Organic Amendments and Elemental Sulfur Stimulate Microbial Biomass and Sulfur Oxidation in Alkaline Subtropical Soils

Abstract

Sulfur deficiency arising due to intensive cultivation, use of sulfur free fertilizers and reduction in atmospheric sulfur depositions has become a major issue limiting crop production in many parts of the world. Elemental sulfur could be a good source of available S, but its slow oxidation is a problem for its efficient use as a sulfur fertilizer. Main objective of the study was to assess the effect of organic amendments (OA) and elemental sulfur (ES) on microbial activities, sulfur oxidation and availability in soil. A laboratory incubation experiment was carried out for a 56 days period using two sulfur deficient alkaline soils. Organic amendments (OA), i.e., farmyard manure (FYM), poultry litter (PL) and sugarcane filter cake (SF), were applied (1% w/w) with or without elemental sulfur (ES) at 50 mg kg⁻¹. Application of ES alone or in combination with OA significantly increasedCO₂-C evolution, microbial biomass, and enzyme activities in the soils, except dehydrogenase activity (DHA) which was not affected by ES application. Combined application of OA and ES had a more pronounced effect on microbial parameters compared to ES or OA applied alone. Ratios of dehydrogenase activity-to-microbial biomass C and arylsulfatase activity-to-microbial biomass C were high in ES+FYM and ES+SF treatments, respectively. Elemental sulfur got sufficiently oxidized resulting in significant improvement in plant available S. Relatively more ES was distributed into C-bonded-S than ester bonded-S. Increase in sulfur availability in ES+OA amended soils was the combined function of sulfur oxidation and mineralization processes through improved microbial activity.

1. Introduction

Sulfur (S) is an essential macronutrient required for normal growth and metabolic functions in plants. It is an integral part of many biological and structural compounds of living entities and is directly involved in protein synthesis as a constituent of S containing amino acids like methionine and cysteine [1]. Most of the grain crops absorb sulfur in amounts similar to those of P while the sulfur requirements of oilseed crops are particularly high [2]. Intensive cultivation, use of sulfur-free fertilizers and reduction in atmospheric sulfur depositions are the major causes of sulfur deficiency in agricultural soils [3]. Therefore, use of sulfur fertilizers has become imperative for successful crop production in many parts of the world including South Asia [4]. Most of the chemical fertilizers contain S either as sulfate ions or as elemental sulfur. Sulfate form is readily available to crop plants but being mobile in nature is susceptible to leaching losses particularly in alkaline and sandy soils which inherently have low sulfate retention [5]. Under these conditions, elemental S attains importance as a fertilizer resource due to its high S content, slow S releasing property and low leaching. However, S contained in elemental sulfur is not directly available to plants and requires oxidation to sulfates for absorption by plant roots [6]. Thus, knowledge of sulfur oxidation and its distribution into various forms in soil is important for efficient use of elemental sulfur as a fertilizer.

The microbial oxidation of reduced sulfur compounds (e.g., elemental sulfur) is influenced by soil physico-chemical properties, environmental conditions and the activity of S oxidizing microorganisms in soil [6,7]. Historically, chemoautotrophic oxidizers such as Thiobacillus spp. are considered primarily responsible for microbial oxidation of reduced sulfur compounds in soil [8,9,10,11]. Several groups of heterotrophic microorganisms (bacteria, fungi, actinomycetes) can also oxidize reduced sulfur compounds and prefer neutral to alkaline soil conditions for their optimal activity [12,13]. Since organic C acts as a source of energy for the activity of heterotrophic microorganisms, therefore use of organic amendments have been reported to have beneficial effects on S oxidation in soil [14,15,16].

In nature both organic and inorganic forms of sulfur go through a dynamic network of biogeochemical processes catalyzed by the activities of soil microorganisms [17,18]. Biological mineralization pathway involves oxidation of organic S compounds by soil microorganisms, while the biochemical mineralization comprises hydrolysis of ester sulfates through sulfatases to release S mainly as sulfate for microbial and plant uptake [19,20,21]. The activity and size of soil microbial biomass thus may play a key role in S mineralization and its biogeochemical cycling [22,23,24]. Some previous studies reported a negative correlation between elemental sulfur application and microbial activities in soils. Significant decline in microbial biomass C and the activities of dehydrogenase and arylsulfatase enzymes in soil as the result of repeated application of elemental sulfur [25,26]. However, the reduction in microbial biomass and enzyme activities may result from the deficiency of carbon substrates with applied amendments, particularly in organic matter deficient soils [27]. On the other hand, there are numerous reports on the stimulating effects of organic amendments on soil microbial biomass and activities responsible for nutrients release in soils [28,29]. Manure application as an organic amendment may lead to sustainable and environmentally sound agriculture by stimulating new ideas and directions in animal manure [30]. There is also evidence showing that S oxidation in soil increased linearly with soil microbial biomass [30,31].

