Mineralization of Nitrogen in Soils with Application of Acid Whey at Different pH


Agro-industrial wastes are commonly used as nitrogen sources in agriculture, and its availability depends on the dynamics of nitrogen mineralization. The pH has great effects on soil nitrogen dynamics; thus, we hypothesized that the increase of pH would increase nitrogen mineralization and nitrification. The aim of this study was to evaluate the effects of pH and application of acid whey in nitrogen mineralization and nitrification rates using two types of soils, a hapludult and a eutrudox. In a completely randomized factorial design, N mineralization under different pH levels (4.5; 5.0; 5.5; 6.0; 6.5) was evaluated, with and without whey application, with an equivalent of 40 mg N dm−3. Soils were incubated for 182 days, and throughout this period, eleven evaluations were made to assess N mineralization over time. The hapludult soil had higher nitrate concentrations and the acid whey fertilization increased inorganic nitrogen in both soils. Soil pH did not influence inorganic nitrogen contents, but affected nitrification in both soils. Soil pH levels also resulted in variations on the constant of mineralization, a parameter related to mineralization speed, but without any strong trend. The application of acid whey displayed a satisfactory potential in relation to nitrogen incorporation in both tested soils. Soil pH around 4.5–5.5 is the optimum pH range, because it did not affect the nitrogen supply and decreases nitrification. Results demonstrate that soil pH can be used to avoid nitrification without reducing nitrogen availability to plants.


Wastes generated in agro-industrial activities have long been used as sources of soil organic matter (SOM) and plant nutrients, a practice considered both economically and environmentally sustainable, as it optimizes the use of materials and prevents excessive disposals (Francou et al. 2008; Azeez and Van Averbeke 2010). Among several wastes derived from agricultural activities, a few residues from dairy productions are still underused, such as the acid whey, which represents about one third of all wastes generated in bovine milk processing. In 2017, Brazil produced 35 million tons of cheese (Conab 2017), and for each kg of cheese, there is an average generation of 9 L of whey (Lima and Rocha 2016), resulting in an approximated volume of 7.2 million m3. This waste contains significant amounts of organic carbon and nitrogen, which hampers its disposal and treatment as liquid effluent (Chatzipaschali and Stamatis 2012). Thus, its direct application in the soil becomes particularly attractive, aiming both the input of nutrients (especially N, K, and Ca) and the incorporation of its organic constituents in the SOM (Mantovani et al. 2015).

Organic fertilizations are beneficial to the soil, especially with regard to the possibility of increasing total organic nitrogen (TON) and SOM contents over time, besides supplying nutritional demands of plants (Mantovani et al. 2006; Zhao et al. 2016). However, the input of organic wastes in the soil should be made in adequate amounts, in order to avoid possible losses by lixiviation or even gasification, thus causing soil and groundwater contaminations (Mantovani et al. 2005), due to the mineralization and nitrification of the large amounts of N found in wastes (Basso and Ritchie 2005). Overall, the application of wastes with low C/N ratios (< 20) leads to high mineralization rates, while C/N above 30 results in immobilization of the soil N (Walecka-Hutchison and Walworth 2007). Additionally, the chemical, physical, and microbiological characteristics of fertilized soils are known to dabble the dynamics of N mineralization, which in turn affects N losses and absorption (Schoenholtz et al. 2000; Marzi et al. 2019).

The oxidation of ammonium (NH4+) to nitrate (NO3) (nitrification)—a biochemical process performed by a restrict group of chemolithotrophic microorganisms—controls the availability of NO3 to plants. This process is influenced by several factors, such as type of soil, quantity of added substrate, and adaptation of microbial communities, besides soil pH (Yao et al. 2011). Under high acidity conditions (pH < 5.0), nitrification occurs in reduced rates (Sahrawat 2008), although Gubry-Rangin et al. (2010) reported nitrification in soils with a pH of 4.5. In general, this process occurs in soils with pH ranging from 5.5 to 10, with an optimum around 8.5, while its inhibition is verified in acid soils (pH < 5.0), a fact which is directly related to the groups of active microorganisms (Nugroho et al. 2007) and to the availability of N in the form of free ammonia (NH3) and NH4+.

