DYNAMICS OF MINERAL NITROGEN RELEASE AND SOIL SELF-CLEANING PROCESSES AFTER APPLYING FARMYARD MANURE AND SEWAGE SLUDGE

Krzysztof Gondek¹, Maria Chmiel^2
1 Department of Agricultural Chemistry, Agricultural University of Cracow, Poland
² Department of Microbiology, Agricultural University of Cracow, Poland

ABSTRACT

The study of the dynamics of nitrogen mineral forms release (to soil solution) and changes in the number of bacteria of the family Enterobacteriaceae (Salmonella) was carried out on the base of a model incubation experiment. The research was conducted in three soils: weak loamy sand (I), sandy loam (II) and moderate loam (III). The reaction of the soils used in the experiment decreased gradually, irrespective of applied fertilization and liming. The highest content of N-NH₄⁺ in soils was recorded in initial periods of the study, which was an effect of organic matter mineralization process. An increase in N-NO₃‾ content applied to all the soils tested, and the rate of N-NO₃‾ growth after applying sewage sludge and farmyard manure was the largest in weak loamy sand. Applying the mixture of sewage sludge with peat resulted in a decrease in growth rate of nitrate nitrogen content in the soils tested. Limited uptake of nitrate anions may result in washing out this nitrogen form from the rhizosphere. The population of bacteria of the genus Salmonella decreased to the level indeterminable after 64 days of incubation.

Key words: soil, nitrogen, farmyard manure, sewage sludge, Salmonella.

INTRODUCTION

Returning elements contained in sewage sludge to soil is not only economically justifiable, but it is also essential for restoring and maintaining ecological balance in agrocenoses. Introduction of sewage sludge into soil may restore its lost biological activity and enrich it in nutrients essential for plants [3,13,16].

In scientific literature one can find studies concerning a favourable effect of sewage sludge on soil structure, organic matter content and plant yield [1,4,5,15,24,34,36]. On the other hand, however, the presence of toxic heavy metals and pathogenic microorganisms in sewage sludge, which was reported by Gambuœ [9] as well as Kalisz et al. [14] is alarming. Knowledge of the amount of nitrogen released in the process of organic matter mineralization into soil after applying sewage sludge abundant in this component is also of great importance.

Presently one of the criteria of sewage sludge agricultural management is the content of heavy metals [28], thus the majority of the works published so far have referred to the possibility of using sewage sludge for plant fertilization in terms of soil contamination with toxic trace elements [6,7,11]. The problem of the dynamics of nitrogen release due to the mineralization process and soil biological contamination has been examined considerably less frequently.

In the control of soil environment condition it is essential to determine mineral forms of nitrogen, and to evaluate the occurrence and number of pathogenic organisms, mainly bacteria and the eggs of enteric parasites of people and animals. The application of farmyard manure and sewage sludge results in gradual changes in soil physical, chemical and biological properties, which differently (depending on a granulometric composition) influences an increase in mineral nitrogen content and a decrease in the survival of some pathogens, to which soil is not a specific living environment.

MATERIAL AND METHODS

The studies on the dynamics of release of nitrogen mineral forms (to the soil solution) and changes in number of bacteria of the family Enterobacteriaceae (Salmonella) were made on the basis of a model incubation experiment. The experiment concerning a soil reaction and mineral nitrogen release was conducted in three soils: weak loamy sand (soil I), sandy loam (soil II) and moderate loam (soil III), while the dynamics of the changes in the number of Salmonella was tested only on soil (I) and (III). A scheme of the experiment was the same in all soils and it included: the control (without fertilization), objects fertilized with farmyard manure, fertilized with sewage sludge (A), fertilized with a mixture of sewage sludge (A) and peat, fertilized with sewage sludge (B), fertilized with a mixture of sewage sludge (B) and peat.

Determination of physicochemical properties in the soils and experimental conditions