Alkaline calcareous soils with high base saturation percentage dominate in arid/semi-arid subtropical regions throughout the world and account for almost 60% of the world’s food production [32]. These soils are multi-nutrient deficient and require appropriate nutrient management strategies to address the existing gap between current and potential achievable yield. In addition to primary macronutrients (NPK), deficiency of sulfur has been found to be responsible for poor crop productivity in these soils [33]. Knowledge of sulfur oxidation and its distribution in the alkaline subtropical soils may play an important role for efficient use of elemental sulfur as a fertilizer to attain optimum crop yields [34]. So far there have been minimal studies regarding interactive effects of organic amendments and elemental sulfur on sulfur availability in alkaline subtropical soils. Hence, the present study was conducted with the objectives to, (i) evaluate the effect of organic amendments and elemental sulfur on microbial biomass and activities of enzymes involved in S oxidation in soil, and (ii) elucidate their effect on S oxidation and distribution in soil.

2. Materials and Methods

2.1. Soils and Organic Amendments

Two sulfur deficient soils belonging to the series Missa (Haplic Calcisols, Inceptisol, 33°22′09.1′′ N 72°29′08.5′′ E) and Kahuta (Calcic Luvisols, Alfisol, 33°04′07.1′′ N 73°00′30.7′′ E) were collected from the agricultural fields located in northern Punjab, Pakistan. Composite soil samples of 50 kg each were taken at the plough layer (0–15 cm depth), brought to the laboratory, hand-picked to remove stones, pebbles and plant residues, sieved (<2 mm) in moist condition, homogenized and kept frozen until the incubation started. Subsamples comprising of 500 g of each soil were air-dried, ground, passed through a 2 mm sieve and analyzed for physical and chemical properties such as moisture content, particle size distribution, pH, EC, CaCO₃, CEC, total organic C, total N, Olsen P, extractable K, extractable SO₄²⁻ and AB-DTPA extractable micronutrients (Fe, Cu, Mn, Zn), arylsulfatase activity (ASA) and dehydrogenase activity (DHA). Among the organic amendments used in the study, farmyard manure (FYM) was collected from the cattle manure heap in a farmer’s field at Taxila, poultry litter (PL) was taken from the Poultry Research Institute, Rawalpindi and the sugarcane filter cake (SF) was taken from the industrial dump of the Koh-e-Noor Sugar Mills, Jauharabad, district Khushab. The organic amendments were air dried, ground, homogenized and analyzed for their chemical composition.

2.2. Experiment Details

The frozen soil samples were equilibrated at room temperature, adjusted moisture content to 50% of their WHCs and pre-incubated for 7 days at 25 °C prior to treatment application. Each soil was added to incubation jars at the rate of 600 g jar⁻¹ and the following treatments were applied: (T1) control, (T2) elemental sulfur (ES), (T3) elemental sulfur + farmyard manure (FYM), (T4) elemental sulfur + poultry litter (PL), and (T5) elemental sulfur to sugarcane filter cake (SF). Each organic source was applied at 1% w/w and the elemental sulfur was added at 50 mg kg⁻¹ soil [35]. All the treatments were replicated three times following the completely randomized design (CRD). The lids of the incubation jars were closed air-tight and placed in darkness in a temperature-controlled incubator at 25 °C for the period of 56 days. The CO₂ released was captured in 1 M NaOH solution and estimated by back-titration with 1 M HCl after 1, 2, 3, 5, 7, 10 and 14 days and thereafter weekly. Fifty gram of soil sample was collected from each jar at 0, 14, 28, 42 and 56 days of incubation and analyzed for dissolved organic carbon (DOC), microbial biomass C (MBC), microbial biomass S (MBS), arylsulfatase activity (ASA), dehydrogenase activity (DHA) and available sulfate. Soil samples collected at 0, 28 and 56 days were additionally analyzed for sulfur fractions.

2.3. Analytical Methods

Particle size analyses were performed using a standard hydrometer after pretreatment of soil samples with 1% sodium hexametaphosphate and 250 mL of DI water. Soil pH and electrical conductivity were estimated in a saturated soil paste using calibrated HANNA-212 pH meter and HANNA HI-8033 conductivity meter, respectively. Calcium carbonate was determined by the acid neutralization method as described by Rayan et al. [36]. Cation exchange capacity was determined by saturating the soil with Na⁺, followed by extraction of the saturating cation with Mg²⁺ and measuring Na⁺ flame photometrically [37]. Total organic C was measured after dichromate digestion and total N was estimated by the Kjeldahl method [38]. The 0.5 M NaHCO₃ (pH 8.5) extractable P was determined colorimetrically [39]. Soil microbial biomass C (MBC) and microbial biomass S (MBS) were measured by chloroform fumigation-extraction methods [40]. Fumigated and non-fumigated portions of 10 g soil were extracted with 40 mL 0.5 M K₂SO₄ for MBC and 50 mM NH₄NO₃ for MBS by horizontal shaking at 200 rev min⁻¹ for 30 min and filtered. Microbial biomass C was calculated by measuring the organic C in the extracts as CO₂ by infrared absorption after combustion at 760 °C using a Shimadzu automatic TOC analyzer (Tokyo, Japan) and calculated by using the following formula: Microbial biomass C = E_C/k_EC, Where E_C = (organic C extracted from the fumigated soil)—(organic C extracted from non-fumigated soil) and k_EC = 0.45 [41]. Microbial biomass S was calculated by using the following formula: Microbial biomass S = E_S/k_ES, whereas E_S = (total S extracted from fumigated soil)—(total S extracted from non-fumigated soil) and k_ES = 0.35 [42].

