Relationships Between Carbon Fractions and Soil Nutrients in Organic Cassava Cultivation in the Sandy Soil of Northeastern Thailand

Abstract

This research investigated the interaction between the labile and stable fractions of soil organic carbon (SOC) during the cultivation of organic cassava in sandy soil in northeastern Thailand over a period of five years. We collected surface soil samples (0–30 cm) from this sandy region, utilizing a combination of cow and chicken manure along with dried distilled grains (DDGs) from cassava fermentation for ethanol production, to monitor and compare the effects of continuous mixed organic fertilization on SOC, carbon fractions, soil pH, and nitrogen and phosphorus levels throughout a five-year period of varying land use ages (LUA) to the pre-fertilization state. This study proposed that the use of a combination of organic fertilizers could increase soil organic carbon levels. This study indicates that the continuous application of organic fertilizers over five years does not lead to a significant increase in soil carbon; however, it may result in temporary alterations in different organic carbon fractions. The study showed that the mixed organic fertilization could the increase carbon fractions. Labile carbon (LBC) fraction was at its lowest before fertilization, peaking at LUA 3 and increasing by 5.44–25.50% after organic fertilizer addition. The first year revealed high non labile carbon (NLBC) levels, exceeding 60%, in comparison to the pre-fertilizer period. In the second year, NLBC levels declined to LUA 5, a change that was not statistically significant. After pre-fertilization, the concentration of recalcitrant carbon (REC) did not significantly decrease. Nitrate (NO₃⁻-N) concentrations exhibited no significant fluctuation pre-and post-fertilization. Furthermore, the Bray II-extractable phosphorus (P(B)) decreased (i.e., LUA 1 and 4). The pH levels dropped after the addition of organic fertilizer, particularly in the second year. We found strong positive links between SOC and carbon fractions such as NLBC (r = 0.54 ***) and POXC (r = 0.49 ***). However, neither LBC nor less labile carbon fraction (LLBC) showed any significant correlations with SOC. The negative correlations were observed between ammonium (NH₄⁺-N) and NO₃⁻-N with labile carbon types, such as LBC, LLBC, and POXC, while positive correlations were noted with stable carbon fractions, such as NLBC, and REC. From the application of this organic fertilizer, there are various amounts of organic carbon which cause the following effects: The inclusion of LBC from mixed organic fertilization seems to enhance SOC decomposition rather than accumulation. NLCB may persist in sandy soil for a longer duration than LBC, resulting in the retention of SOC in sandy soil. Our results suggested the implementation of a systematic soil testing strategy to monitor temporal variations in carbon fractions and nutrient levels. Using the right amounts of both LBC and NLBC would improve soil health and help store carbon through organic fertilizers.

1. Introduction

Sandy soils cover approximately 31% of the global land area, spanning around 900 million hectares, primarily in arid and semi-arid regions such as the Sahara, Saudi Arabia, Turkey, northwest China, and western Australia [1,2]. In northeastern Thailand, sandy soils typically consist of a sandy surface layer overlying a loamy to clayey substratum, a stratification caused by mass movement processes that transport clay particles downward, leaving coarser and sandier materials at shallower depths [3]. These soils have inherently low fertility and rely heavily on soil organic carbon (SOC) for nutrient cycling and soil health maintenance [1,2]. Cassava cultivation in such soils benefits from organic amendments such as compost and cassava residue, which have been shown to enhance yield [4]. Proper management of nitrogen, phosphorus, and potassium is essential for maintaining soil fertility and productivity [5]. Organic fertilizers, including manure and crop residues, enhance soil fertility by increasing SOC and total nitrogen, both of which are critical for plant growth [6]. The decomposition of high-quality organic inputs such as groundnut stover occurs rapidly in sandy soils, significantly influencing nutrient cycling [7]. Organic amendments may also induce a priming effect, accelerating the decomposition of native SOC, particularly when high-quality organic matter is added [8,9].

The application of organic fertilizers significantly enhances labile carbon fractions in soil, increasing carbon mineralization and turnover rates [10,11]. Long-term application of organic fertilizers, such as farmyard manure, promotes soil health and fertility by increasing labile carbon pools and improving carbon sequestration [11]. The rate of organic fertilizer application influences the size of these labile carbon pools, with higher application rates leading to greater increases in labile carbon [11,12]. Labile carbon fractions, including dissolved organic carbon (DOC), microbial biomass carbon (MBC), and oxidizable organic carbon (KMnO₄⁻C), are readily available for microbial activity [13]. Before fertilization, labile carbon levels in soil are generally low but increase as organic amendments decompose and release carbon. Soil labile carbon can be stabilized through its integration into stable organic matter, which is facilitated by compost and humic acids [14]. Studies have shown that farmyard manure significantly enhances labile carbon fractions compared with chemical fertilizers alone [11]. Over time, the decomposition of organic matter contributes to increases in labile carbon content [15,16]. The addition of labile organic substrates, such as ¹³C-glucose, can stimulate microbial activity, enhancing SOC mineralization through a positive priming effect [17]. Organic amendments, including manure and crop residues, increase microbial and enzyme activities, leading to higher labile carbon content [11,16]. However, labile carbon is more susceptible to microbial decomposition, resulting in a relative decline in non-labile carbon content [11,18]. While organic fertilization enhances SOC, the increased presence of labile carbon may pose challenges for long-term carbon sequestration due to its rapid decomposition [19]. Environmental changes, such as rising temperatures and fresh carbon inputs, can trigger the priming effect, making stable carbon more vulnerable to decomposition [20].

Intensive agricultural practices often lead to SOC depletion in sandy soils, whereas sustainable management can enhance SOC sequestration [21]. Monoculture systems in northeastern Thailand accelerate soil organic matter degradation and weaken soil aggregation, but continuous recycling of organic residues can restore soil quality [22]. Organic amendments such as manure and biochar can improve SOC levels in sandy soils, but they may also increase CO₂ emissions, which could counteract some carbon storage benefits [23,24]. The quality of organic fertilizers significantly influences soil carbon storage, with high-quality amendments generally enhancing labile carbon fractions, potentially decreasing SOC stability [25]. Pig manure, for example, enhances SOC accumulation but may also reduce stability due to increased labile carbon fractions [25,26]. Organic fertilizers stimulate microbial biomass and activity, accelerating labile carbon decomposition and transformation [15,27]. Additionally, they improve the availability of nutrients, including nitrogen, phosphorus, and potassium, which are essential for plant growth [20]. The soil carbon–nitrogen–phosphorus (C/N/P) stoichiometry plays a key role in nutrient availability and SOC stabilization. Long-term organic fertilization alters these ratios, particularly in topsoil layers, improving SOC sequestration [28]. Distilled dried grains (DDGs) are a valuable nutrient source, providing nitrogen, phosphorus, and potassium [29], as well as magnesium, zinc, and sulfur [30]. DDG amendments can improve microbial respiration by lowering the C:N ratio [31] and, when combined with cattle manure, enhance soil fertility and structure [29,32]. Therefore, integrating organic fertilizers such as chicken and cow manure with DDGs could influence soil carbon dynamics and sequestration in sandy soils.

Soil nitrogen availability, a key factor in crop uptake [33], is largely regulated by nitrogen mineralization, which follows distinct patterns in sandy soils [34,35]. Labile and non-labile carbon fractions respond differently to nitrogen inputs, with labile fractions increasing significantly and promoting nitrogen mineralization by providing more carbon for microbial activity [36]. However, nitrogen mineralization can be decoupled from carbon availability in some fractions, suggesting that nitrogen availability does not always directly correlate with SOC content [37]. Labile carbon inputs can enhance SOC mineralization, which is closely linked to nitrogen availability [38]. Nitrification, a microbial process, can also influence SOC mineralization by altering nitrogen availability and stimulating microbial communities [39].

While organic fertilizers improve fertility, their impact on soil carbon fractions and SOC stability must be carefully managed. Effective organic matter management is crucial for maximizing benefits while minimizing environmental risks. Integrating organic fertilizers with soil amendments tailored for sandy soils and carbon sequestration could enhance soil quality. Therefore, investigating the combined use of organic fertilizers (e.g., chicken and cow manure) with DDGs is essential for understanding their effects on soil carbon dynamics and sequestration in sandy soils, an area that remains underexplored.

