Heterotrophic nitrification in an acid forest soil: isolation and characterisation of a nitrifying bacterium

Soil Biology & Biochemistry 33 (2001) 1403±1409

www.elsevier.com/locate/soilbio

Heterotrophic nitri®cation in an acid forest soil: isolation and characterisation of a nitrifying bacterium E.D.R. Brierley a,*, M. Wood b a

Cran®eld University, Institute of Water and Environment, Silsoe, Bedfordshire MK45 4DT, UK The University of Reading, Department of Soil Science, Whiteknights, Reading RG6 6DW, UK

b

Received 1 December 1999; received in revised form 10 November 2000; accepted 29 November 2000

Abstract Bacteria and fungi were isolated from an acid forest soil in which nitri®cation occurred via a heterotrophic pathway. Enrichment of soil in a 2 liquid inorganic salts medium, containing b-alanine as the sole source of C and N, led to formation of NO2 2 and NO3 . In pure culture, balanine was not a suitable substrate for nitri®cation by any of the isolates, but did support nitri®cation when one bacterium (BD) was cocultured with a fungus. This bacterium was gram-positive and rod-shaped and identi®ed provisionally as an Arthrobacter sp. The bacterium BD was able to nitrify ammonium acetate and, to a lesser extent peptone, in pure culture. Nitri®cation of ammonium acetate was greater in sterile soil solution than in de®ned media of inorganic salts. From a less reduced form of N, a -ketoglutaric oxime supported the highest rates of nitri®cation, but nitri®cation of pyruvic and ketobutyric oximes was at levels lower than from reduced N. The organism was able to survive and nitrify at pH 3, and despite its isolation from a very acidic environment, at pH # 10. It is proposed that a metabolic product of b-alanine produced by a non-nitrifying microorganism provided a suitable substrate for the nitrifying bacterium. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Acid soil; Arthrobacter sp; Heterotrophic nitri®cation

1. Introduction Nitri®cation in environments which provide unfavourable conditions for autotrophic nitrifying bacteria may result from the activity of heterotrophic microorganisms (Killham, 1986). Studies of soil conditions which stimulated or inhibited nitri®cation in a UK acid forest soil indicated that the microorganisms responsible were heterotrophs (Brierley et al., 2001b). There is no selective enrichment or isolation method for heterotrophic nitrifying microorganisms. Media supporting growth of these species must contain organic carbon in addition to nitrogen; such media permit the development of a large proportion of the soil micro¯ora, most of which are unable to oxidise the N. Peptone (Eylar and Schmidt, 1959; van Goole and Schmidt, 1973) and ammonium sulphate (Johnsrud, 1978) have been used as sources of reduced N to investigate the acceptability of organic and inorganic N sources for heterotrophic nitri®cation. Since Doxtader and Alexander (1966) identi®ed a possible role * Corresponding author. Tel.: 144-1525-863145; fax: 144-1525863344. E-mail address: e.d.r.brierley@cran®eld.ac.uk (E.D.R. Brierley).

for b-alanine in the heterotrophic nitri®cation pathway of Aspergillus ¯avus, amino acids have also been used as a source of organic N (Hatcher and Schmidt, 1971; Stroo et al., 1986). Castignetti and Gunner (1980) identi®ed oximes as possible intermediates in heterotrophic nitri®cation. The aim of this work was to understand the microorganisms responsible for nitri®cation in an acid forest soil by isolating pure cultures of nitrifying organisms and characterising these isolates in terms of C and N substrates and pH tolerance using pure cultures and mixed cultures. All the above forms of N were used in these studies.

2. Materials and methods 2.1. Sampling site The sampling area, Ironhill (grid reference SU854298) lies in the Liphook forest, on the Hampshire/Sussex border, at an altitude of approximately 130 m above sea level. The soil was a humoferric podsol of the Shirrel Heath series (Jarvis et al., 1984). The L F H horizons were rotovated with the Ah horizon (and in some areas with the Ea horizon) to a depth of 0.2 m. The soil, vegetation and ®eld treatments

