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Fate of urea and ureaformaldehyde nitrogen in a one-year laboratory incubation with Douglas-fir forest floor
Soil Bid. Biochem.
Pergamon
Vol. 28, No. Copyright
PII: s0038-0717(%)00148-4
0
lO/ll, pp. 1407-1415, 1996 1996 Elsevier Science Ltd
Printed in Great Britain. All rights reserved 0038-0717/96$15.00+ 0.00
FATE OF UREA AND UREAFORMALDEHYDE NITROGEN IN A ONE-YEAR LABORATORY INCUBATION WITH DOUGLAS-FIR FOREST FLOOR T. AARNIO*,
K. MCCULLOUGH
and J.A. TROFYMOW
Pacific Forestry Centre, Canadian Forest Service, Victoria, British Columbia, V8Z lM5, Canada (Accepted 27 May 1996) Summary-In a 1 y laboratory incubation of a Douglas-fir forest floor (FH) the effects of two different kinds of organic N compounds, fast-release urea (U) and slow-release ureaformaldehyde (UF), on N transformations were studied. Compounds labelled with 15N were used to follow the mineralization and distribution of added N in the following pools: extractable NH&N, (NO; + NOTFN, soluble organic N, microbial biomass N and total N in the soil residue. The effects of U and UF on microbial activity (CO2 production), microbial biomass (FE and SIR) and on the numbers of autotrophic nitrifiers (MPN) were also studied. The pattern of transformation of N was quite different. In the U-treated soils the added N contributed mostly to the exchangeable NH: pool, whereas in the UF-treated soils the highest amount of the added N was found in the soil residue. In the U-treated soils the amount of NH: was constant throughout the experiment, but the 15Nin it was diluted by mineralization of native organic N. In the UF-treated soils the accumulation of exchangeable NH: started slowly and increased steadily. However, the atom%“N excess in the NH: pool stayed constant, as it did in the soil residue. This unchanged 15N enrichment of NH; indicates formation of a UF-humic complex. Higher atom%15N excess in the UF-treated soils in the exchangeable NH: pool (2.8%) than in the soil residue (1.5%) suggests also that the UF-N entered the active organic N pool in the soil. The results presented here help to explain earlier field observations, where UF was shown to improve the N status of forest soil, and the applied N was retained in an available N pool. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
In coniferous forests fertilization is used to increase the amount of available N for stand growth. However, the efficiency of traditional fertilizers, such as urea, is low due to volatilization, leaching, denitrification and immobilization (Morrison and Foster, 1977). Uptake by crop trees is only 5-20% of added N and most of it seems to occur during the first year (Melin and Nommik, 1988; Hulm and Killham, 1990; Preston and Mead, 1994). One possible means to improve the synchrony between availability and uptake by trees is the use of slow-release fertilizers as suggested by Mahendrappa and Salonius (1974). Ureaformaldehyde (UF), a condensation product of urea (U) and formaldehyde, is a slow-release organic nitrogen fertilizer, which is mineralized by microbial activity (Fuller and Clark, 1947; Corke and Robinson, 1966). UF has mainly been used on greens, lawns and in greenhouses (Kaempffe and Lunt, 1967; Alexander and Helm, 1990). *Author for correspondence. Department of Bioscience, Division of &neral Microbiology, P.O. Box 56, 00014 University of Helsinki, Finland. fax: 358-o-708 59262. e-mail: [email protected].
Previous results from forest trials indicate that UF is retained in the soil organic horizon and is available for plant uptake but does not increase the risk for NOT leaching. UF has a long-term positive effect (up to 14 y) on the availability of N in acid forest soil (Martikainen et al., 1989; Aarnio and Martikainen, 1995a). The improved availability of NH: did not lead to enhanced nit&cation as it did in the U-treated soils (Martikainen, 1984, 1985; Aamio and Martikainen, 1995, 1996). UF has not shown any negative effects on natural decomposition activity in soil (Fuller and Clark, 1947; Martikainen et al., 1989). In contrast, U has repeatedly been shown to cause a long-term decrease in CO2 production in poor acid forest soil (B&Ith et al., 1981; Martikainen et al., 1989; Nohrstedt et al., 1989). Our aim was to elucidate the behaviour of these two N sources and the initial transformations of applied N in soil. A l-year long laboratory incubation of a forest soil fertilized with “N-U and “N-UF was conducted and the distribution of “N between various N pools monitored. Effects of U and UF on C mineralization and on nitrification activity were also studied.
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T.
1408
Aarnio
MATERIALS AND METHODS
rr/.
Volatilization losses of N were estimated using glass microfibre filter (Whatman GF/F, 5.5 cm) traps, which had 0.5 ml of 2.2 N H3P04 containing 25 ml glycerine I-‘. These were put into five jars of each treatment for the first 8 days. Filters were extracted thoroughly with distilled water and the extracts were analysed for total N. In the U-treated soils less than 0.2% and in the UF-treated soils less than 0.1% of the amount of added N was found in the traps. Therefore it was concluded that in this study loss of N through volatilization of NH3 was negligible. This was expected since incubation conditions (low T, high moisture and acid soil) do not favour NHs formation.
Site and sampling Organic soil (FH) material was gathered from a 50-y-old coastal Douglas-fir (Pseudotsuga menziesii (Mirab.) France) stand growing in Humo-Ferric Podzols on Vancouver Island (site described by Nason et a/., 1990). Bulked fresh FH soil was homogenized by hand, sieved (3 mm) and stored in polyethylene bags at +4”C for 8 days before use. Soil properties Soil pH was measured in soil--water suspensions (1:2, v/v). Total N was determined by automatic combustion on a LECO FP-228 nitrogen analyzer. Total C was determined by combustion using a LECO CR12 cabon analyzer. Ash% was determined by ignition (600°C for I2 h). Available P was determined by the Bray 1 Method utilizing an acid fluoride extractant and subsequent colorometric analysis for POi~--P. Exchangeable Ca' +, Mg*+ and K + were determined on unbuffered NH&I leachates of the soil by Inductively Coupled Plasma Emission Spectrometry (ICP). An estimation of cation exchange capacity (CEC) was determined on the extracted soil using the alcohol wash, NaCl replacement procedure with subsequent distillation and titration to quantify retained NH,f ions. Chemical properties of the soil before incubation are shown in Table 1. Experimental
ef
CO1 production and microbial biomass At each sampling after mixing a 10 g subsample of soil was taken from each jar and was incubated overnight at +22”C. Then basal respiration was measured for 3 h by an automated continuous flowthrough respiration apparatus based on infra-red gas analysis (Setala et al., 1995). Substrate induced respiration (SIR) (80 mg glucose g-’ dw of soil) was used to estimate microbial C (Anderson and Domsch, 1978; Setala et al., 1995). Microbial biomass N was estimated by the fumigation-extraction method (FE) (Ocio and Brookes, 1990). At each sampling a 4 g subsample of soil from each jar was fumigated with ethanol-free chloroform (J.T. Baker Inc.) at +22“C for 24 h. After chloroform was removed the fumigated and unfumigated samples were extracted with 0.5 M K7S04 (shaken for 30 min at 200 rev min-‘). Soil suspensions were filtered (0.5 M KzS04 washed glass microfibre filters, Whatman FF/A) and the extracts were stored at -18°C until analysis for C and N. Preliminary extraction of samples was not done before measuring FE biomass N, a procedure which has been suggested when high background inorganic N may interfere with biomass measurement (Widmer et al., 1989). However, as all species of N were measured in the extracts separately, only values of organic N before and after fumigation were used for biomass N calculations. The fumigated, extracted soils with filters (residue soil) were rinsed with 0.5 M KzS04, dried at + 70°C. ground in a Wiley mill (850 pm) and stored at room temperature until N analysis. For samplings after 24 h and 1 week, similar NH1 traps as described above, were placed into a desicator while fumigation was carried out. No
design
To study the immediate effects of urea (U) and ureaformaldehyde (UF) on forest floor. 30 g of fresh FH soil was weighed into 500 ml open mouthed Mason jars. Treatments consisted of: control (C), urea (U) and ureaformaldehyde (UF). Soil moisture content was adjusted to 60% of water holding capacity (WHC) and maintained during the experiment. Prior to the N additions samples were kept at + 14°C for 6 d, then labelled U (8.878 atom%“N excess) and UF (9.947 atom%15N excess) powders (prepared by Kemira Ltd., Finland) were added in the amount of 1100 pg N g-’ fresh soil (equivalent to 150 kg N ha-‘). Treatment and control samples were each mixed thoroughly to ensure homogenity. The samples were incubated in the dark at + 14°C and aerated weekly, a fan was used for efficiency. From each treatment a set of five jars were taken at 24 h, I, 6. 15, 45 and 54 wk.