This study aims to evaluate the effects of combining organic manure fertilizers with DDGs on SOC content and sequestration in sandy soils. Specifically, it examines SOC fractionation, microbial activity, and soil physicochemical properties before and after fertilization. The research hypothesizes that using a mix of organic fertilizers would raise SOC levels. By stabilizing SOC in sandy soil from an organic cassava plantation, this study explores the mechanisms of SOC stabilization, carbon fractionation, and their interactions with nitrogen and phosphorus availability in sandy soil. Understanding these interconnections is crucial for improving soil carbon stability and fertility in sandy soils.

2. Materials and Methods

2.1. Study Site

The study was carried out on an organic cassava (Manihot esculenta) plantation located in Ubonrachathani (UBR), Amnat Charoen (AMC), and Yasothon (YST) provinces in northeastern Thailand. The age of this land use was between 0 and 5 years old. Ubon Ratchathani (at coordinates 15.2490° N, 104.8521° E), Yasothon (at coordinates 15.8025° N, 104.4770° E), and Amnat Charoen (at coordinates 15.43° N, 104.67° E) are in the northeastern region of Thailand, which is part of the Khorat Plateau, with an elevation of 116 m above sea level (Figure 1). The landscape is influenced by past tectonism, with variations in bedrock and fault characteristics affecting the topography and channel profiles [40]. This region features gently rolling low hills, which contribute to its unique topography [41]. The precipitation and relative humidity significantly influence tree growth, particularly during the dry-to-wet transition season from April to June [42].

Weather: From January to April, precipitation is modest; it then rises steadily in May, peaks in September, and then drops dramatically from October to December. The difference is most noticeable between September and May. The average annual temperature is 27–28 °C. The average minimum and maximum temperatures are 22.5–22.8 °C and 33.2–33.5 °C. April has the highest summer temperatures of up to 40 °C. June–October rains lower the temperatures to 25–33 °C and increase the humidity. Winter is from November to February. Some areas may fall as low as 14–18 °C in the mornings [43]. Heat waves prompted record April temperatures of 41 °C in all three provinces in 2019, according to Climate Highlights (2017–2023) [43]. Historically of this study soil, all farmers cultivated crops, including cassava, rice, and garlic, using traditional methods with chemical fertilizers. To cultivate organic monoculture cassava, farmers must restore soil previously treated with pesticides by allowing it to remain fallow for a minimum of one year. Upon the farmer’s readiness, they should begin planting using chemical-free cuttings, organic fertilizers, biological pest and disease control measures, and chemical-free weed management. This process guarantees that cassava production adheres to 100 percent organic agricultural requirements. Maintenance while planting includes organic fertilization during the first 30 days, the use of mixed organic fertilizers at three months, weed management by weeding and mulching, and regular plot inspections to avert insect-borne illnesses such as cassava mosaic disease or mealybugs. The cassava tubers may be harvested when they reach 8 to 12 months in age.

The quantity of organic fertilizer and soil amendment usage: This study collected historical data on the amount of fertilizer application through the lens of organic fertilization. The main organic fertilizers are animal manure, such as chicken manure, cow manure, and dried distilled grains (DDGs), which are mixed together. The amount of these organic fertilizers varies, and the quantities are different. The fertilization pattern comprised a combination of DDGs and local animal dung, including cow and chicken manure, applied continuously for a duration of 1 to 5 years, in contrast to the absence of organic fertilization. The DDGs used in this area come from the use of cassava for ethanol production. It is a material left over from the ethanol production process, especially in the step of removing water from the waste after fermentation of ethanol from cassava. Then, it is dried in the open air to reduce the amount of toxic substances before being added to the organic cassava plantation of this area. While the animal manure used by farmers is chicken manure and cow manure, both of which are available for sale in the area and nearby. Local livestock raisers provide these manures.

We collected data concerning the intensity of organic fertilization according to certain management strategies. The variability in organic fertilization practices among farmers highlights the diverse land-use age (LUA). Collectively, these data only illustrate the average amount of organic fertilizer practices. For a more precise study, the quality of local farmers’ practices was also addressed. Individual farmers always mixed chicken manure and cow manure as nutrient sources for cassava plantations and used DDGs to amend the soil with the same amount every year. We conducted a survey and interviewed local farmers who cultivated cassava in a contract farming system to gather information about the land-use history for the soil and site studies. We then combined this information with secondary data, such as the land-use area and soil type, to facilitate soil sampling. Data on the combinations of organic fertilizer management in the study area were collected to determine the patterns of mixed organic fertilizer application from interviews with the farmers who owned the plots (totaling 168 plots) where we collected soil samples. The details of the organic fertilizer management in the different periods of organic fertilizer application are presented in terms of the average amount of organic fertilizer. It was observed that the pattern of organic fertilizer application was similar in each year of organic fertilizer application in each area (

Table 1. Information on the combinations used in organic fertilizer management.

Land-Use Age (Year)	Information on the Combinations Used in Organic Fertilizer Management
LUA5	These soils only received DDGs, varying, on average, 811, 681, 594, 4420, and 4351 kg ha−1 year−1 from 2018 to 2022.
LUA4	These soils received an average combination of cow manure, chicken manure, and DDGs (i.e., 1389, 3125, and 4410 kg ha−1 year−1, respectively) from 2019 to 2022.
LUA3	These soils received an average combination of chicken manure and DDGs (i.e., 2500 and 625–833 kg ha−1 year−1, respectively) from 2020 to 2022.
LUA2	These soils received an average combination of cow manure, chicken manure, and DDGs (i.e., 13,021, 2600, and 572 kg ha−1 year−1, respectively) from 2021 to 2022.
LUA1	These soils received an average combination of chicken manure and DDGs (i.e., 888 and 636 kg ha−1 year−1, respectively) in 2022.
LUA0	The soil and land underwent pre-organic fertilization and entered a period of transition.

). It is also clear that LUA5 only used DDG. The rates of organic fertilizer application depend on the experiences from previous times that were recommended by government and non-government organizations and mainly on the budgets of their farmers. Moreover, these organic fertilizers (i.e., cow and chicken manure and DDG) are necessary purchases from various nearby sources within this area.

2.2. The Study’s Experimental Design

The study’s experimental design aimed to assess how using continuous organic fertilizer impacts soil carbon fractions and their connections with nitrogen (N) and phosphorus (P), and in turn, the soil organic carbon (SOC) content across six different land-use ages (Table 1). After continuously applying organic fertilizer mixtures, we evaluated the carbon fractions and nutrient levels in the soils and compared them to pre-fertilization.

We also determined how these soil properties changed over time. The study covered a period from pre-organic fertilization (zero) to five years. The study examined 251 plots that were used at the beginning of organic cassava cultivation and during the transition phase LUA0). These plots were then compared with those that were utilized before converting to organic fertilizer for one year (LUA1), followed by two, three, four, and five years (LUA2,3,4 and 5), in that sequence (Table 1). These land-use measures were implemented since 2018.

2.3. Soil Sampling and Sample Distribution

The soil sampling procedure was planned by examining the number of years that the cassava plantation had been organically farmed and by using soil series maps, along with other supported soil classifications. The soil samples collected in April 2024 from representatives of each land-use age were samples that had been continuously fertilized with organic fertilizers. Soil samples were collected using an auger at a depth of 0–30 cm from study sites with different durations of organic fertilization (0–5 years). The numbers of samples per land-use age were as follows: 0 years (36 samples), 1 year (60 samples), 2 years (58 samples), 3 years (56 samples), 4 years (28 samples), and 5 years (13 samples) (Table 1, Figure 1). The number of samples varies depending on the number of farmer plots. Samples were air-dried, sieved, and underwent chemical and soil particle analysis.

2.4. Methodology for Soil and Organic Fertilizer Analysis

2.4.1. Soil Organic Carbon (SOC)

Soil organic carbon (SOC) was assessed by sieving 2 mm of dry soil combined with K₂Cr₂O₇, heating the mixture to 130 °C for 30 min, and permitting it to rest overnight [44]. An FeSO₄ solution was utilized for titration, following the procedure established in [45].