0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00045-1

1404

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

have been described in full by Brierley et al. (2000a). Scots pine, Sitka spruce and Norway spruce were planted at 1 m 2 spacing; this enabled the establishment of 1 m 2 quadrats, for the study of N cycling. Soil samples were taken from the rotovated layer to a depth of 0.1 m. The soil pH values were in the range 3.6±4.1 in water and 3.0±3.2 on 0.01 M calcium chloride (5 g soil 25 ml 21 solute). 2.2. Isolation using enrichment cultures In the absence of a selective enrichment method, the isolation procedure adopted was that of Stroo et al. (1986). Samples were taken, on two occasions, from sample plots which showed nitri®cation potential and sites with no nitri®cation potential (Brierley et al., 2001b). Samples of approximately 0.5 g fresh soil were added to duplicate 50ml conical ¯asks containing 20 ml of a sterile medium of inorganic salts and 0.1% ®lter sterilised b-alanine (as the sole source of C and N); the pH was adjusted to 4.5 by the addition of ®lter sterilised H2SO4(aq). The ¯asks were incubated at 258C on a rotary shaker at 100 rpm. Every two days spot tests for total oxidised-N (nitrite and nitrate) were made on approximately 0.1 ml medium using the Griess-Ilosvay method (Keeney and Nelson, 1982). When the test proved positive for total oxidised-N a 1 ml aliquot of the enrichment cultures was transferred to fresh medium. This procedure of transfer to fresh medium was repeated once more when spot tests again proved positive. Pure isolates were obtained from the liquid b-alanine medium by plating onto a b-alanine agar; the composition was identical to the liquid medium with the addition of 1% agar (Oxoid Bacteriological Agar No. 1) and with either 1.0 mg ml 21 streptomycin or 0.5 mg ml 21 penicillin or 0.1 mg ml 21 cycloheximide added. The resulting isolates of fungi and bacteria were tested for their ability to produce nitrite or nitrate by inoculation into duplicate 50-ml conical ¯asks containing 20 ml glucose±peptone, glucose±ammonium acetate, or b-alanine media incubated at 258C on a rotary shaker. After 6 weeks of incubation, these cultures were plated onto antibiotic-free b-alanine agar in order to con®rm purity, and the medium was centrifuged and analysed colorimetrically for ammonium and total oxidised-N and for change in pH. All results were expressed relative to uninoculated, incubated medium. 2.3. Mixed cultures of isolates Bacterium BD was inoculated into duplicate ¯asks of balanine medium alone or in combination with each of the other microbial isolates, and incubated also as described above. After 6 weeks the cultures were centrifuged and the amount of oxidised-N in the medium was measured. 2.4. In¯uence of pH on nitri®cation The in¯uence of pH on the nitrifying capacity of bacterial isolate BD was assessed in glucose±peptone medium

described by Eylar and Schmidt (1959) and in a similar glucose±ammonium acetate medium. The initial pH was adjusted to values of 3, 4, 5, 6, or 7 using H2SO4(aq) or NaOH(aq). 2.5. In¯uence of carbon source on nitri®cation Bacterium BD was cultured alone and co-cultured with a fungus (isolated from the same soil sample) in the salts medium of Doxtader and Alexander (1966) containing the following C sources: b-alanine, glucose, acetate (as sodium acetate and ammonium acetate), citrate (as trisodium citrate and triammonium citrate), ethanol, methanol, methylamine, trimethylamine, and formate (as ammonium formate). Pyruvic, ketoglutaric, and ketobutyric oximes, which had been synthesised from the potassium salts of pyruvic, a -ketoglutaric, and a -ketobutyric acids, using the method of Quastel et al. (1952), were also used. The C was supplied at 1 mg C ml 21 and N was adjusted using ammonium sulphate, to provide 0.3 mg N ml 21, except for the media containing oximes, to which there was no addition of N. The pH of the media was adjusted to 5.5 with NaOH(aq). Culture conditions were as described above. After 6 weeks, the levels of total oxidised-N and NH1 4 were measured, and the ®nal pH recorded. 2.6. In¯uence of substrate concentration on nitri®cation Bacterium BD was cultured in the salts medium of Verstraete and Alexander (1972a) and of Doxtader and Alexander (1966). The media were of slightly different composition but the latter approximated to a ten-fold dilution of the former. Similarly, ten-times the quantity of C and N was provided in the former medium than in the latter. Sodium acetate or tri-sodium citrate supplied 3 or 0.3 mg C ml 21, and ammonium sulphate provided 1 or 0.1 mg N ml 21, respectively. The initial pH was adjusted to 7 with NaOH(aq) (as neutral conditions had been shown to favour nitri®cation by the isolate BD). 2.7. Arthrobacter selective medium An arthrobacter selective medium, described by Hagedorn and Holt (1975), was tested for its ability to support the growth of the microbial isolates derived from the enrichment procedure described above. In addition, this medium was used to conduct bacterial counts from dilutions of soil collected from 1 m 2 quadrats which had been characterised for N mineralisation potential (Brierley et al., 2001a). Duplicate 1 g samples of soil were diluted in 99 ml sterile water, shaken for 5 min and allowed to settle for 5 min. One ml was diluted into 49 ml sterile water which was again shaken and allowed to settle. From these suspensions (10 22 and 5 £ 10 23 dilutions) duplicate 1 ml samples were used to prepare pour plates of arthrobacter selective medium.

