Soil Science Society of America Journal

Soil Sci Soc Am J doi102136sssaj2014040146 Received 14 Apr 2014 Corresponding author (michaelkaiseruni-kasselde) copy Soil Science Society of America 5585 Guilford Rd Madison WI 53711 USA All rights reserved No part of this periodical may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or any information storage and retrieval system without permission in writing from the publisher Permission for printing and for reprinting the material contained herein has been obtained by the publisher

Influence of Calcium Carbonate and Charcoal Applications on Organic Matter Storage in Silt-Sized Aggregates Formed during a Microcosm Experiment

Soil Chemistry

Increasing exploitation of soil resources under changing climatic conditions re-quires the development of sustainable land management strategies that ensure food and energy production while maximizing soil organic matter (SOM)

storage The application of charcoal (Lehmann 2007) and most recently liming (Fornara et al 2011) have been suggested as options to promote soil aggregation and organic matter retention However only few studies with contrasting results have analyzed the effects of charcoal application on aggregate dynamics in soil (George et al 2012 Pronk et al 2012 Sun and Lu 2014)

Silt-sized aggregates (2ndash53 mm) can store up to 55 of the total soil organic C (SOC) in agricultural soils (Virto et al 2008 Moni et al 2010) The OM occluded in silt-sized aggregates has been shown to have mean residence times between 77 yr (Virto et al 2010 aggregates 20ndash50 mm) and 275 yr (Monreal et al 1997 aggregates lt50 mm) Organic matter associated with such aggregates is involved in (i) nutrient supply on intermediate time scales (ie 10ndash100 yr) and (ii) long-term C storage (ie gt100 yr) Both factors are critical for the productivity of sustainable agricultural systems long-term C storage is also important for mitigating effects of anticipated climate change

The addition of CaCO3 is a routine agronomic practice to raise pH values of acid soils (pH lt 55) for crops that have a pH optimum just below (many cereals) or at pH

Michael Kaiser Life amp Environmental Sciences GroupUniv of California-Merced4225 N Hospital RdAtwater CA 95301

Dep of Environmental ChemistryUniv of KasselNordbahnhofstr 1a 37213 WitzenhausenGermany

Teamrat A GhezzeheiLife amp Environmental Sciences GroupUniv of California-Merced4225 N Hospital RdAtwater CA 95301

Markus KleberDep of Crop and Soil ScienceOregon State Univ3050 SW Campus WayCorvallis OR 97331 Inst of Soil Landscape ResearchLeibniz-Center for Agricultural Landscape Research (ZALF) Muumlncheberg Eberswalder Str 84 15374 Muumlncheberg Germany

David D MyroldDep of Crop and Soil ScienceOregon State Univ3050 SW Campus WayCorvallis OR 97331

Asmeret Asefaw BerheLife amp Environmental Sciences Group Univ of California-Merced 4225 N Hospital RdAtwater CA 95301

Silt-sized aggregates (2ndash53 mm) can store a high percentage of organic matter (OM) in agricultural soils This study aimed to determine whether additions of charcoal and CaCO3 may enhance the retention of organic C (OC) and total N (Nt) in silt-sized aggregates We used artificial soil mixtures without a silt com-ponent (89 sand 10 clay 1 OM) to emulate sandy soils with little natural structure Charcoal andor CaCO3 were added and the resulting mixtures were incubated for 16 wk in the dark The newly formed silt-sized fraction was separat-ed and analyzed for OC and Nt concentrations and characterized using FTIR and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) Compared to the control treatment CaCO3 addition had no positive effects on C and N retention in the silt-sized fraction (17ndash20 g kgminus1 OC 015ndash017 g kgminus1 Nt) whereas the silt-sized fraction from treatments with charcoal additions showed significantly higher OC and Nt concentrations (50ndash56 g kgminus1 OC 031ndash085 g kgminus1 Nt) Silt-sized fractions from the charcoal treatments also showed a signifi-cant increase in the proportion of C=O groups These initial results justify more detailed investigations into the improvement of the structure and nutrient reten-tion of sandy soils by charcoal and CaCO3 applications

Abbreviations BC black C EDS energy dispersive X-ray spectroscopy MO microorganisms Nt total N OC organic C OM organic matter SOC soil organic C SEM scanning electron microscopy SOM soil organic matter

Published September 9 2014

∆ Soil Science Society of America Journal

7 (alfalfa [Medicago sativa L]) Raising the pH typically increases bacterial abundance and activity (Fornara et al 2011) This increased activity may lead to the production of microbially derived adhesives (eg extracellular polymeric substances cell wall residues) that promote aggregate formation At the same time liming adds Ca2+ ions to induce the formation of OMndashcationndashmineral interactions and subsequent aggregation processes (Wuddivira and Camps-Roach 2007 Majzik and Tombaacutecz 2007)

Charcoal is a C-rich solid material produced by combusting biomass in an oxygen-limited environment The chemical and physical characteristics of manufactured charcoal strongly depend on production conditions and feedstock (Keiluweit et al 2010) Charcoal produced at low to intermediate pyrolysis temperatures can be expected to promote aggregation processes because it contains ionizable carboxyl groups (minusCOOH reg minusCOOminus) which may engage in bonding interactions with suitable reaction partners such as positively charged soil minerals ( Joseph et al 2010) Additionally charcoal provides habitat and energy (ie volatile organic compounds) for the soil microorganisms (Zimmerman 2010) that may be involved in producing aggregate forming adhesives

