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Sep. 06, 2025 





 
 
 
 
 
 
 

 
 
 
 

 

MARIA GABRIELA DE OLIVEIRA ANDRADE 

 

 

 

 

 

 

 
INTERAÇÃO DA CORREÇÃO DA ACIDEZ DO SOLO COM NITROGÊNIO NO 

ACÚMULO DE CARBONO ESTÁVEL NO SOLO 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Botucatu  

2024 
 

  



  

  



 

MARIA GABRIELA DE OLIVEIRA ANDRADE 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

INTERAÇÃO DA CORREÇÃO DA ACIDEZ DO SOLO COM NITROGÊNIO NO 

ACÚMULO DE CARBONO ESTÁVEL NO SOLO 

 
 
 
 

 
Thesis presented to the Faculty of 

Agronomic Sciences of UNESP - Botucatu 

for the degree of Doctor of Agronomy. 

 
Advisor: Prof. Dr. Ciro Antonio Rosolem 

 
 
 
 
 
 
 
 
 
 
 
 
 

Botucatu  
2024 



  

  



 
 

 

 

 

 



  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

To my beloved parents Waldirene and Eduardo, 
 and my sister Maria Fernanda. 

 
  



  

 

  



ACKNOWLEDGMENTS 

 
To God, for His infinite kindness in allowing me to reach this achievement. 

To my beloved parents, Maria Waldirene Oliveira and Eduardo Andrade, for their love, 

prayers, sacrifices, and unconditional support throughout all these years. Thank you 

for living my dream with me. 

To my sister, Maria Fernanda Andrade, for her love and constant comfort; this 

achievement is largely for you. 

To Prof. Dr. Ciro A. Rosolem, my heartfelt gratitude for your guidance, teaching, 

patience, and for believing in my work. It is an honor to be your advisee and a member 

of your team. 

To my dear friend and partner, Carlos Felipe dos Santos Cordeiro, thank you for the 

emotional support, encouragement, and assistance whenever needed. 

I am eternally grateful to the other members of my family who have always supported 

me, especially Marcelo Pachoal, Kamila Fernanda, and Maria Cecília Sobriho. 

To friends and collaborators Jorgiani de Avila, Amanda Duim, Emanueli Possas, 

Jolinda Mercis, Luciana Prieto, Amana Ferraresi, Fabrício Vivieiro, Luan Alves, and 

Bianca Sekiya, my sincere thanks for the friendship and collaboration. 

To my undergraduate advisor, Prof. Sebastião Ferreira, thank you for the 

encouragement that motivated me to pursue a doctorate, allowing me to reach this 

point. 

Special thanks to my supervisor during my exchange program, Prof. Alan 

Franzluebbers, for his encouragement and support in developing part of this study. 

To the staff of the Graduate Section and the Department of Production and 

Improvement, especially Professors Juliano Calonego and Carlos Crusciol, my deep 

gratitude for their support. 

To the team at Fazenda Lageado, FCA – UNESP, thank you for the assistance with 

agricultural inputs and field activities, essential for obtaining high-quality results. 

To all who contributed directly or indirectly to this stage of my life, I extend my deepest 

gratitude. 

To the São Paulo Research Foundation (FAPESP), my sincere thanks for the financial 

support through the doctoral scholarship (Process nº 2020/07559-7) and the 

BEPE/FAPESP scholarship for the overseas research internship (Process nº 

2022/10355-0). 



  

  



 

 

ABSTRACT 
 

The effect of the direct seeding system on the accumulation of carbon (C) in the 

soil is well known. The amount of labile C in the soil is related to the quantity and 

quality of straw in the system. However, the accumulation of stable C, linked to soil 

minerals, is more interesting due to its greater resilience. It is known that 

conservation production systems improve soil aggregation and that smaller 

aggregates accumulate stable C, which, in turn, depends on the nitrogen (N) 

present in the system and the presence of cationic bridges. Although the effect of 

each of these factors is known, little is known about the interaction between them. 