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

2.8. Nitri®cation in soil solution Soil samples were collected from the 1 m 2 sample plot from which the nitrifying bacterium BD had been isolated. The soil solution was extracted by centrifugation at 6000 rpm in te¯on tubes using a Europa 24M centrifuge, refrigerated at 58C (Menzies and Bell, 1988). The solution was ®lter-sterilised and amended with 1.0% ®lter-sterilised ammonium acetate solution (but neither buffer nor additional nutrients were added). Duplicate samples were inoculated with bacterium BD or other bacterial isolates having colony morphology similar to isolate BD. After 6 weeks, samples were analysed for ammonium and total oxidised-N.

3. Results 3.1. Isolation using enrichment cultures Of the four samples taken from different quadrats and used as inocula for the b-alanine enrichment cultures, only soil from one of the nitrifying quadrats caused the formation of nitrite or nitrate. After 6 weeks the medium contained in excess of 10 mg ml 21 total oxidised-N. One of the other soils tested had previously exhibited nitri®cation potential during incubation of soils in the laboratory (Brierley et al., 2001a). From this mixed enrichment culture ten fungal isolates and six bacterial colonies were obtained which grew on antibiotic-supplemented b-alanine agar media. When tested for the ability to nitrify in glucose-peptone medium the microorganism which produced most oxidised-N, most rapidly, was a bacterium (BD). The ®nal concentration after 6 weeks incubation was only 0.4 mg ml 21 total

1405

oxidised-N. However, when ammonium acetate was used instead of peptone, as the source of N, bacterium BD produced up to 2.5 mg total oxidised-N ml 21, whereas no other isolate produced in excess of 0.3 mg ml 21 total oxidised-N (Table 1). No oxidised-N was detected in the media before 12±14 days growth. When the isolates were screened for their ability to nitrify in pure culture, in the balanine medium, less than 0.1 mg ml 21 total oxidised-N was produced by the isolates. The growth of all the microorganisms in all these media resulted in an increase in pH values $7. The isolation procedure was repeated 9 months later, using fresh soil samples from the same 1 m 2 sample plots. Enrichment of soil from the same sample plot as previously was alone in resulting in nitri®cation. On this occasion, six fungal isolates and six bacterial isolates were obtained from the enrichment culture. One bacterium exhibited similar colony morphology and nitrifying capability in pure culture to the nitrifying bacterium isolated previously. 3.2. Mixed culture of isolates Mixed cultures obtained from the enrichment of soil in balanine medium resulted in the formation of oxidised-N but pure cultures in this medium could not produce oxidised-N (Table 1). Either the microorganism responsible for the nitri®cation had not been isolated or nitri®cation in this medium could only be induced by the recombination of isolates. This was investigated by pairing one bacterial isolate (BD) of proven nitri®cation ability with each of the other isolates and culturing in b-alanine medium. Production of oxidised-N was always greater in mixed cultures than when the organisms were grown separately. The highest levels of nitri®cation were attained when the bacterium

Table 1 Total oxidised nitrogen (mg N ml 21), following 6 weeks incubation, of bacteria (B) and fungi (F), in three media (mean of two observations, corrected for uninoculated controls). In the medium containing b-alanine isolates were grown alone or in combination with bacterium BD Isolate

FA FB FC FD FE FF FG FH FI FJ BA BB BC BD BE BF

C and N source in media Glucose±peptone (pure culture)

Glucose±ammonium acetate (pure culture)

b-Alanine (pure culture)

b-Alanine (mixed culture with bacterial isolate BD)

0 0 0.1 0 0 0 0 0 0 0.1 0.1 0.1 0 0.4 0 0

0.1 0.1 0.1 0.1 0 0.1 0.1 0.1 0.1 0.2 0.2 0.3 0.1 2.4 0.1 0.1

0 0 0.1 0.1 0.1 0 0 0.1 0 0.1 0 0.1 0.1 0.1 0.1 0

0.8 0.1 0.2 0.1 0.4 0 0.1 0.2 0.2 0.4 0.2 0 0.1 0.1 0.1

1406

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

Table 2 Nitri®cation (mg oxidised N ml 21) in two media, following 6 weeks incubation, of a nitrifying bacterium (BD), at a range of initial pH values (means of two observations, corrected for uninoculated controls) Glucose-peptone medium

