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An easy incubation method for measuring nitrogen mineralization from soils and organic residues
Teixeira T. et al.

An easy incubation method for measuring nitrogen
mineralization from soils and organic residues

Teixeira T. ,Kokkonen A., Cordovil C.M.d.S. Instituto Superior de Agronomia, DQAA,
Contact: Prof. Claudia M.d.S. Cordovil, Instituto Superior de Agronomia, TU Lisbon, Dep. Environmental
Chemistry DQAA, Tapada da Ajuda, 1349-017 Lisboa, Portugal, +351 213653424, cms@isa.utl.pt

EXECUTIVE SUMMARY
The growth of the population in the last century, and the acknowledgment that the growth will continue, lead to various
problems. One of the problems is the need to produce enough food to the entire population, witch will lead to an
excessive use of the land, and to its impoverishment in nutrients and organic matter, soil physical conditions
degradation, increasing problems with plagues and diseases, and environmental pollution due to fertilizers applications,
among others (Cordovil, 2004). Other problem is the increasing amount of residues produced by the population. These
residues may be faced as a product that might help increase the soil value in organic matter, nutrients, water holding and
erosion resistance, and at the same time solving the problem of what to do with them.
Nitrogen (N) is an essential element to all organisms’ survival and growth, being present in essential compounds such
as proteins, nucleic acids and other molecules, and it’s a very large part of organic residues composition.
The application of organic residues to agricultural soils as a source of nitrogen (N) needs a better understanding of the
processes involving N organic compounds mineralization. A good prediction of the amounts of N mineralized from the
residues is an interesting issue, and also a valuable tool for the sustainable and rational use of these sources of nutrients
to plants growth, while preserving the environment.
Having all that into consideration, the objective of the present work is to know more about nitrogen mineralization, and
finding if it’s possible to predict its availability from organic residues applied to soil.
A quick and easy to perform waterlogged incubation experiment was developed, to investigate mineralization of
nitrogen compounds in several different soils, with and without application of different organic residues (poultry
manure and secondary pulp mill sludge). Soils tested were differed in texture, organic matter content as well as in pH. A
mild solubilising agent (H2O) was used to extract easily mineralizable N. After, the extraction suspensions were further
incubated at 37ºC for 10 days and sampled over this period. Mineral N (NH4-N and NO3-N) was determined in all the
soils tested. Both soils and soil – residues mixtures were analysed after 0, 2, 5 and 10 days.
N mineralization curve was well adjusted to polynomial equations and was better fitted for soil with secondary pulp
mill sludge mixture. The r-squared values of the polynomial trend lines fitted to the results of the experiment showed
good values. The less favourable results were obtained for soil with poultry manure, and this effect was particularly
visible in soils with lower sand content. When secondary pulp mill sludge was added to the soils, all r-squared values
were excellent.
The highest mineralization percentages were observed in soils with the highest organic matter (OM) contents, showing
the presence of significant amounts of easily mineralizable compounds. Soils that were poorer in OM, had a low
mineralization potential during the time of the experiment, as expected, due to the more recalcitrant nature of the
organic matter itself. The amounts of N mineralized from soil organic matter (SOM), were more related to OM content
of soils than to the texture itself. Sand and clay contents were poorly correlated to the N mineralization potential in all
the soils tested. The addition of organic residues to the soil enhanced the N mineralization potential as expected.
The correlation between the r-squared and the final amount of N, was only favourable for soil-secondary pulp mill
sludge mixture. It was also possible to encounter a correlation between the initial amount of N (Nkj) and the N
mineralization evolution, being the best result once again for soil-secondary pulp mill sludge mixture.
This simple incubation procedure was efficient, on the simulation of the release of the easily mineralisable organic N,
both from soil and residues. However, further studies are to be made, in order to understand the N mineralization from
this and other residues more deeply.

ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 1

Teixeira T. et al.