The objective was to study the effect of applying limestone and agricultural gypsum, 

associated or not with nitrogen fertilization, on soil aggregation, accumulation of C 

and humic substances, relating the accumulation of stable C with the classes of 

aggregates in a production system with soybeans and corn intercropped with 

Tanzania grass, in the second harvest. The field experiment was installed in 

Botucatu-SP, in 2016, and the data presented are from 2020 to 2022. The 

treatments were: control (without application of limestone and gypsum), limestone, 

and limestone + gypsum, with combined doses of N of 0, 80, 160, and 240 kg ha-

1, applied annually to corn. Soybeans were grown in the spring/summer and corn 

in the fall/winter of each year. The combination of limestone, gypsum, and nitrogen 

fertilizer led to a 13% higher total C stock and a 20% higher total N stock in the 0-

60 cm soil profile compared to the control. Liming + N reduced fulvic acids (FA) 

and humic acids (HA), but the application of gypsum mitigated this negative effect. 

The application of lime, gypsum, and N increased humin levels, mainly on the soil 

surface. Thus, the best strategy to increase C storage and stabilization in humid 

tropical soils in double soybean-corn cultivation was the combined use of 

limestone, gypsum, and nitrogen fertilizer applied to corn, achieving greater C 

storage in larger aggregates. The application of limestone and gypsum resulted in 

lower carbon mineralization compared to the control under N deficiency. N 

mineralization was greater when limestone + gypsum was applied together with N. 

Liming and nitrogen fertilization significantly increased nitrate levels in the soil, 

with no differences between liming and liming + plaster. Greater microbial activity, 

carbon mineralization, and nitrification were observed when N was applied to the 



  

soil. Furthermore, it was observed that the application of liming and liming + 

gypsum played a substantial role in the processes involving C mineralization in 

soils, with greater effects on smaller aggregates. Liming management and gypsum 

application can extend these benefits to deeper soil layers, resulting in increased 

soil fertility in subsurface layers. Soil management using limestone + gypsum 

proved to be a promising practice in increasing the productivity of soybeans and 

corn. Grains, especially at a lower dose of N (80 kg ha-1) or in the absence of 

nitrogen fertilizer. This result demonstrates the positive impact of liming and 

gypsum management on the N-use efficiency of crops, thus reducing dependence 

on nitrogen fertilization in acidic soils. Productivity gains were directly linked to the 

physical (greater stability and diameter of aggregates) and chemical (pH and 

macronutrient content) improvements in the soil provided by limestone + gypsum 

management. Therefore, it is concluded that the synergism of soil correction 

practices with liming, gypsum, and nitrogen fertilization applied to second-crop 

corn intercropped with forage species resulted in greater grain productivity, 

increased soil fertility, C sequestration, and storage, especially in larger 

aggregates. 

 
Keywords: nitrogen fertilization; soil aggregates; liming; Glycine max L; Zea mays 
L. 
 

 
 
 
 
 
 

 
 
 
  



RESUMO 
 

É bem conhecido o efeito do sistema de semeadura direta no acúmulo de carbono 

(C) no solo. A quantidade de C lábil no solo tem relação com a quantidade e 

qualidade de palha do sistema. Entretanto, o acúmulo de C estável, ligado aos 

minerais do solo, é mais interessante devido à sua maior resiliência. Sabe-se que 

sistemas de produção conservacionistas melhoram a agregação do solo, e que 

agregados menores acumulam C estável, o que, por sua vez, depende do 

nitrogênio (N) presente no sistema e da presença de pontes catiônicas. Embora o 

efeito de cada um desses fatores seja conhecido, pouco se sabe sobre a interação 

entre eles. Objetivou- se estudar o efeito da aplicação de calcário e gesso agrícola, 

associados ou não à adubação nitrogenada, na agregação do solo, acúmulo de C 

e substâncias húmicas, relacionando o acúmulo de C estável com as classes de 

agregados em um sistema de produção com soja e milho consorciado com capim 

Tanzania, em segunda safra. O experimento de campo foi instalado em Botucatu-

SP, no ano de 2016, e os dados apresentados são de 2020 a 2022. Os 

tratamentos foram: controle (sem aplicação de calcário e gesso), calcário, e 

calcário + gesso, com doses combinadas de N de 0, 80, 160, 240 kg ha-1, aplicadas 