Glucose±ammonium acetate medium

Initial pH

Net NO2 2 (mg N ml 21)

Net NO2 3 (mg N ml 21)

Net pH increase a

Net NO2 2 (mg N ml 21)

Net NO2 3 (mg N ml 21)

Net pH increase a

3 4 5 6 7

0.2 0.2 0.2 0.1 0.2

0.3 0.3 0.3 0.2 0.2

5.3 4.4 4.5 3.4 2.4

0.2 0.2 0.4 0.5 0.5

0.1 0.3 0.6 1.3 1.9

5.2 4.0 3.2 2.4 1.5

a

The pH of uninoculated controls rose by 0.4±0.7.

was paired with a fungal isolate; the maximum amount was 0.8 mg ml 21 total oxidised-N (Table 1). 3.3. In¯uence of pH on nitri®cation Bacterium BD was able to tolerate a broad pH range: growth was recorded in both glucose±peptone and glucose±ammonium acetate media across a wide pH range. At an initial pH of 3, growth was observed in only one of the duplicate cultures, for both media, and similarly, in only one of the cultures, in the ammonium acetate medium at pH 4. However, experimentation into the effect of C sources on nitri®cation indicated that the bacterium was tolerant of pH , 3, if acidic metabolic products gradually reduced the pH of media. Growth in both glucose± peptone and glucose±ammonium acetate media resulted in a ®nal pH in the range 8.0±9.5 irrespective of initial pH (Table 2). Using different media (to study the response to substrate concentration), growth at pH 10 was recorded. The production of oxidised-N in glucose±peptone medium was unaffected by pH but in glucose±ammonium

acetate medium increased with increasing initial pH (Table 2). Growth in the colourless glucose±ammonium acetate medium resulted in the formation of an unidenti®ed yellow pigment. 3.4. In¯uence of carbon source on nitri®cation With the exception of ammonium formate all of the C sources tested supported growth, including single C compounds. However, in pure culture, only the salts of organic acids and the oxime of ketoglutaric acid supported nitri®cation (Table 3). The latter compound resulted in the formation of up to 21.6 mg ml 21 total oxidised-N, approximately an order of magnitude higher than with any other substrate. However, the oximes of pyruvic and ketobutyric acids resulted in the formation of only 0.6 and 0.5 mg ml 21 total oxidised-N, respectively. When a -ketoglutarate (a precursor of the oxime) was provided as the C source, oxidation of ammonium resulted in 1.4 mg ml 21 total oxidised-N. This was substantially less than from the

Table 3 Nitri®cation (mg oxidised N ml 21) of ammonium by a nitrifying bacterium (BD) in pure culture and co-cultured with a fungus (isolated from the same soil sample) in an inorganic salts medium (initial pH 5.5) with different compounds as the sole source of organic C (means of two observations, corrected for uninoculated controls) Carbon source

Bacterium (BD)

Bacterium (BD) and fungus (FA) 21

b-alanine Ammonium acetate Tri-ammonium citrate Ethanol Glucose Methanol Ammonium formate Methylamine Trimethylamine Pyruvic oxime Ketoglutaric oxime Ketobutyric oxime Pyruvic acid Ketoglutaric acid Ketobutyric acid

Net oxidised-N (mg N ml )

pH®nal

Net oxidised-N (mg N ml 21)