1 INTRODUCTION
Nitrogen (N) is an essential element to all organisms’ survival and growth, and it is present in essential compounds such
as proteins, nucleic acids and other molecules. N is a 79% part of our atmosphere but, despite that, for most organisms it
is only obtained from other sources. This means that molecular nitrogen in the atmosphere cannot be used directly by
either plants or animals, and needs to be converted into mineral nitrogen compounds, or "fixed," in order to be used by
living organisms. This means that plant and animal production depend mostly on the N pool in the soil and its
availability.
On the other hand, the increasing world population, is giving rise to the need for more food producing, in particular
vegetables and cereals, in quantity and quality enough to supply man’s needs. It is therefore necessary to supply more
and more nutrients to the soil in order to achieve the production levels required to reduce world hunger. Having this into
consideration is easy to understand that the intensive and somewhat indiscriminate growing use of commercial
fertilizers will not be environmentally sustainable. In fact, the excessive application of N, as mineral fertilizers, may
lead to many environmental problems such as nitrate pollution of water resources, amongst others.

• Air pollution as far as fertilizers are concerned, arises mostly from fertilizers industries that discharge SO2, N2O,
NOx and NH3 to the atmosphere. NOx air pollution contributes to acidifying nitrate deposition. Nitrate deposition
changes water and soil chemistry and can also results in excess nitrogen in ecosystems, which can cause changes in
vegetation, loss of biodiversity, and increased greenhouse gas emissions.

• Soil pollution has to do with the amount of fertilizer applied to soil as well as how it’s applied. Soil reaction can
change, the level of ions in soil solution can rise, and nutrient’s unbalance can take place.

• Water pollution is a very important problem because, as said earlier, nitrogen is present in soils as nitrate ion,
ammonium ion, and as a component of soil organic matter. In all soils, except in very wet or dry soils, the
ammonium form is readily converted into the nitrate form. This nitrate form is completely soluble and not tightly
held into soil particles. Therefore, nitrate can readily leach downward with percolating water and contaminate
groundwater supplies. Surface water can also be contaminated, trough surface runoff that can transport soil,
vegetation and even the fertilizer applied.

• Plant pollution is mainly caused by a luxury consumption of N by the plants. If an excessive amount of fertilizer is
applied, the plant, being able to support high levels of NO3-, will absorb it in excess, and accumulate it in their
tissues. Therefore, some vegetable products can present levels of N that can cause toxicity to the consumers.

Simultaneously, the intensification of agriculture, as well as the development of industry, has been leading to the
increasing production of organic residues such as manure, municipal solid waste, crop and forest waste, food industry
waste, among others (Sims, 1995).
These organic materials need to be recycled in order to prevent, or at least to minimize environmental damage. Their
application to agricultural land can be a good solution to organic residues recycling. They can be added to soils, not
only to increase organic matter content, thus improving the physical nature of the soil, but also to provide several plant
nutrients such as nitrogen. These materials are therefore, a good alternative to commercial fertilizers, but they must be
applied in high quantities, because their nutrients concentration is relatively low. This might become a problem, if the
residues are not produced in the same place where they’ll be applied, because it involves high transportation costs.
Another cost associated, is the need to treat the materials in order to remove all pathogens (Cordovil, 2004).
Despite these disadvantages, there are many environmental benefits in using these materials. The most obvious one,
after the cutback in the use of mineral fertilizers, is the enrichment of the soil in organic matter. Not less important, is
the increase of microbial activity, the decrease of erosion losses, because the residues will protect the soil, and they will
also improve soil physical characteristics (Costa 1999; Varennes, 2003).
The application of organic residues to agricultural soils as a source of nitrogen needs a better understanding of the
processes involving N organic compounds mineralization. A good prediction of the amounts of N mineralized from the
residues is an interesting issue, and also a valuable tool for the sustainable and rational use of these sources of nutrients
to plants growth, while preserving the environment. More than 90% of the nitrogen (N) in soils is bond in the form of


Teixeira T. et al.

organic N compounds. Within the organic N in soils, the available N is considered to be a fraction of easily
mineralizable organic N that can be estimated based on the mineral N released during incubation of soil at an
appropriate temperature. However, incubation procedures are time consuming, and several chemical methods have been
developed as substitutes for incubations by several authors. Thus, the development of a rapid accurate and cost-effective
method for the prediction of N supply both from soil organic matter and the application of organic waste materials is of
great interest.

