Publication n 121 of the International Association of Hydrological Sciences Proceedings of the Anaheim Symposium, December 197G

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1 Publication n 121 of the International Association of Hydrological Sciences Proceedings of the Anaheim Symposium, December 197G NITRIFICATION IN EVERGLADES HISTOSOLS: A POTENTIAL ROLE IN SOIL SUBSIDENCE 1 Robert L. Tate Til Agricultural Research and Education Center, P. 0. Drawer A Belle Glade, Florida Abstract Subsidence of the Everglades Histosols occurs at a rate of approximately 3 cm/year. The major cause of this loss of soil is the oxidation of the organic matter by the microbial community. One of the products of this microbial activity is inorganic nitrogen. Assuming an annual subsidence rate of 3 cm, approximately 1400 kg/ha of nitrogen is released per year. Much of this nitrogen accumulates in the soil as nitrate. The microbial reaction sequence involved in the production of this nitrate is initiated by mineralization of the soil organic matter by the heterotrophic bacteria and fungi. The major nitrogenous product of this process is ammonium. This is the primary step involved in soil subsidence. The ammonium formed through the mineralization is then oxidized by the nitrifying bacteria to nitrate. Two groups of microorganisms are known to participate in this process, heterotrophic and autotrophic nitrifiers. Nitrate can be removed from the soil by denitrification, assimilation by the plant community or microbial community, or run off in the drainage waters. Examination of the microbial community involved in nitrogen metabolism in Histosols suggests that the nitrate concentration found in the soil can be controlled through prudent control of the water table. Raising the water table would decrease the rate of soil subsidence, hence decreasing the amount of inorganic nitrogen formed, as well as inhibit nitrification and stimulate denitrification. Preservation of one natural resource, the organic soils, will help in the preservation of the surrounding lakes and streams. The Everglades Histosols are subsiding at a rate of approximately 3 cm/ year (Stephens and Speir, 1969). Among the reasons proposed for this loss of soil elevation are compaction, shrinkage due to drying, burning, wind erosion and microbial oxidation (Stephens and Speir, 1969). The primary cause must be considered to be microbial oxidation. This aerobic process which commenced upon drainage of the soil accounts for 58 to 73 percent of the observed subsidence (Volk, 1972). These values were obtained by comparing the amount of carbon dioxide produced by microbial oxidation of the soil with the known rate of subsidence of the soils in the field. Among the products of this microbial oxidation of soil organic matter are the inorganic nitrogenous compounds nitrate and ammonium. With the soil organic nitrogen content of 3.5 percent (Davis and Lucas, 1959) in Everglades Histosols, approximately 1400 kg/ha inorganic nitrogen can be expected to be produced by the subsidence of 3 cm of soil. The accumulation of much of this nitrogen as nitrate was demonstrated by Neller (1944) when he observed nitrate accumulation in fallow soil and soil planted to corn. The nitrate concentration was measured in surface (0-15 cm) and subsurface (15-30 cm). Concentrations of 239 and 127 ppm nitrate-nitrogen (dry soil) were found in the surface and subsurface fallow soils, respectively. The growth of the corn resulted in a decrease in the nitrate in the surface soil to 145 ppm. No change was observed in the subsurface sample. Recent studies have demonstrated nitrate- Florida Agricultural Experiment Stations Journal Series No. Ov'l 657