Table I Chemical propertres of the untreated soil” C.E.C.
C%
N%
C!N
Mg’
ash %
+
PO: -P
mg kg-’
cr&, kg-’ 99
K’
35
0.9
39
30
‘Pooled. homogenized soil sample. Values are mean of three determinatmns
8087
481
1072
52
Urea and ureaformaldehyde nitrogen NH3 volatilization occured during fumigation in these organic soils as suggested by Antisari et al. (1990). measurable
Nitrogen and carbon analysis The amounts of NH&N, (NO: + NO:)-N and soluble organic N in the soil extracts (FE) and total N in the soil residues were determined by the semi-
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micro Kjeldahl method (Bremner and Mulvaney, 1982; Preston et al., 1990). The “N enrichment on each sample was analysed as described by Preston et al. (1990) using a Vacuum Generators Sira 9 mass spectrometer. 15N determinations were made on only three of the five replicates. Soluble organic C in the non-fumigated soil extracts was estimated by a wet oxidation diffusion procedure (Snyder and Trofymow, 1984).
47 NH)N
0
Residue-N
C U UF
C U UF
C U UF
C U UF
C U UF
C U UF
24 h
1 wk
6wk
15wk
45 wk
54 wk
Fig. 1. Amount of N in exchangeable m and NO;, soluble organic (erg-N), microbial biomass (Nmic) and soil organic matter (residue-N) pools in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. Within a date, unlabelled bars or bars headed with the same letter denotes treatments that were not significantly different at P = 0.05.
T. Aarnio
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Autotrophic
nitriJiers
A most probable number (MPN) method was used to determine the numbers of autotrophic ammonium and nitrite oxidizers in the soil samples from each incubation period (only three replicate jars of five were used). The modified media of Bhuiya and Walker (1977) were used (Aarnio and Martikainen, 1992; Martikainen, 1985). The MPN tubes were kept for 10 weeks at + 14°C in the dark. The presence of ammonium and nitrite oxidation was checked by a drop test using diphenylene (Rowe et al.. 1977) and Gries-Ilosvay reagents (Alexander and Clark, 1965). Due to insufficient dilutions, some MPN values on week 45 are given as ‘higher than’ ( > ) estimations. Results are presented on a dry matter basis ( 105°C overnight). Statistical
anaIyses
The results were analysed by analysis of variance. and the means were compared using the StudentNeuman-Keuls (SNK) test.
RESULTS
Amount of N in df~~rent N pools Addition of U increased immediately the amount of exchangeable NH; in the soil and a high NH: concentration persisted throughout the 54-week incubation (Fig. I). During the first weeks of incubation exchangeable NH: pool was very low in the UF-treated soils as it was in the untreated soils. At 6-weeks the amount of NH: had increased in the UF-treated soils and continued to increase until a plateau was reached at week 45. Exchangeable NH4f also accumulated in the untreated soils from week 15 on. No NO; was seen within any of the treatments prior to week 45 (Fig. I). At the start the amount of NO; produced was about the same in all soils but by week 54 more NO; was found in the N-treated soils than in the untreated soils. The amount of soluble organic N was highest in the UF-treated soils right from the beginning of the experiment (Fig. 1). A slight increase in organic N occurred in the U-treated soils on week 6. At 54 weeks the amount of soluble N was only about one tenth what it was at 24 h in all the treatments. Urea and UF increased the amount of microbial biomass N (N,,,) immediately after the application (Fig. 1). However, after prolonged incubation the Nmic decreased considerably in both the N-treated and untreated soils, as seen at the 45-week sampling. Nitrogen content in the soil residue (after fumigation and extraction) was highest in the UF-treated soils from day 1 (Fig. 1). In the U-treated soils a slight increase in the N content compared to the
et al
untreated on.
soils was seen at week 6 and from there
A tom % “N excess in N pools The high amount of lSN in the exchangeable NH; pool in the U-treated soils confirms the fact that immediately after application a high proportion (90%) of the NH&N came from the fertilizer (Fig. 2. Table 2). However, during the incubation the proportion of fertilizer derived N (%NdQ in the NH: pool decreased and was only 40% after 54 weeks. In the UF-treated soils the degree of “N in the exchangeable NH: was relatively constant. The atom%15N excess in the accumulated NOJ stayed constant in both the U-treated and UFtreated soils and was at the same degree of enrichindicating that only autotrophic ment as in NH:. nitrification occured (Fig. 2). In the UF-treated soils the atom%15N excess in the soluble organic N was relatively high during the first weeks of the incubation (Fig. 2(c)). Of the extracted organic N 50% originated from the water-soluble fraction of UF, which consists of free urea and short chain polymers of methylene ureas (Table 2. Christianson et al., 1988). In the U-treated soils soluble organic N had low atom%“N excess, but the “N content was somewhat increased at 6-weeks. In both the U- and UF-treated soils atom%15N excess in microbial biomass N (N,,,,) increased for a few weeks, but after week I5 decreased (Fig. 2). At the beginning of incubation the amount of “N immobilized in the soil was very low in the urea treatment (Fig. 2). However, the %Ndff in the soil increased from 4% to 15% during the 54-week incubation (Table 2). Application of UF increased the atom%“N excess in the soil immediately and the content of 15N stayed constant. Soil pH, microbial hiomuss and uctivit? Soil pH increased gradually during the incubation in the untreated and UF-treated soils (Table 3). In the U-treated soils the increase in pH was immediate and the high pH remained throughout the incubation. However, at 54-weeks the soil pH in the Uand UF-treated soils were lower than in the untreated soils. Soil COZ production during the laboratory incubation was highest in the U-treated soils at l-week and 6-weeks (Fig. 3). The production of CO2 in the untreated and the UF-treated soils were essentially the same. Prolonged incubation decreased the CO* production with all the treatments. At 45-weeks the amount of COZ produced was about half what it was at the beginning of the experiment. There was no difference in microbial biomass C (Cm,,) between treatments, but in accordance with temporal change in the CO* production, the amount of Cmic was greatly reduced by the 54-week sampling (Fig. 3).