1.1 Research objectives
The nitrogen mineralization is not yet fully understood. Having in mind that N is one of the most important elements for
crops, it becomes of major importance to study its behaviour.
The growth of the population in the last century, and the acknowledgment that the growth will continue, lead to various
problems. One of the problems is the need to produce enough food to the entire population, witch will lead to a
excessive use of the land, and to its impoverishment in nutrients and organic matter, soil physical conditions
degradation, increasing problems with plagues and diseases, and environmental pollution due to fertilizers applications,
among others (Cordovil, 2004).
Other problem is the increasing amount of residues produced by the population. If we look at these residues as litter, the
problem increases along with the quantities, and the question becomes “what to do with them?”.
But we know that the organic residues are rich in organic matter and elements considered nutrients for plants, so they
may be faced as a products that might help increase the soil value in organic matter, nutrients, water holding and
erosion resistance, and at the same time solving the problem of these organic materials disposal.
The objective of the present work is therefore to know more about nitrogen mineralization, and finding if it’s possible to
predict by the use of a quick and easy method, its availability from organic residues applied to soil.

1.2 Organic Residues
The organic residues chosen for this experiment were:

• Secondary pulp mill sludge (S) – this material is very rich in biodegradable organic matter, an as there are little
alternatives for its improvement, the utilization as fertilizer, is an effective alternative that might resolve
several issues, both environmental and economical (Vasconcelos and Cabral, 1993). This material is mostly
water, biosolids and fibres. Presenting a high content in organic matter, they are rich in N, P and Mg (Cordovil,
2003; Costa, 1993). They have almost all the totality of the elements considered nutrients. The potentially toxic
elements, as metals, are present in these materials in quantities so low that problems are not to be expected as
long as the applications are not excessive (Costa, 1993).

• Poultry manure (PM) – this material is considered one of the best organic fertilizers, due to its low humidity
levels (Moore et al., 1998). Poultry manure contains large amounts of N, P, and K as well as secondary and
trace elements. In addition to providing nutrients for crop production, poultry manure applications build soil
organic reserves. The organic matter benefits crop production as it increases soil water-holding capacity, water
infiltration rates, cation exchange capacity, structural stability, and soil tilt (Moore et al. 1998).

2 METHODOLOGY
A quick and easy to perform waterlogged incubation experiment was developed by Kokkonen et al. (2006), to
investigate the evolution of mineralization of nitrogen compounds in several different soils. In this experiment, this
methodology was used in soils with and without application of two different organic residues (poultry manure and
secondary pulp mill sludge). Soils tested, differed in texture, organic matter content as well as in pH. A mild
solubilising agent (H2O) was used to extract easily mineralizable N.

2.1 Soil and residues
Fifteen different soils and two different residues were used in this experiment. Both soils and residues are representative
of the Portuguese soils and of Mediterranean climates, and of the animal and industrial activities related to the


Teixeira T. et al.

agricultural practices in such region. The soils were collected in regions between the latitudes 37º05’N and 40º71’ N
and respective longitudes 8º06’ W and 7º89’ W. After the gathering of soil samples from 15 different locations in
Portugal with Mediterranean like climate, as referred above, these samples where dried and ground to pass through a 2
mm mesh. Soils were then submitted to a mechanical analysis, to determine the sand, loam and clay fractions, according
to Póvoas and Barral (1992) methodology. Some characteristics were also analysed, namely the pH and the organic
matter content. In table 1 we can see the results of the analysis.

TABLE 1 Characteristics of the soils used in the experiment, including particle size distribution.