2.4.2. Permanganate Oxidizable Carbon (POXC)

POXC, or “active carbon”, was extracted by following the method in [46] and spectrophotometrically analyzed at 550 nm. We introduced 3 g of air-dried soil into a 50 mL centrifuge tube and subjected it to a 0.5 mm sieve using 20 mL of 0.02 M KMnO₄. We rapidly agitated the tubes for 30 min at a rate of 120 revolutions per minute using a reciprocating shaker (HERMEL, HERMLE Labortechnik GmbH, Wehingen (Baden-Württemberg), Germany), and thereafter promoted the oxidation process and sedimentation of particulate matter via centrifugation (HERMEL Z 206 A, HERMLE Labortechnik GmbH, Wehingen (Baden-Württemberg), Germany) for 5 min. Subsequently, 0.1 mL of the sample solution was transferred from the tube to another tube containing 9.9 mL of deionized water to halt the reaction. We used a spectrophotometer (Cecil Aurius Series CE 2021, Cecil Instruments Limited, Cambridge (Cambridgeshire), UK) to measure the absorbance of each sample at 550 nm and calculated the POXC (mg kg⁻¹ soil) [47] correspondingly.

POXC = [0.02 (mol L⁻¹) − (a+ b Abs)] × (9000 mg C mol⁻¹) × (0.02)

(1)

where, 0.02 mol L⁻¹ represents the initial concentration of the KMnO₄ solution, a represents the standard curve’s beginning point, and b represents its steepness. Abs represents the soil sample absorbance, 1 mol of MnO₄ changes 9000 mg of carbon when Mn⁷⁺ becomes Mn⁴⁺, 0.02 L of KMnO₄ solution reacts with the soil, and Wt is the soil weight (kg) employed in the reaction.

2.4.3. Different Concentrations of H₂SO₄ Were Used to Chemically Oxidize Carbon [48] Using an Oxidizable Carbon Fraction Technique

We mixed the soil sample contained in acidic solutions with potassium dichromate according to C content (Yeomans and Bremner, 1988; Maia et al., 2007 [49,50]). The solution employed high H₂SO₄ concentrations and needed no external heating [50]. The dichromate content was consistent as the concentration of H₂SO₄ increased to maximum levels of 9, 12, 18, and 24 mol L⁻¹. According to Chan et al. (2001) [48], the ratios of 0.5:1, 1:1, 2:1 and 4:1 correspond to 18 M H₂SO₄ and 0.167 M K₂Cr₂O₇. LBC (labile carbon fractions), LLBC (less labile carbon fractions), NLBC (Non labile carbon fractions), and REC (Recalcitrant carbon) denote the ratios in Equations (2), (3), (4) and (5), respectively.

Labile carbon; LBC = 6 M − 9 M H₂SO₄ oxidizable C

(2)

Less labile carbon; LLBC = 12 M − 9 M H₂SO₄ oxidizable C

(3)

Non labile carbon; NLBC = 18 M − 12 M H₂SO₄ oxidizable C

(4)

Recalcitrant carbon; REC = 24 M − 18 M H₂SO₄ oxidizable C

(5)

2.4.4. Nutrient Analysis (NH₄⁺, NO₃⁻, Available Phosphorus), pH, and Particle Distribution

Available phosphorus was extracted via the Bray (II) method by utilizing a 0.1 M HCl and 0.03 M NH₄F solution, followed by quantification through spectrophotometry using a Thermo Spectronic Genesys 20 spectrophotometer, Thermo Fisher Scientific Inc., Rochester (New York), NY, USA) [51]. NH₄⁺ and NO₃⁻ content: NH₄⁺ was extracted by 2 M potassium chloride, and measured by observing the color formed when ammonium, chlorine, and Na salicylate reacted. NO₃⁻ was extracted from soil samples using potassium sulfate at a concentration of 0.5 M, and NO₃⁻ determined using Na-nitroprusside to intensify the green hue using a spectrophotometer (Thermo Spectronic Genesys 20) [52]. The P malachite green method was used to determine the phosphorus concentration in new soil by combining sulfuric acid, ammonium molybdate tetrahydrate, malachite green, and polyvinyl alcohol. The absorbance was quantified using a double-beam spectrophotometer (Thermo Spectronic Genesys 20) at a wavelength of 630 nm after 30 min. This method’s sensitivity is enhanced by its ability to detect phosphorus concentrations that are too low for standard methods like the Murphy–Riley method [53], while the pH was determined using diluted water with a 1:2 ratio (Consort C3210 pH meter, Consort bvba, Turnhout, Belgium) [54]. The soil particle size distribution was determined using the hydrometer method [55].

2.4.5. Organic Fertilizer and DDG Analysis

Samples of organic fertilizer were desiccated at 65 °C and taken for further analysis. Carbon was determined through dry combustion using a muffle furnace (Thermolyne Type 4800 Furnace; Barnstead international, Boulevard, CA, USA) at 550–600 °C for 24 h [44], while the pH was determined using deionized water with a ratio of 1:10 (Consort C3210 pH meter) [54]. The percentage of nitrogen content was determined using the Kjeldahl method [54,56], while the determination of P and K was carried out using the wet digestion method based on the mixing of HNO₃, H₂SO₄, and HClO₄ acids [57]. Potassium was quantified using an atomic absorption spectrophotometer (SavantAA GBC, Cecil Instrumentation Services Ltd., Cambridge, UK). The phosphorus concentrations were quantified using a spectrophotometer (Thermo Spectronic Genesys 20). The C/N ratio was determined by dividing %C by %N.

2.4.6. Nutrients in Organic Fertilizer and DDGs via Water Extraction

A subsample of dried solid manure was assessed with a 1:5 water extract (w:v) for organic amendments and shaken for 30 min. We used a paper filter Whatman No. 5 to eliminate the particles, and the residual solution was diluted as necessary for the colorimetric analysis of NH₄⁺ and NO₃⁻ [58,59] and of soluble organic carbon with heat and a dicromate reagent [60]. Phosphorus concentrations were quantified using a spectrophotometer (Thermo Spectronic Genesys 20) [61].

2.5. Statistical Analysis

Statistical analyses were performed using JASP 0.19.3. One-way ANOVA was conducted to evaluate the effects of organic fertilizer according to age (LUA) on soil nutrients and carbon fractions. The results were compared using the Tukey HSD (honestly significant difference) method, and the reported format for homogenous groups was used; the averages and standard errors are presented. Pearson’s correlation was calculated and illustrated, with differences in the heatmaps being considered significant at p < 0.05, <0.01, and <0.001; then, a heatmap diagram was created to illustrate the relationships.

2.6. Writing the Manuscript by Generative AI

We use generative AI to support the writing of this manuscript for grammar checkers, paraphrasers, summaries, and searches for relevant references.

3. Results

3.1. The Nutrient Content of Organic Fertilizer and DDGs as Soil Amendments

Chicken manure exhibited a neutral to slightly alkaline pH, with a statistically significant difference among treatments (p = 0.037) (

Table 2. The nutrient content and chemical properties of organic fertilizers made of chicken and cow manure, and dried distilled gains (DDGs).