anualmente ao milho. A soja foi cultivada na primavera/verão e o milho no 

outono/inverno de cada ano. A combinação de calcário, gesso e adubação 

nitrogenada levou a um estoque total de C 13% maior e um estoque total de N 

20% maior no perfil de solo de 0-60 cm em comparação com o controle. A calagem 

+ N reduziu ácidos fúlvicos (AF) e ácidos húmicos (AH), mas a aplicação de gesso 

mitigou esse efeito negativo. A aplicação de calcário, gesso e N aumentou os 

teores de humina, principalmente na superfície do solo. Assim, a melhor estratégia 

para aumentar o armazenamento e estabilização de C em solos tropicais úmidos 

no duplo cultivo soja-milho foi o uso combinado de calcário, gesso e adubação 

nitrogenada aplicada ao milho, alcançando maior armazenamento de C em 

agregados maiores. Aaplicação de calcário e gesso resultou em menor 

mineralização de carbono comparado ao controle sob deficiência de N. A 

mineralização de N foi maior quando calcário + gesso foi aplicado junto com N. A 

calagem e a adubação nitrogenada aumentaram significativamente os níveis de 

nitrato no solo, sem diferenças entre calagem e calagem + gesso. Foi observada 

maior atividade microbiana, mineralização de carbono e nitrificação quando N é 



  

aplicado no solo. Além disso, observou-se que a aplicação de calagem e calagem 

+ gesso desempenhou um papel substancial nos processos que envolvem a 

mineralização de C nos solos, com maiores efeitos em agregados menores. O 

manejo de calagem e aplicação de gesso pode estender esses benefícios para 

camadas mais profundas do solo, resultando em um aumento da fertilidade do 

solo nas camadas subsuperficiais. O manejo do solo utilizando calcário + gesso 

mostrou-se uma prática promissora no aumento da produtividade de grãos de soja 

e milho, especialmente na menor dose de N (80 kg ha-1) ou na ausência da 

adubação nitrogenada. Este resultado demonstra o impacto positivo do manejo da 

calagem e gessagem na eficiência de uso de N das culturas, reduzindo assim a 

dependência da adubação nitrogenada em solos ácidos. Os ganhos de 

produtividade foram diretamente ligados à melhoria física (maior estabilidade e 

diâmetro de agregados) e química (pH e teores de macronutrientes) do solo 

proporcionadas pelo manejo de calcário + gesso. Portanto, conclui-se que o 

sinergismo das práticas de correção do solo com calagem, gessagem e adubação 

nitrogenada aplicadas no milho segunda safra consorciado com espécie forrageira 

resultou em maior produtividade de grãos, incremento da fertilidade do solo, 

sequestro e armazenamento de C, especialmente em agregados de maior 

tamanho. 

 
Palavras-chave: adubação nitrogenada; agregados de solo; calagem; Glycine max 
L; Zea mays L. 

  



LIST OF ILLUSTRATIONS 
 
Chapter 1 - SOIL AMENDMENT AND N FERTILIZATION STRATEGIES TO 

IMPROVE C SEQUESTRATION AND STORAGE IN SOIL 
AGGREGATES 

Figure 1 - Soil C stocks in soil aggregate classes and depths as affected by 
no correction (Cont.), lime only, lime + gypsum (L+G) and N 
fertilization. Different underscored capital letters above bars show 
significant differences (P< 0.05) between treatments within a N 
level and soil aggregate class summed across depths. Different 
capital letters within bars show significant differences between 
treatments within a N fertilizer treatment, aggregate class, and 
soil depth increment. Different lowercase letters within bars show 
significant differences between N rates within corrective 
treatment, aggregate size class, and soil depth………………….. 30 

Figure 2 - Soil N stocks in soil aggregate classes and depths as affected by 
no correction (Cont.), lime only, lime + gypsum (L+G) and N 
fertilization. Different underscored capital letters above bars show 
significant differences (P< 0.05) between treatments within a N 
level and soil aggregate class summed across depths. Different 
capital letters within bars show significant differences between 
treatments within a N fertilizer treatment, aggregate class, and 
soil depth increment. Different lowercase letters within bars show 
significant differences between N rates within corrective 
treatment, aggregate size class, and soil depth……………........... 31 

