Science of the Total Environment 684 (2019) 682–693

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Long-term fertilization and manuring with different organics alterstability of carbon in colloidal organo-mineral fraction in soils of varyingclay mineralogy

Ruma Das a,⁎, T.J. Purakayastha a, Debarup Das a, Nayan Ahmed a, Rahul Kumar a, Sunanda Biswas a, S.S. Walia b,Rohitashav Singh c, V.K. Shukla d, M.S. Yadava e, N. Ravisankar f, S.C. Datta a

a Division of Soil Science and Agricultural Chemistry, ICAR-Indian Agricultural Research Institute, New Delhi 1100 12, Delhi, Indiab Department of Agronomy, Punjab Agricultural University, Ludhiana 141004, Punjab, Indiac Department of Agronomy, GB Pant University of Agriculture and Technology, Pantnagar 263145, Uttarakhand, Indiad Department of Agronomy, Jawaharlal Nehru Krishi Viswa Vidyalaya, Jabalpur 482004, Madhya Pradesh, Indiae Department of Agronomy, Birsa Agricultural University, Ranchi 834006, Jharkhand, Indiaf Division of Integrated Farming System Management, ICAR-Indian Institute of Farming Systems Research, Modipuram 250110, Uttar Pradesh, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Long-term fertilization and manuringaltered the C stability in COMF.

• Amorphous Fe, Al oxides positively in-fluenced the C stability in COMF.

• Fertilization and manuring decreasedthe average crystallite size of illite.

• NPK + GM was most effective in in-creasing C stability in COMF.

⁎ Corresponding author at: ICAR-Indian Agricultural ReE-mail address: rumadas13@gmail.com (R. Das).

https://doi.org/10.1016/j.scitotenv.2019.05.3270048-9697/© 2019 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 February 2019Received in revised form 7 May 2019Accepted 21 May 2019Available online 23 May 2019

Editor: G. Darrel Jenerette

Majority of organic matter is bound to clay minerals to form stable colloidal organo-mineral fraction (COMF) insoil. Stability of carbon (C) in COMF is crucial for long-term C sequestration in soil. However, information on theeffect of long-term fertilization and manuring with various organic sources on C stability in such fraction in soilswith varying claymineralogy is scarce. The present studywas, therefore, carried out to assess the effect of thirty-one years of continuous fertilization andmanuringwith different organics on C-stability in COMF extracted froman Inceptisol, a Vertisol, a Mollisol, and an Alfisol. The treatments comprised of control (no fertilization), 100%NPK (100% of recommended N, P and K through fertilizer), 50% NPK+ 50% of recommended N supplied througheither farm yard manure (FYM) or cereal residue (CR) or green manure (GM). The stability of C (1/k) in COMFwas determined from desorption rate constant (k) of humus-C by sequential extraction and correlated with ex-tractable amorphous Fe-Al-Si-oxides, and crystallite size of illite minerals. Long-term fertilization and manuringwith the above sources of organic altered the contents of amorphous Fe-Al-Si-oxides, and decreased the crystal-lite size of illite in all the soil orders. Fifty percent substitution of fertilizer N by various organics significantly

Keywords:Amorphous Fe-Al-Si-oxidesCarbon stabilityColloidal organo-mineral fractionClay minerals

search Institute (IARI), New Delhi 110012, India.

683R. Das et al. / Science of the Total Environment 684 (2019) 682–693

increased C-stability in COMF by 27–221% (mean 111%) over full dose of NPK (100% NPK). Smectite dominatingVertisol exhibited highest stability of C followed by the Mollisol, the Inceptisol and the Alfisol. Stability of such Cin soil was correlated positively with the amount of amorphous Fe and Al oxides but negatively with crystallitesize of illite (r = −0.46, P b 0.01). Application of NPK + GM or NPK + FYM in Inceptisol, Vertisol and Mollisoland NPK + GM or NPK + CR in Alfisol emerged as the best management practices for higher stabilization of Cin COMF for long-term C sequestration.

© 2019 Elsevier B.V. All rights reserved.

Crystallite sizeLong-term fertilization

1. Introduction

Soil organic matter (SOM) is an important component of global car-bon (C) cycle. The most significant contribution of SOM towards miti-gating global warming is through C-sequestration that reduces the netCO2 emission to atmosphere. The small sized (b20 μm) soil particles,such as silt and clay, can retain higher amounts of organic C as comparedto larger particles, thus play an important role in C-stabilization(Homann et al., 2007; Lorenz et al., 2008). It is well known for decadesthat organic matter (OM) in soil is mainly associated with minerals inthe form of organo-mineral complexes of varying stability (Jung,1943). The SOM interacts with the reactive mineral surfaces by hydro-gen (H)-bonding, van der waals attraction, hydrophobic interactions,polyvalent cation bridging, and ligand exchange (von Lützow et al.,2007; Koegel-Knaber and Kleber, 2011; Schmidt et al., 2011), and inturn gets protection from decomposition by various agents. The extentof such protection of C fromdecomposition largely depends on themin-eral make-up of clay particles (Laird et al., 2001). Clayminerals not onlystabilize SOM, but also decrease the potential of SOM loss by respiration,erosion and leaching. The stabilization and subsequent accumulation oforganic C (OC) in soil are influenced by the type of phyllosilicate clays aswell as the iron oxides in soil (Saidy et al., 2013). Clay minerals espe-cially amorphous or poorly crystalline minerals of soil are very impor-tant for OM stabilization due to their charge characteristics, smallparticle size and large surface area (Torn et al., 1997; Kleber et al.,2005; Mikutta et al., 2005; von Lützow et al., 2007; Schneider et al.,2010; Wen et al., 2018). However, the extent of protection providedto SOM by interaction with minerals varies as different clay mineralshave different specific surface areas and charge characteristics (Huang,1990; Robert and Chenu, 1992). Some previous reports indicated thatamorphous iron oxides are, perhaps, the most important for SOM-stabilization due to higher sorption capacity for SOM as compared toother metal oxides and phyllosilicates (Lalonde et al., 2012; Grüneberget al., 2013; Chatterjee et al., 2013; Zhao et al., 2017; Wen et al.,2018). Different soil orders have different dominating clay mineralsand bridging cations in clay-humus complex (Ahmed et al., 2002),hence similar management practices might have dissimilar impacts onC-stabilization by clay minerals in different soil orders.