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Urea and ureaformaldehyde nitrogen
NH;-N 8
t 3
4
zr 8
0
c
8
NOi-N
41
$ ae
4
1
Residue-N
U UF 24 h
U UF
U UF
U UF
U UF
U UF
1 wk
8wk
15wk
45 wk
54 wk
Fig. 2. “N enrichment in exchangeable NH: and NOT, soluble organic (org-N), microbial biomass (N,,+) and soil organic matter (residue-N) pools in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. To calculate Nmtc mean values before and after fumigation were used.
quotient metabolic The (qCO2 = KS CO2 - Cmg-’ C,i,h-‘), which is considered to reflect both changes in available C as well as changes in microbial metabolic state (Bradley and Fyles, 1995), followed the pattern of the CO2 production (Fig. 3). Due to similar temporal decrease both in the C02-production and the Cmic the qCO2 did not change with time except for the U-treated soil.
The amount of soluble organic C differed greatly within treatments (Fig. 3). However, at 6-weeks the highest amount was clearly in the U-treated soils. At week 15 the values were as low as after 24 h, but by week 54 the amount of soluble carbon had increased considerably in all the treatments. The number of autotrophic nitrifiers showed great variation between samples within a treatment (Table 4). Low numbers of nitrite oxidizers were
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T. Aarnio er al. Table 2. The proportmn of ferttlizer derived N (%NdfQ in different N pools of soil 24 h
I wk
92.0 (1.58) 0 4.37 4.08 (0.17) 0 0 51.9 (7.63) 17.0 (5.29)
X9.0 (0.56) 0 2 53 6.44 (O.YS) 0 0 52.X (8.6X) 12.4 (2.67)
Incubation time Treatment
u
UF
6 wk
15 wk
45 wk
54 wk
55.9 (1.81) 0 I2 3 13.9 (0.44) 32.7 ( I .4X) 0 0 14.7 (3.18)
43.8 (0.81) 37.1 7.86 15.0 (0.49) 27.8 (3 13) 24.8 3 94 16.9 (I 14)
38.2 (2.32) 36.9 (1.13) 0 15.0 (0.68) 30.0 (0.40) 29.5 (3.39) 3.37 17.5 (2.31)
N pool NH; N NOj -N Org-N Residue N” NH; -N NOT-N Or-N Residue N
65.7 0 20.x (1.37) Il.2 (0.41) 2.5.6 (5.22) 0 65.6 (2.86) 13.‘) (I 49)
Mean of three replications. Standard dewations m parentheses (not shown when only two replicas). U = urea: UF = ureaformaldehyde. a Residue N includes microbial biomass
detected from the beginning in all soils. The presence of ammonium oxidizers was first detected at 6-weeks and then only in the N-treated soils. At 15weeks the number of nitrifiers had increased. as well as the number of ammonium oxidizers in the untreated soils. These increases continued throughout subsequent samplings.
DISCUSSION
Microbial biomuss und activit? In accordance with earlier observations U caused an immediate increase in pH and the subsequent release of soluble C stimulated a temporary increase in CO2 production by existing microbial populations (Fig. 3, Table 3) (Ogner, 1972; Baath et d., 1981; Foster et al., 1985b). However, increased activity did not increase C,,, (Fig. 3), but did enhance immobilization of N (Fig. I). At 45-weeks there was a clear reduction in the amount of microbial biomass and in CO1 production in all soils (Figs 1 and 3). Prolonged incubation of soil without external inputs leads to nutrient deficiencies in the system. Reduced availability of oxidizable C restricts the activity of heterotrophic microorganisms. The collapse of the microbial population was a probable cause for the increase in the amount of soluble organic C seen at 45 and 54-weeks (Fig. 3). At this stage the released C did not enhance microbial activity, probably due to changes in population structure or dormancy. A similar increase was not seen in the amount of soluble organic N, which indicates tighter immobilization of organic N compounds into humus (Fig. 1).
iii Irun~fbrmations The pattern of N transformation was quite different with the two fertilizers. In the U-treated soils the added N contributed mostly to the exchangeable NH: pool (Figs I and 2, Table 2). The enhancement in microbial activity by U (Fig. 3) increased the mineralization of soil organic matter which was seen as a continuous dilution of “N in the NH: pool. The gradual increase in lSN in the microbial biomass and in the soil residue indicates that ureaN entered the soil organic N pool via immobilization by microbes (Fig. 2). Neither the “N results nor total N concentration in the soil residue suggest rapid chemical fixation of NH3 as suggested by Foster c’t a/. (1985a) and Schimel and Firestone (1989). It has been shown that chemical fixation of N can be important in U-treated forest floor, exceeding the amount of N immobilized by microbes (Foster et al., 1985a). Results from field and laboratory studies should be compared with some caution. The lack of plant uptake and leaching during a laboratory incubation of soil increase the accumulation of exchangeable NH: (Foster ef al., 1985b). In the UF-treated soils most of the added N was found in the soil residue (Fig. I). At the beginning of the experiment it is expected for most of UF is still undegraded. Atom%“N excess in soil residue stayed constant throughout the incubation (Fig. 2). The soluble fraction of UF was quickly immobilized by biomass. The accumulation of NH: from UF started slowly as time was needed for a suitable community of ammonifying organisms to develop (Fig. 1) (Corke and Robinson, 1966; Carter et al., 1986). Although the amount of NH: increased steadily the atom%‘5N excess in the NH: pool
Table 3. SolI pH (H,O) Incubation time
24 h
I wk
6 wk
15 wk
45 wk
54 wk
4.4 (0.08) 6.4 (0.17) 4.5 (0.09)
4.5 (0.02) 6.4 (0.15) 4.6 (0.05)
4.8 (0.18) 6.3 (0.13) 5.1 (0.11)
5.1 (0.03) 6.4 (0.04) 5.7 (0.05)
5.8 (0.56) 7 0 (0.26) 6.1 (0.7)
6.5 (0.16) 5.7 (0.78) 5.7 (0.88)
Treatment
c U UF
Mean of five replications. Standard deviation in parentheses, C = untreated: U = wea: UF = ureaformaldehyde
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Urea and ureaformaldehyde nitrogen
81%nic
C U l;F
C U UF
C U UF
C U UF
C U UF
C U UF
24 h
1 wk
6wk
15wk
45 wk
54 wk
Fig. 3. Basal respiration rate (CO*-C), microbial biomass C(C,i,J, metabolic quotient (qCO2) and soluble organic C (org-C) in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. Within a date, unlablled bars or bars headed with the same letter denotes treatments that were not significantly different at P = 0.05.