Soil material for the research was taken from arable fields used for agricultural purposes according to agronomic recommendations, situated in the south of Poland (10 km to the west of Cracow), from the layer 0-20 cm. The soil material collected was brought to an air-dry condition, thoroughly mixed and sieved through a sieve of 1 mm mesh diameter. In dried and sieved samples of initial soils granulometric composition was determined by the Casagrande method modified by Prószyński, pH value in KCl solution of 1 mol·dm^-3 concentration – by the potentiometric method, the content of organic carbon after mineralization of samples in potassium dichromate(VI) by the Tiurin method, total nitrogen after sample mineralization in concentrated sulphuric acid(VI) in the open system by the Kjeldahl method using the automatic kit Kjeltec II Plus (Tecator), total sulphur after the oxidation of organic sulphur and its compounds to SO₄^2- with magnesium nitrate solution and mineralization in a muffle furnace for 8 hours at 500°C by the ICP-AES method, hydrolytic acidity in CH₃COONa with a concentration of 1 mol·dm^-3 – by the Kappen method, available forms of phosphorus and potassium by the Egner-Riehm method, and magnesium by the Schachtschabel method. The content of general forms of heavy metals after organic matter mineralization (500°C for 8 h) and solubilization the samples in concentrated acids – nitric(V) and chloric(VII) (2:1) [23,25]. The content of Cd, Cr, Zn, Cu, Pb, Mn, and Ni was determined by the ICP-AES method using the apparatus Jobin Yvon 238 Ultarce, and mercury by the cold steams method using the apparatus Philips PU 9100X equipped with a VP 90 countershaft (Unicam). Chemical analyses were made in accordance with the methodology elaborated by Ostrowska et al. [23] and Page [25], and the results obtained were presented in Table 1.

Before setting up the experiment the soils were gradually saturated to a humidity of 30% maximum soil water capacity. Afterwards sandy loam (soil II) and moderate loam (soil III) were subjected to liming, in order to raise their pH value. The treatment was carried out using chemically pure CaO according to total hydrolytic acidity. Next all the soils (soil I, II and III) were left for about 4 weeks in PVC containers protecting them from humidity loss. After that time, soil weighed portions of 260 g (each soil separately) dry matter were prepared, to which organic materials were introduced. The soil after mixing with organic materials was transferred to PVC containers and placed in an incubator. During the experiment the constant humidity of incubated samples was maintained at a level of 45% ± 0.22 of the maximum soil water capacity, at a temperature of 23.3°C ± 0.03. Each series of the experiment was set up in three replications.

A dose of nitrogen introduced in the form of organic materials was 0.217 g N·kg^-1 of soil dry matter. Phosphorus and potassium were supplemented to the equal level in the soil of all objects, P – 0.196 g and K – 0.230 g·kg^-1 of soil dry matter. Phosphorus and potassium were applied in the form of water solutions of chemically pure salts: P – Ca(H₂PO₄)·H₂O, K – KCl.

Determination of the chemical properties of farmyard manure, sewage sludge and their mixtures with peat

Sewage sludge used in the study derived from two municipal sewage treatment plants (a mechanical biological system) from Krzeszowice (A) and Niepołomice (B) situated in the Małopolska province (in southern Poland). Before sampling, sewage sludge was stabilized, the technology and time of stabilization being different. The sewage sludge used in the research was subjected to oxygen stabilization in separate open chambers, where the process of aeration proceeded continuously at a temperature of ambient environment. The process of aeration lasted 5 days in the case of sludge (A), and 8 days in the case of sludge (B). After that period sewage sludge was dehydrated on filtration plots. The process lasted 2 months for sludge (A), and 3 months for sludge (B). The final step of sewage sludge (A) stabilization was its hygienization using hydrated lime in a dose of 0.15 kg Ca(OH)₂ per kg of sludge dry matter. Sewage sludge (B) after dehydration was transferred to a pile and left. Mixtures of sewage sludge with peat, mixed in a weigh ratio 1:1 (dry weight) were also used in the experiment. An addition of peat was aimed at improving sewage sludge physical properties and diluting the heavy metal content. The object fertilized with farmyard manure was treated as a point of reference for the parameters examined.

Dry matter content was determined in fresh samples of organic materials (105° C for 12 h) and the total nitrogen content after sample mineralization in concentrated sulphuric acid(VI) by the Kjeldahl method. In dried and ground material were determined as follows: the total sulphur content after sample mineralization in nitric acid(V) and oxidation of organic sulphur and its compounds with magnesium nitrate by the ICP-AES method, and the content of ash components after sample mineralization in a muffle furnace (450° C for 5 h) and the ash solubilization in nitric acid(V) (1:2). The phosphorus content was determined by the vanadium-molybdenum method on the Beckman DU 640 spectrophotometer, potassium, sodium and calcium by the FES photometry method, magnesium by the atomic absorption spectrophotometry (AAS) method using the apparatus Philips PU 9100X. Heavy metals (Cr, Cd, Cu, Pb, Ni, Zn) were determined by the ICP-AES method using the apparatus JY 238 Ultrace, and mercury by the cold steams method using the apparatus Philips PU 9100X equipped with a VP 90 countershaft (Unicam).