Arylsulfatase activity was determined by the procedure given by Tabatabai and Bremner [43]. One gram of soil was incubated with 4 mL of 0.5 M acetate buffer (pH 5.8), 0.25 mL toluene and 1 mL of 50 mM potassium p-nitrophenyl sulphate solution at 37 °C for 1 h. One ml of 0.5 M CaCl₂ and 4 mL of 0.5 M NaOH were added and filtered. The yellow color intensity of p-nitrophenol was observed by reading the absorbance at 400 nm by spectrophotometer (Cecil-2000). Dehydrogenase activity was determined using the procedure of Casida et al. [44]. Twenty grams of soil was mixed with 0.2 g of CaCO_3. Six -gram of this mixture was placed in each of the three test tubes. After adding 1 mL of 3% aqueous solution of triphenyl tetrazolium chloride (TTC) and 2.5 mL of distilled water, samples were incubated at 37 °C for 24 h. TRIS buffer was used for dehydrogenase activity. After adding 10 mL of methanol, samples were shaken and filtered. The red color intensity was measured at a wavelength of 485 nm. Methanol was used as a blank. The quantity of triphenyl formazan (TPF) produced was calculated by using TPF standards calibration graph as reference.

Sequential extraction procedure proposed by Morche [45] was followed to extract inorganic sulfur fractions in soils. To 1.0 g soil, 10 mL of deionized water (1:10 w/v) was added, contents were shaken for 1 h on the reciprocal shaker and centrifuged at 10,000× g rpm for 10 min. The clear supernatant was collected for measuring water-soluble plant-available sulfur (S_w). The residual soil in the centrifuge tube was added with 0.032 M NaH₂PO₄ solution (1:10 w/v) and shaken the contents for 1 h on the reciprocal shaker and centrifugation at 10,000× g rpm. The aliquot collected was used to determine weakly adsorbed sulfur (S_ads). Twenty ml of 1 M HCl (1:20 w/v) was added to residual soil, contents were shaken for 1 h and centrifuged at 10,000× g rpm for 10 min, and the supernatant was collected to determine occluded S (S_ocl). Sulfur contents in the sequential extracts were determined by measuring the turbidity [46]. Organic sulfur fractions in soil including ester sulfate and C-bonded S were analyzed following the procedure proposed by Johnson and Nishita [47]. HI-reducible sulfur was determined by treating the soil with a mixture of hydriodic acid, hypophosphoric acid, and formic acid. Resulting H₂S was absorbed in NaOAc-Zn (OAc)₂ solution, and the sulfide was measured as the methylene blue complex with a spectrophotometer at 670 nm. Ester bonded S was calculated from the differences between HI-reducible S and inorganic S. Carbon bonded S was calculated by subtracting HI reducible S from total S contents of the soil. Available SO₄²⁻ in the soil was determined by extracting 5 g of air-dried soil with 25 mL of 15% CaCl₂. 2H₂O solution and determining the SO₄²⁻ contents in extracts by turbidimetric method [46].

The oxidation of elemental S was calculated by the following Equation [48]:

$$\mathrm{S}\mathrm{Oxidized}(\%)=\frac{(\mathrm{Available}{{\mathrm{SO}}_4}^{2-}\mathrm{in}\mathrm{ES}+\mathrm{OA}\mathrm{treated}\mathrm{soil}-\mathrm{Available}{{\mathrm{SO}}_4}^{2-}\mathrm{in}\mathrm{OA}\mathrm{treated}\mathrm{soil})}{\mathrm{Amount}\mathrm{of}\mathrm{ES}\mathrm{added}\mathrm{to}\mathrm{soil}}\times 100$$

S oxidation by heterotrophic microorganisms was calculated by:

$$\mathrm{S}\mathrm{oxidized}(\%)=\frac{(\mathrm{S}\mathrm{oxidized}\mathrm{in}\mathrm{ES}+\mathrm{OA}\mathrm{treated}\mathrm{soil}-\mathrm{S}\mathrm{oxidized}\mathrm{in}\mathrm{ES}\mathrm{treated}\mathrm{soil})}{\mathrm{Amount}\mathrm{of}\mathrm{ES}\mathrm{added}\mathrm{to}\mathrm{soil}}\times 100$$

S oxidation by autotrophic microorganisms was calculated by:

ES oxidized (%) = Total ES oxidized (%) − ES oxidized by heterotrophs (%)

2.4. Statistical Analyses

The experimental data presented in tables are arithmetic means of three replicates and the significance of soil, treatments and sampling days were tested using three-way analysis of variance in which soil was factor 1, treatments factor 2 and sampling days were factor 3. Differences among the treatment means were determined by using the Tukey’s HSD test at 0.05% level of significance. All the statistical analyses were performed using Statistix 8.1 software.

3. Results

3.1. Characteristics of Soils and Organic Amendments

The Missa soil used in this experiment was silt loam and the Kahuta soil was sandy loam in texture (

Table 1. Characteristics of soils and organic amendments used in the study.