Table 4. Numbers (MPN g-’ dry soil) of ammonium and nitrite oxidizers in the soil Incubation time
Treatment
NH;
oxidizers
% (min - max) 1 wk
6wk
1s wk 45 wk
54 wk
C U UF C U UF C U UF C U UF C
NOT oxidizers % (min
- max)
0 0 0 0
14 62 358 > 10’S IX 10s > 10’ >I06 > 10’ 7x 106 2X 10s 4x10’
(O-14) (o-186) (O-l x lo3 (6x 109 >2x 10’ (O-2 x 10’) (5 X 106- > 2 X 10’ (8 x lo’- > 2 x IO’) (2 X 106- > 2 x 10’) (254-2 x 10’ (2 x IO’-3 x 108) (2 X lo’-5 x 10’)
Mean (% of three replications, minimum (min) and maximum (max) values are given in parentheses. ‘Insufficient dilution, see materials and methods. C = untreated; U = urea; UF = ureaformaldehyde.
33 39 34 162 14 36 955 s x IO4 IS0 > IO6 3 X 104 sx 10s 326 1x lo* 2x 10s
(O-100) (O-100) (O-100) (104-196) (O-41) (O-66) (O-3 X 103) (O-7 x IO’) (o-270) (17- >2 x IO’) (O-8 x 10’) (1 x IO>-1x 106) (213-456) (1 x 104-3x 108) (2 x IO’-5 x 106,
1414
T. Aarnio et al
stayed constant (Fig. 2). The soluble organic N pool was not big enough to be a sole source of NH: but mineralization from soil residue also contributed (Fig. 1). This unchanged “N enrichment of NH: could indicate formation of some kind of UF-humic complex in soil. Besides, the atom%15N excess was only 1.5% in the soil residue, but 2.8% in the exchangeable NH;, which suggests that the UF-N entered the active mineralizable organic N pool in the soil. The partly protective role of humic substances has been shown before. Binding with orfactors ganic matter was one of the main controlling the bioavailability of xenobiotics in aquatic environments (Kukkonen and Oikari, 1991). Also, organic compounds were degraded more slowly in soil when mixed with humic acid than when mixed with other soil constituents such as kaolinite or illite (Knabel et al.. 1994). It is assumed that in this artificial system only after complete decomposition of the UF-humus complex would 15N in the NH4f be diluted by mineralization of native organic N. This being the case, it seems that the duration of present experiment was not sufficiently prolonged. In addition to the decrease in microbial activity after prolonged incubation, low temperature ( + 14°C as used here) can delay the mineralization process of UF (Basaraba, 1964; Hadas and Kafkafi. 1974; Sasson, 1979) as can the increase in pH. The mineralization of UF is favoured by acidity and is greatly reduced in neutral soil (Basaraba. 1964; Nommik, 1967; Tlustos and Blackmer. 1992). Delayed decomposition due to low temperature in boreal coniferous forest soil could partly explain the long-term effect of UF on soil N status reported by Martikainen et al. (1989). The formation of a UF-humic complex in the active N pool of soil, as we have suggested, could explain earlier observations of positive, long-term effects of UF (in contrast to U) on soil N availability (Martikainen er N/.. 1989). The portion of urea-N which is not taken up rapidly after application is susceptible for incorporation in relatively stable, poorly-mineralizable organic forms in forest floor (Johnson ef ul., 1980; Nason and Myrold, 1992). It has also been shown that a significant fraction of newly immobilized N in soil is transformed to unavailable components of microbial tissues such as fungal melanins (He et al., 1988). NitriJication The soil studied has naturally low nitrification activity. Low pH and low availability of NH; are considered to be the main factors limiting autotrophic nitrifiers in acid forest soils (Martikainen, 1985). The increase in pH and of NH4f after U application had no immediate effect on the number of autotrophic ammonium oxidizers (Table 4). In our study limited availability of C seemed to be among
the key factors giving the autotrophic nitrifiers a competitive advantage over heterotrophic microbes. As shown earlier, nitrifiers are successful competitors under conditions of stationary or declining population of heterotrophs (Hart et al., 1994). After prolonged incubation the number of nitrifiers also increased in the untreated soils, although higher nitrate accumulation was maintained in the N-treated soils (Fig. I). There was no difference between the slow-release UF and U in the time needed for nitrifiers to proliferate (Table 4). The results presented here confirm our earlier observations that it is possible to select and develop a fertilizer that improves, for a prolonged period, the N status of forest soil and to ensure applied N is retained in the mineralizable N pool. Acknowledgemenrs-We warmly Pert0 thank Drs Martikainen, Caroline Preston and Aino Smolander and also Outi Priha MSc, for their constructive criticism and valuable comments throughout the study. We thank Ann van Niekerk for analysing soil properties and Dan Dunway for graphics. This work was supported by the Finnish Academy of Science through a grant to T. Aarnio.
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Fuller W. H. and Clark K. G. (1947) Microbiological studies on urea-formaldehyde preparations. Soil Science Society of America Proceedings
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Hadas A. and Kafkafi U. (1974) Kinetics of the mineralization of ureaform as influenced by temperature. Soil
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Science 118, 1621.
Hart S. C., Nason G. E., Myrold D. D. and Perry D. A. (1994) Dynamics of gross nitrogen transformations in an old-growth forest: the carbon connection. Ecology 75, 880-891. He X. -T., Stevenson F. J., Mulvaney R. L. and Kelley K. R. (1988) incorporation of newly immobilized “N into stable organic forms in soil. Soil Biology and Biochemistry
20, 75-8
1.
Hulm S. C. and Killham K. (1990) Response over two growing seasons of a Sitka spruce stand to 15N-urea fertilizer. Plant and Soil 124, 65-72. Johnson D. W., Edwards N. T. and Todd D. E. (1980) Nitrogen mineralization, immobilization, and nitrification following urea fertilization of a forest soil under field and laboratory conditions. Soil Science Society of America Journal 44, 610-616.
Kaempffe G. C. and Lunt 0. R. (1967) Availability of various fractions of urea-formaldehyde. Journal of Agricultural and Food Chemistry l&967-97 1. Knabel D. B., Federle T. W., McAvoy D. C. and Vestal J. R. (1994) Effects of mineral and organic soil constituents on microbial mineralization of organic compounds and Environmental in a natural soil. Applied Microbiology
60, 45004508.
Kukkonen I. and Oikari A. (1991) Bioavailability of organic pollutants in boreal waters with varaying levels of dissolved organic material. Water Research 25, 455-463. Mahendrappa M. K. and Salonius P. 0. (1974) Ammonia volatilization from black spruce raw humus treated with normal and controlled-release urea. Soil Science 117, 117-119. Martikainen P. J. (1984) Nitrification in two coniferous forest soils after different fertilization treatments. Soil Biology and Biochemistry
16, 577-582.
Martikainen P. J. (1985) Numbers of autotronhic nitrifiers and nitrification‘in fertilized forest soil. Soi Biology and Biochemistry
17, 245-248.
Martikainen P. J., Aamio T., Taavitsainen V.-M., Piiivinen L. and Salonen K. (1989) Mineralization of
carbon and nitrogen in soil samples taken from three fertilized pine stands: Long-term effects. Plant and Soil 114, 99-106.
Melin J. and Nommik H. (1988) Fertilizer nitrogen distribution in a Pinus sylvestris/Picea abies ecosystem, Central Sweden. Scandinavian Journal of Forest Research 3, 3-15.
Morrison I. K. and Foster N. W. (1977) Fate of urea fertilizer added to a boreal forest Pinus banksiana Lamb. stand. Soil Science Society of America Journal 41, 44448.