We hypothesized that a combined application of charcoal and CaCO3 amplifies aggregation because OM containing reactive negatively charged functional groups (R-COOminus) and potentially binding Ca2+ cations are added to the soil at the same time Results of several studies indicate that reactions between charcoal particles and Ca2+ may promote long-term storage of charcoal in soils (Clough and Skjemstad 2000 Czimczik and Masiello 2007) The formation of protective aggregates occluding charcoal and Ca2+ is assumed to be one of the underlying processes (Chia et al 2010 2012) However mechanistic information about the effects of CaCO3 andor charcoal application on aggregation processes at the silt-sized scale and the dynamics of associated OM is scarce

Determining the combined vs independent effects of charcoal and CaCO3 additions on soil variables relevant to soil ecology and land management is complicated by the history of land management environmental conditions andor anthropogenic disturbance at any given study site Therefore several authors have conducted microcosm experiments in the laboratory using artificial mixtures of known organic andor mineral compounds to control the complexity of influencing factors (Watts et al 2005 Pronk et al 2012 Wei et al 2012) This approach has been effective in enabling researchers to manipulate for example environmental microbial and mineral characteristics and to study the respective effects on aggregation processes on different scales At the same time the influence of factors such as land use history and small-scale soil heterogeneities can be reduced Here we used a similar approach to determine how combined vs independent application of CaCO3 andor charcoal control aggregation processes at the silt-sized scale

To the best of our knowledge there are no studies analyzing the effects of combined charcoal and CaCO3 application on aggregation dynamics at the silt-sized scale Therefore the

objectives of our study were to determine the influence of environmental and anthropogenic variables on (i) the formation of silt-sized aggregates and (ii) the amount and composition of OM associated with such aggregates for an artificial sandy soil Soils dominated by sand can be found at agricultural sites worldwide and typically exhibit very little aggregation This might negatively affect their productivity because of the positive effects of aggregates on for example OM content soil water holding capacity and biological activity Charcoal additions have been shown to improve cation exchange capacity and available water holding capacity most efficiently in coarse and medium textured soils ( Jeffery et al 2011) The environmental variable tested in this initial experiment was the microbial activity (ie sterilized vs inoculated) The anthropogenic factors tested were CaCO3 andor charcoal addition

MATERIALS AND METHODSArtificial Soil Mixtures

We prepared a silt-free starting mixture composed of 89 sand 10 clay and 1 SOM to mimic a sandy soil Other compounds and their relative proportions used for creating the artificial soil mixture are given in Table 1 All compounds were industrially manufactured and commercially available The added charcoal was produced by slow pyrolysis at 300degC from a mixture of different hardwood shavings including maple aspen choke cherry and alder (Sarkhot et al 2012) Low-temperature charcoal was used because it has been shown to increase micro-bial activity due to higher amounts of bioavailable C than high temperature char (ie gt500degC) (Deenik et al 2010) Before adding the char to the soil mixtures the charcoal was homog-enized in a ball mill (two runs for 2 min) to achieve an average particle size below 53 mm (confirmed using SEM) which mimics the naturally occurring degradation of charcoal in soils as a pre-requisite for the formation of silt-sized aggregates

The different compounds used to create the mixtures (200 g in total) were filled in 250-mL high-density polyethylene (HDPE) bottles before 2 g (1 ww) charcoal (equivalent to 10 t haminus1 0ndash10 cm assuming a dry bulk density of 1 t mminus3) andor 04 g (02 ww) CaCO3 (powder) (equivalent to 2 t haminus1 0ndash10 cm assuming a dry bulk density of 1 t mminus3) were added The amounts of charcoal and CaCO3 represent rates in the lower range usually applied to agricul-tural soils (charcoal Chan et al 2008 Jones et al 2011 CaCO3 liming Farhoodi and Coventry 2008 Flower and Crabtree 2011) We used three replicates for each factorial combination except for the CaCO3 + Charcoal treatment where six replicates were prepared (see below) The resultant experimental design thus included the follow-ing four treatments (i) no additions (Control) (ii) CaCO3 addition (Ca) (iii) charcoal addition (Char) and (iv) CaCO3 and charcoal ad-dition (Char + Ca) After initial homogenization by hand shaking the samples were sterilized using an electron beam radiation dose of 6 Mrad (NUTEK Corporation Hayward CA) Subsequently we placed the samples for 24 h on a rocking and rolling mixer (STR9 TECHNE Bibby Scientific US Burlington NJ) to ensure a thor-ough homogenization