Long-term cultivation with different nutrient supply options involv-ing the addition of chemical fertilizers with or without organics mightdifferentially affect the stability of C present in a soil as a whole, and es-pecially that present in the organo-mineral fraction (Schulten andLeinweber, 2000). Chemical fertilization alone indirectly increased OCcontent in soils through enhanced crop biomass production (Yadavet al., 2017; Ghosh et al., 2018a). Compared to no fertilization and chem-ical fertilization, continuous application of organic manure to soil fur-ther improved soil organic C (SOC) accumulation and storage (Ghoshet al., 2018b; Wen et al., 2019). Such an increased C storage is due tothe direct C input from manure and indirectly from increased biomassproduction (Maillard and Angers, 2014). Again, the nature of organicsapplied to the soil also has a bearing on soil C build-up and long-termstorage (Mandal et al., 2007; Ghosh et al., 2012; Yadav et al., 2017;Sarkar et al., 2018). The different biochemical constituents of organicamendments affect the mineralization (Mohanty et al., 2013) owing tothe change on microbial community, hence, stability of C in soil (vonLutzow et al., 2006; Baumann et al., 2009). The C:N ratio of organics

associatedwith availability of N per unit of C and determine the amountof substrate C addition to soils (Hessen et al., 2004;Manzoni et al., 2012;Dannehl et al., 2017). An increase in C content of soil due to long-termaddition of farm yard manure (FYM), crop residue (CR) or green ma-nure (GM) was observed by earlier workers (Bandyopadhyay et al.,2010; Das et al., 2017; Yadav et al., 2017), but similar information onC-stability of COMF in soils of widely differing mineralogy is scarce.The stability of C can be determined by the rate constant of dissociationof C from the soil by using sodium hydroxide sodium pyrophosphate asan extractant (Datta et al., 2015; Lungmuana et al., 2018). Very fewstudies were conducted to know the stability of C directly from the ex-tracted colloidal organo-mineral fraction (COMF) from soil. Moreover,knowledge on factors affecting the C-stability of COMF is also lacking.Datta et al. (2015) examined the stability of humus-C in two wheatbased systems bydesorption of adsorbed humus on COMFusing sodiumhydroxide sodium pyrophosphate solution. Lungmuana et al. (2018)determined the C-stability in bulk soil following the above mentionedprocedure. It has already been showed by previous workers that inte-grated nutrientmanagement is beneficial for long-termC sequestration.On the other hand, the proportion of different clay minerals could alsobe affected by long-term fertilization and manuring. However, there isscarcity of information on the long-term effect of fertilization and ma-nuring on stabilization of organic C in organo-mineral fraction of soilsvarying in clay mineral make up. Besides, little knowledge is availableon the impact of various organics on organic C stabilization in soil. Thepresent investigation was, therefore, carried out to address the follow-ing questions:

1. Whether fertilization and manuring for years together affect the sta-bility of C in COMF of different soil orders?

2. To what extent do the amorphous Fe-Al-Si-oxides and phyllosilicateclayminerals affect C stability in COMF in soils of varyingmineralog-ical make-up?

To address the above questions, the present investigation was car-ried out with the following objectives: i) to study the effect of long-term application of fertilizers alone or in combination with different or-ganics on C stability in COMF in Mollisol, Vertisol, Inceptisol, and Alfisoland (ii) to find out the role of amorphous and crystalline clay mineralson stability of C in COMF.

2. Materials and methods

2.1. Experimental sites

Four on-going long-term experiments (LTEs) continuing since1983–84 under the aegis of All India Coordinated Research Project onIntegrated Farming Systems (AICRP-IFS) of Indian Council of Agricul-tural Research (ICAR), located at Ludhiana (Inceptisol), Jabalpur(Vertisol), Pantnagar (Mollisol) and Ranchi (Alfisol) of India were cho-sen for the present study. Locations, elevations, climatic variables, soiltypes and existing cropping systems of the four sites are given inTable 1. Important physico-chemical properties of surface (0–15 cm)soils of those sites at the beginning of the experiments are briefed inTable 2.

Table 1Site details of the long-term experiments (LTEs) selected for the study.

Experimentalsite

Latitude Longitude Elevation (m,above meansea level)

Mean annualtemperature (°C)

Mean annualrainfall(mm)

Agro-climatic region Croppingsystem

Soiltaxonomy

Minimum Maximum

Ludhiana 30° 56′ N 75° 52′ E 247 5.7 40.6 500 Trans-Gangetic Plains region Rice-wheat Typic HaplusteptJabalpur 23° 10′ N 79° 57′ E 412 22.8 31.9 1386 Central Plateau and Hills region Rice-wheat Typic HaplustertPantnagar 29° 08′ N 79° 05′ E 244 7.3 37.4 1383 Western Himalayan region Rice-wheat Aquic HapludollRanchi 23° 17′ N 85° 19′ E 120 16.7 31 1450 Eastern Plateau and Hills region Maize-wheat Typic Haplusalf

684 R. Das et al. / Science of the Total Environment 684 (2019) 682–693

2.2. Details of the field experiments and treatments

Rice-wheat sequence was followed every year in Ludhiana, Jabalpurand Pantnagar whereas, maize-wheat sequence was followed in Ranchisince the initiation of the experiments. Rice and maize were grown inmonsoon season (July–October) without any irrigation and wheat wasgrown in rabi (winter) season (November–April) with 4–5 irrigationseach of 6 cm depth. All the LTEs have 12 treatments laid out incompletely randomized block design, from which 5 treatments (eachwith 3 replications), viz. control, 100% NPK, NPK + FYM, NPK + CR,and NPK + GM were chosen for the study. Treatment details for mon-soon and winter seasons are provided in Tables 3 and 4. In the NPK+ CR treatment, wheat straw was applied in Ludhiana, Jabalpur,Pantnagar, and paddy straw in Ranchi. In the NPK + GM treatment,the applied greenmanurewas sesbania (Sesbania aculeata) at Ludhiana,sunhemp (Crotalaria juncea) at Jabalpur, green-gram (Vigna radiata) atPantnagar, and pongamia (Pongamia pinnata) at Ranchi. All these treat-ments were applied inmonsoon, while in winter season only N, P and Kfertilizerswere applied. The C andN content of different organic sourcesare presented in Table 5.