The application of acid whey may lead to significant increases of mineral N in the soil, and this nutrient input may cause high lixiviation rates of NO3, in case it is not absorbed by roots, since this ion is not adsorbed by the negative charges of the soil’s solid phase (Monaghan et al. 2013). Concurrently, reductions of soil pH due to acid whey fertilization may lead to reduced nitrification rates, bypassing the problems of groundwater contamination by reducing NO3 losses, but it should decrease N mineralization. Considering the potential use of acid whey as an organic fertilizer, and the factors involving N nitrification and leaching, the hypothesis of this study was that the application of acid whey may contribute to the N supply and nitrification rates, which may be lower as the soil acidifies. In this sense, this study aimed at evaluating the effects of soil pH and acid whey fertilization on the rates of N mineralization and nitrification in a sandy loam and sandy clay soils.

Materials and Methods

Soil and Acid Whey Sampling and Characterization

The experiment was conducted under controlled conditions, in the Soil Fertility Laboratory of Unesp/FCAV in Jaboticabal, SP, Brazil. The used soils had different textures, being one of sandy loam texture, kaolinitic-gibbsitic Argissolo Vermelho-Amarelo (PVA), equivalent to hapludult, and one of sandy clay texture, ferric-kaolinitic, Latossolo Vermelho (LV), equivalent to eutrudox, according to Embrapa (2013) and the Soil Survey Staff (2014), respectively. The PVA was collected in Pindorama, SP, Brazil in a native forest area, while the LV was sampled in Jaboticabal, SP, Brazil, in a Pinus sp. forest area, cultivated for more than 30 years. Approximately 30 kg of each soil were sampled within the 0–20 cm layer. Samples were air-dried, sieved (4 mm), and stored in a well-ventilated storage room. Subsamples were collected for chemical analysis (pH, SOM, exchangeable cations, acidity) performed as the methods described in Raij et al. (2001), soil texture (Camargo et al. 2009) and bulk density; these data are shown in Table 1.

Table 1 Chemical characteristics, texture, and bulk density of the soils used for incubation experiments

The acid whey used in this experiment was provided by an industry located in Poços de Caldas, MG, Brazil, and stored in a refrigerator at 4 °C. A subsample was collected in order to obtain its chemical parameters: pH, NH4+, and NO3 levels (Cantarella and Trivelin 2001), total N (Tedesco et al. 1995), and organic C (Brasil 2007), as well as P, K, Ca, Mg, S, Cu, Zn, Fe, and Mn contents (Carmo et al. 2000). The obtained results were dry matter 8%, pH 3.7; total N 710 mg L−1; NH4+ 27.72 mg L−1; NO3 2.25 mg L−1; organic C 11900 mg L−1; C/N ratio 17.0; P 790 mg L−1; K 1330 mg L−1; Ca 1220 mg L−1; Mg 66 mg L−1; S 41 mg L−1; Cu 0.03 mg L−1; Zn 1.79 mg L−1; Fe 0.53 mg L−1; and Mn 0.14 mg L−1.

Experimental Design

In a completely randomized design, N mineralization of the acid whey under different pH levels was evaluated in a 2 × 2 × 5 factorial setup, with three replicates. The factors were two soil types (PVA and LV), the application rate of acid whey aiming to supply 0 and 40 mg N dm−3 (80 kg N ha−1), and five pH CaCl2 levels (4.5, 5.0, 5.5, 6.0, and 6.5). In order to increase soil’s pH to the pre-established values, a neutralization curve of soil acidity was determined by means of the application of CaCO3 doses, in the presence and absence of whey; therefore, the used CaCO3 doses were calculated based on neutralization curves. The acid whey dosage was calculated based on its fraction of mineralization (FM), which is calculated considering that not all N from the whey will be mineralized, and is performed according to the Cetesb normative P4.230 (CETESB 1999). The FM is the percentage of the total N which was mineralized during the incubation.