wwwsoilsorgpublicationssssaj ∆

Experimental Setup of the Aggregation Experiment

The homogenized samples were filled in mason jars (baked at 400degC for 12 h) The samples were adjusted to a matric potential of minus03 MPa using a 14-strength Hoagland so-lution (FISHER SCIENTIFIC Waltham MA) The Hoagland solu-tion was sprayed on the surface of the samples To determine the influence of microbial activity on aggregation processes three replicates of the Char + Ca samples were incubated with soil microorganisms (MO) resulting in a fifth treatment of Char + Ca + MO For incubation (at 20degC) we prepared inoculum by diluting 100 g of soil from a local organically man-aged farm (T amp D Willey Farm) in 500 mL of distilled water and shak-ing the mixture for 30 min The sus-pension was filtered (Whatman No 1 Fisher Scientific Waltham MA) (Epstein and Lockwood 1984) and 2 mL of the filtrate were added to the samples This should have supplied an active microbial community that maintained most of its diversity and allowed for adequate op-portunity of bacterial and fungal growth The previously added Hoagland solution contained a complete mixture of macro- and micronutrients needed to support microbial growth and mainte-nance (Myrold 1994) The microcosms were covered with an Al cap to minimize exchange with the open air and kept in the dark for 16 wk (at 20degC) To maintain constant moisture conditions the samples were weighed every 2 wk and any deficit was balanced by adding the respective amount of Hoagland solution

Separation of Silt-Sized AggregatesAfter 16 wk we separated newly formed water-stable silt-

sized aggregates by wet sieving and sedimentation Afterward the separated aggregates were characterized by elemental analy-ses (C and N) SEM EDS and FTIR spectroscopy For the wet-sieving step according to the method of Six et al (2000) 150 g of field-moist soil were placed on a 53-mm sieve and submerged for 5 min in water The sample was then sieved by moving the sieve gently 3 cm vertically 50 times in 2 min to separate the frac-tion gt53 mm From the fraction lt53 mm we separated the clay fraction (lt2 mm) by sedimentation (Moni et al 2010) The sedi-mentation time was determined using Stokesrsquo law and adjusted based on SEM observations This latter step was essential because the validity of Stokes law is based on assumptions for spherical shape and homogenous density of the targeted material which is not realistic for soil particle or aggregate fractions Based on preliminary tests and SEM analyses we found that a sedimenta-

tion time of 60 min was needed to achieve separation of the 2- to 53-mm fraction which is corroborated by the SEM images de-picted in Fig 1 (method described below) Not aggregated and floating organic particles (eg charcoal) were skimmed off The soil recovery rate for the sieving treatment was between 94 and 99 After sedimentation the 2- to 53-mm fractions were dried in the oven at 40degC weighed and stored in glass vials for further analyses The mass of C N and aggregates is reported with refer-ence to 105degC dry samples

Chemical and Microscopic Characterization of the 2- to 53-mm Fraction

The pH value of the artificial soil mixtures after the 16-wk incubation was determined in the supernatant by mixing a 5-g sample with 50 mL of distilled water and allowing the suspen-sion to settle for 5 min (Accumet basic AB15 Fisher Scientific Waltham MA) The OC concentrations of the artificial soil mix-tures were calculated based on the amounts (g compound kgminus1 mixture) and total C concentrations of the organic compounds used for mixing the artificial soils (Table 1) The total C and N (Nt) concentrations of the compounds and the 2- to 53-mm frac-tions were determined by dry combustion on an elemental com-bustion system (ECS 4010 CHNS-O Elemental COSTECH Valencia CA) The total C concentrations of the 2- to 53-mm fractions were considered as OC Given the pH values of pound53 carbonate (CO3)-C amounts in the CaCO3 amended size-frac-tions were considered to be negligible because of the removal of CO3ndashC in the course of wet-sieving and sedimentation (ie sep-aration of the solid and the aqueous phase containing H2CO3

Table 1 Characteristics manufacturer and relative proportions of the individual compounds used to compose the artificial bulk soil mixtures

Compound of the soil mixture Manufacturer Proportion in

Total sand (gt53 mm) 89bulk mixture

Coarse sand (gt500 mm) Laguna Clay Companydagger 35total sand

Medium sand (gt250 and lt500 mm) Laguna Clay Companydagger 55total sand

Fine sand (gt53 and lt250 mm) Laguna Clay Companydagger 10total sand

Total clay (lt2 mm) 10bulk mixture

Al-oxide (Al2O3) SkySpring NanoMaterialsDagger 1total clay

Fe-oxide (FeOOH) Sigma-Aldrichsect 22total clay

Kaolinite (Al2O3 times 2SiO2 times 2H2O) Sigma-Aldrichsect 60total clay

Montmorillonite ((NaCa)033(AlMg)2(Si4O10)(OH)2 times nH2O) Fisher Scientificpara 368total clay

Total soil organic matter 1bulk mixture

Cellulose microcrystalline (C12H20O10) C 427 (plusmn014) Acros Organics 40total SOM

Lignin C 635 (plusmn061) N 059 (plusmn003) CN 108 (plusmn7) Sigma-Aldrichsect 25total SOM

Starch (C6H10O5) C 398 (plusmn020) Fisher Scientificpara 20total SOM

Casein C 485 (plusmn065) N 136 (plusmn021) CN 4 (plusmn001) Acros Organics 10total SOM

Stearic acid (CH3(CH2)16COOH) C 766 (plusmn056) Sigma-Aldrichsect 5total SOM

Additive

CaCO3 C 117 (plusmn005) Fisher Scientificpara 02bulk mixture

Charcoal C 68 (plusmn029) N 039 (plusmn001) CN 174 (plusmn1) Commercial biochardaggerdagger 1bulk mixturedagger La Puente CA Dagger Houston TX sect Saint Louis MO para Waltham MA Geel Belgium daggerdagger Charcoal Gardens (httpwwwbuyactivatedcharcoalcomcharcoal_gardens accessed 21 Sept 2010)