Indicator	Amendments.	Number of Samples	Median	Mean	F-Value	p-Value	Std. Deviation	Minimum	Maximum
pH	Chicken	10	7.25	7.14 a	4.39	0.037	0.42	6.49	7.63
pH	Cow	2	7.91	7.91 ab			0.09	7.84	7.98
pH	DDF	3	8.5	8.11 b			1.01	6.97	8.87
C (%)	Chicken	10	30.50	29.80 b	40.20	<0.001	4.80	19.00	36.00
C (%)	Cow	2	10.50	10.50 a			7.77	5.00	16.00
C (%)	DDF	3	4.39	4.35 a			0.86	3.48	5.20
N (%)	Chicken	10	3.38	3.49 b	17.17	<0.001	1.12	1.73	5.10
N (%)	Cow	2	0.36	0.37 a			0.05	0.34	0.41
N (%)	DDF	3	0.31	0.37 a			0.12	0.30	0.51
C/N ratio	Chicken	10	8.85	9.05 a	10.82	0.002	1.89	6.5	11.5
C/N ratio	Cow	2	27.90	27.90 b			16.97	15.9	39.9
C/N ratio	DDF	3	11.60	11.98 a			2.01	10.19	14.16
P (%)	Chicken	10	1.21	1.22 b	70.02	<0.001	0.18	0.97	1.56
P (%)	Cow	2	0.105	0.11 a			0.04	0.08	0.13
P (%)	DDF	3	0.16	0.20 a			0.08	0.15	0.3
K (%)	Chicken	10	3.71	3.82 ns	3.82	0.052	1.43	2.22	6.98
K (%)	Cow	2	0.39	0.39 ns			0.23	0.23	0.55
K (%)	DDF	3	0.58	2.13 ns			2.93	0.30	5.50

Note: The same lower letters in the mean of individual organic amendments were not significantly different with various p values. The ns indicated that there was no statistical significance.

). For carbon content, chicken manure had the highest average, followed by cow manure and DDG. These differences were highly significant (p < 0.001), indicating that chicken manure contains much more carbon than the other two amendments. In terms of nitrogen, chicken manure again had the highest, while both cow manure and DDGs had almost the same very low values, with significant differences confirmed by a p-value of less than 0.001 (Table 2). The C/N ratio significantly differed among the amendments (p = 0.002), with cow manure having the highest ratio, followed by DDGs and chicken manure. Phosphorus content also showed highly significant differences (p < 0.001), with chicken manure containing much more phosphorus than cow manure and DDGs. However, potassium content did not differ significantly among the amendments (p = 0.052) (Table 2).

Nutrient and chemical properties of water extraction from organic fertilizers: The results showed significant differences in soluble carbon content among amendments, with chicken manure having a higher mean value of 613.13 mg kg⁻¹ compared to cow and DDG. Chicken manure also had the highest mean value of 50.04 mg kg⁻¹ (

Table 3. The nutrient concentration and chemical properties of chicken manure, cow manure, and dried distilled grains (DDG) via water extraction.

Indicator	Organic Amendments	Number of Sample	Mean	F-Value	p-Value	SD	SE	Minimum	Maximum
soluble C (mg kg−1)	Chicken	4	613.13 b	12,134.89	<0.001	9.44	4.72	600.00	622.50
	Cow	4	16.88 a			3.75	1.88	15.00	22.50
	DDG	4	16.88 a			3.75	1.88	15.00	22.50
H2PO4 (mg kg−1)	Chicken	4	50.04 c	12,425.43	<0.001	0.74	0.37	49.18	50.97
	Cow	4	7.69 b			0.20	0.10	7.45	7.93
	DDG	4	5.38 a			0.16	0.08	5.18	5.56
NH4+ (mg kg−1)	Chicken	4	25.93 a	29.51	<0.001	7.75	3.87	16.30	35.20
	Cow	4	61.25 b			10.00	5.00	48.60	71.00
	DDG	4	25.83 a			3.10	1.55	21.40	28.50
NO3− (mg kg−1)	Chicken	4	338.10 ns	0.84	0.461	19.03	9.52	321.29	364.87
	Cow	4	351.18 ns			9.27	4.63	338.72	361.14
	DDG	4	338.10 ns			19.03	9.52	321.29	364.87
pH	Chicken	4	7.06 ns	0.68	0.528	0.02	0.01	7.03	7.08
	Cow	4	7.16 ns			0.23	0.11	7.03	7.50
	DDG	4	7.08 ns			0.03	0.01	7.05	7.11

Note: The same lower letters in the mean of individual organic amendments were not significantly different with various p values. SE and SD were represented as standard errors and standard deviations, respectively. The ns indicated that there was no statistical significance.

), suggesting it is a richer immediate source of plant-available phosphorus. Ammonium content showed a reversed trend, with cow manure having the highest value at 61.25 mg kg⁻¹. Nitrate content did not differ significantly among the three treatments, with mean values around 338 to 351 mg kg⁻¹ (Table 3). pH values showed no statistically significant differences between amendments, with chicken manure at 7.055, cow manure at 7.16, and DDG at 7.08b (Table 3).

3.2. Organic Fertilization’s Effects on the Soil’s Physical Properties, Various Carbon Fractions, and N and P Changes

The distribution of soil texture components—sand, silt, and clay—varied across different land-use ages (LUAs), indicating changes in land-use intensity over time. Sand content varies across different LUAs, with LUA 4 having the highest sand content (858.708 g kg⁻¹), followed by LUA 3 and LUA 5 (

Table 4. The distribution of sand, silt, and clay particles measured in grams per kilogram (g kg⁻¹) across different land-use ages.

Particle Size (g kg−1)	LUA	Valid	Mean	F-Value	p-Value	Std. Error	Std. Deviation	Minimum	Maximum
Sand	0	108	821.6 a	6.576	<0.001	6.1	63.4	661.6	921.6
Sand	1	192	836.7 ab			5.3	72.9	661.6	961.6
Sand	2	135	817.9 a			5.5	63.6	681.6	921.6
Sand	3	197	845.9 b			3.9	55.4	681.6	981.6
Sand	4	83	858.7 b			5.9	54.1	741.6	961.6
Sand	5	38	845.4 ab			10.4	64.2	721.6	941.6
Silt	0	108	98.9 ab	F = 3.690	p = 0.003	5.4	55.8	5.6	245.6
Silt	1	192	97.7 a			4.2	57.7	25.6	245.6
Silt	2	135	105.3 b			4.4	50.8	25.6	225.6
Silt	3	197	90.0 ab			3.3	45.9	5.6	205.6
Silt	4	83	78.4 a			6.0	55.0	5.6	205.6
Silt	5	38	81.9 ab			8.3	51.3	25.6	185.6
Clay	0	108	79.5 b	F = 3.422	p = 0.004	4.5	46.9	12.8	172.8
Clay	1	192	65.6 ab			3.2	44.0	12.8	192.8
Clay	2	135	76.8 ab			3.5	40.7	12.8	152.8
Clay	3	197	64.2 a			2.8	38.9	12.8	132.8
Clay	4	83	62.9 a			3.6	32.8	12.8	132.8
Clay	5	38	72.8 ab			6.7	41.1	12.8	132.8

Note: The same lower letters on the individual organic amendment were not significantly different with various p values.

). The lowest sand content was 817.896 g kg⁻¹. Silt content also varies, with LUA 2 having the highest mean value (85.240 g kg⁻¹), while LUA 5 has the lowest (81.916 g kg⁻¹). Clay content also varies across LUA ages, with the highest recorded at LUA 0 (79.467 g kg⁻¹). This meant that sand dominated the soil composition or coarse soil (Table 4).

The soil organic carbon (SOC) content significantly increased after one year of organic fertilizer application, rising from 20.24 g kg⁻¹ (pre-fertilization) to 25.49 g kg⁻¹ (F = 2.599, p = 0.026). However, continued organic fertilizer application resulted in a gradual decline in SOC levels, though they remained higher than the initial values. SOC peaked in the first year, increasing by 25.93% compared with the pre-fertilization levels, followed by a slight decline in subsequent years. However, these were not significant compared with pre-fertilization (Figure 2).

Carbon fractions: The water-soluble carbon (WSC) content varied significantly across LUAs (F = 2.468, p = 0.031), with the highest levels being observed in LUA 3. WSC increased by 12.01–22.64% but showed no significant differences compared with the pre-fertilization soil levels. Apart from the fifth year, the WSC content remained consistently higher than the pre-fertilization levels (Figure 3A). Permanganate-oxidizable carbon (POXC) also exhibited notable changes over time (F = 3.807, p = 0.002). Initially, POXC significantly increased in response to organic fertilizer application but began to non-significantly decline in the second year compared with LUA 0 (Figure 3B).