pH®nal

0 2.5 2.6 0 0 0 0 0 0 0.6 21.6 0.5 0 1.4 0

7.7 8.7 8.1 4.1 4.1 4.7 5.5 5.7 5.5 8.0 6.8 8.6 5.7 5.8 5.8

0.7 2.8 2.7 0 0 0 0 0 0 0.4 15.7 0.5 0 1.1 0

8.0 8.3 8.0 2.9 2.7 5.0 7.3 5.4 5.6 8.2 6.9 8.6 5.5 5.6 5.6

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

oxime and approximately half that from acetate or citrate (Table 3). The change in the pH of media was strongly in¯uenced by the C source (Table 3). Metabolism of glucose, methanol and ethanol, resulted in acidi®cation of the media whereas acetate and citrate made the medium more alkaline. The latter C sources supported nitri®cation whereas the former did not. This was not simply the effect of pH however as balanine also resulted in a pH increase of the media but this was an unsuitable substrate for nitri®cation in pure culture. When co-cultured with a non-nitrifying fungal isolate from the same soil, oxidised-N was produced from b-alanine, con®rming the earlier observation (Table 1), but co-culturing did not increase nitri®cation by the bacterium with any other C source. 3.5. In¯uence of substrate concentration on nitri®cation A ten-fold dilution of C, N and mineral salts resulted in only a 20% reduction in total oxidised-N (2.9 mg N ml 21 c.f. 2.3 mg N ml 21). This was true for C supplied as acetate or 2 citrate. In both cases the ratio of NO2 2 :NO3 was approximately 1:2. The pH of all inoculated media rose during incubation, reaching a pH of 10 in the higher substrate medium (containing 3 mg C ml 21 and 1 mg N ml 21). 3.6. The use of an Arthrobacter-selective medium Bacterial isolate BD was provisionally identi®ed as a coryneform rod, possibly an Arthrobacter sp. (NCIMB, personal communication),, therefore, the medium of Hagedorn and Holt (1975) which is selective for arthrobacters was used to investigate the spatial distribution of this organism at the ®eld site. The nitrifying bacterium BD exhibited pale pink pigmented, creamy colonies on this medium, therefore, all colonies of this characteristic appearance were assumed to be nitrifying bacteria. This was con®rmed by testing for nitri®cation potential in pure culture. In samples of soil from 42 plots (each 1 m 2 and investigated by Brierley et al., 2001a) 21 showed nitri®cation potential when incubated in the laboratory. The plate counts indicated that the heterotrophic nitrifying bacterium described here was present at a population density of .100 cfu g 21 in eight of these nitrifying soils, and in seven soils which did not show nitri®cation potential. 3.7. Nitri®cation in soil solution The highest rates of nitri®cation from a reduced N source were achieved in soil solution, amended with ammonium acetate. The nitrifying organisms isolated using the enrichment procedure produced 3.8 and 3.3 mg ml 21 total oxidised-N respectively. Seven similar colonies (presumed to be the same nitrifying bacterium) isolated on the arthrobacter selective medium from soils with and without nitrifying potential produced 2.5±4.5 mg N ml 21 total oxidised-N (Table 4).

1407

4. Discussion It is generally believed (Killham, 1986) that the micro¯ora of acid forest soils is dominated by the fungi and it is assumed, therefore, that nitri®cation may be attributed to the heterotrophic activity of these microorganisms. Whilst it is thought unlikely that a heterotrophic nitri®er will ever be isolated with a nitrifying potential per unit biomass comparable to that found amongst the chemoautotrophs, Killham (1986) suggests that the large heterotrophic biomass may more than offset the low nitri®cation per unit of biomass. Nitri®cation was shown to be favoured by neutral or slightly alkaline media. This illustrates the problem of establishing the ability of heterotrophs to nitrify under acid ®eld conditions. Remacle (1977) isolated a nitrifying fungus of the genus Aspergillus from an acid soil and showed that the optimum pH for mycelium production was 6.5 and nitrate production reached a maximum at pH 8.3. However, Lang and Jagnow (1986) found that the optimum pH for nitri®cation by Verticillium lecanii was 3.5 and Stroo et al. (1986) isolated a strain of Absidia cylindrospora from an acid forest soil which produced nitrite and nitrate at pH values ranging from 4.0 to 4.8. De Boer et al. (1992) proposed that both acid-tolerant and acid-sensitive ammonium oxidising bacteria were responsible for nitrate producing in an acid forest soil saturated with N. The possible role of b-alanine as a key intermediate in the heterotrophic nitri®cation pathway of Aspergillus ¯avus was proposed by Doxtader and Alexander (1966), hence its use in the isolation procedure of Stroo et al. (1986). Enrichment of Liphook soils in the medium was successful: oxidised-N was formed in amounts similar to those detected by Stroo et al. (1986). Similar numbers of bacterial and fungal colonies were isolated. However, when pure cultures were tested for their nitri®cation capability in the b-alanine medium, all Table 4 Total oxidised nitrogen (mg N ml 21) in sterile soil solution amended with ammonium acetate, inoculated with nine bacterial isolates, and incubated for 6 weeks (mean of two observations). The bacteria were obtained either by enrichment of soil in a b-alanine medium (on two separate occasions) or by plating a soil dilution onto an arthrobacter selective medium. Nitri®cation potential in soil was determined by Brierley et al. (2000a) Nitri®cation potential of soil from which bacterium was isolated

Method of isolating bacterium

Total oxidised-N (mg N ml 21)

1 1 1 1 1 2 2 2 2

Enrichment 1 a Enrichment 2 Plating Plating Plating Plating Plating Plating Plating Uninoculated control

3.8 3.3 3.0 3.8 3.4 2.6 4.2 3.3 4.5 0.4

a

Bacterium BD.