∆ Soil Science Society of America Journal

and HCO3minus previously dissolved in the pore water) The absence

of CO3ndashC was supported by the lack of effervesce on addition of 4 N HCl to the 2- to 53-mm fractions The OC and Nt con-centrations of the 2- to 53-mm size-fraction was referenced to the weight of 105degC dry samples

Shifts in organic matter composition were assessed using FTIR spectroscopy (Tensor 27 BRUKER Ettlingen Germany) The silt-size fractions were examined after drying at 40degC with-out any further pretreatment The spectra were recorded in the range of wave numbers from 400 to 4000 cmminus1 at a resolution of 4 cmminus1 To obtain single spectra 200 scans were performed All spectra were baseline corrected using the same subroutine from the OPUS 72 software (Bruker Ettlingen Germany)

Spectral information was parameterized following the method of Ellerbrock et al (2005) Briefly signal heights in the ldquoaliphaticrdquo region of the FTIR spectrum (Capriel et al 1995 Capriel 1997) representing-CH stretching at wavenumbers 2928 plusmn 20 cmminus1 and 2856 plusmn 20 cmminus1 (Band ldquoArdquo) were added up and related to the peak height of the broad signal at 1625 plusmn 20 cmminus1 (Band ldquoBrdquo) The latter signal has been taken in the past to represent C=O functional groups in SOM (Kaiser et al 2012 Ellerbrock and Gerke 2013) The magnitude of this peak tends to vary as a function of the degree of oxidative de-composition (which adds carboxyl groups to the decomposing substrate) and as a function of peptidendashprotein concentration (the amide C=O stretch or amide I peak occurs at 1645 cmminus1) For this reason peak height in the 1625 plusmn 20 cmminus1 region can be considered a useful proxy for (i) the abundance of ionizable carboxyl groups and the resulting cation exchange capacity of the organic matter in the sample (ii) the polarity and hydrophilic-ity of the organic matter in the sample and (iii) the abundance of proteinaceous microbial debris By relating the ldquoC=Ordquo region (B) to the ldquoaliphaticrdquo region (A) of the FTIR spectrum we thus obtain a numerical parameter ldquoBArdquo that allows a semiquantita-tive comparison of samples while being mechanistically mean-ingful The BA ratio was scaled with the OC concentrations

(g OC kgminus1 fraction) to derive a quantitative estimate of the functional group contribution to the concentration of total OM (Kaiser et al 2008)

To examine the size and structure of silt-sized aggregates ma-terial from the 2- to 53-mm fractions separated from the Control and Char + Ca + MO samples were mounted onto Al stubs sputtered with Ag and scanned with a SEM (Quanta 200 FEI Hillsboro OR) Material (not sputtered) from the 2- to 53-mm fractions of the Char + Ca + MO samples was additionally ana-lyzed by EDS (Genesis EDAX Mahwah NJ) to gain information about the elemental composition on the micrometer scale

StatisticsData sets for the 2- to 53-mm fraction (dry mass OC con-

centration and proportion of SOC BA ratio BAOC concen-tration three replications for each of the five treatments) were analyzed with one-way ANOVA (SigmaStat version 35 Systat Software Inc Richmond CA) The normality of the data was tested with the KolmogorovndashSmirnov procedure and the ho-mogeneity of variances with Levenersquos test Bonferronirsquos t test was used for pairwise comparisons of the different treatments The degrees of freedom were 4 between groups and 9 for the residual Significant treatment effects are given for p pound 005 and p pound 01

RESULTSThe charcoal showed a pH value of 92 and after the 16-wk

incubation the pH values of the bulk soil mixtures ranged between 4 (Control) and 53 (Char + Ca) (Table 2) The pH changes are in line with observations from liming experiments under field conditions (Farhoodi and Coventry 2008 Flower and Crabtree 2011) and from laboratory incubation studies using similar charcoal application rates (Novak et al 2009 Yuan and Xu 2011) The OC concentrations of the bulk mixtures were between 46 and 108 g kgminus1 and the Nt concentrations ranged from 014 to 017 g kgminus1 (Fig 2) The dry masses of the silt-sized fractions

Fig 1 Scanning electron microscopy (SEM) images of the 2- to 53-mm fractions separated from the bulk mixtures of the Control (a)ndash(c) and the Char + Ca +MO (d)ndash(h) treatments (Control no application of charcoal or CaCO3 Char + Ca + MO incubated and addition of charcoal and CaCO3)

wwwsoilsorgpublicationssssaj ∆

(g fraction kgminus1 mixture) were between 59 g kgminus1 (Char + Ca + MO) and 102 g kgminus1 (Char) (Table 2)

The OC concentration of the silt-sized fractions was lowest for the Control sample (174 g kgminus1) and largest for the Char + Ca + MO sample (561 g kgminus1) (Fig 2) The same pattern was observed for the Nt values (015 and 085 g kgminus1 respectively) The concentrations of OC and Nt in the silt-sized fraction represented between 31 (Char + Ca + MO) and 47 (Char) of bulk OC concentrations and between 10 (Control) and 29 (Char + Ca + MO) of bulk Nt concentrations (Fig 2) (the percentages were calculated under consideration of the dry mass amounts and the OC or Nt concentrations of the silt-sized fractions) The CN ratios varied between 66 for the silt-sized fraction of the Char + Ca + MO treatment and 162 for the respective fraction of the Char treatment