2.3. Collection and analyses of soil samples

Soil (0–15 cm) samples were collected after the harvest of wheatduring 2014–15 crop cycle, from five randomly chosen spots in eachreplicated plot with a core sampler. Five sub-samples from each plotwere pooled together to represent a replication of a particular treat-ment. Soil sampleswere dried in air, ground bywooden pestle andmor-tar, and passed through a 2-mm sieve before analyses.

2.3.1. Clay mineralogySoil samples from 3 replicated plots of each treatment were pooled

together to study the clay mineralogy. Separation of clay particles (b2μm) was done by the procedure detailed by Jackson (1985). Clay sam-ples were subjected to four treatments, viz. (i) magnesium (Mg)saturated-air dried (Mg-air), (ii) Mg-saturated and glycerol solvated(Mg-gly), (iii) potassium (K) saturated-air dried (K-air), and (iv) K sat-urated and heated at 550 °C for 2 h (K-550). The X-ray diffractograms ofthe differentially treated clayswere recorded using a Philips diffractom-eter (X-ray generator: PW-1729, diffractometer control: PW-1710,

Table 2Important physico-chemical properties of surface (0–15 cm) soil of the four LTE sites at the be

Soil property Inceptisol (Ludhiana) Vertisol

Sand (%) 54 28Silt (%) 28 19Clay (%) 18 53Texture Sandy loam ClaypH (1:2.5) 8.15 7.54EC (1:2.5) (dS m−1) 0.32 0.48Organic C (g kg−1) 3.1 6.0Available N (kg ha−1) 143 238Available P (kg ha−1) 11.0 8.6Available K (kg ha−1) 101 287

Philips, Holland) with Ni-filtered Cu-Kα radiation at a scanning speed1.5°2θ min−1. The X-ray diffractograms of Mg-gly clays weredecomposed to get the component peaks of the observed peaks(Datta, 1996). Clay minerals were identified and categorised based onthe observations of earlier researchers (Moore and Reynolds, 1997;Barre et al., 2008; Datta et al., 2015; Das et al., 2019). Peak positionsspread over 4.5 to 5.1°2θ with c-axis spacing ~1.8 nm were consideredas smectite (category-1). Adjacent to smectite, there was 1 or 2 morepeaks ranging from 5.2 to 6.2°2θ. Such peaks could be due to the pres-ence of chlorite, hydroxyl-interlayered minerals (HIMs), vermiculiteor smectite rich mixed layer minerals interstratified with illite or chlo-rite or other hydroxyl-interlayered minerals. All these were categorisedinto a single group andnamed interstratified-smectite (Si) (category-2).Presence of chlorite and HIMs were confirmed after heating the K-saturated clays at 550 °C, where only chlorite peak got reinforced near1.4 nm (Dixon and Jackson, 1960), and the peaks of HIMs were shiftedto near 1 nm. Peaks between 8.3 and 8.8°2θ with spacing near 1 nmwere considered as illite (category-3). Illite peaks were classified intotwo groups i.e. poorly crystallized illite (PCI) with peaks between 8.3and 8.7, and well crystallized illite (WCI) with peaks between 8.7 and8.8. Adjacent to illite peak there was some more peaks in the range of6.6 to 8.3 and 9.2 to 10.3°2θ, which could be attributed to the presenceof illite rich illite/smectite or illite/chlorite interstratified minerals.These minerals were collectively grouped as interstratified illite (Ii)(category-4). The peak positions at 12.3 to 12.4°2θ with spacing near0.7 nm were assigned to kaolinite (category-5). Peaks adjacent to thatof kaolinite (from 10.5 to 12.3°2θ) were considered as interstratified ka-olinite (Ki) (category-6). These were mainly kaolinite rich minerals in-terstratified with illite or smectite as smectite shows second orderdiffraction at 9.9°2θ. The above mentioned six categories of clay min-erals were further clubbed into broader groups as (i) SRM i.e. smectiteand smectite richminerals (categories 1 and 2), (ii) IRM i.e. illite and il-lite rich minerals (categories 3 and 4); and (iii) KRM i.e. kaolinite andkaolinite rich minerals (categories 5 and 6).

Semi-quantification of minerals present in the clay particles weredone from X-ray diffractograms of Mg-gly samples as per Gjems(1967), Datta et al. (2015) and Paul et al. (2017). The average particlediameter of illite was also computed from peak broadening. Broadeningof peak at 1 nm mainly occurs due to crystallite size and lattice strainapart from that caused by instrumental factors (Klug and Alexander,

ginning of the experiments.

(Jabalpur) Mollisol (Pantnagar) Alfisol (Ranchi)

32 5539 2229 23Silty clay loam Sandy clay loam7.3 6.50.35 0.1014.2 4.2280 25514.5 14.2120 195

Table 3Treatment details of the LTEs.

Treatment Monsoon (Rice/Maize) Winter (Wheat)

Control No fertilizer or manure No fertilizer or manure100% NPK 100% of recommended rate of

NPK through fertilizers100% of recommended rate ofNPK through fertilizers

NPK + FYM 50% of recommended rate ofNPK through fertilizers +50%N through FYM

100% of recommended rate ofNPK through fertilizers

NPK + CR 50% of recommended rate ofNPK through fertilizers +50%N through wheat straw

100% of recommended rate ofNPK through fertilizers

NPK + GM 50% of recommended rate ofNPK through fertilizers +50%N through green manure

100% of recommended rate ofNPK through fertilizers

Table 5Total N and C content (% on dry weight basis) in organic sources used in those LTEs.

Organic source C (%) N (%)

FYM 27.3 ± 7.1⁎ 0.75 ± 0.21Crop residueRice straw 38.9 ± 8.2 0.50 ± 0.11Wheat straw 40.8 ± 5.1 0.64 ± 0.15

Green manureDhaincha 49.8 ± 9.2 3.10 ± 0.42Green-gram 43.3 ± 9.6 2.12 ± 0.11Sunhemp 42.3 ± 4.1 2.24 ± 0.21Karanj 48.1 ± 6.1 2.36 ± 1.15

⁎ Mean ± standard error of mean (SEM).

