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Inhibition of nitrification by potassium ethyl xanthate in soil and in liquid culture
Soil Bio/. Eiochem. Vol. 17, No. 2, pp. 229-233, 1985 Printed in Great Britain. All rights reserved
0038-0717/85 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
INHIBITION OF NITRIFICATION BY POTASSIUM ETHYL XANTHATE IN SOIL AND IN LIQUID CULTURE SUSAN
Department
of Microbiology,
E. UNDERHILL*
Marischal
College,
and J. I.
University
PROSSER
of Aberdeen,
Aberdeen,
Scotland
AB9 1AS
(Accepted 30 October 1984) Summary-Inhibition of nitrification by potassium ethyl xanthate has been investigated in liquid batch culture and in soil, using axenic cultures of Nitrosomonus and Nitrobacter. In all cases the major effect was induction of a prolonged lag rather than a reduction in specific oxidation rates, although this was also found for Nitrosomonas at high xanthate concentrations. Inhibition of both ammonium and nitrite oxidation was reduced in soil, because of immobilization of xanthate or its breakdown products at the soil surface. However, in liquid culture nitrite oxidation was more sensitive while ammonium oxidation was more sensitive in soil. It is proposed that this also results from accumulation of xanthate, or its breakdown products at the soil surface. Xanthate was shown to have a bacteriostatic, rather than bactericidal effect, but xanthate-treated cells exhibited a significantly longer lag when transferred to xanthate-free medium, emphasizing the need for caution when using the most probable number method in such studies.
INTRODUCTION
but with some variation between different herbicides. None of these studies involved axenic cultures and interpretation of results is therefore complicated by the possibility of microbial breakdown of added compounds, selection of different nitrifier strains in different situations and by undefined interactions with other organisms. These problems have been eliminated in the present study by the use of axenic cultures of nitrifying bacteria inoculated into sterile soil and liquid medium.
Under certain conditions, application of ammoniumbased fertilizers is followed by significant oxidation of ammonium to nitrate. As well as increasing rates of loss of N from the soil, through leaching or denitrification of nitrate, unacceptable levels of nitrate may be reached in run-off waters. To prevent this, nitrification inhibitors may be applied along with such fertilizers (Parr, 1973). The most commonly used of such inhibitors is N-Serve (nitrapyrin) (Goring, 1962), which inhibits the cytochrome oxidase component involved in ammonium oxidation (Campbell and Aleem, 1965), but the use of carbon disulphide based inhibitors has recently been investigated (Ashworth et al., 1977,1979, 1980). The most efficient of these is potassium ethyl xanthate which is oxidized chemically in soil, releasing carbon disulphide which is a strong inhibitor of nitrification at levels which have little effect on other microbial processes. Rodgers and Ashworth (1982) distinguished between bactericidal and bacteriostatic actions of several inhibitors including potassium ethyl xanthate which they found to be bacteriostatic. They observed complete inhibition of nitrification by potassium ethyl xanthate at concentrations of 10 mgl-‘, in aqueous culture, but no evidence of bactericidal effects. At 10 mg l-‘, nitrification was inhibited for 14 days. In soil, Ashworth et al. (1979) reported inhibition for 6 weeks at a concentration of 315 mg kg-’ soil. Ratnayake and Audus (1978) studied the effects of a range of herbicides on several measures of activity and growth of nitrifying bacteria in liquid culture and in soil reperfusion systems. They also report reduced sensitivity to inhibition in soil,
MATERIALS AND METHODS
Soil isolates of Nitrosomonas europaea and Nitrobatter sp. were kindly supplied by Dr R. M. MacDonald, Rothamsted Experimental Station, Harpenden, England. Nitrosomonas was routinely subcultured in 100ml inorganic medium (Skinner and Walker, 1961) contained in 250 ml Erlenmeyer flasks fitted with side-arms half-filled with aqueous sodium carbonate solution (5% w/v). This facilitated aseptic neutralization of acid formed during ammonium oxidation. Nitrobacter was routinely cultured in Skinner and Walker’s medium with (NH,),SO, replaced by NaNO, (1.42 g l-‘, equivalent to 410 pg NOT-N ml-‘). Both were incubated at 27°C on a New Brunswick Scientific Rotary Incubator. Growth was assessed by spot tests for NH: (Nesslers reagent) and NO; and NO; (Griess llosvay’s reagents I and II). Cultures were checked regularly for contamination by microscopic examination and by inoculation onto nutrient agar plates which were then incubated at 27°C for 3-4 weeks. Liquid culture Nitrosomonas and Nitrobacter were grown at 27°C in 250 ml Erlenmeyer flasks containing 150 ml Smith and Hoare’s (1968) medium with 50 pg NH:-N ml-’ as (NH,)$Od and 50 pg NOT-N ml-’ as NaNO,
*Present address: Department of Chemical Engineering, The Polytechnic of Wales, Pontypridd, Mid Glamorgan, CS37 IDL, U.K. 229
230
SUSAN
E.