The BA ratios derived from the FTIR spectra of the silt-sized fractions are given in Fig 3 and increase in the order Ca lt Control lt Char lt Char + Ca lt Char + Ca + MO After scaling of BA ratios with the respective OC concentration of the silt-sized fractions (BAOC) we found a similar order compared to the BA ratios but the differences between samples with charcoal addition and the Control and Ca samples were more pronounced Because of the 25 to 32 times higher OC concentrations the scaling approach increased the differences between the silt-size fractions of noncharcoal and charcoal treatments from factors between 12 and 29 for the BA ratios to factors between 34 and 58 for the BAOC data Despite maximum effort to keep the incubation vessels and contents sterile samples with charcoal addition showed visible signs of microbial activity at the end of the 16-wk incubation in form of mold patches for the Char Char + Ca and Char + Ca + MO treatments

DISCUSSIONNone of the different treatments we tested showed an

increase in the dry mass of the silt-size fraction compared to the Control We take this as an indication that the CaCO3 andor charcoal application had no positive effects on the dry mass of the silt-sized aggregates (Table 2) The dry mass of the 2- to 53-mm aggregate fraction might also be influenced by single silt-sized particles that can be contained even in industrially manufactured clay minerals (Pronk et al 2012)

One explanation for the observed significant decrease in the dry mass of the silt-sized fraction of the Char + Ca + MO treatment compared to Control (Table 2) can be the formation of macroaggregates gt 2 mm (Char + Ca + MO 261 g kgminus1 (plusmn048) Control no aggregates gt 2 mm detected) In the Char + Ca + MO treatment charcoal and clay-sized particles Ca ions and microbially derived compounds likely acted as binding agents towards macroaggregate formation This confirms data from Sun and Lu (2014) who detected positive effects of charcoal (lt250 mm 500degC) addition to samples from a soil with 43 clay content on the amount of aggregates gt 2 mm for application rates of 27 to 4 These effects were detected for biochar from straw and waste water sludge but

not for biochar from woodchips and an application rate of 13 indicating an influence of the added amount and the feedstock

Table 2 Mean values (n = 3) and standard errors (in parentheses) of the pH of the differently treated bulk soil mixtures the dry mass and the CN ratio of the silt-sized fractions separated from the bulk mixtures of the different treatments

Treatmentdagger pH Dry mass CN ratio

(g fraction kgminus1 soil mixture)Control 40 (plusmn005) 89 (plusmn15) ABDagger absect 118 (plusmn15) B bCa 45 (plusmn01) 87 (plusmn15) AB b 116 (plusmn8) B bChar 42 (plusmn005) 102 (plusmn14) A a 162 (plusmn6) A aChar + Ca 53 (plusmn01) 82 (plusmn54) B b 121 (plusmn4) AB bChar + Ca + MO 51 (plusmn02) 59 (plusmn44) C c 66 (plusmn3) C cdagger Control no application of charcoal or CaCO3 Ca addition of CaCO3 Char

addition of charcoal Char + Ca addition of charcoal and CaCO3 Char + Ca + MO incubated and addition of charcoal and CaCO3 MO microorganisms

Dagger Within columns means followed by different capital letters are significantly different at the 005 probability level

sect Within columns means followed by different lowercase letters are significantly different at the 01 probability level

Fig 2 Concentrations of organic C (OC) (above) and total N (Nt) (below) for the bulk mixtures and the 2- to 53-mm fractions of the analyzed treatments (first y axis) (Control no application of charcoal or CaCO3 Ca addition of CaCO3 Char addition of charcoal Char + Ca addition of charcoal and CaCO3 Char + Ca + MO incubated and addition of charcoal and CaCO3) and proportions of the OC and Nt concentrations of the 2- to 53-mm fractions relative to the OC and Nt concentrations of the bulk mixtures (second y axis) (mean values for n = 3 error bars are standard errors) Means followed by different capital letters are significantly different at the 005 probability level and by different lowercase letters at the 01 probability level

∆ Soil Science Society of America Journal

material on macroaggregate formation In contrast our results differ from findings of Pronk et al (2012) who did not observe positive effects of charcoal (63ndash200 mm) application on gt2-mm aggregate formation in artificial soil mixtures (lt7 clay) despite an application rate of 2 instead of 1 as used in our study The findings of our study and these of Pronk et al (2012) and Sun and Lu (2014) demonstrate that the amount and the nature of charcoal used (eg feedstock pyrolysis temperature particle size content of functional groups) andor mineral characteristics such as the clay content are determining factors for aggregate size

Measuring only dry mass without considering the OC concentrations may not always be the most appropriate way to evaluate changes in aggregate-associated OM because both charcoal and soil OM have low density relative to the mineral constituents This is confirmed by greater OC concentrations in the silt-sized fractions separated from the samples with charcoal addition compared to the Control and the Ca treatments (Factor 2ndash3 Fig 2) The finding suggests an increase in the OC concentration due to incorporation of charcoal into silt-sized aggregates The latter was also shown by Brodowski et al (2006) who found for arable grassland and forest soils maximum concentrations of charcoal derived black C (BC) in particle size fractions lt 53 mm Brodowski et al (2006) postulated that

an enrichment of BC in the aggregate interior could actively contribute to the formation and stabilization of microaggregates which in turn might stabilize BC there which is supported by our data However microbial activity or the addition of CaCO3 seems to have no amplifying effects on the amount of charcoal derived OC associated with silt-sized aggregates