1408

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

failed to produce detectable quantities of oxidised-N. Therefore, either an organism responsible for nitri®cation in the enrichment culture was not isolated, or b-alanine is not a suitable substrate or intermediate in the heterotrophic nitri®cation pathway of the responsible organism. A bacterium (BD) was isolated which in glucose±peptone and glucose±ammonium acetate media produced quantities of nitrite and nitrate in excess of the other isolates, and at concentrations comparable to heterotrophic nitri®ers isolated from other forest soils (Lang and Jagnow, 1986). It is likely, therefore, that this bacterium was responsible for the nitri®cation in the enrichment culture. There was, however, counter evidence: the incidence of this bacterium in sample plots did not correlate with nitri®cation potential of soils, from the same plots, determined by laboratory incubation. A possible explanation lies in the observation that, in pure culture, b-alanine was an unsuitable intermediate for nitri®cation, but enrichment cultures of mixed populations of eukaryotic and prokaryotic microorganisms produced substantially more oxidised-N. This suggested that a product of a non-universal metabolic pathway of b-alanine provided either a key intermediate organic N compound or a speci®c C source, which provided the acceptor molecule for the synthesis of such an intermediate. If no organism in the b-alanine enrichment culture was capable of forming a suitable intermediate then nitri®cation did not proceed. This is possible because of the small amount of soil inoculum used. The demonstration of nitri®cation potential in soils which failed to nitrify in enrichment culture may also be explained if there is an alternative source, such as from vegetation, of the key intermediate compound or acceptor molecule. Nitri®cation did not begin until after 2 weeks of growth. It has been proposed that heterotrophic nitri®cation which occurs after growth has ceased is associated with cell lysis (Marshall and Alexander, 1962; Witzel and Overbeck, 1979). It is unlikely, therefore, that heterotrophic nitri®cation provides energy and, therefore, it is not essential for growth. Wood (1988) proposed a mechanism for nitri®cation in which hydroxyl radicals are formed; these are a potent oxidising and hydroxylating agent. Potentially, the factor of greatest signi®cance in determining heterotrophic nitri®cation of ammonium is the C source. There is no convincing explanation why acetate and citrate supported nitri®cation of ammonium while several other C-sources did not, despite supporting growth. Witzel and Overbeck (1979) showed that acetate was the preferred C source for nitri®cation of ammonium by Arthrobacter sp. However, unlike the ®ndings of Witzel and Overbeck (1979) and of Verstraete and Alexander (1972a), the 2 accumulation of NO2 2 and NO3 was not proportional to the initial concentration of acetate or citrate. The possibility that hydroxylamine might be an intermediate in an inorganic pathway of the oxidation of ammonia to nitrite has been proposed by a number of authors, such as Amarger and Alexander (1968); Verstraete and Alexan-

der (1972b). Lees and Quastel (1946) worked on the hypothesis that hydroxylamine formed might be bound as an oxime and organisms capable of producing nitrite from oximes have been reported (Jensen, 1951). Although oximes do occur in both plant and animal tissues (Yamafugi et al., 1950) there is no evidence that oximes might be produced in considerable amounts during humus degradation (Lang and Jagnow, 1986). Nitri®cation of oximes by the bacterium BD in this study was of interest because only the oxime of ketoglutarate supported nitri®cation at a rate comparable with the highest reported rates of nitri®cation by Arthrobacter sp. (Verstraete, 1975). Oxime-N is at a higher oxidation state than the N tested with the other C sources and is not, therefore, directly comparable. Pyruvic and ketobutyric oximes supported growth but resulted in the formation of relatively small amounts of oxidised-N. The possibility that mutagenic and toxic products of heterotrophic nitri®cation might be used in response to competitors has been raised by Verstraete (1975). The antibiotic activity of hydroxamic acids (such as aspergillic acid) has been established and the toxic or mutagenic properties of hydroxylamine and nitrite might be useful against competitors (Waid, 1975). This might explain why oxidised-N was formed in b-alanine medium in mixed or paired cultures but not in pure cultures. The addition of soil extract to media has been used to enhance nitri®cation by heterotrophic and autotrophic soil microorganisms, by providing them with some growth factor (Stroo et al., 1986; De Boer et al., 1992). In this study, nitri®cation was greater in soil solution to which ammonium acetate was added, than in de®ned media. This may have been due to the provision of some unknown growth factor or nutrient. Alternatively, it could have been because the organisms cultured in soil solution had been freshly isolated from soil samples whereas those grown in de®ned media had been sub-cultured several times. The nitrifying bacterial isolate was a coryneform rod and similar to Arthrobacter sp. in a number of characteristics and it was shown to be capable of growth on the arthrobacter selective medium of Hagedorn and Holt (1975). It exhibited an ability to utilise single-carbon compounds as its sole source of energy; an ability rarely exhibited amongst Gram-positive or Gram-variable bacteria but of which Arthrobacter sp. are capable. However, the typical morphological life cycle of Arthrobacter sp., consisting of a marked change in form during growth (Cacciari and Lippi, 1987), was not observed. The morphogenic cycle may depend on both the species and the cultural conditions but the media employed in this investigation have been used in previous studies of Arthrobacter sp. for which the changes of cell form have been observed (Verstraete and Alexander, 1972a). The ability of Arthrobacter sp. to nitrify has been recognised since Gunner (1963) observed that the genus Arthrobacter may be included among those few select genera