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1974). In the present study, the full width at half maximum intensity(FWHM) was considered as a measure of peak broadening and thebroadening caused by particle size (Bsize) was computed by the follow-ing equation:

Bsize ¼ FWHM−Binstrument

where, FWHM is the experimentally obtained full width at halfmaxmium intensity (for illite peak), and Binstrument is the instrumentalbroadening. The FWHM of the 1 nm peak obtained from a silt sizedmica particle was considered as equal to Binstrument. The average diame-ter of illite particles (Nd) present in the soil clays was computed fromthe following equation given by Scherrer (1918):

Nd ¼ kλBsize cosθ

where, k is a dimensionless shape factor (whose value was consideredas 0.9 in the present study), λ is the wavelength of Cu-Kα radiation(0.154184 nm), θ is the Bragg angle (in radians), d is the c-axis spacingof the mineral under consideration (here, illite), and N is the effectiveaverage number of illite layers in a crystallite (i.e. the ordered domains).

2.3.2. Colloidal organo-mineral fraction (COMF)Separation of COMF frombulk soilwas done following themethod of

Datta et al. (2015). Briefly, 20 g soil along with 200 mL distilled waterwere taken in a stainless steel beaker and the mixture was stirred for15 min with a mechanical stirrer. The suspension was then subjectedto ultrasonic vibration for 5 min to further disperse the micro-aggregates. The dispersed suspension was entirely transferred to a2.5-L bottle and distilled water was added up to the neck. The suspen-sion in the bottle was kept undisturbed for 8 h, after which upper10 cm portion, containing COMF having diameter b 2 μm as per Stokes'law (Jackson, 1985), was siphoned out. Again distilled water was addedto the suspension in the bottle up to the neck, and the same procedure,asmentioned above,was repeated until the upper 10 cm the suspensionbecame clear after 8 h of settling time. The extracted suspensions

Table 4Recommended fertilizer dose of different crops in LTEs.

Location/soil type Cropping system

Ludhiana (Inceptisol)Rice (Oryza sativa L.), cv. PR-116 (Monsoon seasWheat (Triticum aestivum L.), cv. PBW-343 (Win

Jabalpur (Vertisol)Rice (Oryza sativa L.), cv. MR-219 (Monsoon seaWheat (Triticum aestivum L.), cv. GW-273 (Win

Pantnagar (Mollisol)Rice (Oryza sativa L.), cv. PR-113 (Monsoon seasWheat (Triticum aestivum L.), cv. PBW-343 (Win

Ranchi (Alfisol)Maize (Zea mays L.), cv. M-9000 (Monsoon seasWheat (Triticum aestivum L.), cv. DWR-162 (Wi

containing COMF (b2 μm) were pooled together, and concentrated byrepeated centrifugation and decantation. After centrifugation the con-centrated suspension was freeze dried in a lyophilizer (Labconco,USA) (Kaiser and Guggenberger, 2003) and stored for the determina-tion of contents of total organic C (Schollenberger, 1927) and amor-phous Al-Fe-oxides and Si-oxides (McKeague and Day, 1966) presentin the COMF. Stability of C in COMFwas determined by a humus desorp-tion experiment (Datta et al., 2015; Lungmuana et al., 2018). For this,0.1 g of freeze dried COMF was taken in a polypropylene centrifugetube and 25 mL of sodium hydroxide sodium pyrophosphate (0.1 Mwith respect to each) solution was added. The contents were shakenfor 2 h at the speed of 200 rpm on a rotary shaker, and then centrifugedfor 10min at 8000 rpmwith a centrifuge (Remi R-24). The supernatantwas separated and a fresh solution of 25 mL sodium hydroxide sodiumpyrophosphate was added followed by shaking the contents again for2 h, centrifugation and decantation. The same process was repeatedsix times. At the end of each 2-hours shaking, the supernatant contain-ing the desorbed humus was removed to prevent any re-adsorption ofhumus on to the clay surfaces, which otherwise would affect the rateof humus release. The C concentrations in the first extracts were deter-mined by dichromate oxidation in acid medium as described bySchollenberger (1927). Absorbance of 440 nm wavelength by the ex-tracts was also determined, and a linear regression relationship wasestablished between C concentration and absorbance at 440 nm. Atlater stages of humus desorption, C concentration, being relativelysmall, was obtained from absorbance values using the linear regressionrelationship. From the C present in the supernatants extracted at differ-ent times, the cumulative humus-C desorbed per unit quantity of COMFwas computed, and the samewas subtracted from the original C contentof the COMF to get the clay-humus C remaining at time t (Ct). The valuesof Ct and t were fitted to a first-order equation:

Ct ¼ C0e−kt

where, C0 is the original C content of the COMF, and k is the desorptionrate constant of humus-C.

100% recommended fertilizer dose (kg ha−1)

N P K

on) 120 30 30ter season) 120 60 30son) 120 60 40ter season) 120 60 40on) 120 40 –ter season) 120 40 –on) 100 22 21nter season) 100 22 21

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Control 100%NPK NPK+FYM NPK+CR NPK+GM

Fig. 2. Carbon stability of colloidal organo-mineral fraction (COMF) in different soils undervarying nutrient supply options. Column followed by a different letter in the same soilorder are significantly different at p ≤ 0.05 by Duncan's multiple range test. Error barsindicate standard error of mean (SEM).

686 R. Das et al. / Science of the Total Environment 684 (2019) 682–693

2.4. Statistical analysis

To determine the effect of treatments on total organic C content andC-stability in COMF, analysis of variance (ANOVA) was carried out.Duncan's multiple range test (DMRT) was used to compare the treat-ment means of total organic C content and C-stability in COMF. Pearsoncorrelation was computed to study the effect of clay mineralogy on C-stability in COMF. All these statistical analyses were done followingthe methodology described by Gomez and Gomez (1984) using SPSS21.0.

3. Results

3.1. Total organic C (TOC) in colloidal organo-mineral fraction (COMF)

Total organic C (TOC) content in COMF was the highest in Mollisol(21.6–24.2 g kg−1, mean 22.9 g kg−1) followed by Inceptisol(14.1–19.8 g kg−1, mean 17.9 g kg−1), Vertisol (12.1–17.0 g kg−1,mean 15.3 g kg−1) and Alfisol (11.2–15.0 g kg−1, mean 13.7 g kg−1)(Fig. 1). Application of inorganic fertilizers alone or in combinationwith organic manure significantly increased the TOC content in COMFover control. Averaged over the sites, the increases in TOC contents ofCOMF over control were 12, 22, 18 and 24% under 100% NPK, NPK+ FYM, NPK + CR and NPK + GM, respectively. Irrespective of soiltypes, NPK + GM recorded the highest TOC followed by NPK + FYMand NPK + CR, except for Alfisol, where NPK + FYM recorded thehighest TOC in the COMF.