Fresh samples of sewage sludge and stable manure intended for microbiological tests were collected to aseptic containers using a sterile botanical knife. A representative sample of organic material (10 g) was treated with 90 cm³ of physiological solution and shaken. Next successive dilutions of the material tested were made and 1 cm³ of each was inoculated on selective agar medium. The plates were incubated at 37^oC for 48 hours, and then bacterial colonies were counted. The results were expressed in CFU of Salmonella in 1 g of material dry matter (105°C for 12 h). The study was carried out in three replications. A parasitological analysis (determination of parasite eggs of the genera Ascaris sp., Toxocara sp. and Trichuris sp.) was made after drying the organic material for 24 hours at room temperature and sieving through a sieve of 2 mm mesh diameter. A sample of 100 g was treated with 100 cm³ NaOH of 5% concentration, then mixed and left for 1 hour. Afterwards the material was shaken for 10 minutes, and then centrifuged (1500 rotations · min^-1 for 5 min). After centrifuging, the solution was decanted, and the remains were treated with a saturated solution of sodium nitrate, mixed and re-centrifuged fourfold (2500 rotations · min^-1 for 2 min). After each centrifuging the surface layer of a solution (2 cm³) was decanted. The material obtained was filtered, and then observed under the microscope at a medium magnification. Species affiliation of eggs was determined on the basis of their morphological features. The analyses were carried out in accordance with the methodology elaborated by Krzywy [18], and the results of the research are presented in Table 2.

Periodic chemical analyses in incubated material

Periodic analyses in the incubated material were carried out after 3, 8, 16, 32 and 64 days from starting the experiment. The number of bacteria of the genus Salmonella was determined in fresh samples of the soil material which were subjected to microbiological analysis before 24 hours from sampling. The analysis was made by the method applied previously for sewage sludge and farmyard manure. Mineral nitrogen (N-NH₄⁺, N-NO₃‾) was extracted from fresh soil using the 1% AlK(SO₄)₂ · 12 H₂O solution at room temperature for 1 hour maintaining a soil to solution ratio of 1:2.5. Mineral nitrogen in the filtrate was determined by the calorimetric method on the spectrophotometer Backman DU 640: ammonia nitrogen (N-NH₄⁺) using Nessler’s reagent, and nitrate nitrogen (N-NO₃‾) with phenyldisulfonic acid [26]. In dried material (105°C for 12 h), pH was determined with a potentiometer in KCl suspension of 1 mol·dm^-3 concentration. The analysis was conducted in accordance with the methodology elaborated by Panak [26].

Data analysis

Analyses of the soil material from the experiment were carried out in three replications, and analyses of organic materials and samples of initial soil material – in two replications. A laboratory sample with known parameters was added to each series of the material analyzed and a result was considered reliable if a relative standard deviation (RSD) did not exceed 5%. The results obtained were analyzed statistically according to a constant model with fertilizing as a factor. One-factor analysis of variance was applied in statistical calculations, and the significance of differences was estimated using the Duncan test, at the significance level p < 0.05 [31].

RESULTS

The pH value of the soils tested

Fertilization applied resulted in a significant diversification of the pH value of the soils used in the experiments at particular dates (Table 3). In the initial period of the experiment (till day 8) a slight pH decrease in weak loamy sand (soil I) and sandy loam (soil II) was observed at all the objects. More pH regression (till the eighth day of the experiment) was found in moderate loam (soil III). Until day 64 of the experiment a gradual soil acidifying (soil I) and (soil II) was recorded. This was not the case with moderate loam (soil III), in which an increase in the pH value was recorded from day 32. In all the soil formations the lowest pH value was found at the objects fertilized with sewage sludge (B) and its mixture with peat, and at the object fertilized with a mixture of sewage sludge (A) with peat. The pH reduction was statistically significant for the soils of the objects fertilized with farmyard manure as well as the soils of the control.