E.D.R. Brierley, M. Wood / Soil Biology & Biochemistry 33 (2001) 1403±1409

reported capable of oxidising N to nitrate. The nitri®cation ability of Arthrobacter sp. has been observed amongst isolates from soil (Tate, 1977), sewage sludge (Verstraete and Alexander, 1972a,b), and lake water (Witzel and Overbeck, 1979). Arthrobacters have been reported to be intolerant of acid conditions; reports indicated that they were inhibited in soils of pH #5 (Cacciari and Lippi, 1987). Hagedorn and Holt (1975) observed that the percentage of bacterial colonies represented by arthrobacters were negatively correlated with soil acidity. However, Arthrobacter sp. were commonly isolated from a pine forest (Goodfellow, 1968) and from acid litter of Sitka spruce (Goodfellow and Dawson, 1978). Such observations raise the possibility of protected microhabitats. The activity of heterotrophic organisms does not necessarily make the continued occupation of such sites untenable, as protons may be removed by the ammoni®cation of organic N at the same time as they are generated by the oxidation of reduced N. We conclude, therefore, that the heterotrophic bacterium isolated in these studies possesses many of the characteristics associated with Arthrobacter sp., and it is likely to be important in the production of nitrate in this soil. Acknowledgements The research was sponsored by the Natural Environmental Research Council with collaborative funding from the Central Electricity Generating Board. The authors acknowledge, with gratitude, this ®nancial support.

References Amarger, N., Alexander, M., 1968. Nitrite formation from hydroxylamine and oximes by Pseudomonas aeruginosa. Journal of Bacteriology 95, 1651±1657. Brierley, E.D.R., Shaw, P.J.A., Wood, M., 2001. Nitrogen cycling and proton ¯uxes in an acid forest soil. Plant and Soil 229, 83±96. Brierley, E.D.R., Wood, M., Shaw, P.J.A., 2001. In¯uence of tree species and ground vegetation on nitri®cation in an acid forest soil. Plant and Soil 229, 97±104. Cacciari, I., Lippi, D., 1987. Arthrobacters: successful arid soil bacteria. A review. Arid Soil Research and Rehabilitation 1, 1±30. Castignetti, D., Gunner, H.B., 1980. Sequential nitri®cation by an Alcaligenes sp. and Nitrobacter agilis. Canadian Journal of Microbiology 26, 1114±1119. De Boer, W., Tietma, A., Klein Gunnewiek, P.J.A., Laanbroek, H.J., 1992. The chemolithotrophic ammonium oxidizing community in a nitrogensaturated acid forest soil in relation to pH-dependent nitrifying activity. Soil Biology and Biochemistry 24, 229±234. Doxtader, K.G., Alexander, M., 1966. Nitri®cation by heterotrophic soil microorganisms. Soil Science Society of America Proceedings 30, 351±355. Eylar, O.R., Schmidt, E.L., 1959. A survey of heterotrophic microorganisms from soil for ability to form nitrite and nitrate. Journal of General Microbiology 20, 473±481. Goodfellow, M., 1968. Properties and composition of the bacterial ¯ora of a pine forest soil. Journal of Soil Science 19, 154±167.