Nason G. E. and Myrold D. D. (1992) Nitrogen fertilizers: Fates and environmental effects in forests. In Forest Fertilization: Sustaining and Improving Nutrition and Growth of Western Forests (H. N. Chappell, G. F.
Weetman and R. E. Miller, Eds), pp. 67-81. Institute of Resources Conttrib. 73. University of Forest Washington, Seattle. Nason G. E., Pluth D. J., Hardin R. T. and McGill W. B. (1990)Dynamics of foliar N in Douglas-fir after spring and fall application of ammonium nitrate and urea. Canadian Journal of Forest Research 20, 1515-I 523. Nohrstedt H.-G., Arnebrant K., B&h E. and Sijderstrom B. (1989) Changes in carbon content, respiration rate, ATP content, and microbial biomass in nitrogen-fertilized pine forest soils in Sweden. Canadian Journal of Forest Research 19, 323-328.
Nommik H. (1967) Mineralization nitrogen in urea-formaldehyde Agriculturre Scandinavica
and availability of Acta compounds.
17, 33-38.
Ocio J. A. and Brookes P. C. (1990) An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterization of the biomass that develops. Soil Biology and Biochemistry
22, 6855694.
Ogner G. (1972) The composition of a forest raw humus after fertilization with urea. Soil Science 113, 440447. Preston C. M. and Mead D. J. (1994) Growth response and recovery of “N-fertilizer eight growing seasons after application to lodgepole pine in British Columbia. Forest Ecology and Management
65, 219-229.
Preston C. M., Marshall V. G., McCullough K. and Mead D. J. (1990) Fate of “N-1abelled fertilizer aDDlied on _ snow at two forest sites in British Columbia. Canadian Journal of Forest Research 20, 1583-l 592. Rowe R., Todd R. and Waide J. (1977) Microtechnique for MPN analysis. Applied and Environmental I
Microbiology
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33, 675-680.
Sasson P. (1979) Evaluation of ureaform activity in soil. Soil Science 12B, 285-290. Schimel J. P. and Firestone M. K. (1989) Inorganic N incorporation by coniferous forest floor material. Soil Biology and Biochemistry
21, 4146.
Setala H., Marshall V. G. and Trofymow J. A. (1995) Influence of micro- and macro-habitat factors on collembolan communities in Douglas-fir stumps during forest succession. Applied Soil Ecology. 2, 227-242. Snyder J. D. and Trofymow J. A. (1984) A rapid accurate wet oxidation diffusion procedure for determining organic and inorganic carbon in plant and soil samples. Communications 587-597.
in Soil Science and Plant Analyses
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Tlustos P. and Blackmer A. M. (1992) Release of nitrogen from ureaform fractions as influenced by soil pH. Soil Science Society of America Journal 56, 1807-1810.
Widmer P., Broolces P. C. and Parry L. C. (1989) Microbial biomass nitrogen measurements in soils containing large amounts of inorganic nitrogen. Soil Biology and Biochemistry
21, 865-867.
Pergamon
Vol. 28, No. Copyright
PII: s0038-0717(%)00148-4
0
lO/ll, pp. 1407-1415, 1996 1996 Elsevier Science Ltd
Printed in Great Britain. All rights reserved 0038-0717/96$15.00+ 0.00
FATE OF UREA AND UREAFORMALDEHYDE NITROGEN IN A ONE-YEAR LABORATORY INCUBATION WITH DOUGLAS-FIR FOREST FLOOR T. AARNIO*,
K. MCCULLOUGH
and J.A. TROFYMOW
Pacific Forestry Centre, Canadian Forest Service, Victoria, British Columbia, V8Z lM5, Canada (Accepted 27 May 1996) Summary-In a 1 y laboratory incubation of a Douglas-fir forest floor (FH) the effects of two different kinds of organic N compounds, fast-release urea (U) and slow-release ureaformaldehyde (UF), on N transformations were studied. Compounds labelled with 15N were used to follow the mineralization and distribution of added N in the following pools: extractable NH&N, (NO; + NOTFN, soluble organic N, microbial biomass N and total N in the soil residue. The effects of U and UF on microbial activity (CO2 production), microbial biomass (FE and SIR) and on the numbers of autotrophic nitrifiers (MPN) were also studied. The pattern of transformation of N was quite different. In the U-treated soils the added N contributed mostly to the exchangeable NH: pool, whereas in the UF-treated soils the highest amount of the added N was found in the soil residue. In the U-treated soils the amount of NH: was constant throughout the experiment, but the 15Nin it was diluted by mineralization of native organic N. In the UF-treated soils the accumulation of exchangeable NH: started slowly and increased steadily. However, the atom%“N excess in the NH: pool stayed constant, as it did in the soil residue. This unchanged 15N enrichment of NH; indicates formation of a UF-humic complex. Higher atom%15N excess in the UF-treated soils in the exchangeable NH: pool (2.8%) than in the soil residue (1.5%) suggests also that the UF-N entered the active organic N pool in the soil. The results presented here help to explain earlier field observations, where UF was shown to improve the N status of forest soil, and the applied N was retained in an available N pool. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
In coniferous forests fertilization is used to increase the amount of available N for stand growth. However, the efficiency of traditional fertilizers, such as urea, is low due to volatilization, leaching, denitrification and immobilization (Morrison and Foster, 1977). Uptake by crop trees is only 5-20% of added N and most of it seems to occur during the first year (Melin and Nommik, 1988; Hulm and Killham, 1990; Preston and Mead, 1994). One possible means to improve the synchrony between availability and uptake by trees is the use of slow-release fertilizers as suggested by Mahendrappa and Salonius (1974). Ureaformaldehyde (UF), a condensation product of urea (U) and formaldehyde, is a slow-release organic nitrogen fertilizer, which is mineralized by microbial activity (Fuller and Clark, 1947; Corke and Robinson, 1966). UF has mainly been used on greens, lawns and in greenhouses (Kaempffe and Lunt, 1967; Alexander and Helm, 1990). *Author for correspondence. Department of Bioscience, Division of &neral Microbiology, P.O. Box 56, 00014 University of Helsinki, Finland. fax: 358-o-708 59262. e-mail: [email protected].
Previous results from forest trials indicate that UF is retained in the soil organic horizon and is available for plant uptake but does not increase the risk for NOT leaching. UF has a long-term positive effect (up to 14 y) on the availability of N in acid forest soil (Martikainen et al., 1989; Aarnio and Martikainen, 1995a). The improved availability of NH: did not lead to enhanced nit&cation as it did in the U-treated soils (Martikainen, 1984, 1985; Aamio and Martikainen, 1995, 1996). UF has not shown any negative effects on natural decomposition activity in soil (Fuller and Clark, 1947; Martikainen et al., 1989). In contrast, U has repeatedly been shown to cause a long-term decrease in CO2 production in poor acid forest soil (B&Ith et al., 1981; Martikainen et al., 1989; Nohrstedt et al., 1989). Our aim was to elucidate the behaviour of these two N sources and the initial transformations of applied N in soil. A l-year long laboratory incubation of a forest soil fertilized with “N-U and “N-UF was conducted and the distribution of “N between various N pools monitored. Effects of U and UF on C mineralization and on nitrification activity were also studied.
1407
T.
1408
Aarnio
MATERIALS AND METHODS
rr/.