Soils	pH	EC dS m−1	CEC (cmol kg−1)	Ca CO3	TOC	WHC	Textural Class				Total N	Olsen P	NH4OAc K	Available SO42−	AB-DTPA
							Sand	Silt	Clay	Class					Zn	Fe	Cu	Mn
				%							(g kg−1)		(mg kg−1)
Missa	7.5	0.31	6.10	13.4	0.47	36	37.5	54	8.5	Silt loam	6.2	79	105	9.4	2.2	2.8	0.8	23.8
Kahuta	7.2	0.92	5.08	5.3	0.33	30	47.5	34	18.5	Sandy loam	6.7	84	76	7.7	2.7	1.4	0.7	26.2
Organic amendments			TOC (%)			Moisture %	TOC/TN	TOC/TP	TOC/TS	Total N		Total P	DOC	Total S %	(mg kg−1)
										(g kg−1)
Farmyard manure (FYM)			22.4			7.9	13.9	4.8	133		1.3	3.6	21.2	0.13	2.5	63.4	0.4	7.6
Poultry litter (PL)			31.8			41.2	11.1	2.5	138		2.9	12.7	24.7	0.23	18.5	5.6	0.7	1.5
Sugarcane filter cake (SF)			28.6			12.2	14.0	2.9	92.3		2.1	9.7	15.9	0.31	0.7	5.0	0.7	1.1

). Both the soils were non-saline, moderately alkaline and calcareous in nature with pH of 7.5 and 7.2, and the CaCO₃ contents of 13.4 and 5.3 percent, respectively. The cation exchange capacity of Missa soil was relatively higher than that of Kahuta soil. Both the soils were deficient in plant-available sulfur (<10 mg kg⁻¹ soil), and low in organic matter with total organic C contents less than 0.5 percent. The soils were also deficient in total N, available P and extractable K. AB-DTPA extractable Fe was deficient in both the soils whereas the extractable Mn, Cu and Zn were above the permissible range described by Soltanpour [49]. The three organic amendments i.e., FYM, PL, and SF differed in their chemical composition. Moisture content was higher in PL as compared to FYM and SF. Total organic carbon, total N, total phosphorus and total zinc contents were higher in PL while the total iron and total manganese contents were higher in FYM as compared to the other two amendments. Total S was high in SF, followed by PL and FYM. The ratio of total organic C to total S was lowest in SF, whereas the ratio of total organic C to total N, and total organic C to total P was lowest in the P.

He et al.studied the distribution of P forms in 23 poultry litter samples and investigated impacts of relatively less labile Po in poultry litter [50], which was isolated in the hydroxide and acid fractions and suggested that sequential fractionation coupled with phosphatase hydrolysis provides the tool to quantitatively monitor the transformations of these organic P species in various environments.

3.2. Soil Microbial Parameters

The interactive effects of elemental sulfur (ES) and organic amendments on soil respiration are presented in

Table 2. Main effects of soils, treatments, and their interaction on rate of CO₂-C evolution, ΣCO₂-C and ratio of ΣCO₂-C/MBC.

Main Effects	Rate of CO2-C Evolution (mg kg−1 soil day−1)	ΣCO2-C (mg kg−1 soil)	ΣCO2-C/MBC
Soils (S)
Missa (S1)	44.01 a	2464.6 a	10.85 b
Kahuta (S2)	36.78 b	2059.7 b	12.88 a
HSD	0.62	34.63	0.31
Treatments (T)
Control (T1)	23.36 e	1308.2 e	11.19 cd
ES (T2)	25.73 d	1440.9 d	10.78 d
ES+FYM (T3)	38.71 c	2167.6 c	11.77 bc
ES+PL (T4)	59.86 a	3352.3 a	12.18 b
ES+SF (T5)	54.32 b	3041.7 b	13.41 a
HSD	1.41	78.76	0.69
Soils (S) × Treatments (T)
S1 × T1	24.67 h	1381.6 h	9.59 d
S1 × T2	27.86 g	1560.4 g	9.61 d
S1 × T3	43.13 e	2415.4 e	11.44 c
S1 × T4	65.43 a	3664.0 a	11.79 bc
S1 × T5	58.96 b	3301.5 b	11.82 bc
S2 × T1	22.05 i	1234.8 i	12.78 b
S2 × T2	23.59 hi	1321.4 hi	11.96 bc
S2 × T3	34.28 f	1919.8 f	12.09 bc
S2 × T4	54.29 c	3040.6 c	12.57 bc
S2 × T5	49.68 d	2782.0 d	14.99 a
HSD	2.36	132.02	1.17
CV	1.99	1.99	3.37

FYM: Farmyard manure; PL: Poultry litter; SF: Sugarcane filter cake. Letter(s) indicate significant differences at p < 0.05 within the column (HSD test). Data are mean, n = 3.

. The rate of CO₂-C and ΣCO₂-C evolution differed significantly with the soil type, and was higher in Missa than the Kahuta soil, while the ratio of ∑CO₂-C to MBC was significantly lower in the Missa soil. Organic amendments caused a considerable increase in CO₂-C evolution from both the soils, the highest rate of CO₂-C and the ΣCO₂-C evolution has been observed in PL amended soils. The ratio of ΣCO₂-C to MBC was significantly lower in ES, compared to the FYM, SF and PL amended soils.