The labile carbon (LBC) fraction was at its lowest before fertilization, significantly peaked at LUA 3 (F = 3.913, p = 0.002), and did not significantly decrease at LUA 4–5 compared with LUA 0. The amount of less labile carbon (LLBC) showed significant differences (F = 4.011, p = 0.001) and increased by 4.30–12.67% after adding organic fertilizer, peaking at an LUA 3–4 before decreasing at an LUA 5; however, it was still about 5.09% higher than LUA 0 (Figure 4A,B).

The non-labile carbon (NLBC) fraction varied significantly across LUAs (F = 3.468, p = 0.004). The highest NLBC levels were recorded in the first year, showing an increase of over 60% compared with pre-fertilization. However, from the second year onward, the NLBC levels gradually declined (31.57–179.52%) (Figure 5A). Recalcitrant carbon (REC) exhibited significant variations (F = 3.003, p = 0.011), with the concentrations steadily decreasing (32.83–224.71%) following organic fertilization (Figure 5B). However, these were not significant compared with pre-fertilization.

Availability of nitrogen: Following organic fertilizer application, the soil ammonium (NH₄⁺) concentrations initially decreased, showing a downward trend in the first two years but with no significant recovery by the fifth year. Nitrate (NO₃⁻) levels declined significantly across LUAs (F = 2.677, p = 0.021), particularly in the last three years, remaining lower than the pre-fertilization levels (Figure 6A,B). The NO₃⁻ levels originally peaked during the second year of organic fertilizer treatment but exhibited a declining trend thereafter, whereas the NH₄⁺ concentrations remained comparatively steady. While NH₄⁺ concentrations remained relatively low, NO₃⁻ levels fell following the third year of organic fertilizer application and exhibited a declining trend thereafter (Figure 6A,B). The NH₄⁺-N levels dropped by 3.15–4.94% after LUA 0. However, all the NH₄⁺-N concentrations after fertilizer addition did not differ from NH₄⁺-N in LUA 0. After applying organic fertilizer, the amount of NO₃⁻-N in the soil went down (1.45–18.27%) in most LUAs, but it went up in LUA 2 (2.30%) (Figure 6A,B). However, our results did not show a significant difference before and after fertilization.

pH and availability of and P: Figure 7A Bray-extractable phosphorus (P(B)) showed a significant difference among the different LUAs (F = 5.699, p < 0.001), At LUA 1, 3, and 4, P(B) concentrations were lower (16.38–22.77%) compared with pre-fertilizer. Figure 7B shows the malachite-extractable phosphorus (P(M)), which did not show significant differences among the different LUAs (F = 0.887, p = 0.489), and the trend of P(M) compared with pre-fertilization is demonstrated. Figure 7C indicates that soil pH also showed significant variation among the different LUAs (F = 3.171, p = 0.008). Therefore, the pH levels fell (1.17–6.68%) after the application of organic fertilizer, particularly in the second year.

3.3. The Relationship Between Soil Carbon Fractions and Some of the Effects of the Availability of Nutrients on Carbon Stability

The relationships between labile and stable carbon fractions and SOC:

The relationships between carbon fractions (Figure 8) showed that NLBC and POXC were positively related to SOC. This was demonstrated by Pearson’s (r) coefficients of 0.54 *** and 0.49 ***, respectively. However, neither LBC nor LLBC showed any significant correlations with SOC. We found strong positive links between SOC and carbon fractions such as NLBC (r = 0.54 ***) and POXC (r = 0.49 ***), as well as positive correlations between POXC and NLBC (r = 0.32 ***). Additionally, we observed strong positive correlations between LBC and LLBC (r = 0.94 ***). There were negative correlations between LBC, LLBC, and NLBC (r = −0.62 *** and −0.62 ***, respectively) and between LBC, LLBC, and REC (r = −0.74 *** and −0.79 ***, respectively) (Figure 8). Together, these images show how different parts of the soil carbon interact with each other. They show how different parts affect soil fertility and soil organic carbon cycling. They also show different trends in soil organic carbon.

The relationships between the labile and stable carbon fractions with NH₄⁺, and NO₃⁻: The strongest positive correlation was observed between POXC and SOC (r = 0.488 ***), reinforcing POXC as a key indicator of organic matter content. Conversely, a negative correlation was observed between POXC and NH₄⁺ (r = −0.244 ***) and a positive correlation between POXC and NO₃⁻ (r = 0.095 **), suggesting that microbial activity led to a reduction in ammonium (Figure 8). Similarly, LBC showed negative correlations with NH₄⁺ (r = −0.184 ***) and NO₃⁻ (r = −0.074 *), while LLBC showed negative correlations with NH₄⁺ and NLBC at −0.216 *** and −0.621 ***, respectively. On the other hand, NLBC showed positive correlations with NH₄⁺ (r = 0.092 **) and NO₃⁻ (r = 0.105 **), and REC showed positive correlations with NH₄⁺ (r = 0.15 ***) and NO3- (r = 0.104 **) (Figure 8). These results underscore the significance of labile carbon (as LBC, LLBC, and POXC) and recalcitrant carbon fractions (as NLBC) in monitoring alterations in soil organic matter, as they are associated with the concentrations of NH₄⁺ and NO₃⁻ (Figure 8). Negative correlations were observed between NH₄⁺ and NO₃⁻ with labile carbon types, such as LBC, LLBC, and POXC, while positive correlations were noted with stable carbon fractions, such as NLBC, and REC.

The relationships between the carbon fractions and availability of P and pH in relation to SOC content: A correlation matrix illustrating the relationships among soil chemical properties, including the labile carbon fractions, phosphorus (P), and pH, is presented. The correlation matrix presents pairwise correlation coefficients, with blue indicating positive and red indicating negative correlations. Negative correlations were observed for LBC with P (B) and P(M) (r = −0.079 * and r = −0.099 **, respectively), while LLBC was negatively correlated with P(B) and P(M) (r = −0.095 ** and r = −0.101 **, respectively) (Figure 9). The findings indicate a relationship between LBC and LLBC, since both represent significant forms of labile carbon that have access to phosphorus. Comprehending these interactions is essential for regulating soil organic matter and nutrient cycling in the organic fertilization of sandy soil.

4. Discussion

4.1. The Carbon Fractions with the Combinations of Animal Manure with DDGs

Soil carbon is a crucial component of soil health that influences fertility, structure, and water retention. In organic farming systems, particularly in cassava (Manihot esculenta) cultivation in tropical sandy soils, the use of cassava ethanol residue (DDGs) as a soil amendment has gained interest due to its potential benefits for soil quality and agricultural productivity. Our results indicate that a combination of organic fertilizer and DDGs provided essential soil nutrients, particularly NH₄⁺, NO₃⁻, H₂PO₄⁻, while maintaining a neutral pH, which is beneficial for organic cassava cultivation in low-yield environments such as tropical sandy soils [32]. DDG amendments also significantly affected soil carbon concentrations in sandy soils, with higher carbon accumulation being observed in the studied soils following supplementation with DDG [62]. Furthermore, the addition of 20% DDGS from corn reduced the C:N ratio of potting mix from 90:1 to 24:1 and soil from 23:1 to 10:1. Microbial respiration doubled in potting mix and soil at 3 and 14 days due to the lower C:N ratio [31]. Our study found that the soil carbon content was 43.6 g kg⁻¹, while the water-soluble carbon content was 0.113 g kg⁻¹. Zhang et al. [63] reported a much higher carbon content in DDGs, reaching up to 470 g kg⁻¹. The lower carbon content in our study may be attributed to the reduction in volatile organic compounds under open-field conditions.

The use of organic fertilizer, such as composted materials or high-quality organic amendments, typically results in more stable soil carbon levels [64,65]. Various organic amendments, such as pig manure and corn straw, have different impacts on soil organic carbon (SOC) stability, with higher-quality materials generally leading to increased SOC accumulation [25]. These effects are particularly pronounced in systems receiving high-nitrogen inputs from organic amendments [66]. Manure and other organic amendments significantly enhance SOC, including various carbon fractions such as loss-on-ignition carbon, Walkley–Black carbon, permanganate-oxidizable carbon (POXC), and microbial biomass carbon (MBC) [18]. Additionally, manure improves specific forms of soil carbon, including cold- and hot-water-extractable carbon, microbial biomass carbon, and dissolved organic carbon [67]. Doubling the application rate of organic fertilizers increases different carbon fractions, including light-fraction carbon, particulate organic matter, and mobile humic acid [18]. These fractions are more sensitive to changes in soil management practices compared with total organic carbon [11]. Manure, in particular, has been shown to be more effective than chemical fertilizers in maintaining and enhancing labile carbon pools [68]. The application of organic fertilizers also boosts soil enzyme activities, which are linked to the decomposition of organic matter and nutrient cycling. This rise in enzyme activity further helps to improve labile carbon fractions [15,69]. Farmyard manure, in particular, significantly increases labile carbon in soils, enhancing microbial biomass and enzyme activities, with application rates influencing the size of labile carbon pools [15,16].