3.2. C stability in colloidal organo-mineral fraction (COMF)

Averaged over the treatments, the C-stability in COMF was thehighest in Vertisol, followed by Mollisol, Inceptisol and Alfisol (Fig. 2).Irrespective of soil types, values of C-stability of COMF in the treatmentscombining inorganic fertilizers and organics (NPK + FYM, NPK + CRand NPK+ GM) were significantly higher than the treatment receivingno fertilizer or manure (i.e. control) or that receiving only inorganic fer-tilizers (i.e. 100% NPK). Partial substitution of fertilizers by organics re-sulted in 46 to 294% (mean 139%) and 27 to 221% (mean 111%)increases in C-stability of COMF over control and 100% NPK, respec-tively. Among the treatments receiving organics along with fertilizers,C-stability of COMF in Inceptisol, Vertisol and Mollisol were in theorder of NPK + FYM ≥ NPK + GM N NPK + CR, whereas in Alfisol, theorder was NPK+ GM ≥ NPK+ CR ≥ NPK + FYM. The mean magnitudeof increases over the 100% NPK being 70, 32 and 72% in Inceptisol, 138,49 and 142% in Vertisol, 221, 50 and 188% inMollisol and 27, 28 and 51%in Alfisol with NPK+ FYM, NPK+ CR, and NPK+GM respectively. Thespecific stability, i.e. the C-stability per unit of TOC content of COMFwas

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TO

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Control 100%NPK NPK+FYM NPK+Straw NPK+GM

Fig. 1. Total organic carbon content of colloidal organo-mineral fraction (COMF) indifferent soils under varying nutrient supply options. Column followed by a differentletter in the same soil order are significantly different at p ≤ 0.05 by Duncan's multiplerange test. Error bars indicate standard error of mean (SEM).

the highest in Vertisol (38%) followed by Alfisol (26%), Inceptisol (22%)and Mollisol (19%). Irrespective of soil types, the highest specific stabil-ity was recorded with NPK+GM and NPK+ FYM treatments followedbyNPK+CR treatment, except in Alfisol, where, theNPK+CR andNPK+ GM treatments recorded higher stability than the NPK + FYM treat-ment. The specific stability was always higher with 50%NPK+organicsover the control as well as 100%NPK fertilized soils withmean values of61 and 58%, respectively.

3.3. Amorphous Fe, Al, Si oxide (Fe-Al-Si-ox) in colloidal organo-mineralfraction (COMF)

Averaged over the treatments, the respective contents of amorphousFe-oxide (Fe-ox), Al-oxide (Al-ox) and Si-oxide (Si-ox) in COMF were14.8, 5.55 and 0.56 g kg−1 in Inceptisol; 4.96, 5.63 and 0.55 g kg−1 inVertisol; 16.7, 6.24 and 0.56 g kg−1 in Mollisol; and 15, 6.30 and0.55 g kg−1 in Alfisol (Table 6). Application of 50% NPK along with or-ganics significantly increased the amount of Fe-ox in each of the soilsover 100% NPK and control. Irrespective of soil types, Fe-ox contentwas the highest under NPK + GM and lowest under control. Substitu-tion of 50% of recommended N through GM also significantly increasedthe Al-oxide content over control in Inceptisol and Vertisol; and overcontrol as well as 100% NPK in Mollisol and Alfisol. In Inceptisol andVertisol, all the treatments receiving inorganic fertilizers alone or incombination with organics significantly increased the Al-ox contentover unfertilized-control. In Inceptisol, Si-ox content under NPK + CRtreatment was statistically at par with that under control, whereas, inVertisol, Si-ox content was significantly higher with application of 50%of recommended NPK along with organics over the control. However,among the three oxides, Si-ox content did not vary significantly in 50%NPK + organics treatments. In Mollisol, Si-ox content was lowest withNPK + FYM treatment. In Alfisol, Si-ox content was relatively higherunder the control treatment than the NPK+ CR treatment. On an aver-age, the relative increases with NPK + GM, NPK+ CR, NPK + FYM and100% NPK treatments over unfertilized-control were 30, 27, 22 and 12%for Fe-ox; 30, 28, 22 and 13% for Al-ox; and 13, 13, 3 and− 2% for Si-ox,respectively.

3.4. Clay mineralogy of soils

Clays in Inceptisol of Ludhianaweremainly dominated by IRM (illiteand illite rich minerals) (60–73%, mean 69%), and also contained KRM(kaolinite and kaolinite rich minerals) (18–29%, mean 21%), and SRM(smectite and smectite rich minerals) (9–11%, mean 10.5%) (Figs. 3, 4and Table 7). In Vertisol, soil clays contained SRM (45–53%, mean49%), IRM (17–43%, mean 35%), along with some KRM (10–30%, mean

Table 6Effect of long-term fertilization and manuring on the amount of amorphous Fe, Al and Si oxides (g kg−1) in colloidal organo-mineral fraction (COMF).

Am-Fe-ox Am-Al-ox Am-Si-ox

Inceptisol Vertisol Mollisol Alfisol Inceptisol Vertisol Mollisol Alfisol Inceptisol Vertisol Mollisol Alfisol

Control 12.9c 3.27d 14.6c 13.1d 4.48b 4.20b 5.28c 6.05b 0.46b 0.51c 0.51b 0.62ab100%NPK 13.8bc 4.73c 16.0bc 14.8c 5.48a 5.90a 6.04bc 5.11b 0.62a 0.53bc 0.51b 0.42cNPK + FYM 15.8a 5.64ab 16.6ab 15.7bc 6.18a 5.85a 6.20b 6.21b 0.66a 0.58ab 0.47b 0.45cNPK + CR 16.3a 5.24bc 17.7ab 16.7ab 6.11a 6.25a 6.57ab 6.63ab 0.47b 0.54abc 0.69a 0.68aNPK + GM 15.1ab 5.91a 18.4a 17.6a 5.49a 5.98a 7.10a 7.52a 0.57a 0.59a 0.65a 0.58bMean 14.8 4.96 16.7 15.6 5.55 5.63 6.24 6.30 0.56 0.55 0.56 0.55

Values are means of three replicates across locations for each soil order. Am-Fe-ox, Am-Al-ox, and Am-Si-ox were amorphous Fe-oxides, amorphous Al-oxides, and amorphous Si-oxides,respectively. Means followed by a different letter are significantly different at p ≤ 0.05 by Duncan's multiple range test for each soil order.

