 Fig. 3. Variation in isotope values of (a) C (δ13C, ‰) and (b) N (δ15N, ‰) in size-fractionated particles as

874 a function of redox oscillations. The error bars represent standard deviations in stable isotope values

875 among replicates.

876

877

878

879
 Reducing half-cycle (RHC) Oxidizing half-cycle (OHC)
5

C-C/C=O
4
3
2
1
0
3 NNP Fine colloid Particulate
C-C/C-O

0
5
C-O/C=O

4
3
2
1
0
Initial 1st RHC 1st OHC 2nd RHC 2nd OHC 3rd RHC 3rd OHC
soil Treatment
880

881 Fig. 4. Ratios of atomic % of different C functional groups in size-fractionated particles plotted by redox

882 oscillations. The error bars represent standard deviations among replicates.

883

884

885

886

887

888
 6 NNP t = 0.17 x C (atom%) - 4.87

Thickness, t (nm)
Fine colloid
r2= 0.74
4 Particulate
(a)

t = 0.07 x C (atom%) - 0.46

2
r2 = 0.80 t = 0.03 x C (atom%) + 0.06
r2 = 0.93
0
0 10 20 30 40 50 60 70
C (atom %)
600
Csurface (XPS)(mg g-1)

(b)
Csurface (XPS) = 1.46Cbulk (IRMS) + 93.31
400
Pearson's r = 0.92

200 -- 2.3-100 nm 100-450 nm 450-1000 nm

Intercept 137 56 169
(1:1) Slope 1.27 1.73 0.35
Pearson's r 0.79 0.56 0.04
0
0 100 200 300 400 500 600
Cbulk (IRMS)(mg g-1)
Reducing half-cycle (RHC) Oxidizing half-cycle (OHC)
NNP Fine colloid particulate
5
Csurface/Cbulk

4
(c)
3

2
Initial 1st RHC 1st OHC 2nd RHC 2nd OHC 3rd RHC 3rd OHC
889 Soil Treatment

890 Fig. 5. (a) Regression plots between the layer thickness (t, nm) of the SOM coatings on the mineral

891 surfaces and the surface C (atom %) content in different size fractions measured by XPS; (b) relationship

892 between surface C concentration (Csurface) measured by XPS and bulk C concentration (Cbulk) measured by

893 IRMS; (c) variation in C surface enrichment factor (Csurface/Cbulk) in different size fractions as a function of

894 redox oscillations. Surface C concentration was estimated from atomic % and molecular weight of all

895 elements assuming hydrogen has a negligible contribution on a weight basis.

896
897
898

899  Redox oscillations may influence colloid (1-1000 nm) sized organo-mineral associations
900  NNP, fine colloid, and particulate fractions were analyzed using IRMS and XPS
901  NNP and fine colloidal C constituted up to respective 9% and 22% of the COC
902  Operational definition of DOM (cutoff size of 450 nm) lumps significant amount of COC
903  Microbial metabolites are the pervasive SOM components in larger fractions
904

905 Declaration of interests

906

907 ☒ The authors declare that they have no known competing financial interests or personal relationships
908 that could have appeared to influence the work reported in this paper.

909

910 ☐The authors declare the following financial interests/personal relationships which may be considered
911 as potential competing interests:
912

913
914
915

916

917