The dissolved organic C (DOC) also varied significantly (p < 0.05) with the soils, higher in Missa than the Kahuta soil (

Table 3. Main effects of soils, treatments, sampling days and their interaction on DOC, MBC, MBS, dehydrogenase activity, arylsulfatase activity and ratios of MBC/MBS, DHA/MBC and ASA/MBC in soils. All the treatments showing different letters are significantly different (p < 0.05).

Main Effects	DOC (mg kg−1 soil)	MBC (mg kg−1 soil)	MBS (mg kg−1 soil)	DHA (mg INF kg−1 soil)	ASA (µg p-Nitrophenol kg−1 s−1)	MBC/MBS	DHA/MBC	ASA/MBC
Soils (S)
Missa (S1)	107.1 a	219.9 a	4.19 a	9.24 a	2.19 a	56.05 a	0.047 a	0.010 b
Kahuta (S2)	76.8 b	157.0 b	3.02 b	7.10 b	1.73 b	54.59 b	0.042 b	0.012 a
HSD	0.75	2.99	0.03	0.11	0.025	1.18	0.0001	0.0003
Treatments (T)
Control (T1)	67.1 d	119.8 e	1.87 e	4.62 d	0.99 d	64.59 a	0.040 d	0.0086 c
ES (T2)	65.1 e	135.8 d	2.08 d	4.69 d	1.04 d	65.68 a	0.036 e	0.0079 d
ES + FYM(T3)	85.5 c	182.8 c	3.85 c	10.25 b	2.29 c	49.39 c	0.057 a	0.0131 a
ES + PL (T4)	131.0 a	274.2 a	4.65 b	11.83 a	2.59 b	54.98 b	0.046 b	0.0118 b
ES + SF(T5)	111.2 b	229.8 b	5.58 a	9.47 c	2.89 a	41.95 d	0.044 c	0.0126 a
HSD	1.66	6.62	0.07	0.24	0.052	2.61	0.0021	0.0006
Sampling days (D)
0 day	102.1 a	184.7 c	2.92 e	4.80 e	0.99 e	61.98 a	0.029 d	0.0061 d
14 day	80.5 d	247.6 a	4.77 a	7.13 d	1.44 d	54.28 b	0.029 d	0.0060 d
28 day	90.7 c	192.7 b	3.99 b	11.84 a	2.54 b	50.42 c	0.050 c	0.0126 c
42 day	94.7 b	165.4 d	3.27 c	9.37 b	2.63 a	55.08 b	0.055 b	0.0152 a
56 day	91.8 c	152.0 e	3.07 d	7.71 c	2.22 c	54.81 b	0.059 a	0.0141 b
HSD	1.66	6.62	0.07	0.24	0.052	2.61	0.0021	0.0006
Analysis of variance (p-value)
S	0.0000	0.0000	0.0000	0.0000	0.0000	0.0551	0.0000	0.0000
O	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
D	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
S × O	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
S × D	0.0001	0.8903	0.0000	0.0000	0.0000	0.0001	0.0000	0.0000
O × D	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0001	0.0000
S × O × D	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0003	0.0000

). Among the organic amendments, PL caused more increase in DOC than the ES, FYM and SF amendments in the order: PL > SF > FYM > ES. Contrary to the MBC and MBS, the DOC was significantly higher at 0 days of incubation (DOI) in both the control and the treated soils, declined sharply till 14 DOI, and then increased gradually at 28 and 42 DOI to become stable at 56 DOI.

The microbial biomass carbon (MBC) and microbial biomass sulfur (MBS) contents also differed significantly between the soils and were higher in Missa than the Kahuta soil irrespective of organic amendments addition (Table 3). The organic amendments had a substantial prompting effect on above-mentioned soil microbial parameters. However, the increase in soil MBC was highest in PL amended soils, while the MBS was found higher in the SF amended soils (Figure 1a–d and Figure 2a–d). During the whole incubation, the higher increase in the MBC and MBS was observed at 14 DOI, which later decreased gradually until the incubation ended.

The activities of soil enzymes i.e., arylsulfatase activity (ASA) and dehydrogenase activity (DHA) varied widely among the soils being higher in Missa compared to the Kahuta soil (Table 3). Organic amendments combined with elemental sulfur significantly increased ASA and DHA in the soils in comparison with the elemental sulfur alone which showed a non-significant increase in case of DHA as compared to control. DHA was highest in the PL+ES amended soils whereas ASA was observed highest in SF+ES amended soils, but the overall trend of increase varied as PL+ES > FYM+ES > SF+ES > ES for DHA and PL > SF > FYM > ES for ASA. Both the enzyme activities increased gradually from the start of incubation. DHA reached its peak at 28 DOI while the ASA at 42 DOI and later on decreased till the end of incubation.

The ratios of microbial biomass C to microbial biomass S (MBC/MBS), dehydrogenase activity to microbial biomass C (DHA/MBC), and arylsulfatase activity to microbial biomass C (ASA/MBC) also varied significantly in both the soils (Table 3).