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16%). Clays in Mollisol contained more or less similar amounts of IRM(31–45%, mean 40%) and KRM (32–45%, mean 38%), along with rela-tively lower amounts of SRM (17–24%, mean 21%). In Alfisol clay parti-cles contained 33–50% (mean 44%) IRM and 50–67% (mean 56%) KRM,while the amounts of minerals under SRM category were negligible.

3.5. Crystallite size of illite

Average size of illite particles varied from 15.9 to 37.9, 14.2 to 159,5.2 to 266, and 3.5 to 114 nm in Inceptisol, Verisol, Mollisol, and Alfisol,respectively (Table 8). Averaged over soil types, the mean crystallitesizes of illite particles were 124, 48.8, 32.5, 20.4 and 10.3 nmunder con-trol, 100%NPK,NPK+FYM,NPK+CRandNPK+GM, respectively. Thehighest crystallite size of illite was found under control in all the soilsexcept Alfisol, where the highest crystallite size of illite was recordedunder 100% NPK. On an average, NPK + GM showed lower crystallite

Control

100% NPK

NPK+FYM

NPK+CR

NPK+GM

4 9 14 19

Inte

nsity

Diffrrac�on angle (°2θ)

Incep�sol

Control

100% NPK

NPK+FYM

NPK+CR

NPK+GM

4 9 14 19

Inte

nsity

Diffrrac�on angle (°2θ)

Mollisol

Fig. 3. Simple XRD profiles of Mg-gly clay of differe

size of illite as compared to the other treatments. Considering all foursoil orders, the crystallite size of illite and C-stability in COMFwere neg-atively correlated (−0.46, P b 0.01).

3.6. Relationships of C-stability with contents of amorphous Fe, Al and Si ox-ides of colloidal organo-mineral fraction (COMF)

Positive and significant correlation was obtained in each of the sitesbetween the C-stability and amorphous Fe, Al and Si oxides of COMF(Table 9). Among the oxides, amorphous Fe-ox showed the highest cor-relation with C-stability in all soil types. The correlation between amor-phous Al-ox and C-stability was significant in each of the sites exceptIncpetisol, where the correlation was positive but not significant. Thecorrelation between amorphous Si-ox and C-stability was positive butnot significant except in Vertisol, where C-stability was significantlyand positively correlated with all the amorphous oxides.

Control

100% NPK

NPK+FYM

NPK+CR

NPK+GM

4 9 14 19

Inte

nsity

Diffrrac�on angle (°2θ)

Ver�sol

Control

100% NPK

NPK+FYM

NPK+CR

NPK+GM

4 9 14 19

Inte

nsity

Diffrrac�on angle (°2θ)

Alfisol

nt soils under varying nutrient supply options.

Fig. 4. Decomposed XRD patterns of Mg-gly clay of different soils under control and NPK + GM.

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asetal./Science

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ent684(2019)

682–693

Fig.

4(con

tinue

d).

689R. Das et al. / Science of the Total Environment 684 (2019) 682–693

Table 7Approximate quantities of minerals (%) in the clay fractions of different soils under differ-ent long-term fertilization and manuring.

Smectite andsmectite richminerals (SRM)

Illite and Illiterich minerals(IRM)

Kaolonite andkaolinite richminerals (KRM)

InceptisolControl 11 71 18100%NPK 11 60 29NPK + FYM 9 73 19NPK + CR 11 69 20NPK + GM 11 71 18

VertisolControl 53 17 30100%NPK 50 37 13NPK + FYM 47 43 10NPK + CR 49 39 12NPK + GM 45 41 14

MollisolControl 24 31 45100%NPK 21 45 34NPK + FYM 20 42 38NPK + CR 17 40 43NPK + GM 24 44 32

AlfisolControl – 50 50100%NPK – 47 53NPK + FYM – 47 53NPK + CR – 33 67NPK + GM – 42 58

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4. Discussions

4.1. Content and stability of C in colloidal organo-mineral fraction (COMF)under different nutrient management options

The content of TOC in COMF as well as its stability is very importantfor long-term C sequestration. Thirty-one years of fertilization with orwithout manuring generally increased the TOC content of COMF overcontrol in all the soils (Fig. 1). Long-term application of fertilizers

Table 8Effect of long-term fertilization andmanuring on integral breadth and the average crystal-lite size of illite.

Samplesites

Treatments °2θ d(001)nm

Integralbreadth

Crystallitesize(nm)

Inceptisol Control 8.80 1.005 0.410 37.9100%NPK 8.80 1.005 0.597 20.1NPK +FYM

8.75 1.011 0.703 15.9

NPK + CR 8.80 1.005 0.488 27.7NPK + GM 8.77 1.009 0.638 18.1

Vertisol Control 8.88 0.995 0.246 159100%NPK 8.74 1.012 0.756 14.2NPK +FYM

8.71 1.016 0.579 21.0

NPK + CR 8.76 1.009 0.735 14.8NPK + GM 8.70 1.016 0.753 14.5

Mollisol Control 8.81 1.004 0.229 266100%NPK 8.81 1.004 0.369 46.9NPK +FYM

8.78 1.007 0.342 56.9

NPK + CR 8.85 1.000 0.434 34.6NPK + GM 8.84 1.001 1.732 5.20

Alfisol Control 8.74 1.012 0.444 33.2100%NPK 8.74 1.012 0.270 114NPK +FYM

8.74 1.011 0.416 36.2

NPK + CR 8.68 1.019 0.480 4.50NPK + GM 8.70 1.017 2.545 3.50

alone or fertilizers with organics maintained much higher biomassyields (Annual report-AICRP on IFS, 2014–15) over control resulting ingreater root biomass C inputs and this could be one of the reasons forgreater accumulation of TOC in COMF in soils. However, the extent of in-creasewas greater in integrated nutrientmanagement than the sole ap-plication of fertilizers. This could be attributed to the additional C inputsfrom manures (FYM) and crop residues and green manure (GM)(Bandyopadhyay et al., 2010; Das et al., 2017; Ghosh et al., 2018a). Sig-nificant increases in C content of bulk soils by application of FYM, strawand GM were already reported by other researchers (Bandyopadhyayet al., 2010; Ghosh et al., 2012; Yadav et al., 2017). With the existingknowledge, our study added new information on OC accumulation inthe relatively stable clay-humus C pool due to integrated nutrient man-agement in soils of varying mineralogical make-up. Such effect wasmost prominent in treatments with GM or FYM in Inceptisol, Vertisoland Mollisol, and with GM or CR in Alfisol.