2 nitrogen concentrations in surface samples of fallow soil collected in January, 1976, of 320 ppm. In July, 1976, variation of the nitrate concentration with depth of Pahokee muck was examined. The nitrate concentration varied with depth with 456, 176, 149, and 83.6 ppm found at the 0-18, 18-28, and cm depths, respectively. The nitrate found deep in the profile was apparently in part formed in_ situ since nitrifying bacteria were found in high concentrations at these depths (Tate, unpublished data). The moisture content of the soil has an effect on the observed nitrate content. Hortenstine and Forbes (1972) examined the nitrate content of soil water from a swampy area and an adjacent drained, newly cleared, unfertilized field. The nitrate-nitrogen concentration area was 8 fold higher in the drained area than in the swamp. Approximately 48 ppm nitrate-nitrogen was detected in the soil water at a 60 cm depth from the drained soil as compared to approximately 6 ppm from the swamp at a comparable depth. Neller (1944) also observed a decrease in nitrate with elevated water tables. With a water table of approximately 30 cm, 145 ppm nitrate-nitrogen was detected in the top 15 cm of soil. Lowering the water table to about 60 cm resulted in an increase in the nitrate concentration to 260 ppm. This indicates a direct relationship between subsidence rate and nitrate production. The rate of subsidence is inversely, linearly proportional to the depth to water table (Stephens, 1969). Thus, an increase in the depth to water table by 50 percent resulted in nearly a 50 percent increase in the nitrate detected. This solubilization of the nitrogen as a result of subsidence is of great importance to the agriculturalist since it allows growth of crops on Histosols generally in the absence of exogenously added nitrogenous fertilizer. The environmentalist also finds an interest in this nitrate since it is produced in concentrations above those needed or used by the crop (Neller, 1944). Thus, a potential for nitrogenous enrichment of waters adjacent to and flowing from Histosols exists. As a result of the problems and benefits of this nitrogen, the microbial involvement in nitrogen metabolism in Histosols has been examined. The microbial processes leading to and affecting the accumulation of nitrate in Histosols are presented in Fig. 1. The nitrogen is converted from the organic to the inorganic state by the process of mineralization. This is the step primarily relating to soil subsidence since the nitrogen is converted from an insoluble, chemically complexed form into a soluble form which can then be lost from the soil system. The primary product of this solubilization is ammonium. Some of this ammonium can be returned to the soil organic matter pool by the microbes and plants. These organisms use the ammonium in the synthesis of new cellular material. Upon the death of the organism the newly synthesized cellular material is cycled back into the soil organic matter. The ammonium formed from mineralization is further metabolized by the microcommunity to nitrate by a process called nitrification. Nitrite serves as an intermediate in this oxidative pathway. Since most of the organisms involved use ammonium as the primary substrate, one of the limiting factors in the formation of nitrate in the Histosol is the amount of ammonium available for oxidation. One exception to this will be discussed later. Nitrate can be removed from the soil by denitrification. In this process, the nitrate is reduced via nitrite to atmospheric nitrogen (dinitrogen). Because of the importance of the latter two processes, nitrification and denitrification, to the nitrate concentration found in the soil, they will be discussed in greater detail. Nitrification: Nitrification is the biological oxidation of nitrogen from a reduced to a more oxidized state (Alexander, 1965). This aerobic process is primarily accomplished by the autotrophic nitrifier. These 658

3 NITRIFICATION (OXIDATION) NITRATE REDUCTION MINERALIZATION IMMOBILIZATION ORGANIC NITROGEN Fig. 1: Nitrogen Transformations in Histosols organisms gain their energy from the oxidation of nitrogen, hence the name nitrifier, while using carbon dioxide as a carbon source. The microbes are classified as autotrophs since they only use inorganic compounds as their sources of carbon and energy. Since the oxidation of nitrogen is the sole source of energy for these organisms, their presence in the soil sample indicates that they are functioning in the production of nitrate (Alexander, 1965). The more prevalent autotrophic nitrifiers are classified in the bacterial genera Nitrobacter and Nitrosomonas. Nitrosomonas oxidize ammonium to nitrite while the Nitrobacter complete the oxidation by oxidizing the nitrite to nitrate. The energy yielded to the microorganism depends upon the portion of the reaction completed. Oxidation of ammonium to nitrite yields 65.2 to 84 kcal per mole of ammonium. The oxidation of nitrite yields 17.5 