3.3. Available Sulfur and Sulfur Fractions

Available sulfate represented by CaCl₂ extractable sulfate contents was significantly higher in the Missa soil (16.5 mg kg⁻¹ soil) compared to the Kahuta soil (14.2 mg kg⁻¹ soil) (Figure 3). Elemental sulfur and organic amendments caused a significant increase in the available sulfate in both the soils in the order: ES+SF > ES+PL > ES+FYM > ES. The concentration of available sulfate increased gradually in the amended soils from 0 DOI till 42 DOI followed by a slight increase till the end of incubation (56 DOI).

Missa had significantly higher contents of total S and different S fractions compared to the Kahuta soil (

Table 4. Main effects of soils, treatments, sampling days and their interaction on sulfur fractions in soils.

Main Effects	Water Soluble-S (mg kg−1 soil)	Adsorbed-S (mg kg−1 soil)	Occluded-S (mg kg−1 soil)	Carbon Bonded-S (mg kg−1 soil)	Ester-Bonded-S (mg kg−1 soil)	Organic S (mg kg−1 soil)	Total S (mg kg−1 soil)
Soils (S)
Missa (S1)	13.8 a	3.89 a	25.1 a	60.1 a	26.16 a	86.2 a	146.95 a
Kahuta (S2)	11.8 b	2.21 b	19.3 b	54.1 b	17.40 b	71.55 b	133.63 b
HSD	0.14	0.09	0.20	0.65	0.38	0.55	1.31
Treatments (T)
Control (T1)	6.9 e	1.83 e	19.7 e	45.6 e	18.4 d	64.07 e	89.2 e
ES (T2)	9.4 d	2.09 d	22.4 b	51.5 d	19.8 c	71.31 d	137.5 d
ES+FYM(T3)	13.1 c	2.98 c	20.9 d	58.5 c	21.9 b	80.41 c	147.0 c
ES+PL (T4)	16.7 b	3.70 b	21.8 c	63.7 b	24.7 a	88.39 b	158.1 b
ES+SF (T5)	18.1 a	4.67 a	25.9 a	66.2 a	24.1 a	90.28 a	169.7 a
HSD	0.31	0.19	0.45	1.46	0.85	1.22	2.91
Sampling days (D)
0 day	8.8 c	2.69 c	24.0 a	56.9 b	21.9 b	78.8 b	148.2 a
28 day	14.3 b	3.00 b	21.6 b	58.1 a	23.0 a	81.1 a	136.7 b
56 day	15.3 a	3.46 a	20.9 c	56.3 b	20.4 c	76.7 c	136.0 b
HSD	0.21	0.13	0.29	0.96	0.56	0.67	1.93
Analysis of variance (p-value)
S	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
O	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
D	0.0000	0.0000	0.0000	0.0001	0.0000	0.0000	0.0000
S × O	0.0000	0.0000	0.0000	0.0277	0.4400	0.0418	0.0076
S × D	0.0202	0.0000	0.0000	0.0000	0.0000	0.5299	0.7377
O × D	0.0000	0.0000	0.2833	0.0000	0.0075	0.0000	0.0000
S × O × D	0.4159	0.2161	0.0061	0.0035	0.2199	0.0036	0.9837

FYM: Farmyard manure; PL: Poultry litter; SF: Sugarcane filter cake. Letter (s) indicate significant differences at p < 0.05 within the column (HSD test). Data are mean, n = 3.

). Application of elemental sulfur and organic amendments significantly affected the total S and S fractions in both the soils. Among inorganic S fractions, increase in the water-soluble and adsorbed S fractions followed the trend: ES+SF > ES+PL > ES+FYM > ES, while for the occluded S, the increasing order was: ES+SF > ES+PL > ES > ES+FYM. Carbon bonded S dominated among the organic S fractions in both the soils and was significantly higher in ES+SF, followed by ES+PL, ES+FYM and ES treatments. The ester-bonded S fraction was at par in both ES+PL and ES+SF treatments, followed by ES+FYM and ES. Among inorganic S fractions, water soluble-S and adsorbed-S increased gradually throughout the incubation and were highest at 56 DOI, whereas the occluded-S was higher in the beginning and decreased gradually till the end of incubation. Organic fractions (carbon bonded S and ester bonded S) and total organic S increased gradually to reach at the peak at 28 DOI and later on decreased till the end of incubation (56 DOI).

3.4. Sulfur Oxidation

The percent oxidation of the ES was slightly higher in Kahuta soil than the Missa soil (Figure 4). Approximately 9% of the applied ES was oxidized to available sulfate during 56 days of incubation. Organic amendments significantly increased sulfur oxidation in both the soils compared to ES alone. During the initial phase of incubation, i.e., up to 14 DOI, there was a rapid increase in S oxidation in PL amended soil, followed by SF and FYM treatments. Overall, 10 to 13.69% ES was oxidized in the Missa, and 11.2 to 13.94% of the ES was oxidized in the Kahuta soils amended with organic amendments during 56 days of incubation.

The maximum increase in S oxidation occurred in SF treated soils and the minimum in FYM treated soils. Contribution of heterotrophic microorganisms to ES oxidation ranged from 1.56 to 2.97% in Missa, and 2.19 to 4.50% in Kahuta soil, while that of the autotrophic microorganisms varied from 6.29 to 7.68% in Missa and 6.56 to 6.56% in Kahuta soils.