Figure 3 - Fulvic acid in soil aggregate classes and depths as affected by no 
correction (Cont.), lime only, lime + gypsum (L+G) and N 
fertilization.   Lowercase letters compare the effect of N rates in 
each soil correction management. Asterisks show differences of 
soil correction management on each soil layer……………………. 32 

Figure 4 - Humic acid in soil aggregate classes and depths as affected by no 
correction (Cont.), lime only, lime + gypsum (L+G) and N 
fertilization. Lowercase letters compare N rates in each soil 
correction management. Asterisks show the effects of soil 
correction on each soil layer………………………………………… 33 

Figure 5 - Humin in soil aggregate classes and depths as affected by no 
correction (Cont.), lime only, lime + gypsum (L+G), and N 
fertilization. Lowercase letters compare the effect of N rates in 
each soil correction management. Asterisks show the effects of 
soil correction on each soil layer.................................................... 34 

Figure 6 - Humic substances in soil aggregate classes and depths as 
affected by no correction (Cont.), lime only, lime + gypsum (L+G) 
and N fertilization. Lowercase letters compare the effect of N rats 
in each soil correction management. Asterisks show the effects of 
soil correction on each soil layer…................................................. 35 

 

 
 
 
 



  

Chapter 2 - IMPROVING SOIL FERTILITY, AGGREGATE STABILITY, AND YIELD 
IN A DOUBLE-CROPPED SYSTEM: SYNERGY OF NITROGEN, LIME, 
AND GYPSUM 

Figure 1 - Precipitation and monthly maximum and minimum temperatures 
during the soybean and maize harvests in 2020/2021 and 
2021/2022.…………………………………………………..………… 47 

Figure 2 - Soybean and maize grain yield, depending on soil correction and 
nitrogen fertilization. Capital letters compare N doses in each soil 
correction management. Lowercase letters compare soil correction 
management at each N dose….………………….............................. 52 

Figure 3 - Aggregate stability index, depending on soil correction and 
nitrogen fertilization. Capital letters compare N doses in each soil 
correction management. Lowercase letters compare soil correction 
management at each N dose.…………………………..………...…. 56 

Figure 4 - Geometric mean diameter, depending on soil correction and 
nitrogen fertilization. Capital letters compare N doses in each soil 
correction management. Lowercase letters compare soil correction 
management at each N dose.……………………………..………….. 57 

Figure 5 - Weighted average diameter, depending on soil correction and 
nitrogen fertilization. Capital letters compare N doses in each soil 
correction management. Lowercase letters compare soil correction 
management at each N dose.……………………………….............. 58 

Figure 6 - pH and calcium (Ca) content in the soil, depending on soil 
correction and nitrogen fertilization. Capital letters compare N 
doses in each soil correction management. Lowercase letters 
compare soil correction management at each N dose.…................ 59 

Figure 7 - Phosphorus (P) and sulfur (S) contents in soil, depending on soil 
correction and nitrogen fertilization. Capital letters compare N 
doses in each soil correction management. Lowercase letters 
compare soil correction management at each N dose……………. 60 

Figure 8 - Magnesium (Mg) and potassium (K) contents in soil, depending on 
soil correction and nitrogen fertilization. Capital letters compare N 
doses in each soil correction management. Lowercase letters 
compare soil correction management at each N dose……………… 61 

Figure 9 - Organic matter contents in soil, depending on soil correction and 
nitrogen fertilization. Capital letters compare N doses in each soil 
correction management. Lowercase letters compare soil correction 
management at each N dose………………………………………..… 62 

 
 
 
 
 

 
 
 
 
 
 
 

 
 
 



Chapter 3 - SOIL CARBON MINERALIZATION: A CLOSER LOOK ON THE ROLE 
OF AGGREGATES, PH ALLEVIATION AND N APPLICATION 

Figure 1 - Soil test biological activity as a function of soil amendments (lime – 
L and lime + gypsum – L+G) and nitrogen fertilization in different 
aggregates sizes (from A to E) and the average of all aggregates 
(F). Uppercase letters compare soil correction amendment at each 
N dose. Lowercase letters compare the N rate within each soil 
amendment..………………………………………………………........ 78 