to 20.0 kcal per mole (Alexander, 1965). Based on the growth yields of Nitrosomonas europaea and Nitrobacter agilis in culture, Alexander et al. (1960) estimated that 2 x 10 Nitrosomonas and 4 x 10 Nitrobacter cells are needed to form 1.0 mg nitrate-nitrogen. Likely, the most significant limiting factor for the functioning of these organisms in Histosols, aside from the existance of ammonium the primary substrate, is the presence of oxygen. These organisms are obligately aerobic and thus, would not be expected to be found in swampy soils or flooded muck. Herlihy (1973) found few Nitrobacter and no Nitrosomonas in undrained peat. After draining and cultivation, the number of Nitrosomonas and Nitrobacter increased to 1.3 x 10 and 3.3 x 10 /g dry soil, respectively. Recently, several heterotrophic bacteria and fungi have been shown to oxidize nitrogenous compounds, both inorganic and organic, to nitrite and/or 659

4 nitrate (Eylar and Schmidt, 1959; Doxtander and Alexander, 1966; Gunner, 1963; Marshall and Alexander, 1962 and Odu and Adeoye, 1970). Verstraete and Alexander (1972) isolated an Arthrobacter sp. which oxidized ammonium to hydroxylamine, a bound hydroxylamine, a hydroxamic acid, a substance thought to be a primary nitro compound, as well as nitrite and nitrate. The bound hydroxylamine was identified as 1-nitrosoethanol. These workers also demonstrated that hydroxylamine, 1-nitrosoethanol, nitrite and nitrate were formed in samples of natural waters and soils amended with acetate and ammonium. This suggests that heterotrophic nitrification can occur in natural ecosystems (Verstraete and Alexander, 1973). Since these organisms gain most, if not all, of their energy from the oxidation of organic compounds, their existance in soil does not imply that they are nitrifying. Indeed, it has been shown that at least in culture the nitrate is formed in the culture after the growth of the microorganism. This suggests that the oxidation of ammonium is of no benefit to the heterotrophic nitrifier (Obaton et al., 1968). Both types of nitrifier have been demonstrated in Pahokee muck (Tate, 1977). Samples of fallow.muck collected in November,, 1975, contained 3.3 x 10 Nitrobacter, 1.8 x 10 Nitrosomonas and 4.1 x 10 heterotrophic nitrifying arthrobacter/g dry soil. The nitrate concentration in the soil sample was 318 ppm. Considering the above relationship between population of nitrifiers and the amount of nitrate formed, the population of autotrophic nitrifiers found in this sample was less than 0.1 percent of that necessary to yield the measured quantity of nitrate. This suggests that other organisms were responsible for at least some of the nitrate formed. One potential candidate for the nitrification is the large population of heterotrophic nitrifiers. As indicated previously, the presence of the heterotrophic nitrifier is not an indication of its being active in nitrification. Thus, several other studies were carried out to test this possibility (Tate, 1977). One indication of whether an organism is participating in a particular reaction is to observe the changes in the population when the substrate for the reaction is added. The population of an organism gaining benefit from the activity would be expected to increase. Thus, soil was amended with acetate and/or ammonium and the nitrifier population assayed. Ammonium stimulated the growth of the autotrophic nitrifier but not the heterotroph, whereas the addition of ammonium and acetate resulted in the same increase in the autotrophic nitrifiers accompanied by a four fold increase in the population of heterotrophic nitrifiers. Comparable concentrations of nitrate were produced with both treatments. In both cases, insufficient populations of autotrophic organisms were developed to account for the nitrate produced. As a further test of the function of the heterotrophic nitrifier, soil was amended with ammonium acetate plus sufficient 2-chloro-6-(trichloro-methyl) pyridine to inhibit nitrate formation but not prevent it. 2-Chloro-6-(trichloro-methyl) pyridine is a specific inhibitor of autotrophic nitrification (Goring, 1962). It was hoped that by partially inhibiting nitrate production it would be possible to determine if part of the nitrate formed resulted from the heterotrophs. The autotrophic nitrifier population was inhibited, as expected, while the heterotrophic organisms were totally uninhibited, again suggesting that the heterotrophs were indeed responsible for some of the nitrite found in the soil. A final test of this possibility involved the inoculation of sterile Pahokee muck with an Arthrobacter sp. isolated from muck. This organism produced nitrite but not nitrate when grown in culture. Nitrite was produced by this organism in natural soil and in soil amended with acetate and/or ammonium. This suggests that indeed the heterotrophic nitrifier is functioning in these soils. 660