Volatilization losses of N were estimated using glass microfibre filter (Whatman GF/F, 5.5 cm) traps, which had 0.5 ml of 2.2 N H3P04 containing 25 ml glycerine I-‘. These were put into five jars of each treatment for the first 8 days. Filters were extracted thoroughly with distilled water and the extracts were analysed for total N. In the U-treated soils less than 0.2% and in the UF-treated soils less than 0.1% of the amount of added N was found in the traps. Therefore it was concluded that in this study loss of N through volatilization of NH3 was negligible. This was expected since incubation conditions (low T, high moisture and acid soil) do not favour NHs formation.
Site and sampling Organic soil (FH) material was gathered from a 50-y-old coastal Douglas-fir (Pseudotsuga menziesii (Mirab.) France) stand growing in Humo-Ferric Podzols on Vancouver Island (site described by Nason et a/., 1990). Bulked fresh FH soil was homogenized by hand, sieved (3 mm) and stored in polyethylene bags at +4”C for 8 days before use. Soil properties Soil pH was measured in soil--water suspensions (1:2, v/v). Total N was determined by automatic combustion on a LECO FP-228 nitrogen analyzer. Total C was determined by combustion using a LECO CR12 cabon analyzer. Ash% was determined by ignition (600°C for I2 h). Available P was determined by the Bray 1 Method utilizing an acid fluoride extractant and subsequent colorometric analysis for POi~--P. Exchangeable Ca' +, Mg*+ and K + were determined on unbuffered NH&I leachates of the soil by Inductively Coupled Plasma Emission Spectrometry (ICP). An estimation of cation exchange capacity (CEC) was determined on the extracted soil using the alcohol wash, NaCl replacement procedure with subsequent distillation and titration to quantify retained NH,f ions. Chemical properties of the soil before incubation are shown in Table 1. Experimental
ef
CO1 production and microbial biomass At each sampling after mixing a 10 g subsample of soil was taken from each jar and was incubated overnight at +22”C. Then basal respiration was measured for 3 h by an automated continuous flowthrough respiration apparatus based on infra-red gas analysis (Setala et al., 1995). Substrate induced respiration (SIR) (80 mg glucose g-’ dw of soil) was used to estimate microbial C (Anderson and Domsch, 1978; Setala et al., 1995). Microbial biomass N was estimated by the fumigation-extraction method (FE) (Ocio and Brookes, 1990). At each sampling a 4 g subsample of soil from each jar was fumigated with ethanol-free chloroform (J.T. Baker Inc.) at +22“C for 24 h. After chloroform was removed the fumigated and unfumigated samples were extracted with 0.5 M K7S04 (shaken for 30 min at 200 rev min-‘). Soil suspensions were filtered (0.5 M KzS04 washed glass microfibre filters, Whatman FF/A) and the extracts were stored at -18°C until analysis for C and N. Preliminary extraction of samples was not done before measuring FE biomass N, a procedure which has been suggested when high background inorganic N may interfere with biomass measurement (Widmer et al., 1989). However, as all species of N were measured in the extracts separately, only values of organic N before and after fumigation were used for biomass N calculations. The fumigated, extracted soils with filters (residue soil) were rinsed with 0.5 M KzS04, dried at + 70°C. ground in a Wiley mill (850 pm) and stored at room temperature until N analysis. For samplings after 24 h and 1 week, similar NH1 traps as described above, were placed into a desicator while fumigation was carried out. No
design
To study the immediate effects of urea (U) and ureaformaldehyde (UF) on forest floor. 30 g of fresh FH soil was weighed into 500 ml open mouthed Mason jars. Treatments consisted of: control (C), urea (U) and ureaformaldehyde (UF). Soil moisture content was adjusted to 60% of water holding capacity (WHC) and maintained during the experiment. Prior to the N additions samples were kept at + 14°C for 6 d, then labelled U (8.878 atom%“N excess) and UF (9.947 atom%15N excess) powders (prepared by Kemira Ltd., Finland) were added in the amount of 1100 pg N g-’ fresh soil (equivalent to 150 kg N ha-‘). Treatment and control samples were each mixed thoroughly to ensure homogenity. The samples were incubated in the dark at + 14°C and aerated weekly, a fan was used for efficiency. From each treatment a set of five jars were taken at 24 h, I, 6. 15, 45 and 54 wk.
Table I Chemical propertres of the untreated soil” C.E.C.
C%
N%
C!N
Mg’
ash %
+
PO: -P
mg kg-’
cr&, kg-’ 99
K’
35
0.9
39
30
‘Pooled. homogenized soil sample. Values are mean of three determinatmns
8087
481
1072
52
Urea and ureaformaldehyde nitrogen NH3 volatilization occured during fumigation in these organic soils as suggested by Antisari et al. (1990). measurable
Nitrogen and carbon analysis The amounts of NH&N, (NO: + NO:)-N and soluble organic N in the soil extracts (FE) and total N in the soil residues were determined by the semi-
1409
micro Kjeldahl method (Bremner and Mulvaney, 1982; Preston et al., 1990). The “N enrichment on each sample was analysed as described by Preston et al. (1990) using a Vacuum Generators Sira 9 mass spectrometer. 15N determinations were made on only three of the five replicates. Soluble organic C in the non-fumigated soil extracts was estimated by a wet oxidation diffusion procedure (Snyder and Trofymow, 1984).
47 NH)N
0
Residue-N
C U UF
C U UF
C U UF
C U UF
C U UF
C U UF
24 h
1 wk
6wk
15wk
45 wk
54 wk
Fig. 1. Amount of N in exchangeable m and NO;, soluble organic (erg-N), microbial biomass (Nmic) and soil organic matter (residue-N) pools in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. Within a date, unlabelled bars or bars headed with the same letter denotes treatments that were not significantly different at P = 0.05.
T. Aarnio
1410
Autotrophic
nitriJiers
A most probable number (MPN) method was used to determine the numbers of autotrophic ammonium and nitrite oxidizers in the soil samples from each incubation period (only three replicate jars of five were used). The modified media of Bhuiya and Walker (1977) were used (Aarnio and Martikainen, 1992; Martikainen, 1985). The MPN tubes were kept for 10 weeks at + 14°C in the dark. The presence of ammonium and nitrite oxidation was checked by a drop test using diphenylene (Rowe et al.. 1977) and Gries-Ilosvay reagents (Alexander and Clark, 1965). Due to insufficient dilutions, some MPN values on week 45 are given as ‘higher than’ ( > ) estimations. Results are presented on a dry matter basis ( 105°C overnight). Statistical
anaIyses
The results were analysed by analysis of variance. and the means were compared using the StudentNeuman-Keuls (SNK) test.