The accumulation as well as stability of carbon in COMF varied sig-nificantly in soil under 31 years of fertilization and manuring. Thoughthe application of recommended dose of NPK fertilizers significantly in-creased the C concentration in COMF extracted from Inceptisol andMollisol, but no significant difference in C stability was noticed with100% NPK when compared with unfertilized-control in all the four soilorders. Addition of GM or FYM in Inceptisol, Vertisol and Mollisol, andGM or CR in Alfisol along with NPK fertilizers resulted a sharp rise inC-stability of COMF over either control or 100% NPK treatment (Fig. 2).C-stability was significantly higher in treatments combining inorganicfertilizers and organics than 100% NPK which can be explained by thefact that the C-compounds originating from the added organics tendto form more stable organo-mineral complex than the C-compoundsemerging from root biomass. Mandal et al. (2007) also reported thatthe application of FYM or compost can stabilize C in soil to the tune of1.6 times more compared to its absence. Added organics facilitated theassociation of OM with amorphous oxides in soils and increase the sta-bility of C (Wang et al., 2019). Addition of organic along with inorganicfertilizers for years together also changed the biochemical compositionof SOM by increasing the amine substances and decreasing the polysac-charides (Wang et al., 2019) which increase their cation exchange ca-pacity and eventually provide more binding sites and higher SOCstabilization in soil (Souza et al., 2017). Similar finding was also re-ported by earlier researchers (Yadav et al., 2017; Ghosh et al., 2018a;Wen et al., 2019). Among the organics, FYMandGMweremore efficientthan CR in improving the C-stability of COMF, except for Alfisol, whereGMandCR being at par imparted higher stability. Such enhanced effectsof FYM and GM on C-stability could be due to their narrower C:N ratio(20–30:1) than straw (80:1) as the former two are likely to containmore easily decomposable materials compared to the later one(Dannehl et al., 2017). Narrow C:N ratio is associated with higher avail-ability of N per unit of C, which increases the C use efficiency by soil mi-crobes and eventually larger amount of substrate C could be added toand retained in soils (Hessen et al., 2004; Dannehl et al., 2017). WiderC:N ratio of wheat straw might be responsible for immobilization of Nand lower N availability, leading tomore evolution of CO2 and lesser in-corporation of substrate-C to the soil (Manzoni et al., 2012; Yadav et al.,2017). For this reason C-stability under NPK + CR was mostly lower ascompared to either FYM or GM. Higher C retention in soil with the

Table 9Pearson correlation coefficients (r) between C stability and amorphous Fe, Al and Si oxidesof colloidal organo-mineral fraction in different soils.

Am-Fe-ox Am-Al-ox Am-Si-ox

Inceptisol 0.659⁎⁎ 0.385 0.508Vertisol 0.868⁎⁎ 0.596⁎ 0.756⁎⁎

Mollisol 0.666⁎⁎ 0.578⁎ 0.327Alfisol 0.885⁎⁎ 0.695⁎⁎ 0.147

⁎ Significant at P = 0.05.⁎⁎ Significant at P = 0.01.

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application of GM and FYMover cereal strawwas reported by some ear-lier researchers (Singh et al., 1991; Mahmoodabadi and Heydarpour,2014; Dannehl et al., 2017). The higher stabilization of organic C in silt+ clay fractions under 100% NPK + GM (Bandyopadhyay et al., 2010)and under 100% NPK, 150% NPK and 100% NPK + FYM (Sukumaranet al., 2016) was also reported.

4.2. Amorphous Fe-Al-Si-oxides and C-stability

The objective of our studywas to assess the stability ofmineral asso-ciated C which was performed by desorption of humus from colloidalorgano-mineral fraction (COMF). It was presumed that humus being in-herently recalcitrant (Schneider et al., 2010), having stronger linkages(chemical bonding) with clay minerals (Kleber et al., 2005; Mikuttaet al., 2005; von Lützow et al., 2007) also could impart higher biologicalstability. Recently, the traditional approach of classical “humificationmodel” was criticized and it was reinforced that SOM is a continuumof progressively decomposed organic compounds (Lehman andKleber, 2015). Even we agree with the theory of Lehman and Kleber(2015), humus in spite of being a progressively decomposed compoundmight form stronger linkages with clay, imparting higher biological sta-bility. Amorphous and poorly crystalline minerals have high chelationcapacity and form covalent linkages with organic matter through theirfunctional groups (Lutzow et al., 2006; Kleber et al., 2007; Schneideret al., 2010; Wang et al., 2019). Such interaction protects the organic Cfrom oxidative attack in soils (Mikutta et al., 2005). Application of or-ganics along with mineral fertilizers significantly increased the contentof amorphous Fe-oxides in COMF. Oxidation of organic matter createsreduction microsites in soil where it solubilizes Fe through reductionand Fe2+ concentration increases (Lindsay, 1991). Another mechanismof increased Fe2+ concentration is through the solubilisation of Fe by thereleased reducing agent and different organic acids from the increasedroot biomass as well as the production of siderophores by increasedmi-crobial activity within the rhizosphere (Lindsay, 1991). Wang et al.(2019) observed an increase in non-crystalline Fe fractions in soil after37 years of manuring with or without chemical fertilizers. The signifi-cant positive relationship between the amorphous Fe-oxides and C sta-bility in COMF in all soil orders can be explained by the increasedbinding sites with increase in poorly crystalline Fe minerals (Wanget al., 2019; Wen et al., 2018, 2019).

Not only the amorphous Fe-oxide but also amorphous Al-oxideplayed an important role for C stabilization in COMF because amor-phous Al-oxides was also reported to protect SOC in red soil by alteringthe binding properties of organic ligands (Wen et al., 2014). Therefore,there was a concomitant increase in clay-humus stability in NPK+ GM treatment due to higher extractable Fe, Al and Si. Our result isin conformity with other researchers (Torn et al., 1997; Mikutta et al.,2005; Yu et al., 2012; Yadav et al., 2017) who also reported that thema-nure application promotes the formation of non-crystalline mineralsand improves the binding capacity of soil minerals with OM. Contrarily,sole application of inorganic NPK fertilizes did not influence the con-tents of amorphous Fe, Al and Si-oxides as compared to control andtherefore, caused a lower C-stability in COMF.