5 The functioning of the heterotrophic nitrifier in these soils would have an impact on soil subsidence as well as water quality. These organisms could produce nitrate directly from the organic matter without having to rely on other microorganisms to produce the ammonium. Therefore the organisms would be participating directly in the process of soil subsidence. Another interesting side light to the function of the heterotroph in nitrification involves a public health problem. Verstraete and Alexander (1972) have demonstrated that one of the products of heterotrophic nitrification is 1-nitrosoethanol. Although the toxicological properties of this specific compound are not known, 1-nitrosoethanol is related to the carcinogenic N-nitrosamines which can be formed in nature (Ayanaba et al., 1973; Tate and Alexander, 1974). Denitrification: Denitrification is the major natural biological process for removing nitrogen from these Histosols. For this discussion, denitrification will be defined as the biological reduction of nitrite and nitrate to volatile gases, dinitrogen and nitrous oxide. The microorganisms involved are heterotrophs which use the nitrogen as a terminal electron acceptor. The organisms are not obligatorily linked to the process; thus, the presence of denitrifiers in a soil sample, as it was with the heterotrophic nitrifier, does not indicate nitrifying activity. As a result of the basic nature of the process, two conditions would limit the extent of the reaction in Histosols. The first is the presence of oxygen. Broadbent and Stojanovic (1952) demonstrated that in mineral soils collected in New York denitrification was inversely proportional to the partial pressure of oxygen. They did though observe appreciable denitrification under fully aerobic conditions. The second limiting factor involves the source of electrons for the reductive step. The organism must have a source of metabolizable carbon for the denitrification to occur. Of the two conditions, oxygen is likely the major limiting factor in denitrification in Histosols. 2 Populations of 3.3 x 10 denitrifiers/g dry muck were found in surface (0-15 cm) muck. The muck samples were collected in November, Comparable populations were found in the subsurface samples (46-60 cm). This is interesting since it would be expected that as the depth increased the oxygen would become limiting, which would result in the stimulation of the denitrifier population. If some other factor such as carbon were limiting the population size, then a change in the oxygen supply, such as that expected by the increased depth in the soil, would not have an effect. To test this possibility the oxygen content of muck was decreased by flooding the soil. The Pahokee muck was flooded for two weeks prior to the measurement of the denitrifiers. A 100 fold increase in the denitrifiers in the flooded soil was observed. In control soil incubated under natural aerated conditions, no change in the population was observed. Thus, the limiting of oxygen content of the soil air resulted in a significant increase in the denitrifiers. This implies that oxygen was not decreased sufficiently in the samples from cm listed above to induce the denitrifier population. Further evidence that the denitrifiers were not active in these soils is the large concentrations of nitrate detected. Apparently, the denitrifiers are inhibited sufficiently to allow the nitrate to accumulate. The nitrate is then available to the plant community or to be washed into the surrounding waters. Conclusions: Nitrate accumulation in Histosols is both beneficial and detrimental. The benefits to the agriculturalist are unquestioned. Unfortunately, the nitrate is produced by the soil microorganisms at a rate greater than is needed by the crop. This poses a potential threat to the regional water quality. Examination of the microorganisms responsible for 661