UNDERHILL
respectively. Potassium ethyl xanthate was added aseptically as 1 ml of a filter sterilized solution following autoclaving of the medium at 120°C for 15 min to give final concentrations in the range O-8 pgml-‘. Flasks were inoculated with 1 ml of fully grown cultures of either Nitrosomonas or Nitrobacter, previously subcultured at least twice in Smith and Hoare’s medium. Samples were removed daily (Nitro somonas) or twice daily (Nitrobacter) and were analyzed for NH:, NO, and NO; using a Technicon Autoanalyser II. Samples were also plated onto nutrient agar and incubated at 27°C to check for contamination by heterotrophs. Contaminated flasks were discarded. Viable cell numbers of Nitrosomonas were estimated by the most probable number (MPN) method (Alexander, 1965). A l/l0 dilution series was constructed and five 100~1 samples from each dilution were mixed with 1.50~1 Skinner and Walker’s (1961) medium in the wells of microtitre plates. Plates were incubated for up to 7 weeks at 27°C with water lost by evaporation replaced where necessary. Growth was assessed periodically by change of the medium indicator from red to yellow, as a result of acid production accompanying NH: oxidation. After 7 weeks, spot tests for NO; were carried out on all wells. Soil incubation studies Air-dried soil was adjusted to pH 7.6 by addition of Ca(OH), and 80 g portions were autoclaved for 30 min at 120°C in 2.50 ml Erlenmeyer flasks. Each flask was then supplied, aseptically, with 20ml of a sterile solution containing 50,ug NH:-N ml-’ as (NH,),SO, sulphate or 5Opg NOT-N ml-’ as NaNO,, 1 ml of a fully grown culture of Nitrosomonas or Nitrobacter, prepared as described above, and 1 ml of the appropriate filter sterilized potassium ethyl xanthate solution, giving final concentrations in the range O-80 pg ml-‘. The soil was then mixed to ensure even distribution of solutions and cells. Three flasks were prepared for each treatment and each flask was weighed regularly with water lost through evaporation being replaced. Soil samples of approximately 2.5 g were removed daily and twice daily for Nitrosomonas and Nitrobacter respectively. Each sample was then weighed accurately and NO; and NO; concentrations estimated on a Technicon Autoanalyser following extraction by shaking for 30 min in 1 M KC1 (2.5 g: 12.5 ml) followed by filtering. When necessary, samples were stored at - 15°C. Samples were also checked for contamination by heterotrophs as described above and contaminated flasks discarded. Rate of carbon disulphide production CS, production from potassium ethyl xanthate in soil and in liquid medium was measured using Conway units as described by Ashworth et al. (1979). Evolution from liquid culture was determined in 1 ml samples removed regularly from Smith and Hoare’s (1968) medium containing potassium ethyl xanthate and shaking at 27°C. The sample was placed in the inner compartment of a Conway unit and 2 ml KOH in ethanol (10% w/v) was added to the outer compartment. The units were sealed, incubated for 5 h, when
and J. I.
PROSSER
excess KOH was neutralized with acetic acid and the solution titrated with 20mM iodine in potassium iodide, with sodium starch glycollate as indicator. Potassium ethyl xanthate solution was removed from the inner well between titrations. Evolution from soil was measured by placing 5 g of air dried soil, with pH and moisture content adjusted as described above, in the inner compartment. This was then mixed with 1 ml of a 32% w/v potassium ethyl xanthate solution in water. KOH in ethanol was placed in the outer well and carbon disulphide evolution measured as described above, except that 5 mM iodine was used to increase sensitivity. The solution in the outer compartment was renewed after each measurement while the same xanthate-soil matrix was used for each determination. RESULTS
In liquid or soil culture, growth was assessed by NO; (Nitrosomonas) or NO; (Nitrobacter) production, following a lag period. For Nitrosomonas, growing in liquid culture, the lag period was followed by an exponential increase in NO; concentration and a deceleration phase, due to a reduction in pH which prevented full conversion of NH: (Fig. 1). The reduction in pH was noted as a change in medium indicator (phenol red) from red to yellow at pH 7.