Nitrogen Mineralization Incubation

The FM values used to calculate the whey’s available N were 56% for PVA and 41% for LV, which were previously assessed in an essay using the same acid whey and soil samples. The application rate was calculated based on the fertilization of 40 mg N dm−3; therefore, the calculated rates resulted in whey dosages of 100 mL dm−3 for PVA and 135 mL dm−3 for LV.

Nitrogen mineralization evaluations were made according to Stanford and Smith (1972), but with adaptations to attend the norm P4.230 (CETESB 1999). The equivalent of 0.2 dm3 of soil was dry-blended with reagent grade CaCO3 (the mass of CaCO3 calculated for each pH treatment) and acid whey. The CaCO3 doses for the PVA were 0.156, 0.331, 0.506, 0.682, and 0.857 g, and for the LV, 0.188, 0.383, 0.577, 0.772, and 0.967 g, applied to achieve pH levels of 4.5, 5.0, 5.5, 6.0, and 6.5, in both soils, respectively. Then, the whey dosages were added to this mixture and homogenized. Considering the total volume of the prepared soil, 10 cm3 were used to determine the initial concentrations of NH4+ and NO3, while portions of 40 cm3 were blended with 20 cm3 of washed sand and then transferred to leaching columns. In the column, the blend soil was placed between layers of glass wool and moistened with deionized water, aiming to reach 70% of the soil’s water retention capacity, and incubated in a BOD-type chamber at 28 °C for 182 days.

The soil mineral N was extracted from the columns by percolation of 200 mL of a 0.01 mol L−1 KCl solution at 14, 28, 42, 56, 70, 84, 98, 112, 126, 154, and 182 days after the beginning of incubation. The leachate was collected, and the NH4+ and NO3 contents were determined by steam distillation (Cantarella and Trivelin 2001). After extract collection, 25 mL of a nutrient solution (without N) were added to the columns, followed by the vacuum application (0.067 MPa), in order to eliminate the excess of the nutrient solution and maintain an adequate moisture in the soil.

Soil N Mineralization Kinetics Modeling

The values of N mineralization in each treatment over time were adjusted to the exponential regression equation of the first order kinetics (Stanford and Smith 1972), according to Eq. 1. With the value of the constant of mineralization, it was possible to determine the half life time (T1/2), i.e., the necessary time to mineralize half of the potentially mineralizable N (N0), by means of Eq. 2.

$$ {N}_{\mathrm{m}}={N}_0\times \left(1-{\mathrm{e}}^{-k\mathrm{t}}\right) $$

where Nm is the mineralized inorganic N (mg dm−3); N0 is the potentially mineralizable N (mg dm−3); and k is the constant of mineralization (day−1).

$$ {T}_{1/2}=\frac{\ln 2}{k} $$

where T1/2 is the half life time (days) and k is the constant of mineralization (day−1).

Data were submitted to Shapiro-Wilk and Levene tests to evaluate the normality of residuals and homoscedasticity of variances, respectively. In order to assess the effect of the tested factors in the accumulated levels of soil Ni and NO3 (released during incubation), the data were submitted to analysis of variance (ANOVA), and the mean values of the factors soil and whey were compared by Tukey’s test (p < 0.05). Additionally, the effect of pH was evaluated by means of a regression analysis. The adjustments of mineralized N values as a function of time were also evaluated by means of ANOVA. Statistical analysis was performed with the aid of the Agroestat (Barbosa and Maldonado Júnior 2015) and Microsoft Excel software.