6 this nitrate production suggests two means of limiting the accumulation. These are to decrease the subsidence rate, hence the amount of inorganic nitrogen produced, and to increase denitrification. Previous work has amply demonstrated that prudent control of the water table will decrease the subsidence rate. The characteristics of the microbial community suggest that elevated water table will also decrease nitrification and increase denitrification through the creation of an anaerobic environment. The nitrification will also be decreased through the reduction of the supply of the primary substrate, ammonium. Thus, the preservation of one resource, the organic soils, can lead to the protection of a second, or lakes and streams. References Alexander, M., 1965, Denitrifying Bacteria: in (C. A. Black, ed.) Methods of Soil Analysis, pt. 2. Chemical and Microbiological Properties: American Society of Agronomy. Madison, Wi., p Alexander, M., 1965a, Nitrification: in (W. V. Bartholomew and F. E. Clark, ed.) Soil Nitrogen: American Society of Agronomy, Inc., Madison, Wi., p Alexander, M., K. C. Marshall, and P. Hirsch, 1960, Autotrophy and Heterotrophy in Nitrification: Trans. Int. Congr. Soil Sci., 7th. v. 2, p Ayanaba, A., W. Verstraete, and M. Alexander, 1973, Formation of Dimethylnitrosamine, a Carcinogen and Mutagen, in Soils Treated with Nitrogen Compounds: Soil Sci. Soc. Am. Proc. v. 37, p Broadbent, F. E., and B. F. Stojanovic, 1952, The Effect of Partial Pressure of Oxygen on Some Nitrogen Transformations: Soil Sci. Soc. Am. Proc. v. 16, p Davis, J. F., and R. E. Lucas, 1959, Organic Soils, Their Formation, Distribution, Utilization and Management: Special Bulletin 425, Michigan Ag. Exp. Sta., East Lansing, Mi. Doxtander, K. G., and M. Alexander, 1966, Nitrification by Heterotrophic Soil Microorganisms: Soil Sci. Soc. Am. Proc. v. 30, p Eylar, 0. R., and E. L. Schmidt, 1959, A Survey of Heterotrophic Microorganisms from Soil for Ability to for Nitrite and Nitrate: J. Gen. Microbiol, v. 20, p Goring, C. A. I., 1962, Control of Nitrification by 2-chloro-6-(trichloromethyl) pyridine: Soil Sci. v. 93, p Gunner, H. B., 1963, Nitrification by Arthrobacter globiformis: Nature (London) v. 197, p Hirlihy, M., 1973, Distribution of Nitrifying and Heterotrophic Microorganisms in Cutover Peats: Soil Biol. Biochem. v. 5, p Hortenstine, C. C. and R. B. Forbes, 1972, Concentrations of Nitrogen, Phosphorus, Potassium and Total Soluble Salts in Soil Solution Samples from Fertilized and Unfertilized Histosols: J. Environ. Qual. v. 1, p Marshall, K. C. and M. Alexander, 1962, Nitrification by Aspergillus flavus: J. Bacteriol. v. 83, p Neller, J. R., 1944, Influence of Cropping, Rainfall, and Water Table on Nitrates in Everglades peat: Soil Sci. v. 57, p Obaton, M., N. Amarger, and M. Alexander, 1968, Heterotrophic Nitrification by Pseudomonas aeruginosa: Arch. Microbiol, v. 63, p Odu, C. T. I., and K. B. Adeoye, 1970, Heterotrophic Nitrification in Soils: A Preliminary Investigation: Soil Biol. Biochem. v. 2, p

7 Stephens, J. C., 1969, Peat and Muck Drainage Problems: J. Irrig. and Drainage Div. Paoc. Am. Soc. Civil Eng. v. 95, p Stephens, J. C. and W. H. Speir, 1969, Subsidence of Organic Soils in the U. S. A.: Extrait de la Publication No. 89. Colloque de Tokyo. Assoc. Internationale D'Hydrologie Scientific p Tate, R. L., 1977, Nitrification in Histosols: A Potential Role for the Heterotrophic Nitrifier: Submitted for Publication, Appl. Environ. Microbiol. Tate, R. L. and M. Alexander, 1974, Formation of Dimethylamine and Diethylamine in Soil Treated with Pesticides: Soil Sci. v. 118, p Verstraete, W., and M. Alexander, 1972, Heterotrophic Nitrification by Arthrobacter sp.: J. Bacteriol. v. 110, p Verstraete, W., and M. Alexander, 1973, Heterotrophic Nitrification in Samples of Natural Ecosystems: Environ. Sci. Technol. v. 7, p Volk, B. G., 1972, Everglades Histosol Subsidence 1. C0 Evolution as Affected by Soil Type, Temperature, and Moisture: Soil Crop Sci. Soc. Fl. Proc. v. 32, p Waksman, S. A., and K. R. Stevens, Contribution to the Chemical Composition of Peat: V. The Role of Microorganisms in Peat Formation and Decomposition: Soil Sci. v. 28, p

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