Content of Fe-oxide showed better correlation with clay-humus Cstability than other oxides at each of the sites indicating that Fe-oxidesare the most important among different amorphous oxides in influenc-ing C-stability in COMF. This could be the reason behind significant pos-itive correlations between contents of hydrous Fe-oxides and SOC asdocumented by earlier studies (Kleber et al., 2005; Wiseman andPuttmann, 2006; Spielvogel et al., 2008). Amorphous Al-oxides alsoshowed significant and positive correlation with stability of clay-humus C, but was not as important as Fe-oxides. Amount of amorphousSi-oxides failed to show any significant correlationwith stability of clay-humus C, except in Vertisol where the proportion of Si-oxides amongthe amorphousminerals was comparatively higher than the other sites.

4.3. Clay mineralogy and C-stability of colloidal organo-mineral fraction(COMF)

The stability of C in COMF was the highest in Vertisol and lowest inAlfisol. The presence of greater amount of SRM (49%) in Vertisol couldbe the reason behind this, as smectites have a strong affinity for soilhumus (Figs. 3, 4 and Table 7). Smectite-containing soils and sedimentshave often been found to contain elevated amount of organic matter(Ransom et al., 1998; Kennedy et al., 2002). Because smectites havelarge surface area, they can strongly bind organic molecules via –OHgroups present on surfaces of smectite particles (Chotzen et al., 2016).Smectites also have a huge internal surface area, so it can retain organicmatter in the interlayer spaces by intercalation (Theng, 1979; Ovesenet al., 2011). Besides smectite, IRM is also important in maintaining C-stability of COMF because of relatively higher surface area (than KRM)and higher amphoteric properties in the broken edges that tend to re-tain more organic C (Six et al., 2002; Wiseman and Puttmann, 2006).The Mollisol also contained 20% SRM and 50% IRM and recorded secondhighest stability of clay-humus C. In Inceptisol, 67% of clay mineralswere composed of IRM which also might have contributed to the en-hanced stability in COMF. Thus the adsorption of humus on 2:1 clayminerals occursmainly on the large basal surfaces; electrostatic interac-tions and interactions such as hydrogen bonding and cation bridging ac-count for the formation of organic coatings on the clay particles (Suttonand Sposito, 2006; Wang et al., 2012). Negligible amount of SRM anddominance of KRM inAlfisol led to lowC-stability in COMF. Total surfacearea of kaolinite is much lower than the expanding 2:1minerals, and itsinterlayers are not accessible by foreignmolecules. For these reasons, C-stability of COMF inAlfisolwas relatively low as compared to other soils.Ahmed et al. (2002) observed that clays belonging to Vertisol form thestrongest clay-humic acid bond due to the presence of SRM mineralsand weakest clay-humic acid bond in Alfisols due to kaolinitic minerals.In kaolinitic Alfisol, clay-humus linkage is through hydrogen or othermonovalent cation bridges which is generally very weak in nature caus-ing lower C-stability in COMF (Ahmed et al., 2002). Saidy et al. (2013)also found that the type of phyllosilicate clays affect the sorption andsubsequent accumulation of organic C in soil and they reported thaton mass basis sorption of OC was highest in smectite followed by illiteand kaolinite.

Long-term application of fertilizers alone or in combination with or-ganics mostly led to a decline in the crystallite size of illite as comparedto unfertilized-control (Table 8). Considering the four soil orders to-gether, C-stability in COMF showed negative correlation (r = −0.46, Pb 0.01) with average crystallite size of illite, implying that lower crystal-lite size of illite leads to greater stability of clay-humus C. Smaller sizedillite minerals provide higher surface area for more extensive interac-tions with humus, resulting in higher stability. The relationship be-tween crystallite size and C-stability at each individual site was notmuch prominent due to small sample size. In general, concentrationsof organic matter in soils and sediments correlate well with those offine-grained minerals (Baldock et al., 2004). Increase in poorly crystal-line minerals due to the manure application was also reported earlierby many researchers (Yu et al., 2012; Wang et al., 2019; Wen et al.,2019). This might be the reason of higher stability of clay-humus CunderNPK+FYM,NPK+CRandNPK+GMtreatmentswhere the par-ticles of illite were smaller.

5. Conclusions

Long-term application of fertilizers and organic sources namely,farmyard manure (FYM), crop residue (CR) and green manure (GM)significantly altered the C stability in colloidal organo mineral fraction(COMF), changed the contents of amorphous Fe-Al-Si-oxides and de-creased the crystallite size of illite in all the soil orders. Substitution of50% N-fertilizer by either GM or FYM in Inceptisol, Vertisol and Mollisoland GM or CR in Alfisol emerged as the best management practice for

692 R. Das et al. / Science of the Total Environment 684 (2019) 682–693

higher stabilization of C for long-term C sequestration. Among the foursoils, Vertisol showed the highest stability of C in COMF, followed byMollisol, Inceptisol and Alfisol. The amorphous-Fe-Al and Si-oxideshad a positive influence on C stability in different soil orders. Out ofthese three oxides, amorphous Fe-oxide appeared to be themost impor-tant for C sequestration. Crystallite size of illite contributed negatively tothe C stability in COMF across the soil orders. The present study gives aninsight into the COMF as affected by different nutrientmanagement op-tions in four soils of widely varying mineralogical make-up, therebyexpanding the possibilities for further exploring the role of clay sizedminerals for effective storage of C in different soil orders across theglobe.

Acknowledgements

The authors are highly indebted to the Deputy Director General(DDG), Natural Resource Management (NRM), Indian Council of Agri-cultural Research (ICAR) for providing necessary permission to collectsoil samples from long-term experiments of ICAR-AICRP on IntegratedFarming Systems (IFS). The authors are also grateful to the Director,ICAR-Indian Agricultural Research Institute (IARI) and Head, Divisionof Soil Science and Agricultural Chemistry, ICAR-IARI for providing nec-essary facilities to carry out the work. Authors are grateful to the anon-ymous reviewers for their valuable suggestions to improve themanuscript.

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