According to other studies, long-term organic farming has been shown to promote carbon stabilization, with increases in both labile and recalcitrant SOC pools [70]. The presence of stable and labile fractions in soil is closely linked to the ability of organic fertilizers to retain and regulate carbon [71]. Organic additives such as farmyard manure and compost enhance soil fertility by maintaining a balance between labile and recalcitrant carbon pools [69]. The progressive decomposition of organic materials, which is facilitated by enzymatic activity, enables prolonged carbon sequestration over time [72] and influences the soil structure, increasing macroaggregate formation and improving overall soil stability [18]. The ability of organic fertilizers to improve SOC stabilization is particularly relevant in sandy soils, where organic matter is more susceptible to decomposition and loss [11]. A significant increase in SOC levels within only the first year suggests that organic fertilizers’ initial benefits may diminish without continuous management [20]. Therefore, using a mix of organic materials like chicken and cow manure and DDG could boost the labile and stable carbon fractions in sandy soils, and the presence and alternation of the stable and labile fractions in soil are closely linked to the ability of organic fertilizers to retain and regulate carbon in soil [71], especially within the first year.

4.2. Relationship Between the Soil Carbon Fraction and SOC According to the Mechanisms of Decomposition and Stabilization

Our research showed that there was not much labile carbon at first before applying organic fertilizer. This study demonstrated that the application of mixed organic fertilizer and DDGs initially increased both the labile and stable carbon fractions within the first one to three years LUA3, but a decline was observed in the fourth and fifth years, suggesting that the fertility boost may have been temporary. We showed that there were strong positive links between SOC and carbon fractions such as NLBC. However, neither LBC nor LLBC demonstrated any significant correlations with SOC.

Carbon mineralization in agricultural soils is significantly accelerated by the introduction of exogenous organic carbon. Organic fertilizers promote microbial biomass growth, enhancing carbon mineralization and turnover rates [8]. This microbial activity is crucial for maintaining soil fertility [73], with the presence of non-dominant bacterial species influencing carbon mineralization processes [73]. The labile carbon fraction is essential in enhancing microbial activity, enzyme production, and carbon turnover [11,16]. According to Bongiorno [74] et al. (2019), permanganate-oxidizable carbon (POXC), a type of labile carbon, changed the most quickly in the first year. The addition of labile carbon from organic fertilization can stimulate the decomposition of more stable soil organic carbon (SOC) by enhancing microbial activity. This is because labile carbon serves as an energy source for microbes, which in turn accelerates the breakdown of more resistant carbon compounds in the soil [75]. Labile carbon inputs from mixed organic fertilization could alter the composition and activity of soil microbial communities. For instance, they can increase the abundance and activity of bacteria and fungi, which are crucial for the decomposition process. This microbial response is often independent of microbial growth dynamics, suggesting that extracellular enzyme activity plays a significant role in priming [76]. The priming effect and subsequent mineralization can be influenced by temperature. The impact of labile carbon on decomposition varies with its concentration. Low concentrations can stimulate decomposition, while high concentrations may inhibit it due to microbial substrate preference or negative priming effects [77]. Frequent inputs of labile carbon can lead to stronger priming effects compared to infrequent inputs. This is due to continuous stimulation of microbial activity and enzyme production, which enhances SOM decomposition [76]. Moreover, in our study of tropical soil, the high temperatures could enhance the priming effect, leading to increased CO₂ production from SOC decomposition [77]. However, other types, such as water-soluble carbon (WSC), the labile carbon fraction (LBC), and the less labile carbon fraction (LLBC), changed more slowly and over time from this study. This also triggers catabolic responses in microbial communities, shifting soil organic matter decomposition towards nitrogen-rich components [78]. In addition, in our study, adding labile carbon from organic fertilization over and over again in sandy soils (figuratively, more than once) because there might be fewer priming effects than adding it all at once, especially in soils that were not very fertile [79], which showed that this effect led to a trend in higher SOC compared with the pre-fertilization level that found in the LUA 5.

These more labile carbon fractions are preferentially decomposed by soil microbes in the beginning, leaving behind more recalcitrant carbon [11,18]. Conversely, organic fertilizers also improve the protection of labile carbon against biodegradation by integrating it into stable humic substances, particularly when mature compost or humic acids are applied [14]. This stabilization process further enhances long-term soil carbon retention. However, the sensitivity of this effect varies among different soils and is influenced by factors such as soil fertility and microbial biomass [80]. We would like to consider that the magnitude of the priming effect can vary depending on factors such as the type of labile carbon, the presence of other organic inputs, and environmental conditions [81].

While organic fertilization can enhance SOC accumulation in the short term, it may also decrease the stability of SOC over time, particularly in surface soil layers [67]. Although organic fertilization can improve carbon stocks in the soil, the reduction in recalcitrant carbon fractions may affect the long-term carbon sequestration potential [82]. However, the type and quantity of organic fertilizers should be carefully managed to avoid potential negative impacts on soil carbon stability [16]. According to the decrease in recalcitrant carbon and the type organic fertilizer [83,84], continuous application without proper practices may decrease the recalcitrant fractions due to increased microbial activity and organic matter turnover [82].

Although our study observed a small increase in non-labile carbon (NLBC) after only one year, the contents then non significantly decreased over 3–4 years later. This reduction in recalcitrant carbon fractions due to organic fertilization leads to increased soil respiration rates, as more carbon is mineralized and released as CO₂ [15]. The increased microbial activity enhances the breakdown of stable carbon compounds, thereby reducing the recalcitrance of soil organic carbon [25], such as cellulase and β-glucosidase, facilitating the decomposition of complex carbon compounds, and leading to a decrease in recalcitrant carbon fractions [79]. Organic fertilization alters the chemical composition of SOC by increasing labile carbon fractions (e.g., O-alkyl C) and reducing stable carbon fractions (e.g., aromatic C). This shift in carbon chemistry makes the carbon more accessible to microbial degradation [67]. Therefore, organic fertilization could increase soil microbial activity, leading to soil decomposition and mineralization. This reduces recalcitrant carbon pools, converting more carbon into labile forms [83,84]. Our research showed that organic fertilizers, such as manure mixed with DGG, did not significantly change stable carbon fractions, but increased soil microbial activity via these carbon fractions.

Our results showed positive relationships of POXC with LBC and NLBC, which is considered the most sensitive labile carbon fraction in organic fertilizer in sandy soil. POXC provides insights into the total organic matter content, nutrient availability, soil structure, and microbial biomass activity [74]. POXC is used to measure labile carbon in soils, as it is more sensitive to management practices and environmental changes than recalcitrant carbon [85]. It is closely related to other labile carbon fractions, such as particulate organic carbon (POC) and microbial biomass carbon [74]. POXC is not a purely labile carbon fraction, as it shows high reactivity with lignin, a more complex and less labile compound, challenging the assumption that it only measures labile carbon [86]. POXC’s composition aligns with that of labile carbon [86], challenging the assumption that it only measures it. This study shows that using mixed organic fertilizers greatly affected soil carbon types, especially by boosting soil microbes’ activities due to the presence of easily available carbon while keeping the amount of stable carbon the same. From this study, LUA 5 shows that the accumulation of both labile and recalcitrant carbon pools enhances soil carbon content. The impact of labile carbon on decomposition varies with its concentration. Low concentrations could stimulate decomposition, as mentioned previously, while high concentrations may inhibit it due to microbial substrate preference or negative priming effects [77]. While NLBC is recalcitrant organic carbon, characterized by its resistance to decomposition, it contributes to long-term carbon storage in soils. This part includes substances like alkyl carbon, which are stable and do not break down easily because of their chemical structure [87]. Moreover, the presence of recalcitrant carbon enhances soil aggregation, particularly macro-aggregation, which is vital for SOC stabilization. This is because recalcitrant carbon parts, like methoxyl/N-alkyl C, help create stable soil clumps, which protect SOC from being broken down by microbes [88]. Therefore, using a mix of organic materials from this study could increase both of the carbon fractions, especially LBC and NLBC components, in sandy soils. These amendments boost/maintain the amount of SOC by helping to build up stable carbon parts, which are important for storing carbon over a long time. We observed shifts in the labile and non-labile carbon fractions, highlighting the need for strategic organic fertilizer application to sustain soil health and carbon sequestration. We suggested that the addition of LBC from mixed organic fertilization seems to enhance SOC decomposition more than accumulation. NLCB may persist in sandy soil for a longer duration than LBC, resulting in the retention of SOC in the sandy soil of this study.