After 182 days of incubation, higher NO3 and Ni contents were observed in PVA in comparison with LV (Table 2). When applying the equivalent of 40 mg dm−3 of N in both soils as acid whey, it was possible to observe that NO3 contents increased in both soils. Regarding total Ni concentration, the acid whey application resulted in increase of 8.5% in PVA, as well as 18.3% in LV, when compared with control (Table 2). The low increase of Ni concentration is related to the low net soil N mineralization and presence of immobilization (Fig. 1).

Table 2 Values of mineralized NO3 and Ni accumulated after incubation in function of soil type, acid whey dosages, and soil pH (mean ± standard error)
Fig. 1

Net N mineralization in soils amended with acid whey at different pH. PVA sandy loam hapludult and LV sandy clay eutrudox

Soil pH did not influence total Ni contents, although it exerted an effect in NO3 contents (Table 2), which presented a quadratic fitting, which allows the calculus of the maximum point at pH 7.1 (Fig. 2). Also, the parameters of the equations of the adopted model to assess N mineralization were different in each soil pH level, suggesting it modified the dynamic of N mineralization (Table 3).

Fig. 2

Quadratic regression between pH CaCl2 and nitrate accumulated after 182 days of incubation (F = 24.05**)

Table 3 First-order model fitting results of cumulative N mineralization in different soils fertilized with acid whey

Regarding N mineralization, it was possible to verify that all data adjustments to the Stanford and Smith (1972) model were significant according to the variance analysis (Table 3). The calculated means of N0 and k in both soils without acid whey were 282.8 and 0.010 in PVA, and 216.8 and 0.011 in LV, respectively. The values of k and T1/2 ranged from 0.007 and 22 to 0.031 and 98 days, respectively (Table 3). Generally, the whey application increased the k values of both soils. The application of acid whey resulted in a decrease of N0, in the PVA (Table 3). The average values of N0 and k were 248 and 0.017 for the PVA soil, while in the LV, 214 and 0.017, respectively (Table 3). As for soil pH values, it caused great variations on the regression parameters, but no strong trend was observed; however, in the acid whey treated soils, at pH level of 5.5, the highest k values were observed (Table 3).

After 182 days of incubation, the mean FM values were 31.6% and 47.0% in PVA and LV, respectively (Table 3). These values vary greatly with soil pH, but without a well-defined tendency. In PVA, the FM varied from − 6.4% at a pH level of 4.5 (evidencing net N immobilization) to 54.9% (evidencing net N mineralization). The highest FM in PVA and LV was observed in pH 5.5 and 4.5, respectively (Table 3).


The higher N mineralization in the PVA soil occurred because this soil had lower clay content in comparison with LV, which allowed higher aeration. As the soils evaluated in this study had similar concentrations of TOC, the lower aeration provided by the microstructure of the LV led to a lower N mineralization, as suggested by Ros et al. (2011).

The acid whey average net N mineralization (difference between mineralized N in fertilized and non-fertilized treatments) was 22.5 and 41.5 mg N dm−3 for PVA and LV, respectively. These are considered low values, in comparison with biogas slurry, gelatin sludge, or pig slurry (Terhoeven-Urselmans et al. 2009; Guimarães et al. 2012; Zhao et al. 2016). A rapid mineralization was expected, because the acid whey had a low C/N rate; however, this waste is composed of carbohydrates (especially lactose), minerals, and globular proteins, in which most of the N are contained (Liao and Mangino 1987). The carbohydrates are rapidly mineralized providing energy to the microbial biomass, this added to the presence of mineral N in the whey (4.2% of total N) resulted in net N mineralization at the initial phase of incubation (Marzi et al. 2019). This phase was followed by a slow one, with low net mineralization and immobilization (Fig. 1). The acid whey proteins (mainly immunoglobulins) are difficult to mineralize, seen that they can be strongly adsorbed to soil electric charges, being protected from the soil microbial action. Also, these proteins may be harmful to soil microbes, reducing its activity (McLaren et al. 1958; Quiquampoix et al. 1993; Tapp et al. 1994), and consequently reducing the mineralization rates of the N provided by acid whey.