I 203
I
I
I
400
600
600
I loo0
Time (h)
Fig. I. Effect of potassium ethyl xanthate concentrations of Opgml-’ (01, 1pgml-’ (01, 2pgml-’ (Cl), 3figrnl-’ (a) and 8 pgml-’ (A) on growth of Nitrosomonasin liquid culture.
f ‘9 r
30-
E ?t
2.0 -
3
lo-
g 5 "c
oo-
8 I
-1.0 -
Time (h)
Fig. 2. Effect of potassium ethyl xanthate concentrations of Opg ml-’ (0) and 8 pgml-’ (0) on growth of NitroSOM~~~S in soil.
231
Inhibition of nitrification by xanthate
Time
trations, is due to evaporation. In soil (Fig. 2), the greater buffering capacity increased the duration of the exponential phase and allowed complete oxidation of added NH: to NO;. Although NHr could have been lost by volatilization, the initial concentration was such that it was in excess, until depleted by growth of Nitrosomonas, and any volatilization would not affect either the duration of the lag period or the specific oxidation rate. For Nitrobacter the exponential phase continued until NO; was exhausted in both soil and liquid culture (Figs 3 and 4). Xanthate concentrations are expressed as pg ml-’ of soil solution. This allows a more meaningful comparison with concentrations in liquid culture although it was not possible to quantify precisely the extent of absorption and immobilization of xanthate by the soil and the consequent effect on its concentration in the soil solution. A concentration of 1 pgml-’ is equivalent to a concentration of 0.26 pg g soil-‘. The effect of xanthate was therefore assessed by its effect on the duration of the lag and the specific rate of product formation, termed the specific oxidation rate. The latter was calculated from the slope of a semi-logarithmic plot of product concentration vs time during the exponential phase. The duration of the lag was calculated as the time when the gradient during the exponential phase intercepted the initial product concentration. Three replicates were used for each treatment. Where possible results were calculated from plots of means of these replicates. Variability where lag periods were prolonged, however, necessitated calculation from plots of single replicate points. The effects of a range of xanthate concentrations on lag periods and specific oxidation rate are summarized in Table 1. In soil the specific rate of oxidation by Nitrosomorws in the absence of xanthate was greater than that in liquid culture, although the length of the lag was increased. The reverse was found for Nitrobacter with soil oxidation rates and lag times 71 and 19% respectively of those found in liquid culture. In liquid culture xanthate greatly increased the duration of the
(h)
Fig. 3. Effect of potassium ethyl xanthate concentrations of Opgml-’ (0) 2pgml-’ (0) and 8 pgml-’ (IJ) on growth of Nitrobacter in liquid culture. f ‘d z
15 r
2,o -0 5 E m I?
-1.0 0
20
40
60
80
100
120
Time I h) Fig. 4. Effect of potassium ethyl xanthate concentrations of 0 pg ml-’ (0) and 80 pg ml-’ (0) on growth of Nitrobacter
in soil.