4. Discussion

Microbial biomass and activity parameters, i.e., soil respiration, microbial biomass C and microbial biomass S were higher in the Missa soil compared to the Kahuta soil which might be related to variation in the total organic C (TOC) contents of these soils [51]. Since both the soils were highly deficient in organic matter (<0.5%), therefore a slight variation in organic C availability in these soils, exerted a prominent effect on their microbial properties. Application of elemental sulfur alone promoted microbial biomass and arylsulfatase activity in the soils under sulfur deficient conditions (i.e., <10 mg kg⁻¹) [52,53,54]. Oxidation of elemental sulfur to sulfate increased the availability of sulfur to starving soil microbial populations and thus promoted the growth of soil microbial biomass and soil respiration. Stimulating effect of elemental sulfur on soil microbial populations was also observed by others. Filipek-Mazur et al. [24] and Yang et al. [55] found a strong positive effect of elemental sulfur on the growth and multiplication of soil microorganisms. Similarly, Chapman [56] observed a significant increase of the population of autotrophic sulfur oxidizing microorganisms (Thiobacillus spp.) after elemental sulfur application. However, a few studies also reported a significant decline of soil microbial biomass and heterotrophic microbial activities due to acidification resulting from sulfur oxidation in soil [57,58,59,60], but this effect was mainly observed when elemental sulfur was applied under acidic soil conditions. The oxidation of S⁰ results in H⁺ generation during the process. However, the degree of acidification varies depending on the amount of applied S⁰ and the soil buffering capacity [55]. Contrary to the above reports on acidic soils, our study showed a significant increase in the size and activity of soil microbial biomass in the soils due to the elemental sulfur addition. In alkaline soils used in this study, the strong acidifying effect of sulfur oxidation was counterbalanced by the initial high pH of the soils, thus no negative effect of sulfur oxidation was observed on soil microbial parameters.

Combined application of elemental sulfur and organic amendments had a more stimulating effect on soil microbial parameters (microbial biomass C and respiration) than the elemental sulfur alone. It is a common observation that organic amendments promote the growth and multiplication of heterotrophic microorganisms in soil by providing labile organic C as an energy source to the soil microorganisms. Although the organic amendments significantly increased CO₂-C and ΣCO₂ evolution from the soils, the effect of PL was more prominent than the other two organic amendments (SF and FYM). The higher contents of total organic C and dissolved organic C in PL, and its lower C/N and C/P ratios make the PL easily degradable organic substrate in soil consequently resulting in more release of CO₂-C from the soil amended with this source. Soil microbial biomass S also increased with the addition of elemental sulfur alone or in combination with organic amendments. As observed for microbial biomass C, the effect was more pronounced when organic amendments were applied along with elemental sulfur. Higher microbial biomass carbon and phosphorus in soil were recorded with application of poultry litter enriched compost [61]. The increase in microbial biomass S in sulfur treated soils might also agree with the ability of the heterotrophic soil microorganisms especially the fungi to accumulate sulfur when the available sulphate (SO₄²⁻) levels are high in soil, as due to oxidation of elemental sulfur [62]. It has been reported that some fungi were able to oxidize inorganic sulfur compounds and thus were capable of playing an important role in sulfur oxidation [62]. Notably, in all the studies on sulfur-oxidizing fungi, organic compounds were supplemented into the culture media along with thiosulfate [63], and concluded that organic compounds, especially the organic C, were indispensable to the sulfur oxidation of fungi. Soil fungi have high sulfate requirements, and thus they accumulate higher quantities of sulfur available in the soil [64,65]. This is the reason that the addition of organic amendments containing higher amounts of oxidized and total sulfur along with complex organic C such as the sugarcane filter cake in the present study led to higher incorporation of sulfur into microbial biomass.

The combined application of organic amendments with elemental S may had significantly increased dehydrogenase and arylsulfatase enzyme activities in both the soils because of the addition of C substrate and the intra- and extracellular enzymes contained in the added organic amendments [66]. As dehydrogenase enzyme is responsible for organic matter oxidation in soil and its activity is amplified by the addition of organic substrates, thus soil treatment with easily degradable organic substrate containing high C contents may exhibit high dehydrogenase activity as observed in PL amended soil. On the other hand, the treatment where only the elemental sulfur was applied showed little or no increase in dehydrogenase activity in soil at par with the control of the present study. Reason might be the absence of C source that is the main stimulant for the release of dehydrogenase enzyme [26].