Figure 2 - Basal soil respiration as a function of soil amendments (lime – L 
and lime + gypsum – L+G) and nitrogen fertilization in different 
aggregates sizes (from A to E) and the average of all aggregates 
(F). Uppercase letters compare soil correction amendment at each 
N dose. Lowercase letters compare the N rate within each soil 
amendment.….…………………....................................................... 79 

Figure 3 - Net carbon mineralization as a function of soil amendments (lime – 
L and lime + gypsum – L+G) and nitrogen fertilization in different 
aggregate sizes (from A to E) and the average of all aggregates (F). 
Uppercase letters compare soil correction amendment at each N 
dose. Lowercase letters compare the N rate within each soil 
amendment..…………………………..………………………………... 80 

Figure 4 - Net nitrogen mineralization as a function of soil amendments (lime 
– L and lime + gypsum – L+G) and nitrogen fertilization in different 
aggregate sizes (from A to E) and the average of all aggregates 
(F). Uppercase letters compare soil correction amendment at each 
N dose. Lowercase letters compare the N rate within each soil 
amendment..……………………………………………………………. 81 

Figure 5 - Residual inorganic N as a function of soil amendments (lime – L 
and lime + gypsum – L+G) and nitrogen fertilization in different 
aggregate sizes (from A to E) and the average of all aggregates 
(F). Uppercase letters compare soil correction amendment at each 
N dose. Lowercase letters compare the N rate within each soil 
amendment..…………………………..……....................................... 82 

Figure 6 - Residual soil nitrate as a function of soil amendments (lime – L and 
lime + gypsum – L+G) and nitrogen fertilization in different 
aggregate sizes (from A to E) and the average of all aggregates 
(F). Uppercase letters compare soil correction amendment at each 
N dose. Lowercase letters compare the N rate within each soil 
amendment...................................................................................... 83 

Figure 7 - Principal component analysis of soil attributes under C and N 
mineralization assays. STBA: soil-test biological activity; BSR: 
basal soil respiration; RSA: residual soil ammonium; RSN: residual 
soil nitrate; RIN = residual inorganic; NMIN24 = net N 
mineralization; Nitrif = apparent net nitrification.…………………….. 84 

 

  



  

  



LIST OF TABLES 

 

Chapter 1 - SOIL AMENDMENT AND N FERTILIZATION STRATEGIES TO 
IMPROVE C SEQUESTRATION AND STORAGE IN SOIL 
AGGREGATES 

 
Table 1 - Soil chemical and physical attributes and particle size distribution 

before the implementation of the experiment in August 
2016..…………………………………………………………………........ 27 

 
Chapter 2 - IMPROVING SOIL FERTILITY, AGGREGATE STABILITY, AND YIELD 

IN A DOUBLE-CROPPED SYSTEM: SYNERGY OF NITROGEN, LIME, 
AND GYPSUM 

 
Table 1 - Chemical characteristics and particle size of the soil in which the 

experiment was conducted August 201………………………….…... 47 
Table 2 - Average leaf contents of nitrogen, phosphorus, potassium, calcium, 

magnesium, sulfur, boron, iron, manganese, zinc, and copper for the 
2020/2021 and 2021/2022 harvests for soybean and maize crops. 
Capital letters compare N doses in each soil correction management 
(i.e., lines comparison for each column). Lowercase letters compare 
soil correction management at each N dose (i.e., columns 
comparison for each line).….…..................................... 53 

 
 

Chapter 3 - SOIL CARBON MINERALIZATION: A CLOSER LOOK ON THE ROLE 
OF AGGREGATES, PH ALLEVIATION AND N APPLICATION 

 
Table 1 - Soil chemical and physical attributes and particle size distribution 

before the implementation of the experiment. August 2016….…… 75 

 

 
 

  



  

 

 

  



SUMMARY 
 

 GENERAL INTRODUCTION...................................................................... 21 

 CHAPTER 1 - SOIL AMENDMENT AND N FERTILIZATION STRATEGIES 
TO IMPROVE C SEQUESTRATION AND STORAGE IN SOIL 
AGGREGATES1........................................................................................... 