4.3. The Relationship Between Carbon Fractions and Available Nitrogen and Its Effect on Soil Carbon in Sandy Soil

Sandy soils, owing to their unique texture and structure, exhibit distinct patterns of nitrogen mineralization compared with other soil types [34,35]. Soil organic matter serves as a primary nitrogen reservoir, releasing nitrogen through mineralization, which is essential for plant uptake. The conversion of NH₄⁺ into NO₃⁻ via nitrification is enhanced by organic fertilizers, leading to an initial increase in NO₃⁻ levels [89,90]. After adding organic fertilizer, there were NH₄⁺ and NO₃⁻ received from direct organic fertilization (Table 2 and Table 3), and NH₄⁺ might be rapidly converted into NO₃⁻ through nitrification, a process that was often more pronounced with organic fertilizer application [9]. However, there was no significant difference between before and after fertilization, and the addition of fertilizer caused a decrease in NH₄⁺ and NO₃⁻ levels from this.

On the other hand, adding labile carbon speeds the breakdown of SOM [91]. Depending on microbial community responses and nitrogen availability, labile carbon can either enhance or inhibit SOM mineralization [92]. We considered that organic fertilizers may not have significantly high NH₄⁺ levels if the mineralization of organic matter primarily results in NO₃⁻ production rather than NH₄⁺ accumulation [93]. The negative correlation between labile carbon fractions (LBC and LLBC) and NH₄⁺ in our study further supports the negative correlation between NH₄⁺ and NO₃⁻ levels (Figure 8), and shows how the levels of NH₄⁺ and NO₃⁻ interact with these labile carbon fractions, which then affect SOC (Figure 8). The mineralization of organic nitrogen is often decoupled from carbon in these fractions, meaning that nitrogen availability does not always correlate directly with carbon content [37]. The addition of labile carbon through organic fertilizer applications significantly alters nitrification and nitrogen mineralization. Labile carbon sources, such as glucose, can significantly reduce NH₄⁺-N and NO₃⁻-N concentrations in soils due to increased microbial activity [94]. The presence of labile organic carbon can regulate nitrification rates. High-quality labile carbon sources, such as glucose, can inhibit nitrification by outcompeting nitrifiers for NH₄⁺, thereby reducing nitrogen availability for nitrification [95]. Furthermore, labile carbon can induce priming effects, altering organic matter decomposition rates. These effects depend on the carbon-to-nitrogen (C:N) ratio of inputs and vary depending on whether NO₃⁻ or NH₄⁺ is the dominant nitrogen form [91]. Our study found a negative relationship between labile carbon fractions (e.g., LBC and POXC) and NH₄⁺, as well as between NH₄⁺ and NO₃⁻. This suggests that nitrifying bacteria utilize labile carbon as an energy source, independently of soil nitrogen availability [94]. This research has demonstrated that increasing labile carbon levels can reduce the concentrations of NH₄⁺ and NO₃⁻ in soils, indicating a crucial threshold at which nitrogen transformations shift [38,94]. The priming effect can vary based on the quality of the carbon added [96]. However, labile fractions are more readily available to microbial communities, stimulating nitrogen mineralization [36]. This is because these fractions serve as a substrate for microbial activity, accelerating nitrogen cycling [37,97]. Therefore, the amount of labile carbon fractions might be a partial factor controlling the nitrogen mineralization in this study.

We observed a positive relationship between NH₄⁺ and NLBC. (Figure 8). The presence of recalcitrant carbon can lead to shifts in microbial community composition, favoring organisms capable of utilizing these carbon sources. This adaptation can enhance nitrification processes by maintaining microbial activity and enzyme production [20]. We agree that nitrifiers can modify the composition of organic matter by breaking down recalcitrant compounds. This is facilitated by the production of organic compounds, as this helps degrade stable materials, particularly in carbon-limited environments [98]. Low-quality recalcitrant carbon leads to a delayed effect [96]. This delayed effect is associated with the decomposition of recalcitrant carbon compounds, which can influence microbial strategies and enzyme activities [96]. Moreover, the recalcitrant carbon fraction can stimulate heterotrophic nitrification in soils [20], which is consistent with our results (Figure 8). This is because recalcitrant carbon fractions, such as carbonyl and aromatic carbon, are positively correlated with nitrification rates, especially in acidic soils [20]. The presence of recalcitrant carbon can sustain microbial activity over longer periods, potentially enhancing nitrification [20]. Recalcitrant carbon influences the microbial community composition, which, in turn, affects nitrification. It has been shown that adding biochar to soil can increase the number of heterotrophic nitrifiers, such as Trichoderma, Aspergillus, and Penicillium. These organisms are known to help with the nitrification process [99]. Nitrifiers can change the constituents of organic matter by helping to break down aromatic compounds that are difficult to break down. This process is important because nitrifiers help the cycling of organic carbon and nitrogenous compounds in places such as the aphotic ocean layer, where energy and carbon sources are limited [98]. While recalcitrant carbon can initially boost nitrification rates, the long-term sustainability of this effect is uncertain. The stimulatory impact of carbon amendments tends to decrease as the availability of labile carbon declines. In the future, scientists should investigate ways to keep nitrification rates high for long periods of time and how microbial communities might change over time [100]. Conversely, Aumtong [101] et al. (2024) assessed that when soil had low nitrogen input, it led to less nitrogen being available; the scarcity of soil N would hasten the breakdown of recalcitrant C. However, the labile carbon fractions (as LBC) would not break down as fast through “nitrogen mining”.

The availability of these nutrients can modulate the intensity of the priming effect and the rate of nitrification [102]. The priming effect via recalcitrant carbon fraction and subsequent nitrification is also influenced by nutrient availability, particularly that of N. This study highlights the negative relationship between labile carbon fractions and nitrification processes in sandy soils with the application of organic fertilizer by altering the balance of NO₃⁻ and NH₄⁺, while there is a positive relationship between the recalcitrant carbon fraction and the availability of nitrogen. Then, the recalcitrant carbon fraction can be changed by the activity of microbes and the production of enzymes, which, in turn, changes the priming effect [91], i.e., the amounts of NLBC and REC would be decreased, and they might be converted into the LBC fraction later, as mentioned previously in this study.

4.4. The Relationship Between the Carbon Fraction and P in Sandy Soil

Following organic fertilization, the soil’s availability of P in both methods changed because it changes the organic carbon content [103]. This raises the soil’s Olsen–P and biological-based P fractions, making it easier for plants to take up [104]. Organic fertilizers also change the physical and chemical properties of the soil and organic carbon levels [105] and increase the number of microbes in the soil [106]. The availability and transformation of P in sandy soils are influenced by factors such as soil pH, organic carbon content, and the presence of basic cations. These factors play a critical role in determining the lability of P and its interactions with labile carbon fractions [107]. Certainly, increased soil organic matter from organic fertilization can reduce P fixation by blocking P sorption sites on soil minerals, thus, enhancing long-term P availability [108]. Additionally, the presence of labile carbon can influence soil pH, which, in turn, affects P solubility. Our study showed that P availability varied with the application of organic fertilizer, but the P levels in fresh soil (i.e., Malachite method) remained constant. This leads to enhanced growth and activity of P-solubilizing microorganisms, thereby improving soil P availability [85]. This may indicate heterogeneity in the availability or retention of P in the soil after a combination of organic fertilizers. Our study demonstrates that organic fertilization leads to a decrease in soil pH, especially in LUA 2, primarily due to the acidifying effects of organic matter decomposition and the release of organic acids.