Coarse Fine sand Silt Clay Organic
Soil Location pH
sand (%) (%) (%) (%) Matter (%)
1 Pegões 70 17.2 9.9 2.90 0.59 6.1

2 Viseu 9.10 14.9 56.4 19.60 1.65 8.5

3 Vila Viçosa 14.98 42.08 20.51 22.42 1.8 8.2

4 Mira Sintra 21.36 54.76 10.95 12.93 4.16 4.9

5 Bencatel 8.46 38.97 27.14 25.43 0.9 7.9

6 Escoural 1 18.29 49.43 20.25 12.03 3.42 5.8

7 Escoural 2 17.19 56.78 13.29 12.74 2.43 5.9

8 Albufeira 61.24 28.06 4.04 6.66 0.76 8.1

9 Alcácer 80.25 17.66 1.46 0.62 1.19 5.9

10 Pegões 2 56.65 29.45 8.15 5.75 2.25 7.3

11 Montemor 1 35.20 39.46 13.73 11.60 1.63 6.2

12 Reguengos 32.19 42.49 13.55 11.77 3.13 6.5

13 Montemor 2 37.09 41.88 12.78 8.26 1.89 5.5

14 Montemor 3 17.78 50.53 15.47 16.22 2.02 6.0

15 Montemor 4 31.07 40.04 13.65 14.73 1.95 5.0


Teixeira T. et al.

The organic residues studied were also analysed, for some chemical characteristics as can be seen in Table 2.

TABLE 2 Characteristics of the residues used in the experiment.

Poultry Manure Secondary Pulp Mill Sludge
pH (H2O) 8.35 6.75
-1
Dry Matter (g kg ) 856.50 893.20
-1
Organic Matter (g kg ) 765.80 874.80
-1
Total organic C (g kg ) 444.20 507.40
-1
N Kjeldahl (g kg ) 40.13 33.59
-1
N-NH4 (g kg ) 10.48 1.03
N-NO3 (g kg-1) 0.36 0.41
C/N ratio 12.51 12.65

2.2 Development of incubation method
Samples of each one of the dry soils weighing 5 g were placed into 36 bottles, 12 were left with soil alone, 12 were
mixed with 0.085 g of poultry manure and the other 12 were mixed with 0.101g of secondary pulp mill sludge. The
amounts of organic residues added to each one of the soils, corresponded to applications of 170 kg N ha-1. To each one
of the bottles, an amount of 50 mL of distilled water was then added and the air inside was removed. All the bottles
were placed in a shaker for 1 hour, and immediately after the shaking, each suspension was incubated at 37 ºC. After,
the extraction suspensions were further incubated at 37ºC for 10 days and sampled over this period, for mineral N
content.

2.3 Determination of mineral nitrogen
Mineral N (NH4-N and NO3-N) was determined in all the soils tested. Both soils and soil – residues mixtures were
analysed. After 0 (T0), 2 (T1), 5 (T2) and 10 (T3) days, 3 bottles from each soil preparation were analyzed, by adding
3.72 g of KCl(s) to each one and taking them to shake for 1 hour. After shaking, the suspension was centrifuged for 10
minutes at a speed of 3500 rpm, and the supernatant was taken to N determination by segmented flow
spectrophotometry.

3 RESULTS AND DISCUSSION
Figure 1 shows the different amounts of N extracted from the 15 soils alone, in the different sampling times. During the
anaerobic incubation, the mineral N contents of soils raised by an average amount of 40%. Only soil 4, which had a
higher content of organic matter (Table 1), showed a decrease in mineral N present. This was probably the result of an
intense initial mineralization of the soil organic matter (SOM) that could have given raise to immobilization or to
denitrification, due to anaerobic conditions. Soil 6 and soil 8 showed a similar tendency. While in soil 6 this fact can be
hypothised by the presence of a high SOM content, in soil 8, no logical explanation is found for this result. Unless,
denitrification occurred, no immobilization phenomenon was expected, taking into consideration that the SOM content
of the soil was low and no exogenous organic matter was added to this soil treatment. This increases in soil mineral N,
represent variable mineralization of organic N from the SOM of each one of the soils tested. The percentage of N
mineralized from SOM of the soils ranged between 0.5 and 49.5% (data not shown). The highest mineralization
percentages were observed in soils with the highest OM contents, showing the presence of significant amounts of easily
mineralizable compounds. Soils that were poorer in OM, had a low mineralization potential during the time of the
experiment, as expected, due to the more recalcitrant nature of the organic matter itself. In figure 1, it’s clear that the
amounts of N mineralized from SOM, were more related to OM content of soils than to the texture itself. Sand and clay
contents were poorly correlated to the N mineralization potential in all the soils tested.