The lack of pronounced positive effects on aggregation and OM storage solely by the application of CaCO3 might be explained by the fact that the added amount (2 t haminus1) was probably too low to cause such effects (Fornara et al 2011 Zornoza et al 2013) The absence of microbial processed reactive organic compounds might be an additional reason In consequence the majority of Ca2+ ions in the Ca treatment seem to be adsorbed by the negatively charged surfaces of the added montmorillonite layer silicates or in the soil solution but not to be involved in the formation of silt-sized aggregates

The OC associated with the silt-size fraction accounted for 31 to 47 of SOC which is in line with data reported in the literature For three agricultural topsoils the OC associated with the silt-sized fractionaggregates (50ndash2 mm) accounted for 23 to 55 of the SOC (Virto et al 2008 2010 Moni et al 2010) Although the soils analyzed in these studies (Luvisol and Cambisol with 94ndash161 g kgminus1 SOC 14ndash18 clay and 57ndash63 silt in the 0- to 28-cm depth Moni et al 2010) are very different from those in the present work similar patterns in OM storage were observed This suggests high relevance of the silt-sized fraction for OM storage in very sandy soils such as the ones studied here and suggests that the experimental approach chosen here should be transferable to other questions requiring the addition of soil amendments

Because of the microbial activity resulting from the inoculation we expected the amount of N associated with silt-size aggregates to be largest in the Char + Ca + MO treatment Microbial-derived N containing compounds such as peptides and proteins are suggested to be highly reactive towards the soil mineral phase (Kleber et al 2007 Sollins et al 2009) This was supported by the measured Nt concentrations and CN ratios within the silt-size fractions as shown in Fig 2 and Table 2 The distinctly higher Nt concentration and lower CN ratio of the Char + Ca + MO silt-sized fraction compared to the silt fractions separated from the Char and Control treatment indicate a positive effect of charcoal application and microbial activity on N retention in silt-sized aggregates This observation is underlined by the greater proportion of Nt associated with the silt-sized fraction relative to the bulk Nt content Nitrogen in the silt-sized fraction was 10 of total N in the Control treatment vs 30 in the Char + Ca + MO treatment

The greater BA ratios and BAOC values of the silt-sized fractions of the charcoal treatments indicate a general increase in the proportion of either amide-C=O or carboxylic-C=O or both compared to the Control and Ca treatments (Fig 3) due to the addition of low temperature charcoal to the sandy soil The largest BAOC value for the silt-sized fraction of the inoculated Char + Ca + MO treatment suggest an additional influence of microbial

Fig 3 Ratios of absorption band peak heights indicative for functional-C-H groups (Band A) and minusC=O groups (Band B) of organic matter as derived from FTIR spectra of the 2- to 53-mm fractions separated from the bulk mixtures of the analyzed treatments (above) (Control no application of charcoal or CaCO3 Ca addition of CaCO3 Char addition of charcoal Char + Ca addition of charcoal and CaCO3 Char + Ca + MO incubated and addition of charcoal and CaCO3) and the products from multiplying the BA ratios with the organic C concentration of the respective 2- to 53-mm fraction (below) (mean values for n = 3 error bars are standard errors) Means followed by different capital letters are significantly different at the 005 probability level and by different lowercase letters at the 01 probability level

wwwsoilsorgpublicationssssaj ∆

derived amide or carboxylic functionalities The increase in BA ratios and BAOC values would also suggest a corresponding increase in the cation exchange capacity in the fractions that received charcoal and charcoal + CaCO3 compared to Ca and Control Cations such as Ca2+ are able to interact with the minusCOOminus groups and the SEM-EDS revealed Ca to be present in the analyzed silt-sized aggregates (Fig 4) This supports the assumption that Ca2+ derived from CaCO3 application may have acted as binding agent during the formation of silt-sized aggregates Ca2+ ions are also likely to be present at the cation exchange sites of the clay minerals

While we are able to report positive effects of charcoal additions on the structure of sandy soil substrates we are aware that much of this outcome hinges on the choice of charcoal particle size Grinding large batches of chars down to the micrometer scale may be costly and the application of a powdery amendment to agricultural fields may constitute a significant practical challenge However it is conceivable that further research will develop production strategies and formulations that allow the reduction of cost to economically feasible levels especially when considered together with increased agronomic and environmental benefits

CONCLUSIONSIncreased OC and Nt concentrations of the 2- to 53-mm

fractions separated from the mixtures with charcoal and charcoal + CaCO3 additions indicate management derived benefits for the long-term C storage and intermediate N supply in soils by promoting aggregation processes at the silt-sized scale This can be beneficial for maintaining or increasing the ability of soils to retain and supply nutrients on decadal timescales