Arylsulfatase plays an important role in sulfur bioavailability due to its involvement in the mineralization of organic sulfur by carrying out hydrolysis of ester sulfates [67]. In our study, higher activities of arylsulfatase were observed in soils amended with elemental sulfur and organic amendments, particularly the sugarcane filter cake amendment. It might be linked to more oxidizable S and organic S contents of the sugarcane filter cake as compared to other two organic amendments. The arylsulfatase activity increased in the amended soils until the concentration of available sulfate reached a certain level in the soil (i.e., up to 42 DOI). As the sulfate availability increased further during the later stage of incubation (42 to 56 DOI), the arylsulfatase activity decreased. Previously a strong negative correlation was reported between the activity of the arylsulfatase enzyme and the sulfate S concentration in the soil [24]. Siwik-Ziomek [68] observed highest arylsulfatase activity in soils where the mineral and organic fertilizers were applied in combination. Elemental sulfur application alone did not significantly increase arylsulfatase activity in soil compared to the control which is indicative of the inhibiting effect of the applied mineral sulfur on arylsulfatase activity [69]. However, it is not clear from the contemporary findings that the overall increase in soil microbial biomass in response to elemental sulfur application can result in an increase of enzyme activities in soil. Arylsulfatase activity has shown a strong positive correlation with total sulfur and organic C content [70,71] whereas a strong negative correlation with the sulfate content in soils, signifying that availability of carbon is the main factor leading to sulfur fractional conversions in soil. So, it is suggested that the addition of organic substrates with high S contents such as the sugarcane filter cake in combination with elemental sulfur can improve enzyme activity in the soil.

Oxidation of the added elemental sulfur increased in soils with the addition of organic amendments. Thiobacillus spp. were long considered the S oxidizers but they are not frequently present in significant numbers in most S deficient agricultural soils because of unavailability of reduced S compounds. Hence, the role of heterotrophic sulfur oxidizing microorganisms becomes important in this context [12]. Generally, heterotrophic microorganisms rely on energy obtained from organic carbon for their growth and multiplication. The application of organic substrates to the soils might have enhanced heterotrophic microbial community numbers and their activity to improve sulfur oxidation [9]. The prompting effect of organic matter on S⁰ oxidation was also reported in some previous studies [72]. The composition of added organic amendments also variably affects ES oxidation in soils. More oxidation of ES in the PL amended soil during early stages of incubation might be due to high labile organic C and N contents in the amendment, which made it a better, source of energy for the activity of chemoheterotrophic S⁰ oxidizers. At later stages of incubation, a considerable increase in S⁰ oxidation occurred in SF amended soils due to the recalcitrant nature of organic C present in the substrate which remained available for the growth and activity of chemoheterotrophic microorganisms for a longer period of time. Hence the sulfate concentration was eventually higher in SF amended soil as compared to the PL and FYM amendments. The composition of organic matter and its subsequent effect on the intensity of elemental sulfur oxidation was also described by Cifuentes and Lindemann [73] and Wainwright et al. [63]. Alkaline soil conditions in particular favor heterotrophic S⁰ oxidation. Several studies have shown an increase in S⁰ oxidation in alkaline soils or in response to the addition of CaCO₃ under soil conditions [55,64]. It was suggested that under acidic conditions, S⁰ oxidation is driven predominantly by soil fungi [74,75].

Sulfur availability through oxidation and its distribution to subsequent fractions is poorly understood in alkaline calcareous soils in response to combined application of organic amendments and the elemental sulfur as the minimal research data is available. In arable soils, microbial mediated processes involved in sulfur transformations include oxidation, mineralization and immobilization [76]. It is well documented that the elemental sulfur once applied to soil would be taken up by the autotrophic and heterotrophic sulfur oxidizing microorganisms, transformed into intracellular sulfur globules and oxidized to plant available sulfate [77]. In the present study, a significant and consistent increase was observed in water soluble and plant available S fractions in the soils with the progress of incubation. This might be partly due to mineralization of the added organic sulfur and partly because of the oxidation of added elemental sulfur. Saren et al. [78] and Gouravet et al. [79] reported that integrated use of FYM and mineral S increased the soil’s potential for S mineralization due to the favorable effect of FYM on soil microbial activities. The highest adsorbed S fraction during the advanced phase of incubation might be due to increase in SO₄²⁻ adsorption sites in soil because of organic amendments addition. Combined application of ES and organic amendments significantly affected organic sulfur fractions in soil. Both the ester bonded, and carbon bonded S fractions increased gradually until 28 DOI and declined later on up to the end of incubation (56 DOI). The multiple processes governed at the same time could elucidate the trend of increase in initial phase and decline in the later stages. Oxidation of elemental S increased the available S pool that underwent immobilization into the microbial cells to increase the organic S fractions of the soil, as described by Karimizarchi et al. [80]. Besides immobilization, mineralization of the native as well as added organic S sources into available S pools reduced the organic S fractions again [81]. The soils amended with sewage sludge, dustbin waste and poultry litter showed N and S mineralization whereas soil amended with rice straw induce N and S immobilization [82].

5. Conclusions

The study provides evidence of the strong influence of organic amendments and elemental sulfur on microbial activities, sulfur oxidation and bioavailability in alkaline soils. Availability of plant available S from the elemental sulfur, a slow releasing S source, was enhanced by the addition of organic amendments, and was the combined function of sulfur oxidation and mineralization processes. The effect of organic amendments varied depending upon their quality and composition. Total organic carbon, dissolved organic carbon, and total sulfur contents were the main components of organic amendments which significantly influenced sulfur release and oxidation in soil. Among the organic sources, sugarcane filter cake, a complex organic C source, had a superior effect on sulfur availability due to its high total sulfur contents and consistent availability of energy to soil microorganisms. The results of incubation study were further evaluated on mustard crop in field conditions and the similar pattern of results were recorded showing better productivity and crop quality attributes of the crop and improved soil properties (unpublished data).