Teixeira T. et al.

120 soil 1
soil 2
soil 3
100
soil 4
soil 5
80 soil 6
soil 7
mg Nmin/kg soil

soil 8
60
soil 9
soil 10
40 soil 11
soil 12
soil 13
20 soil 14
soil 15

0
0 2 4 6 8 10

-20
Time (days)

FIGURE 1 Amount of Nmin present in each soil at each sampling time (mg N min kg-1 soil).

350

300
soil 1
soil 2
250 soil 3
soil 4
soil 5
mg Nmin/kg soil

soil 6
200
soil 7
soil 8
soil 9
150
soil 10
soil 11
soil 12
100 soil 13
soil 14
soil 15
50

0
0 2 4 6 8 10
Time (days)

FIGURE 2 Amount of Nmin in the mixtures of each soil with poultry manure at each sampling time (mg N min kg-1
mixture).


Teixeira T. et al.

The addition of organic residues to the soil enhanced the N mineralization potential as expected (Figures 2 and 3). In
general, for all the soils tested, between day 5 and day 10, there was an immobilization effect of the N released by
mineralization (Figure 2). In fact, the addition of poultry manure generally leads to high amounts of N mineralized in a
short period of time (Cordovil et al., 2005), due to the high contents in easily mineralizable organic compounds. On the
contrary, when the organic residue tested was pulp mill sludge, almost all the mixtures showed a lower potential for
mineralization (Figure 3). In fact, at the end of the incubation, the amounts of mineral N present in the mixtures was
approximately half the amounts presents when the residues tested was poultry manure. While the poultry manure
mineralized an average of 3.35 kg N ha-1, the pulp mill sludge mineralized only 1.74 kg N ha-1. This represented
respectively 1.97 and 1.02 % of the total Kjeldahl N applied as organic amendment.

200

150 soil 1
soil 2
soil 3
soil 4
soil 5
mg Nmin/kg soil

100 soil 6
soil 7
soil 8
soil 9
50 soil 10
soil 11
soil 12
soil 13
soil 14
0 soil 15
0 2 4 6 8 10

-50
Time (days)

FIGURE 3 Amount of Nmin in the mixtures of each soil with pulp mill sludge at each sampling time (mg N min kg-1
mixture).

Moreover, except on soil 4, all the mixtures of soil and pulp mill sludge, led to an increasing N mineralization
during the time of the incubation (Figure 3). Soil 4 showed a clear immobilization of N during the period of the
experiment.

In table 3 it is possible to see the R-squared values of the polynomial trend lines fitted to the results of the
experiment and showed good r-squared values. The presence of organic residues clearly weakens the fitting of
the data collected for N mineralization during the incubation period to polynomial trend lines. When only soil
was considered, there was no general trend line in SOM mineralization behaviour (Figure 1). On the other hand,
the presence of poultry manure revealed the potential for an initial flush in N mineralization, contrary to the pulp
mill sludge applied (Figures 2 and 3).


Teixeira T. et al.