24 

1.1 Introduction................................................................................................... 24 

1.2 Material and Methods...................................................................................  26 

1.2.1 Characterization of the study área............................................................... 26 

1.2.2 Experimental planning and treatments.........................................................  27 

1.2.3 Soil Sampling and Aggregate Separation..................................................... 27 

1.2.4 Nitrogen and Carbon Analysis..................................................................... 28 

1.2.5 Data analysis...............................................................................................  29 

1.3 Results.........................................................................................................  29 

1.4 Discussion...................................................................................................  35 

1.5 Conclusion...................................................................................................  38 

 References (Chapter 1)................................................................................ 39 

 CHAPTER 2 - IMPROVING SOIL FERTILITY, AGGREGATE STABILITY, 
AND YIELD IN A DOUBLE-CROPPED SYSTEM: SYNERGY OF 
NITROGEN, LIME, AND GYPSUM2…………………………………………. 

44 

2.1 Introduction..................................................................................................  44 

2.2 Material and Methods..................................................................................  46 

2.1.1 Characteristics of the study area.................................................................. 46 

2.2.2 Experimental planning and treatment...........................................................  47 

2.2.3 Crop management.......................................................................................  48 

2.2.4 Leaf analysis................................................................................................  49 

 Soil sampling................................................................................................ 49 

2.2.6 Soil aggregates............................................................................................. 49 

2.2.7 Soil Fertility...................................................... ............................................ 50 

2.2.8 Statistics...................................................... ................................................ 51 

2.3 Results......................................................................................................... 51 

2.3.1 Grain yield...................................................... ............................................ 51 

2.3.2 Leaf nutrient content...................................................... .............................  52 

2.3.3 Aggregates...................................................... ............................................ 56 

2.3.4 Soil fertility...................................................... ............................................. 58 

2.4 Discussion....................................................................................................  62 

2.4.1 Yield, soil fertility, and leaf nutrient content................................................... 62 



  

2.4.2 Soil aggregates...................................................... ..................................... 64 

2.5 Conclusion.................................................................................................... 66 

 References (Chapter 2)................................................................................ 66 

 CHAPTER 3 - SOIL CARBON MINERALIZATION: A CLOSER LOOK ON 
THE ROLE OF AGGREGATES, pH ALLEVIATION AND N APPLICATION3 

73 

3.1 Introduction...................................................................................................  73 

3.2 Material and Methods................................................................................... 75 

3.2.1 Characterization of the studied area............................................................ 75 

3.2.2 Experimental design..................................................................................... 75 

3.2.3 Soil sampling and analysis............................................................................ 76 

3.2.4 C and N mineralization assay........................................................................ 76 

3.2.5 Statistical analysis........................................................................................ 77 

3.3 Results......................................................................................................... 77 

3.3.1 Soil test biological activity............................................................................. 77 

3.3.2 Basal soil respiration................................................................... ................. 78 

3.3.3 Soil carbon mineralization............................................................................. 79 

3.3.4 Soil Nitrogen Mineralization.......................................................................... 80 

3.3.5 Principal Component Analysis...................................................................... 83 

3.4 Discussion..................................................................................................... 84 

3.5 Conclusions................................................................................................... 86 

 References (Chapter 3) ................................................................................ 87 

 CONCLUDING REMARKS........................................................................... 94 

 REFERENCES.............................................................................................. 95 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



21 

 

 
GENERAL INTRODUCTION 

 
 

Soil quality is directly related to carbon (C) content and is influenced by 

management practices such as crop rotation and no-till farming systems. A crucial 

factor for the accumulation of organic carbon in soil is the availability of nitrogen 

(N) (Boddey et al., 2010), as well as the increase in microbial populations in the 

rhizosphere (Cheng et al., 2008). Grass residues with a high C/N ratio persist 

longer in soil due to their low decomposition rate (Teixeira et al., 2014), which 

favors the accumulation of soil organic matter (SOM) and reduces mineralization 

(Shahbaz et al., 2017). Thus, crop rotation and the type of farming system 

influence the accumulation of organic carbon, biological activity, and soil 

aggregation. Carbon protection within stable aggregates inhibits mineralization 

and increases C retention in soil (Cambardella and Elliott, 1992). This carbon can 

be trapped in aggregates and physically protected from microbial activity (Tisdall 

and Oades, 1982), or adsorbed onto mineral surfaces (Janzen, 2015). 