RESULTS
Amount of N in df~~rent N pools Addition of U increased immediately the amount of exchangeable NH; in the soil and a high NH: concentration persisted throughout the 54-week incubation (Fig. I). During the first weeks of incubation exchangeable NH: pool was very low in the UF-treated soils as it was in the untreated soils. At 6-weeks the amount of NH: had increased in the UF-treated soils and continued to increase until a plateau was reached at week 45. Exchangeable NH4f also accumulated in the untreated soils from week 15 on. No NO; was seen within any of the treatments prior to week 45 (Fig. I). At the start the amount of NO; produced was about the same in all soils but by week 54 more NO; was found in the N-treated soils than in the untreated soils. The amount of soluble organic N was highest in the UF-treated soils right from the beginning of the experiment (Fig. 1). A slight increase in organic N occurred in the U-treated soils on week 6. At 54 weeks the amount of soluble N was only about one tenth what it was at 24 h in all the treatments. Urea and UF increased the amount of microbial biomass N (N,,,) immediately after the application (Fig. 1). However, after prolonged incubation the Nmic decreased considerably in both the N-treated and untreated soils, as seen at the 45-week sampling. Nitrogen content in the soil residue (after fumigation and extraction) was highest in the UF-treated soils from day 1 (Fig. 1). In the U-treated soils a slight increase in the N content compared to the
et al
untreated on.
soils was seen at week 6 and from there
A tom % “N excess in N pools The high amount of lSN in the exchangeable NH; pool in the U-treated soils confirms the fact that immediately after application a high proportion (90%) of the NH&N came from the fertilizer (Fig. 2. Table 2). However, during the incubation the proportion of fertilizer derived N (%NdQ in the NH: pool decreased and was only 40% after 54 weeks. In the UF-treated soils the degree of “N in the exchangeable NH: was relatively constant. The atom%15N excess in the accumulated NOJ stayed constant in both the U-treated and UFtreated soils and was at the same degree of enrichindicating that only autotrophic ment as in NH:. nitrification occured (Fig. 2). In the UF-treated soils the atom%15N excess in the soluble organic N was relatively high during the first weeks of the incubation (Fig. 2(c)). Of the extracted organic N 50% originated from the water-soluble fraction of UF, which consists of free urea and short chain polymers of methylene ureas (Table 2. Christianson et al., 1988). In the U-treated soils soluble organic N had low atom%“N excess, but the “N content was somewhat increased at 6-weeks. In both the U- and UF-treated soils atom%15N excess in microbial biomass N (N,,,,) increased for a few weeks, but after week I5 decreased (Fig. 2). At the beginning of incubation the amount of “N immobilized in the soil was very low in the urea treatment (Fig. 2). However, the %Ndff in the soil increased from 4% to 15% during the 54-week incubation (Table 2). Application of UF increased the atom%“N excess in the soil immediately and the content of 15N stayed constant. Soil pH, microbial hiomuss and uctivit? Soil pH increased gradually during the incubation in the untreated and UF-treated soils (Table 3). In the U-treated soils the increase in pH was immediate and the high pH remained throughout the incubation. However, at 54-weeks the soil pH in the Uand UF-treated soils were lower than in the untreated soils. Soil COZ production during the laboratory incubation was highest in the U-treated soils at l-week and 6-weeks (Fig. 3). The production of CO2 in the untreated and the UF-treated soils were essentially the same. Prolonged incubation decreased the CO* production with all the treatments. At 45-weeks the amount of COZ produced was about half what it was at the beginning of the experiment. There was no difference in microbial biomass C (Cm,,) between treatments, but in accordance with temporal change in the CO* production, the amount of Cmic was greatly reduced by the 54-week sampling (Fig. 3).
1411
Urea and ureaformaldehyde nitrogen
NH;-N 8
t 3
4
zr 8
0
c
8
NOi-N
41
$ ae
4
1
Residue-N
U UF 24 h
U UF
U UF
U UF
U UF
U UF
1 wk
8wk
15wk
45 wk
54 wk
Fig. 2. “N enrichment in exchangeable NH: and NOT, soluble organic (org-N), microbial biomass (N,,+) and soil organic matter (residue-N) pools in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. To calculate Nmtc mean values before and after fumigation were used.
quotient metabolic The (qCO2 = KS CO2 - Cmg-’ C,i,h-‘), which is considered to reflect both changes in available C as well as changes in microbial metabolic state (Bradley and Fyles, 1995), followed the pattern of the CO2 production (Fig. 3). Due to similar temporal decrease both in the C02-production and the Cmic the qCO2 did not change with time except for the U-treated soil.
The amount of soluble organic C differed greatly within treatments (Fig. 3). However, at 6-weeks the highest amount was clearly in the U-treated soils. At week 15 the values were as low as after 24 h, but by week 54 the amount of soluble carbon had increased considerably in all the treatments. The number of autotrophic nitrifiers showed great variation between samples within a treatment (Table 4). Low numbers of nitrite oxidizers were
1412
T. Aarnio er al. Table 2. The proportmn of ferttlizer derived N (%NdfQ in different N pools of soil 24 h
I wk
92.0 (1.58) 0 4.37 4.08 (0.17) 0 0 51.9 (7.63) 17.0 (5.29)
X9.0 (0.56) 0 2 53 6.44 (O.YS) 0 0 52.X (8.6X) 12.4 (2.67)
Incubation time Treatment
u
UF
6 wk
15 wk
45 wk
54 wk
55.9 (1.81) 0 I2 3 13.9 (0.44) 32.7 ( I .4X) 0 0 14.7 (3.18)
43.8 (0.81) 37.1 7.86 15.0 (0.49) 27.8 (3 13) 24.8 3 94 16.9 (I 14)
38.2 (2.32) 36.9 (1.13) 0 15.0 (0.68) 30.0 (0.40) 29.5 (3.39) 3.37 17.5 (2.31)
N pool NH; N NOj -N Org-N Residue N” NH; -N NOT-N Or-N Residue N
65.7 0 20.x (1.37) Il.2 (0.41) 2.5.6 (5.22) 0 65.6 (2.86) 13.‘) (I 49)
Mean of three replications. Standard dewations m parentheses (not shown when only two replicas). U = urea: UF = ureaformaldehyde. a Residue N includes microbial biomass
detected from the beginning in all soils. The presence of ammonium oxidizers was first detected at 6-weeks and then only in the N-treated soils. At 15weeks the number of nitrifiers had increased. as well as the number of ammonium oxidizers in the untreated soils. These increases continued throughout subsequent samplings.
DISCUSSION
Microbial biomuss und activit? In accordance with earlier observations U caused an immediate increase in pH and the subsequent release of soluble C stimulated a temporary increase in CO2 production by existing microbial populations (Fig. 3, Table 3) (Ogner, 1972; Baath et d., 1981; Foster et al., 1985b). However, increased activity did not increase C,,, (Fig. 3), but did enhance immobilization of N (Fig. I). At 45-weeks there was a clear reduction in the amount of microbial biomass and in CO1 production in all soils (Figs 1 and 3). Prolonged incubation of soil without external inputs leads to nutrient deficiencies in the system. Reduced availability of oxidizable C restricts the activity of heterotrophic microorganisms. The collapse of the microbial population was a probable cause for the increase in the amount of soluble organic C seen at 45 and 54-weeks (Fig. 3). At this stage the released C did not enhance microbial activity, probably due to changes in population structure or dormancy. A similar increase was not seen in the amount of soluble organic N, which indicates tighter immobilization of organic N compounds into humus (Fig. 1).