The content of mineral forms of nitrogen

In a model incubation experiment, on all soils with the addition of farmyard manure, sewage sludge and their mixtures with peat, the nitrogen content in mineral compounds resulted from a sequence of their overlapping transformations. Some of the products of nitrogen organic compounds mineralization are N-NH₄⁺ and N-NO₃‾. The most increase in the ammonia nitrogen content in relation to soils of the objects fertilized with the other materials, as well as to soils of the control was found in soils fertilized with sewage sludge (A) (Tables 4-6). The increase was statistically significant. However, some differences in reactions of particular soils after applying the sludge were observed. In weak loamy sand (soil I) and moderate loam (soil III), similar relationships existed which consisted in a decrease in the ammonia nitrogen (N-NH₄⁺) content after day 8 of the experiment in relation to the level determined after day 3. After this period the content of ammonia nitrogen increased, which was confirmed by an analysis after day 16 of the experiment. Next a gradual decrease in the N-NH₄⁺ content was recorded. In sandy loam (soil II) in turn, an increase in the ammonia nitrogen content was observed till day 8 of the experiment, and the highest one occurred in the soil from the object fertilized with sludge (A). A decrease in the N-NH₄⁺ content was recorded after day 8 of the experiment. The changes in the nitrate nitrogen (N-NO₃‾) content during soil incubation depended on the fertilization applied and increased gradually; a rate of nitrate release depended on a type of soil. A common regularity was also found which applied to the objects fertilized with sewage sludge and with a mixture of sludge and peat. Soils from the objects fertilized with a mixture of sludge and peat contained less nitrate nitrogen throughout the experiment than the soils fertilized only with sludge. The soils used in the study (soil I and II) were characterized by a similar course of nitrification process, which was relatively gentle till day 16, and then its remarkable growth was observed. This relationship did not apply to the soils from objects fertilized with sludge (B) where the intensity of the process throughout the study was the largest. Moderate loam soil (III) behaved differently after applying fertilization, since after a period of a slight decrease in nitrates (till day 8) their growth was recorded till day 64.

Survival of Salmonella

The aim of the study was also to evaluate soil self-cleaning rate after introducing materials loaded with bacteria of the genus Salmonella (Table 7). The number of colony forming units of Salmonella marked in soil I and III was the largest at the objects where sewage sludge was applied, and decreased in soils of the objects where mixtures of sludge and peat were used. Less amount of bacteria of the genus Salmonella were found in the control soil and in the soil fertilized with farmyard manure. In both soils analyzed a similar regularity was recorded, regarding a gradual decrease in the bacteria number. A rate of the process depended on fertilization applied and a kind of soil. In soils of the objects where stable manure was introduced, a presence of the microorganisms was not recorded as early as after 16 days. After 64 days soils of all the objects were free of Salmonella. Generally the process of bacteria number reduction proceeded faster in moderate loam soil (III) than in weak loamy sand (I).

DISCUSSION

According to Sapek [29], agriculture is a food producing industry and like each industry it requires some material outlays, which are spread to the environment if they are not used for production. Unused fertilizers penetrate into water and groundwater resources causing their contamination. Agriculture can pollute water with bacteria and viruses [12], nitrogen compounds [29,35] and other substances [16]. An intensity of this phenomenon depends on numerous factors, such as a soil agronomic category, that is its granulometric composition, and an intensity and type of the fertilization applied including organic fertilization and fertilization with substances of waste origin such as sewage sludge. Soil pH value is also of great importance for the intensity of processes connected with nitrogen compounds transformations.

In the present study, the pH value of soils used in the experiment decreased gradually, irrespective of fertilization applied and liming. The pH value was relatively higher in the soils of the objects fertilized with farmyard manure and sewage sludge (A), which may suggest a slower rate of soil acidifying. According to Whalen et al. [32] an acid reaction of soil can be corrected not only by liming, but also by organic fertilization – with farmyard manure. Applying cattle manure for soil fertilization (in an incubation experiment), the authors cited found that it had a favourable effect not only on the pH value of fertilized soil, but also improved its buffer properties and increased the amount of mineral forms of nitrogen (N-NH₄⁺, N-NO₃‾). In the present study such a favourable effect of fertilizing with stable manure on soil pH value was not recorded, although the soil acidifying rate at the objects fertilized with farmyard manure was slower than after applying sewage sludge (B) and mixtures of sludge with peat, and a growth in the content of nitrogen mineral forms (particularly N-NO₃‾) in soils resulted from a progressing process of organic matter mineralization. The study by Sienkiewicz [30] indicated that farmyard manure used every second year remarkably increased pH and decreased soil hydrolytic acidity. Different results were published by Wiater [33], indicating that organic fertilizers, including farmyard manure, caused a significant increase in soil hydrolytic acidity in relation to the soil from the control object. The results cited are in accordance with the tendency found on the basis of the present study. Mercik et al. [22] also found a diverse effect of fertilizing with farmyard manure on soil pH and its acidity. This fertilizer applied together with Ca or CaNPK increased the acidification of deeper genetic levels of soil, while it acted the opposite when it was used in conditions of mineral fertilization, but without liming.

