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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 nitrogenmineralization from soils and organic residuesTeixeira 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. EnvironmentalChemistry DQAA, Tapada da Ajuda, 1349-017 Lisboa, Portugal, +351 213653424, cms@isa.utl.ptEXECUTIVE SUMMARYThe growth of the population in the last century, and the acknowledgment that the growth will continue, lead to variousproblems. One of the problems is the need to produce enough food to the entire population, witch will lead to anexcessive use of the land, and to its impoverishment in nutrients and organic matter, soil physical conditionsdegradation, 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. Theseresidues may be faced as a product that might help increase the soil value in organic matter, nutrients, water holding anderosion 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 suchas 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 theprocesses involving N organic compounds mineralization. A good prediction of the amounts of N mineralized from theresidues is an interesting issue, and also a valuable tool for the sustainable and rational use of these sources of nutrientsto plants growth, while preserving the environment.Having all that into consideration, the objective of the present work is to know more about nitrogen mineralization, andfinding 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 ofnitrogen compounds in several different soils, with and without application of different organic residues (poultrymanure and secondary pulp mill sludge). Soils tested were differed in texture, organic matter content as well as in pH. Amild solubilising agent (H2O) was used to extract easily mineralizable N. After, the extraction suspensions were furtherincubated at 37ºC for 10 days and sampled over this period. Mineral N (NH4-N and NO3-N) was determined in all thesoils 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 pulpmill sludge mixture. The r-squared values of the polynomial trend lines fitted to the results of the experiment showedgood values. The less favourable results were obtained for soil with poultry manure, and this effect was particularlyvisible in soils with lower sand content. When secondary pulp mill sludge was added to the soils, all r-squared valueswere excellent.The highest mineralization percentages were observed in soils with the highest organic matter (OM) contents, showingthe presence of significant amounts of easily mineralizable compounds. Soils that were poorer in OM, had a lowmineralization potential during the time of the experiment, as expected, due to the more recalcitrant nature of theorganic matter itself. The amounts of N mineralized from soil organic matter (SOM), were more related to OM contentof soils than to the texture itself. Sand and clay contents were poorly correlated to the N mineralization potential in allthe 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 millsludge mixture. It was also possible to encounter a correlation between the initial amount of N (Nkj) and the Nmineralization 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 fromthis and other residues more deeply.ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 1
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues Teixeira T. et al.1 INTRODUCTIONNitrogen (N) is an essential element to all organisms’ survival and growth, and it is present in essential compounds suchas proteins, nucleic acids and other molecules. N is a 79% part of our atmosphere but, despite that, for most organisms itis only obtained from other sources. This means that molecular nitrogen in the atmosphere cannot be used directly byeither plants or animals, and needs to be converted into mineral nitrogen compounds, or "fixed," in order to be used byliving organisms. This means that plant and animal production depend mostly on the N pool in the soil and itsavailability.On the other hand, the increasing world population, is giving rise to the need for more food producing, in particularvegetables and cereals, in quantity and quality enough to supply man’s needs. It is therefore necessary to supply moreand more nutrients to the soil in order to achieve the production levels required to reduce world hunger. Having this intoconsideration is easy to understand that the intensive and somewhat indiscriminate growing use of commercialfertilizers will not be environmentally sustainable. In fact, the excessive application of N, as mineral fertilizers, maylead 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 theincreasing production of organic residues such as manure, municipal solid waste, crop and forest waste, food industrywaste, among others (Sims, 1995).These organic materials need to be recycled in order to prevent, or at least to minimize environmental damage. Theirapplication to agricultural land can be a good solution to organic residues recycling. They can be added to soils, notonly to increase organic matter content, thus improving the physical nature of the soil, but also to provide several plantnutrients such as nitrogen. These materials are therefore, a good alternative to commercial fertilizers, but they must beapplied in high quantities, because their nutrients concentration is relatively low. This might become a problem, if theresidues 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, isthe increase of microbial activity, the decrease of erosion losses, because the residues will protect the soil, and they willalso 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 theprocesses involving N organic compounds mineralization. A good prediction of the amounts of N mineralized from theresidues is an interesting issue, and also a valuable tool for the sustainable and rational use of these sources of nutrientsto plants growth, while preserving the environment. More than 90% of the nitrogen (N) in soils is bond in the form ofORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 2
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues Teixeira T. et al.organic N compounds. Within the organic N in soils, the available N is considered to be a fraction of easilymineralizable organic N that can be estimated based on the mineral N released during incubation of soil at anappropriate temperature. However, incubation procedures are time consuming, and several chemical methods have beendeveloped as substitutes for incubations by several authors. Thus, the development of a rapid accurate and cost-effectivemethod for the prediction of N supply both from soil organic matter and the application of organic waste materials is ofgreat interest.1.1 Research objectivesThe nitrogen mineralization is not yet fully understood. Having in mind that N is one of the most important elements forcrops, 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 variousproblems. One of the problems is the need to produce enough food to the entire population, witch will lead to aexcessive use of the land, and to its impoverishment in nutrients and organic matter, soil physical conditionsdegradation, 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, theproblem 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 theymay be faced as a products that might help increase the soil value in organic matter, nutrients, water holding anderosion 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 topredict by the use of a quick and easy method, its availability from organic residues applied to soil.1.2 Organic ResiduesThe 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 METHODOLOGYA quick and easy to perform waterlogged incubation experiment was developed by Kokkonen et al. (2006), toinvestigate the evolution of mineralization of nitrogen compounds in several different soils. In this experiment, thismethodology was used in soils with and without application of two different organic residues (poultry manure andsecondary pulp mill sludge). Soils tested, differed in texture, organic matter content as well as in pH. A mildsolubilising agent (H2O) was used to extract easily mineralizable N.2.1 Soil and residuesFifteen different soils and two different residues were used in this experiment. Both soils and residues are representativeof the Portuguese soils and of Mediterranean climates, and of the animal and industrial activities related to theORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 3