As observed for natural soils the OC concentrations of the silt-sized fractions separated from the artificial soil mixtures accounted for 31 to 47 of the bulk OC and showed the largest Nt concentration for the inoculated microbial active treatment (Char + Ca + MO) This indicates that our methodological approach should be applicable to analyze the interactive effects of environmental factors (eg wettingdrying microbial community structure) and charcoal as well as charcoal + CaCO3 applications on characteristics of OM occluded within silt-sized aggregates

ACKNOWLEDGMENTSThe authors wish to thank Tom Willey and TampD Wiley farms in Madera CA for letting us collect samples from their farm and to M Dunlap from UC Merced Imaging and Microscopy Facility for his support with the SEM analysis This work was supported by University of California-Merced faculty start up fund to AAB The contribution of M Kleber was supported by a research fellowship from the Institute of Soil Landscape Research Leibniz-Center for Agricultural Landscape Research (ZALF) Muumlncheberg Germany

REFERENCESBrodowski S B John H Flessa and W Amelung 2006 Aggregate-occluded

black carbon in soil Eur J Soil Sci 57539ndash546 doi101111j1365-2389200600807x

Capriel P 1997 Hydrophobicity of organic matter in arable soils Influence of management Eur J Soil Sci 48457ndash462 doi101046j1365-2389199700098x

Capriel P T Beck H Borchert J Gronholz and G Zachmann 1995 Hydrophobicity of the organic-matter in arable soils Soil Biol Biochem 271453ndash1458 doi1010160038-0717(95)00068-P

Chan KY L van Zwieten I Meszaros A Downie and S Joseph 2008 Using poultry litter biochars as soil amendments Aust J Soil Res 46437ndash444 doi101071SR08036

Chia CH P Munroe SD Joseph Y Lin J Lehmann DA Muller HL Xin and E Neves 2012 Analytical electron microscopy of black carbon and microaggregated mineral matter in Amazonian dark Earth J Microsc 245129ndash139 doi101111j1365-2818201103553x

Chia CH P Munroe S Joseph and Y Lina 2010 Microscopic characterisation of synthetic Terra Preta Aust J Soil Res 48593ndash605 doi101071SR10012

Fig 4 Scanning electron microscopy (SEM) image of an aggregate from the 2- to 53-mm fraction separated from the bulk mixture of the Char + Ca + MO treatment (incubated and addition of charcoal and CaCO3) and energy dispersive (ED) spectra from four different areas (1ndash4) of the scanned aggregate MO microorganisms

∆ Soil Science Society of America Journal

Clough A and JO Skjemstad 2000 Physical and chemical protection of soil organic carbon in three agricultural soils with different contents of calcium carbonate Aust J Soil Res 381005ndash1016 doi101071SR99102

Czimczik CI and CA Masiello 2007 Controls on black carbon storage in soils Global Biogeochem Cycles 21GB3005 doi1010292006GB002798

Deenik JL T McClellan G Uehara JMJ Antal and S Campbell 2010 Charcoal volatile matter content influences plant growth and soil nitrogen transformations Soil Sci Soc Am J 741259ndash1270 doi102136sssaj20090115

Ellerbrock RH and HH Gerke 2013 Characterization of organic matter composition of soil and flow path surfaces based on physicochemical principlesmdashA review In DL Sparks editor Advances in agronomy 121 Elsevier Academic Press San Diego CA p 117ndash177

Ellerbrock RH HH Gerke J Bachmann and MO Goebel 2005 Composition of organic matter fractions for explaining wettability of three forest soils Soil Sci Soc Am J 6957ndash66 doi102136sssaj20050057

Epstein L and JL Lockwood 1984 Effect of soil microbiota on germination of Bipolaris Victoriae conidia Trans Br Mycol Soc 8263ndash69

Farhoodi A and DR Coventry 2008 Field crop responses to lime in the mid-north region of South Australia Field Crops Res 10845ndash53 doi101016jfcr200802013

Flower KC and WL Crabtree 2011 Soil pH change after surface application of lime related to the levels of soil disturbance caused by no-tillage seeding machinery Field Crops Res 12175ndash87 doi101016jfcr201011014

Fornara DA S Steinbeiss NP McNamara G Gleixner S Oakley PR Poulton AJ Macdonald and RD Bardgett 2011 Increases in soil organic carbon sequestration can reduce the global warming potential of long-term liming to permanent grassland Glob Change Biol 171925ndash1934 doi101111j1365-2486201002328x

George C M Wagner M Kuecke and MC Rillig 2012 Divergent consequences of hydrochar in the plant-soil system Arbuscular mycorrhiza nodulation plant growth and soil aggregation effects Appl Soil Ecol 5968ndash72 doi101016japsoil201202021

Jeffery S FGA Verheijen M van der Velde and AC Bastos 2011 A quantitative review of the effects of biochar application to soils on crop productivity using meta-analysis Agric Ecosyst Environ 144175ndash187 doi101016jagee201108015

Jones DL G Edwards-Jones and DV Murphy 2011 Biochar mediated alterations in herbicide breakdown and leaching in soil Soil Biol Biochem 43804ndash813 doi101016jsoilbio201012015

Joseph SD M Camps-Arbestain Y Lin P Munroe CH Chia J Hook L van Zwieten S Kimber A Cowie BP Singh J Lehmann N Foidl RJ Smernik and JE Amonette 2010 An investigation into the reactions of biochar in soil Aust J Soil Res 48501ndash515 doi101071SR10009