Nitrates in soil originate from ammonia nitrogen nitrification [27]. They are also introduced with organic and mineral fertilizers, from which a rate of nitrogen mineral forms release, including nitrates, is considerably larger. It is confirmed by the results obtained by Kucharski and Niewolak [19], in which a considerable growth of the nitrogen content, particularly nitrate, was found in soil fertilized with urea and ammonium sulphate during a similar period of soil incubation as in the present study. The influence of soil intensive fertilization with mineral fertilizers on the N-NO₃‾ content was also confirmed in the research by other authors [17]. In the own study, nitrogen in mineral form was not used, but an increase in the N-NO₃‾ content was also recorded. The research by Gostkowska et al. [10] proved that high doses of mineral fertilizers and applying a herbicide fallow has also an impact on a passive accumulation of N-NO₃‾ ions in soil, which was a result of the lack of biological sorption. Additionally, soil physicochemical properties and use have a modifying effect on nitrification rate [19]. Łoginow and Kaszubiak [20] report that a reduction of the N-NO₃‾ content in soil may result from reduction in temperature and a rise in soil humidity.

It is assumed that plants assimilate nitrates easily, and thus a good plant growth depends on ability to their uptake. Being a process that results in providing plants with this form of nitrogen, nitrification may also have undesirable consequences. While the ammonia cation is adsorbed on soil colloids and it is relatively motionless, the nitrate anion is mobile in soil solution. Washing out of this form of nitrogen can take place mainly in late autumn and winter, when nitrogen concentration in soil coincides with groundwater renovation. The highest value of N-NH₄⁺ in soil was recorded in initial times of the study, and it was an effect of organic matter mineralization [2,21].

The mineralization rate of sewage sludge and farmyard manure applied was the largest in weak loamy sand (soil I), which together with the lack of nitrate anion uptake in this soil may result in washing out this form of nitrogen from the rhizosphere. Thus all strategy of limiting the nitrate wash-out aims at the limitation of their content after finishing the vegetation season. On the other hand, there is a possibility of limitation of nitrification intensity. A rate of this process is to a large extent dependent on the soil pH. A range from pH 6.6 to 8.0 is assumed to be the optimum; while below pH 6.0 nitrification rate decreases [27]. During the study, the pH of particular soils oscillated within a range of acid and slightly acid reaction, with a tendency to decreasing. Nevertheless, the nitrate content in the soil material tested after applying fertilization showed a growing tendency during the research period, and in moderate loam soil (III) at first a decrease (till day 8 of incubation) in the ammonia nitrogen was observed. Fiuczek [8], however, studying nitrogen transformations in peat soil found that the inhibition of nitrate growth occurred after introducing ammonium sulphate and sucrose, but only in the first week of the experiment, and already in the second week an intensity of nitrification increased remarkably, although it was less than in the soil to which only ammonium sulphate was added. The application of a mixture of sewage sludge (A) and (B) with peat also resulted in lowering nitrate nitrogen increase rate in the soils tested.

According to Guan and Holley [12] the survival of pathogenic microorganisms in soil, organic manure and water differ considerably and is specific to particular pathogens. Generally the authors claim that pathogens, including Salmonella, survive longer in an environment with lower temperature and more abundant in water. In conditions of the experiment carried out, the population of Salmonella decreased and there was practically no presence of those organisms recorded after 64 days of incubation. Bacteria of the genus Salmonella are rods of the family Enterobacteriaceae, pathogenic for people and animals. Their survival rate in soil environment is high, but in the course of time the cells die due to unfavourable environmental conditions.

CONCLUSIONS

1. The application of organic materials resulted in a gradual decrease in the soil pH. A rate of soil acidification depended on a type of organic material and soil agronomic category.

2. Highest N-NH₄⁺ content in soils was recorded at the initial time of the study, which was an effect of organic matter mineralization process.

3. An increase in the N-NO₃‾ content was found in all the soils tested and resulted from the advantage of nitrification process over ammonification.

4. The rate of N-NO₃‾ growth after applying sewage sludge and farmyard manure was the highest in weak loamy sand soil (I), which together with the lack of nitrate anions uptake can wash out this nitrogen from form the rhizosphere.

5. The application of the mixture of sewage sludge with peat caused a reduction in a rate of nitrate nitrogen growth in the soils tested. In these materials nitrogen occurred mainly in the form of complex protein compounds, and in a small amount in mineral form.

6. The population of Salmonella decreased to the level indeterminable after 64 days of incubation. A gradual decrease in the population of Salmonella resulted from unfavourable environmental conditions, namely the lack of easily available sources of carbon, unsuitable temperature and the competition of autochthonous organisms better adapted to living in soil environment.

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Accepted for print: 3.11.2006

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