Increasing carbon stocks in tropical soils in humid regions is a challenge, 

especially in more stable fractions such as mineral-associated C and humic 

substances (Cordeiro et al., 2022), since although susceptible to mineralization, 

soil organic carbon can be protected by the mineral matrix (Kögel-Knabner and 

Amelung, 2014). Aggregates formed by the trapping carbon not only physically 

protect organic matter (Tisdall and Oades, 1982) but also influence microbial 

community structure (Hattori, 1988), limiting oxygen diffusion (Sexstone et al., 

1985), regulating water flow (Prove et al., 1990), determining nutrient adsorption 

and desorption (Wang et al., 2001), and reducing surface runoff and erosion 

(Barthe and Roose, 2002). 

In this sense, improving soil aggregation is crucial for increasing organic 

matter physical protection and C stability (Rowley et al., 2018). The application of 

lime and agricultural gypsum improves soil aggregation, as calcium (Ca) and 

magnesium (Mg) act as flocculating agents that stabilize C (Castro et al., 2015; 

Rowley et al., 2018). Gypsum also protects C by providing available Ca, improving 

aggregation with clay and silt, and promoting the growth of fine roots, which are 

sources of recalcitrant compounds (Carvalho et al., 2023). Additionally, gypsum is 

an excellent source of sulfur, essential for biological nitrogen fixation and soybean 



22  

productivity (Almeida et al., 2023; Virk et al., 2022). The application of gypsum 

alone has shown limited benefits for C capture (Walia et al., 2023). Low soil fertility, 

especially nitrogen, phosphorus, and Ca deficiencies, can restrict C capture 

(Mattila et al., 2022). The main impact of lime and gypsum on C capture is related 

to increased biomass production, especially roots (Costa et al., 2021), promoting 

greater C stocks at depth (Araujo et al., 2019). 

In addition, nitrogen fertilization can enhance root growth, facilitating the 

movement of Ca supplied by lime (Foloni et al., 2006; Crusciol et al., 2022). 

Increased C capture is associated with increased crop productivity, especially in 

C4 plants, achieved through soil correction and integrated production systems 

(Siddique et al., 2024). In production systems where soybean and maize are 

intercropped with forage grasses soil aggregation and C and N contents can 

increase (Cooper et al., 2021; Barcelos et al., 2022). However, in these systems, 

lime is applied to the soil surface, and due to the low solubility and mobility of Ca, 

it is common to apply gypsum together with lime to alleviate aluminum toxicity at 

depth and improve root growth (Pivetta et al., 2019). In these systems, N is not 

applied to soybeans due to the high efficiency of biological nitrogen fixation (BNF), 

and N fertilization in maize is usually low, which may result in a negative N balance 

(Rocha et al., 2020; Barcelos et al., 2022). 

Nitrogen deficiency and soil acidity can limit C storage in stable fractions. 

Humic substances, such as humic acid (HA), fulvic acid (FA), and humin, are stable 

components of organic matter, with stability in decreasing order (Tadini et al., 

2022). Mitigating soil acidity with lime increases microbial activity and soil 

respiration, which can result in some C loss. However, in systems with cover crops, 

this has not been reported (Cordeiro et al., 2021). Moreover, the amount of C loss 

is insignificant compared to the higher dry matter production and addition of cations 

that stabilize soil C (Barcelos et al., 2022). The net balance is a greater efficiency 

of C use by the soil microbiota (Barcelos et al., 2021). Thus, the capture and 

humification of soil C may increase with improved soil fertility (Ovchinnikova, 

2018). The lime application increases humic substance levels in the soil's surface 

layer (Mockeviciene et al., 2022), but few studies have examined the combined 

use of lime and gypsum at depth, especially in no-till systems. In conservation 

systems with cover crops, humic C is stored mainly in macroaggregates through 

the physical interlocking of microaggregates with roots. Newly formed and more 



23 

 

labile HAs are found in greater quantities in macroaggregates, while 

microaggregates contain more recalcitrant HAs. However, these effects were only 

found near the soil surface (Ndzelu et al., 2021), and it is unknown whether the 

application of lime and gypsum associated with N fertilization can affect the 

distribution of humic substances in deeper soil layers. Both N and lime initially 

accelerate SOM mineralization due to increased microbial activity, however, the 

more stable soil C fractions are enhanced in conservation systems through C 

physical protection within aggregates (Neff et al., 2002; Castro et al., 2015). 