Changes in soil pH due to organic fertilization can further affect soil microbial community composition, impacting nutrient cycling and overall soil health [109]. Organic fertilizers typically involve nitrogen inputs that undergo nitrification, converting ammonium into nitrate, releasing hydrogen ions, and gradually lowering soil pH over time [110]. The decomposition of organic matter by soil microorganisms results in the production of organic acids, which contribute to soil acidification. This effect is particularly pronounced in soils with high microbial activity, where the breakdown of organic fertilizers leads to the release of acids that lower soil pH [111]. The relationship between soil organic carbon and pH is complex, as organic fertilizers increase SOC levels, which, in turn, influence soil pH. The interaction between SOC and microbial communities has been reported to drive pH fluctuations, as organic amendments alter bacterial community structures and soil biochemical processes [109]. Over time, mixed continuous organic fertilizer application, particularly when combined with other fertilization strategies, can lead to substantial alterations in soil chemical properties, including persistent pH reduction [112], which would be a concern, particularly in sandy soil. However, this study found that higher LBC and NLBC are linked to more phosphorus being available in sandy soil, highlighting how important phosphorus is in this soil, as it slows down the breakdown of soil carbon fraction (i.e., LBC), lowers phosphorus fixation, and might improve long-term phosphorus availability.

Figure 10 illustrates the complex interactions between different forms of soil carbon, nutrient cycling, and the organic fertilizer application of this study. When mixing organic fertilizer is applied, it can lead to an increase and retention of NLBCs, which are a more stable form of carbon that remains in the soil longer. The presence of NLBC contributes to an increase in SOC, which is essential for soil structure, fertility, and microbial activity. NLBC also plays a crucial role in enhancing nitrification—the biological process that converts ammonium (NH₄⁺) into nitrate (NO₃⁻) by supporting microbial communities through the provision of carbon sources and stimulating enzyme production. Furthermore, NLBC helps increase phosphorus (P) availability by reducing its fixation, making it more accessible to plants. As a result, NLBC promotes both nutrient availability and microbial efficiency (Figure 10).

When mixing organic fertilizer is applied, it can lead to an increase of LBC. Labile carbon can enhance microbial activity, accelerating the breakdown of SOC and NLBC and affecting soil microbial dynamics. It can increase the abundance of bacteria and fungi, crucial for decomposition, and can influence temperature sensitivity. This suggests that extracellular enzyme activity plays a significant role in priming. However, problems arise when the level of LBC becomes too high. LBC, being a more reactive and short-lived carbon source, could enhance or inhibit nitrification by creating conditions that reduce the transformation of NH₄⁺ and NO₃⁻. However, the inhibition of nitrification slows down microbial processing and might induce the more decomposition of NLBC, which is counterproductive to soil carbon retention.

Moreover, in the acidic soil, reduced nitrification also contributes to lower available phosphorus and increases soil acidity due to shifts in microbial activity and chemical balances. However, the presence of LBC might prevent phosphorus fixation in acidic soils (Figure 10). To sustain soil fertility and carbon stocks, a balanced approach is required, mixing organic fertilizers in a way that promotes NLBC buildup while preventing the inhibitory effects of too much LBC. We must carefully manage the organic fertilization approach (i.e., timing, organic fertilizer quality, and environmental conditions) to support a healthy carbon-nutrient cycle in these agroecosystems.

This study showed that organic fertilizers, such as combinations of compost, local manure, and DDGs, have a significant effect on both the quantity and stability of SOC. The implications of this study are as follows: Our findings indicate that a routine soil testing framework should be implemented to monitor changes in carbon fractions and nutrient levels over time. Using the right mix of both labile and stable carbon types, along with enough nutrients like NH₄⁺-N, NO₃⁻-N, and P, makes the soil healthier and helps it hold carbon better when using the right organic fertilizers. This method works especially well in sandy soils. We consider complementing this with other mechanisms, such as cations (i.e., Ca²⁺, Mg²⁺, K⁺), and enhancing the soil structure in these situations. According to Jiang [113] et al. (2019), soil organic carbon pools generally show stable characteristics, including reduced carbon emissions and enhanced sequestration. Notably, the NRLB and REC fractions, which are stable carbon components, exhibited the opposite trend in comparison with the labile carbon fractions (i.e., ratio between stable and labile carbon). This study demonstrated that soil texture could explain the lack of an increase in soil carbon after applying organic fertilizer in sandy soils. The sandy soils (Table 4) have a low carbon storage potential due to their coarse texture [114], which could offset the gains in SOC from organic amendments [115]. Understanding the long-term effects of these organic amendments on the stable fraction and SOC stability will be crucial for developing sustainable soil management strategies.

In the future, these relationships should be used to develop organic fertilizer management strategies and to balance soil carbon and nitrogen dynamics. Thus, additional research should focus on optimizing the application rates and combinations of organic amendments to maximize soil health and agricultural productivity. Understanding these interactions is essential for predicting soil carbon fractions, SOC turnover, and nitrogen transformation, all of which have significant implications for organic fertilization in sandy soil. This will help maximize nitrogen availability, thus, ultimately improving soil sustainability in sandy environments. Moreover, other key factors, including cations such as Ca and Mg, are likely involved in explaining soil carbon stabilization in this soil.

5. Conclusions

This research has shown that using organic fertilizers, like local manure (such as chicken and cow) and DDGs, greatly affects the quantity and stability of soil organic carbon fractions, and as a result, the overall SOC was compared between before and after using. The ramifications of this research are as follows: The SOC level did not change with time, as these results found that over the year, the application of organic fertilizer significantly increased SOC, reaching a peak of 25.93% in only the first year LUA 3 showed the highest in WSC; POXC went up in the first year after adding organic fertilizer but started to drop in the second year, and then their amounts decreased in the other LUAs. The LBC fraction was at its lowest before fertilization, peaking at LUA 3 and increasing by 5.44–25.50% after organic fertilizer addition, and then decreasing for LBC and LLBC at LUA 4 and 5. Notably, these labile carbon fractions were significantly higher than pre-fertilization. However, there were no significant differences at other times. The first year revealed high NLBC levels, exceeded 60% in comparison to the pre-fertilizer period. In the second year, NLBC levels declined to LUA 5, a change that was not statistically significant. After pre-fertilization, the concentration of REC did not significantly decrease. Organic fertilization decreased NO₃⁻-N concentrations; nevertheless, no significant variation was seen before and after fertilization. Moreover, the Bray-extractable phosphorus (P(B)) was reduced (i.e., LUA 1 and 4). The pH levels decreased (1.17–6.68%) after the application of organic fertilizer, especially in the second year. Strong positive correlations were identified between SOC and carbon fractions, specifically NLBC with a correlation coefficient of r = 0.54 *** and POXC with r = 0.49 ***. Neither LBC nor LLBC exhibited significant correlations with SOC. Negative correlations were observed between NH₄⁺ and NO₃⁻ with labile carbon types, including LBC, LLBC, and POXC. In contrast, positive correlations were noted with stable carbon fractions, such as NLBC and REC. This study showed that continuous five-year application of organic fertilizers did not significantly increase soil carbon; however, it was able to cause changes in various organic carbon fractions. The presence of both LBC and NLBC fractions would support microbial communities by providing carbon sources and stimulating enzyme production. In particular, NLBC, a stable form of soil carbon, contributes to SOC content and enhances nitrification. However, we suggest that the addition of LBC from mixed organic fertilization appears to increase SOC decomposition more than accumulation. While NLCB may remain in sandy soil longer than LBC, it leads to the remainder of SOC in sandy soil. Moreover, using mixed organic fertilization might enhance the acidity of sandy soils.