iii Irun~fbrmations The pattern of N transformation was quite different with the two fertilizers. In the U-treated soils the added N contributed mostly to the exchangeable NH: pool (Figs I and 2, Table 2). The enhancement in microbial activity by U (Fig. 3) increased the mineralization of soil organic matter which was seen as a continuous dilution of “N in the NH: pool. The gradual increase in lSN in the microbial biomass and in the soil residue indicates that ureaN entered the soil organic N pool via immobilization by microbes (Fig. 2). Neither the “N results nor total N concentration in the soil residue suggest rapid chemical fixation of NH3 as suggested by Foster c’t a/. (1985a) and Schimel and Firestone (1989). It has been shown that chemical fixation of N can be important in U-treated forest floor, exceeding the amount of N immobilized by microbes (Foster et al., 1985a). Results from field and laboratory studies should be compared with some caution. The lack of plant uptake and leaching during a laboratory incubation of soil increase the accumulation of exchangeable NH: (Foster ef al., 1985b). In the UF-treated soils most of the added N was found in the soil residue (Fig. I). At the beginning of the experiment it is expected for most of UF is still undegraded. Atom%“N excess in soil residue stayed constant throughout the incubation (Fig. 2). The soluble fraction of UF was quickly immobilized by biomass. The accumulation of NH: from UF started slowly as time was needed for a suitable community of ammonifying organisms to develop (Fig. 1) (Corke and Robinson, 1966; Carter et al., 1986). Although the amount of NH: increased steadily the atom%‘5N excess in the NH: pool
Table 3. SolI pH (H,O) Incubation time
24 h
I wk
6 wk
15 wk
45 wk
54 wk
4.4 (0.08) 6.4 (0.17) 4.5 (0.09)
4.5 (0.02) 6.4 (0.15) 4.6 (0.05)
4.8 (0.18) 6.3 (0.13) 5.1 (0.11)
5.1 (0.03) 6.4 (0.04) 5.7 (0.05)
5.8 (0.56) 7 0 (0.26) 6.1 (0.7)
6.5 (0.16) 5.7 (0.78) 5.7 (0.88)
Treatment
c U UF
Mean of five replications. Standard deviation in parentheses, C = untreated: U = wea: UF = ureaformaldehyde
1413
Urea and ureaformaldehyde nitrogen
81%nic
C U l;F
C U UF
C U UF
C U UF
C U UF
C U UF
24 h
1 wk
6wk
15wk
45 wk
54 wk
Fig. 3. Basal respiration rate (CO*-C), microbial biomass C(C,i,J, metabolic quotient (qCO2) and soluble organic C (org-C) in incubated soil (C = control, U = urea, UF = ureaformaldehyde). Standard deviation is shown by bars. Within a date, unlablled bars or bars headed with the same letter denotes treatments that were not significantly different at P = 0.05.
Table 4. Numbers (MPN g-’ dry soil) of ammonium and nitrite oxidizers in the soil Incubation time
Treatment
NH;
oxidizers
% (min - max) 1 wk
6wk
1s wk 45 wk
54 wk
C U UF C U UF C U UF C U UF C
NOT oxidizers % (min
- max)
0 0 0 0
14 62 358 > 10’S IX 10s > 10’ >I06 > 10’ 7x 106 2X 10s 4x10’
(O-14) (o-186) (O-l x lo3 (6x 109 >2x 10’ (O-2 x 10’) (5 X 106- > 2 X 10’ (8 x lo’- > 2 x IO’) (2 X 106- > 2 x 10’) (254-2 x 10’ (2 x IO’-3 x 108) (2 X lo’-5 x 10’)
Mean (% of three replications, minimum (min) and maximum (max) values are given in parentheses. ‘Insufficient dilution, see materials and methods. C = untreated; U = urea; UF = ureaformaldehyde.
33 39 34 162 14 36 955 s x IO4 IS0 > IO6 3 X 104 sx 10s 326 1x lo* 2x 10s
(O-100) (O-100) (O-100) (104-196) (O-41) (O-66) (O-3 X 103) (O-7 x IO’) (o-270) (17- >2 x IO’) (O-8 x 10’) (1 x IO>-1x 106) (213-456) (1 x 104-3x 108) (2 x IO’-5 x 106,
1414
T. Aarnio et al
stayed constant (Fig. 2). The soluble organic N pool was not big enough to be a sole source of NH: but mineralization from soil residue also contributed (Fig. 1). This unchanged “N enrichment of NH: could indicate formation of some kind of UF-humic complex in soil. Besides, the atom%15N excess was only 1.5% in the soil residue, but 2.8% in the exchangeable NH;, which suggests that the UF-N entered the active mineralizable organic N pool in the soil. The partly protective role of humic substances has been shown before. Binding with orfactors ganic matter was one of the main controlling the bioavailability of xenobiotics in aquatic environments (Kukkonen and Oikari, 1991). Also, organic compounds were degraded more slowly in soil when mixed with humic acid than when mixed with other soil constituents such as kaolinite or illite (Knabel et al.. 1994). It is assumed that in this artificial system only after complete decomposition of the UF-humus complex would 15N in the NH4f be diluted by mineralization of native organic N. This being the case, it seems that the duration of present experiment was not sufficiently prolonged. In addition to the decrease in microbial activity after prolonged incubation, low temperature ( + 14°C as used here) can delay the mineralization process of UF (Basaraba, 1964; Hadas and Kafkafi. 1974; Sasson, 1979) as can the increase in pH. The mineralization of UF is favoured by acidity and is greatly reduced in neutral soil (Basaraba. 1964; Nommik, 1967; Tlustos and Blackmer. 1992). Delayed decomposition due to low temperature in boreal coniferous forest soil could partly explain the long-term effect of UF on soil N status reported by Martikainen et al. (1989). The formation of a UF-humic complex in the active N pool of soil, as we have suggested, could explain earlier observations of positive, long-term effects of UF (in contrast to U) on soil N availability (Martikainen er N/.. 1989). The portion of urea-N which is not taken up rapidly after application is susceptible for incorporation in relatively stable, poorly-mineralizable organic forms in forest floor (Johnson ef ul., 1980; Nason and Myrold, 1992). It has also been shown that a significant fraction of newly immobilized N in soil is transformed to unavailable components of microbial tissues such as fungal melanins (He et al., 1988). NitriJication The soil studied has naturally low nitrification activity. Low pH and low availability of NH; are considered to be the main factors limiting autotrophic nitrifiers in acid forest soils (Martikainen, 1985). The increase in pH and of NH4f after U application had no immediate effect on the number of autotrophic ammonium oxidizers (Table 4). In our study limited availability of C seemed to be among
the key factors giving the autotrophic nitrifiers a competitive advantage over heterotrophic microbes. As shown earlier, nitrifiers are successful competitors under conditions of stationary or declining population of heterotrophs (Hart et al., 1994). After prolonged incubation the number of nitrifiers also increased in the untreated soils, although higher nitrate accumulation was maintained in the N-treated soils (Fig. I). There was no difference between the slow-release UF and U in the time needed for nitrifiers to proliferate (Table 4). The results presented here confirm our earlier observations that it is possible to select and develop a fertilizer that improves, for a prolonged period, the N status of forest soil and to ensure applied N is retained in the mineralizable N pool. Acknowledgemenrs-We warmly Pert0 thank Drs Martikainen, Caroline Preston and Aino Smolander and also Outi Priha MSc, for their constructive criticism and valuable comments throughout the study. We thank Ann van Niekerk for analysing soil properties and Dan Dunway for graphics. This work was supported by the Finnish Academy of Science through a grant to T. Aarnio.
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