Kaiser M RH Ellerbrock and HH Gerke 2008 Cation exchange capacity and composition of soluble soil organic matter fractions Soil Sci Soc Am J 721278ndash1285 doi102136sssaj20070340

Kaiser M RH Ellerbrock M Wulf S Dultz C Hierath and M Sommer 2012 The influence of mineral characteristics on organic matter content composition and stability of topsoils under long-term arable and forest land use J Geophys Res Biogeo 117G02018 doi1010292011JG001712

Keiluweit M PS Nico MG Johnson and M Kleber 2010 Dynamic molecular structure of plant biomass-derived black carbon (biochar) Environ Sci Technol 441247ndash1253 doi101021es9031419

Kleber M P Sollins and R Sutton 2007 A conceptual model of organo-mineral interactions in soils Self-assembly of organic molecular fragments into zonal structures on mineral surfaces Biogeochemistry 859ndash24 doi101007s10533-007-9103-5

Lehmann J 2007 Bio-energy in the black Front Ecol Environ 5381ndash387 doi1018901540-9295(2007)5[381BITB]20CO2

Majzik A and E Tombaacutecz 2007 Interaction between humic acid and

montmorillonite in the presence of calcium ions II Colloidal interactions Charge state dispersing andor aggregation of particles in suspension Org Geochem 381330ndash1340 doi101016jorggeochem200704002

Moni C C Rumpel I Virto A Chabbi and C Chenu 2010 Relative importance of sorption versus aggregation for organic matter storage in subsoil horizons of two contrasting soils Eur J Soil Sci 61958ndash969 doi101111j1365-2389201001307x

Monreal CM HR Schulten and H Kodama 1997 Age turnover and molecular diversity of soil organic matter in aggregates of a Gleysol Can J Soil Sci 77379ndash388 doi104141S95-064

Myrold DD 1994 Frankia and the actinorhizal symbiosis In RW Weaver JS Angle and PJ Bottomley editors Methods of soil analysis Part 2 Microbiological and biochemical properties SSSA Book Ser 5 SSSA Madison WI p 291ndash328

Novak JM WJ Busscher DL Laird M Ahmedna DW Watts and MAS Niandou 2009 Impact of biochar amendment on fertility of a southeastern coastal plain soil Soil Sci 174105ndash112 doi101097SS0b013e3181981d9a

Pronk GJ K Heister G-C Ding K Smalla and I Koegel-Knabner 2012 Development of biogeochemical interfaces in an artificial soil incubation experiment aggregation and formation of organo-mineral associations Geoderma 189ndash190585ndash594 doi101016jgeoderma201205020

Sarkhot DV AA Berhe and TA Ghezzehei 2012 Impact of biochar enriched with dairy manure effluent on carbon and nitrogen dynamics J Environ Qual 411107ndash1114 doi102134jeq20110123

Six J K Paustian ET Elliott and C Combrink 2000 Soil structure and organic matter I Distribution of aggregate-size classes and aggregate-associated carbon Soil Sci Soc Am J 64681ndash689 doi102136sssaj2000642681x

Sollins P MG Kramer C Swanston K Lajtha T Filley AK Aufdenkampe R Wagai and RD Bowden 2009 Sequential density fractionation across soils of contrasting mineralogy Evidence for both microbial- and mineral-controlled soil organic matter stabilization Biogeochemistry 96209ndash231 doi101007s10533-009-9359-z

Sun F and S Lu 2014 Biochars improve aggregate stability water retention and pore-space properties of clayey soil Z Pflanzenernaehr Bodenkd 17726ndash33

Virto I P Barre and C Chenu 2008 Microaggregation and organic matter storage at the silt-size scale Geoderma 146326ndash335 doi101016jgeoderma200805021

Virto I C Moni C Swanston and C Chenu 2010 Turnover of intra- and extra-aggregate organic matter at the silt-size scale Geoderma 1561ndash10 doi101016jgeoderma200912028

Watts CW WR Whalley PC Brookes BJ Devonshire and AP Whitmore 2005 Biological and physical processes that mediate micro-aggregation of clays Soil Sci 170573ndash583 doi10109701ss0000178206740400c

Wei S W Tan W Zhao Y Yu F Liu and LK Koopal 2012 Microstructure interaction mechanisms and stability of binary systems containing goethite and kaolinite Soil Sci Soc Am J 76389ndash398 doi102136sssaj20110065

Wuddivira MN and G Camps-Roach 2007 Effects of organic matter and calcium on soil structural stability Eur J Soil Sci 58722ndash727 doi101111j1365-2389200600861x

Yuan J-H and R-K Xu 2011 The amelioration effects of low temperature biochar generated from nine crop residues on an acidic Ultisol Soil Use Manage 27110ndash115 doi101111j1475-2743201000317x

Zimmerman AR 2010 Abiotic and microbial oxidation of laboratory-produced black carbon (biochar) Environ Sci Technol 441295ndash1301 doi101021es903140c

Zornoza R Aacute Faz DM Carmona JA Acosta S Martiacutenez-Martiacutenez and A de Vreng 2013 Carbon mineralization microbial activity and metal dynamics in tailing ponds amended with pig slurry and marble waste Chemosphere 902606ndash2613 doi101016jchemosphere201210107