It was expected that increased pH would rise the total amount of mineralized N (Ni) in both soils, due to the increase of microbial activity in more favorable conditions, as suggested by Pietri and Brookes (2008). However, Rousk et al. (2009) observed that in spite of decreases of pH levels (from 8.3 to 4.0), which directly influences the composition of microbial communities present in the soil (especially bacteria and fungi), it did not affect the rate of microbial activity. According to these authors, the rate of microbial activity suffers significant reduction only in highly acidity conditions (pH < 4.5). Therefore, the absence of an effect of pH in Ni contents might be a result of the non-interference of pH in the microbial activity in the soil, which possesses strict relation with N mineralization. However, increases of NO3 levels in function of the intensification of pH were expected, seen that this is one of the main factors involved in nitrification, and this effect is well described in the literature (Sahrawat 2008), increasing the soil nitrification rate up to the pH 7.1, which is a different maximum value from the related literature (Sahrawat 2008).

The N0 values indicate the total amount of N that the soil can make available to plants, within a determined period, defined by the constant k, while N0 is frequently correlated with N absorbed by plants (Cordovil et al. 2007; Yagi et al. 2009); thus, it is used as reference of soil’s N availability. The higher N0 levels in the PVA agree with Ni data discussed above. The decrease of N0 caused by the acid whey application in the PVA is not in agreement with the Ni data (Table 2), but it is explained by the higher k values and lower T½ of the samples with whey application, suggesting these samples had faster N mineralization in the initial incubation phase. This led to underestimation of the values. The effects of soil pH on the regression parameters are difficult to be explained. The influence of pH on both proteins and soil electric charges might be the source of this variability, seen that adsorption to soil protects these proteins from mineralization by soil microorganisms, and it is highly dependent on pH (Schofield 1950; McLaren et al. 1958; Kontopidis et al. 2004).

In the PVA, acid whey had lower FM mean value than the previously established (56 and 41% for PVA and LV), which means that only 31.6% of the N will become available. Although it results in little N available to plants, it can increase of soil total N and avoids losses by leaching. In the LV, FM was higher at the lowest pH level (4.5), an unexpected result, but indicated that the microbial activity was not affected by pH variations. Conversely, in the PVA soil within the same pH range, FM was found negative, because the amount of mineralized N was higher in samples that did not receive whey, evidencing N immobilization in samples that received this waste. The FM seems to suffer a strong oscillation with the pH value (Fig. 1), which is aligned to the previously proposed discussion, due to the globular proteins which present a high variation in the charge balance as a function of pH, protonation and deprotonation of amine, and carboxylic groups of its structure (Quiquampoix et al. 1993; Quiquampoix 2000).


Acid whey presented a satisfactory potential in relation to nitrogen incorporation in the soil, but the efficiency of this incorporation seems to be dependent on soil type and pH. This waste stream can provide N more gradually, avoiding excessive losses by leaching or gasification, thus contributing to soil N buildup. The effects of soil pH in the mineralization dynamics of acid whey remain unclear. However, pH can be used to avoid nitrification, without reducing the N supply to plants, by correcting soil’s acidity up to pH 5.5.


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The authors would like to express their gratitude to the São Paulo Research Foundation (FAPESP) for providing the second author with a master’s scholarship (grant #2009/13450-9).

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Correspondence to Lucas Boscov Braos.

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Braos, L.B., Ruiz, J.G.C.L., Lopes, I.G. et al. Mineralization of Nitrogen in Soils with Application of Acid Whey at Different pH. J Soil Sci Plant Nutr 20, 1102–1109 (2020). https://doi.org/10.1007/s42729-020-00196-z

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  • Dairy residue
  • Nitrogen availability
  • Nitrogen reutilization
  • Nitrification
  • Organic waste