1409

Goodfellow, M., Dawson, D., 1978. Qualitative and quantitative studies of bacteria colonizing Picea sitchensis litter. Soil Biology and Biochemistry 10, 303±307. Gunner, H.B., 1963. Nitri®cation by Arthrobacter globiformis. Nature (London) 193, 1127±1128. Hagedorn, C., Holt, J.G., 1975. Ecology of soil arthrobacters in Clarion-Webster toposeqences of Iowa. Applied Microbiology 29, 211± 218. Hatcher, J.H., Schmidt, E.L., 1971. Nitri®cation of aspartate by Aspergillus ¯avus. Applied Microbiology 21, 181±186. Jarvis, M.G., Allen, R.H., Fordham, F.J., Hagelden, J., Moffat, A.J., Sturdy, R.G., 1984. Soils and their use in South East England. Bulletin 15 of the Soil Survey of England and Wales Soil Survey, Harpenden. Jensen, H.L., 1951. Nitri®cation of oxine compounds by heterotrophic bacteria. Journal of General Microbiology 5, 360±368. Johnsrud, S.C., 1978. Heterotrophic nitri®cation in acid forest soils. Holarctic Ecology 1, 27±30. Keeney, D.R., Nelson, D.W., 1982. NitrogenÐinorganic forms. In: Page, A.L., Miller, R.H. (Eds.). Methods in Soil Analysis Part 2 Chemical and Microbiological Properties. ASA/SSSA, Madison WI, pp. 643±698. Killham, K., 1986. Heterotrophic nitri®cation. In: Prosser, J.I. (Ed.). Nitri®cation: Special Publications of the Society for General Microbiology, vol. 20. IRL Press, Oxford, pp. 117±126. Lang, E., Jagnow, G., 1986. Fungi of a forest soil nitrifying at low pH values. FEMS Microbiology Letters (Ecology) 38, 257±265. Lees, H., Quastel, J.H., 1946. Biochemistry of nitri®cation in soil. Biochemical Journal 40, 803±828. Marshall, K.C., Alexander, M., 1962. Nitri®cation by Aspergillus ¯avus. Journal of Bacteriology 83, 572±578. Menzies, N.W., Bell, L.C., 1988. Evaluation of the in¯uence of sample preparation and extraction technique on soil solution composition. Australian Journal of Soil Research 26, 451±464. Quastel, J.H., Schole®eld, P.B., Stevenson, J.W., 1952. Oxidation of pyruvic acid oxime by soil organisms. Biochemical Journal 51, 278±284. Remacle, J., 1977. Microbial transformations of nitrogen in forests. Oecologia Plantarum 7, 69±78. Stroo, H.F., Klein, T.M., Alexander, M., 1986. Heterotrophic nitri®cation in an acid forest soil and by an acid-tolerant fungus. Applied and Environmental Microbiology 52, 1107±1111. Tate, R.L., 1977. Nitri®cation in Histosols: a potential role for the heterotrophic nitri®er. Applied and Environmental Microbiology 33, 911± 914. van Goole, A.P., Schmidt, E.L., 1973. Nitri®cation in relation to growth in Aspergillus ¯avus. Soil Biology and Biochemistry 5, 259±265. Verstraete, W., 1975. Heterotrophic nitri®cation in soils and aqueous media (a review). Izvestiya Akademii Nauk S.S.S.R., Seriya Biologicheskaya 4, 541±558 (translation). Verstraete, W., Alexander, M., 1972a. Heterotrophic nitri®cation by Arthrobacter sp. Journal of Bacteriology 110, 955±961. Verstraete, W., Alexander, M., 1972b. Mechanism of nitri®cation by Arthrobacter sp. Journal of Bacteriology 110, 962±967. Waid, J.S., 1975. Hydroxamic acids in soil systems. In: Paul, E.A., McLaren, A.D. (Eds.). Soil Biochemistry, vol. 4. Marcel Dekker, New York, pp. 65±101. Witzel, K.P., Overbeck, H.J., 1979. Heterotrophic nitri®cation by Arthrobacter sp. (strain 9006) as in¯uenced by different cultural conditions, growth state and acetate metabolism. Archives of Microbiology 122, 137±143. Wood, P.M., 1988. Monooxygenase and free radical mechanisms for biological ammonia oxidation. In: Cole, J.A., Ferguson, S.J. (Eds.). The Nitrogen and Sulphur Cycles: Symposia of the Society for General Microbiology 42. Cambridge University Press, Cambridge, pp. 219± 224. Yamafugi, K., Kondo, H., Omura, H., 1950. Distribution of oxime in plant and animal tissues. Enzymology 14, 153±156.