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues Teixeira T. et al.agricultural practices in such region. The soils were collected in regions between the latitudes 37º05’N and 40º71’ Nand respective longitudes 8º06’ W and 7º89’ W. After the gathering of soil samples from 15 different locations inPortugal with Mediterranean like climate, as referred above, these samples where dried and ground to pass through a 2mm mesh. Soils were then submitted to a mechanical analysis, to determine the sand, loam and clay fractions, accordingto Póvoas and Barral (1992) methodology. Some characteristics were also analysed, namely the pH and the organicmatter 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 5.0ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 4
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues 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 12.652.2 Development of incubation methodSamples of each one of the dry soils weighing 5 g were placed into 36 bottles, 12 were left with soil alone, 12 weremixed with 0.085 g of poultry manure and the other 12 were mixed with 0.101g of secondary pulp mill sludge. Theamounts of organic residues added to each one of the soils, corresponded to applications of 170 kg N ha-1. To each oneof the bottles, an amount of 50 mL of distilled water was then added and the air inside was removed. All the bottleswere 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 Ncontent.2.3 Determination of mineral nitrogenMineral N (NH4-N and NO3-N) was determined in all the soils tested. Both soils and soil – residues mixtures wereanalysed. After 0 (T0), 2 (T1), 5 (T2) and 10 (T3) days, 3 bottles from each soil preparation were analyzed, by adding3.72 g of KCl(s) to each one and taking them to shake for 1 hour. After shaking, the suspension was centrifuged for 10minutes at a speed of 3500 rpm, and the supernatant was taken to N determination by segmented flowspectrophotometry.3 RESULTS AND DISCUSSIONFigure 1 shows the different amounts of N extracted from the 15 soils alone, in the different sampling times. During theanaerobic incubation, the mineral N contents of soils raised by an average amount of 40%. Only soil 4, which had ahigher content of organic matter (Table 1), showed a decrease in mineral N present. This was probably the result of anintense initial mineralization of the soil organic matter (SOM) that could have given raise to immobilization or todenitrification, due to anaerobic conditions. Soil 6 and soil 8 showed a similar tendency. While in soil 6 this fact can behypothised 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 contentof 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 Nmineralized from SOM of the soils ranged between 0.5 and 49.5% (data not shown). The highest mineralizationpercentages were observed in soils with the highest OM contents, showing the presence of significant amounts of easilymineralizable compounds. Soils that were poorer in OM, had a low mineralization potential during the time of theexperiment, as expected, due to the more recalcitrant nature of the organic matter itself. In figure 1, it’s clear that theamounts of N mineralized from SOM, were more related to OM content of soils than to the texture itself. Sand and claycontents were poorly correlated to the N mineralization potential in all the soils tested.ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 5
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues 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).ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 6
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues Teixeira T. et al.The addition of organic residues to the soil enhanced the N mineralization potential as expected (Figures 2 and 3). Ingeneral, for all the soils tested, between day 5 and day 10, there was an immobilization effect of the N released bymineralization (Figure 2). In fact, the addition of poultry manure generally leads to high amounts of N mineralized in ashort period of time (Cordovil et al., 2005), due to the high contents in easily mineralizable organic compounds. On thecontrary, when the organic residue tested was pulp mill sludge, almost all the mixtures showed a lower potential formineralization (Figure 3). In fact, at the end of the incubation, the amounts of mineral N present in the mixtures wasapproximately half the amounts presents when the residues tested was poultry manure. While the poultry manuremineralized an average of 3.35 kg N ha-1, the pulp mill sludge mineralized only 1.74 kg N ha-1. This representedrespectively 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 mineralizationduring the time of the incubation (Figure 3). Soil 4 showed a clear immobilization of N during the period of theexperiment.In table 3 it is possible to see the R-squared values of the polynomial trend lines fitted to the results of theexperiment and showed good r-squared values. The presence of organic residues clearly weakens the fitting ofthe data collected for N mineralization during the incubation period to polynomial trend lines. When only soilwas 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 pulpmill sludge applied (Figures 2 and 3).ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 7
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues 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 0,98The less favourable results were obtained for soil with poultry manure, and this effect was particularly visible in soilswith 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 lessfavourable ones for the less sandy soils. The best fitting of mineralization curves to the results obtained in incubationexperiments, 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 thesoil with poultry manure mixture (-0,33) and soil alone (0,31) and a better result for soil with secondary pulp millsludge mixture (-0,61). The correlation between the initial amount of N (Nkj) and the r-squared, reveals good results forsoil alone (0,49) and S (-0,56) and not so favourable for PM (-0,13). Correlating the r-squared and the final amount ofN, obtained after 10 days, only in the soil with secondary pulp mill sludge the result was very good (0,69), while theother two mixtures revealed the worse results of all correlations (0,00 for soil and -0,04 for S).4 CONCLUSIONSN mineralization behaviour of the soils and mixtures was well adjusted to polynomial equations and was better fitted forsoil 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.REFERENCESCordovil CMdS 2004. Dinâmica do azoto na reciclagem de resíduos orgânicos aplicados ao solo. Instituto do Ambiente. Lisboa. 56 p.ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 8
    • An easy incubation method for measuring nitrogen mineralization from soils and organic residues Teixeira T. et al.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- 1006Kokkonen 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 70-83Varennes A 2003. Macronutrientes no solo. In: Produtividade dos solos e ambiente. Escolar Editora. Lisboa. Cap 5. Pp. 157-177.ORBIT2008 - 13 - 15th of Oct. 2008, Wageningen, The Netherlands. 9