TABLE 3 Polynomial trend lines R-squared values

2
r
Soil Soil alone Soil + PM Soil + S
1 0,99 0,68 0,98
2 0,68 0,75 1,00
3 0,96 0,64 0,99
4 0,90 0,72 0,69
5 0,90 0,90 0,99
6 0,98 0,73 0,98
7 0,99 0,50 0,99
8 0,71 0,71 0,96
9 0,61 0,74 0,99
10 0,70 0,75 0,92
11 0,93 0,63 0,95
12 0,87 0,86 0,97
13 0,99 0,68 0,99
14 0,99 0,84 0,99
15 1,00 0,80 0,98

The less favourable results were obtained for soil with poultry manure, and this effect was particularly visible in soils
with lower sand content. When secondary pulp mill sludge was added to the soils, all r-squared values were excellent,
all above 0,9, with only one exception for soil 4. Also for soil alone the results were good, being once again the less
favourable ones for the less sandy soils. The best fitting of mineralization curves to the results obtained in incubation
experiments, was already reported to be applicable to soils with high contents of sand (Cordovil et al., 2005, 2007).
Heavier textures tend to give rise to different mineralization patterns.

The correlation between the N mineralization evolution and the initial amount of N (Nkj) reveals mild results for the
soil with poultry manure mixture (-0,33) and soil alone (0,31) and a better result for soil with secondary pulp mill
sludge mixture (-0,61). The correlation between the initial amount of N (Nkj) and the r-squared, reveals good results for
soil alone (0,49) and S (-0,56) and not so favourable for PM (-0,13). Correlating the r-squared and the final amount of
N, obtained after 10 days, only in the soil with secondary pulp mill sludge the result was very good (0,69), while the
other two mixtures revealed the worse results of all correlations (0,00 for soil and -0,04 for S).

4 CONCLUSIONS
N mineralization behaviour of the soils and mixtures was well adjusted to polynomial equations and was better fitted for
soil and secondary pulp mill sludge mixture. Initial N content of soils was also correlated to N mineralization patern.
This simple incubation procedure was efficient, on the simulation of the release of the easily mineralizable organic N,
both from soil and residues. However, a greater potential for N mineralization exists from both residues tested.

5 ACKNOWLEDGEMENTS (IF ANY)
The authors acknowledge Mrs Isabel Carvalho, and Mrs Madalena Simão for technical assistance.

REFERENCES
Cordovil CMdS 2004. Dinâmica do azoto na reciclagem de resíduos orgânicos aplicados ao solo. Instituto do Ambiente.
Lisboa. 56 p.


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Cordovil CMdS 2003. Previsão da disponibilidade de azoto para as plantas a partir da mineralização de resíduos
orgânicos aplicados ao solo. Doutoramento em Engenharia Agronómica. Instituto Superior de Agronomia –
Universidade Técnica de Lisboa. Lisboa. 188 p.
Cordovil, CMdS, Coutinho J, Goss M, Cabral F 2005. Potentially mineralizable nitrogen from organic materials applied
to a sandy soil: fitting the one-pool exponential model. Soil Use Manage. 21, 65-72.
Cordovil C M d S, Coutinho J, Goss M J, Cabral F 2007. Comparison of chemical methods of assessing potentially
available organic nitrogen, from organic residues applied to a sandy soil. Commun. Soil Sci. Plant Anal. 38, 989-
1006
Kokkonen A, Esala M, Aura E 2006. Acceleration of N mineralization by release of enzymes and substrates from soil
mineral particles with phosphates. Soil Biol. Biochem. 38: 504-508.
Moore P A Jr, Daniel T C, Sharpley A N e Wood C W, 1998. Puoltry manure management. In: Agricultural uses of
municipal animal and industrial byproducts. USDA, Agriculture Research Service, Conservation Research
Report 44, pp 60-77.
Póvoas I e Barral M F 1992. Métodos de análise de solos. Comun. IICT Sér. Ciênc. Agrárias n10. 61 p.
Santos J Q 1995 Fertilização e poluição: Reciclagem agro-florestal de resíduos orgânicos. Author’s edition. Pp 70-83
Varennes A 2003. Macronutrientes no solo. In: Produtividade dos solos e ambiente. Escolar Editora. Lisboa. Cap 5. Pp.
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