Nitrogen fertilization affects the dynamics of soil humic substances (Kou et al., 

2022), although this has not been described in tropical climates. 

Crop residue accumulation on the soil surface provides inputs of C and N 

and promotes a zone of higher potential for soil biological activity, measured by C 

and N mineralization under laboratory conditions (Franzluebbers et al., 2018). Soil 

organic carbon is directly associated with total nitrogen, with strong biological links 

(Cleveland and Liptzin, 2007), indicating that any change in organic carbon will 

also affect total soil N, dominated by the organic fraction (Pringle et al., 2014). N 

fertilization accelerates the decomposition of light carbon fractions while stabilizing 

carbon compounds in heavy fractions in soils, as well as it has been shown to 

increase C in the rhizosphere due to higher rhizodeposition and aggregate 

formation (Chu et al., 2007). In addition, when nitrogen is associated with more 

persistent minerals it is less susceptible to microbial mineralization and 

subsequent leaching and gaseous losses (Kelley and Stevenson, 1995). In soils 

cultivated with grasses, which provide lignin-rich residues, N fertilizer application 

increases carbon sequestration (Jones et al., 2009), reducing microbial lignin’s 

activity (Fog, 1988). 

Given the above, to confirm the hypothesis that liming associated with the 

addition of agricultural gypsum and N in a no-till production system can result in a 

greater accumulation of stable C in soils than each soil amendment alone, this 

study was conducted to verify (i) accumulation of C, total N, and humic substances 

in different soil aggregate classes (ii) aggregation and soil fertility in deeper no-till 

production system soil layers and increased crop yield (iii) C stability, C 

mineralization potential, and microbial biomass C in a no-till production system. 

  



 
 
 
 
 
 
 
 
 
 

 
 
Atendendo solicitação do(a) autor(a), o texto 

completo desta tese/dissertação será 
disponibilizado somente a partir de 

06/09/2025 
 

 
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thesis/dissertation will not be available online until 
Sep. 06, 2025 

 
 



94  

CONCLUDING REMARKS 

Soil acidity is one of the most important limitations to agricultural 

production worldwide. Although the primary goal of liming is to overcome soil 

acidity, it also impacts the chemical, biological, and physical properties of soils, 

affecting the entire system. Thus, the combination of lime, gypsum, and nitrogen 

fertilization was effective in improving soil quality and fertility, as well as increasing 

crop productivity. This combination resulted in a significant increase in soil carbon, 

especially in larger aggregates, which play a crucial role in carbon and nitrogen 

storage. Nitrogen fertilization proved essential for maximizing these benefits, 

confirming the importance of nitrogen in regulating soil carbon and nitrogen cycles.  

Additionally, gypsum was fundamental in preventing the reduction of fulvic 

acid under liming and nitrogen fertilization conditions, thereby maintaining soil 

quality. The application of lime and gypsum improved soil aggregation. These 

improvements led to higher crop productivity, especially in water-limited 

environments, where the reduction in soil acidity had a significant impact. 

Another important aspect is the reduction in soil basal respiration observed 

with the application of lime, gypsum, and nitrogen, indicating a decrease in 

biological activity and carbon loss. However, nitrogen deficiency increased carbon 

mineralization, especially in smaller aggregates. These results underline the 

importance of nitrogen in regulating carbon and nitrogen cycles, highlighting that 

the combined application of lime, gypsum, and nitrogen has distinct effects on 

carbon and nitrogen mineralization compared to individual treatments. 

The results demonstrate that the combined application of lime, gypsum, 

and nitrogen fertilization not only improves soil fertility and physical quality but also 

contributes to optimizing agricultural techniques and reducing environmental 

impacts. Thus, we confirm our hypothesis that the combination of these treatments 

is essential for carbon and nitrogen accumulation in the soil. 



95 
 

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