Final pH was 6.5. At a xanthate concentration of 8 p g ml-’ NH: oxidation was completely inhibited. The gradual increase in NO; levels at this concentration, and following growth at lower concen-
Table 1. Specific oxidation rates and lag periods for Nifrowmonas and Nitrobacfer in soil and liquid culture. Each value is the mean of three replicates. Standard errors are less than 10 and 5% of the mean for Nitrosomonas and Nirrobocter respectively. *Represents a significant decrease andt a significant increase in comparison to the control value at the 5Y level. NG indicates no erowth Nitrosomonas
Xanthate concentration (fig ml_‘) 0 0.2 0.8 1.0 2.0 3.0 6.0 8.0
Liquid culture Specific oxidation rate Lag period W’) (h) 0.030 0.026 0.020’ 0.017’ 0.019: 0.014; NG NC
Soil incubation Specific oxidation rate Lag period (h-l) (h)
6.4 6.1 9.3 10.8’ 270* 502; NG NG
0.038 0.037 0.044 0.038 0.030 0.035
29 20 51. 31* 82* 85*
0.038
92’
0.023
16
0.023 0.022 0 021
17 14 1x
Nitrobacter 0.0 0.2 0.8 1.0 8.0 10.0 80.0
0.032 0.032 0.045t NC
85 134’ 5078 NG
SUSAN E. UNDERHILL and J. I. PROWR
232
lag. Above a concentration
of 3 ,ug ml-’ no growth of during the sampling period (7 weeks). No growth of Nitrobacter was observed above a concentration of 0.8 pg ml-‘. Thus, Nitrobacter is more sensitive than Nitrosomonas to xanthate in liquid culture with respect to its effect on the lag period. The specific oxidation rate of Nitrosomonas was significantly reduced above a xanthate concentration of 0.2pgmll’, with inhibition increasing at higher concentrations until the effect on lag period prevented further assessment. The effect on specific oxidation rate of Nitrobacter could only be studied up to 0.8 pg ml-’ but at this concentration specific oxidation rate increased significantly over the control, suggesting a stimulation of NO; oxidation and growth despite an increase in the lag period. In soil the effect of xanthate on both organisms was less marked. There was no effect on specific oxidation rate of Nitrosomonas up to a concentration 8 pg ml-’ while that of Nitrobacter was unaffected at concentrations of 80pgmll’. The duration of the lag for Nitrosomonas increased with increasing xanthate concentration but the effect was less marked than in liquid culture such that growth was observed at all concentrations applied. Xanthate concentrations up to 80 pgml-’ had no effect on the lag period of Nitrobacter. In soil, therefore, Nitrosomonas was more sensitive to xanthate than Nitrobacter and again the major effect was on the lag period rather than the specific rate of oxidation. Rates of evolution of CS, from potassium ethyl xanthate added to both liquid medium and to soil were measured, to dete-mine whether the extent of the lag period merely represented the time when carbon disulphide release ceased (Fig. 5). Evolution from liquid was followed for a longer period because this medium resulted in greatly extended lags. In both cases the rate of evolution of CS2 varied little over the incubation periods described above. Rates of evolution in soil are one order of magnitude less than those in liquid culture. This is thought to be due to immobilization of xanthate or CS, at the soil surface and also to increased resistance to diffusion of CS, by the soil matrix during assay. In no case does this reduced rate of evolution affect the qualitative Nitrosomonas was observed
0.05
differences described above between oxidation rates and lag periods of the different organisms in liquid and soil culture. To assess the effect of potassium ethyl xanthate on the viability of cells during the long lag periods observed, MPN counts were carried out on two cultures of Nitrosomonas incubated for 7 weeks. The first contained Smith and Hoare’s medium with 50 pg NH:-N ml-’ as (NH&SO, and 3 pg ml-’ potassium ethyl xanthate and the second contained Smith and Hoare’s medium with no NH: or xanthate. No growth occurred within 4 weeks in any of the samples taken from dilutions of the xanthate-treated cells while the non-xanthate treated cells gave a count of 2.53 x lo4 cells ml-’ at this time. After 7 weeks, however, xanthate inhibited cultures gave a count of 6.36 x lo6 cells ml-’ (SE 0.68 x 106) while nontreated cells gave 7.13 x 106cellsml(SE 0.41 x 106). These numbers are not significantly different at the 5% level. DISCUSSION
This study allows assessment of the effect of potassium ethyl xanthate on the growth and activity of both NH:- and NO;-oxidizing bacteria in the absence of interactions with other microorganisms. The major inhibitory effect of xanthate is the induction of long lag phases rather than a reduction in growth rate or specific oxidation rate, although this was also observed at some concentrations. Results show quantitative differences from those of other workers. Rodgers and Ashworth (1982) observed a lag of 14 days in liquid culture at a xanthate concentration 10 pg ml-‘, compared to 20 days at 3 pg ml-’ in our study. Ashworth et al. (1979) found a lag of 6 weeks after application of 315 mg kg-’ soil while a lag of 4 days was observed in our soil incubation studies at a concentration of 2.1 mg kg-’ soil. The shorter lags observed here probably result from differences in the experimental systems, in particular different incubation temperatures. Xanthate breakdown is reported by Ashworth et al. (1979) to be strongly temperature dependent. iions are less sensitive Both NH: and NO; o. to xanthate inhibition in son than in liquid culture presumably through immobilization of xanthate or CS, at the soil surface. However, within each of the two experimental systems the different processes are affected to different extents. In liquid culture, Nitrobccter is more sensitive than Nitrosomonas. In soil, Nitrosomonas is inhibited but Nitrobacter is unaffected even at very high xanthate concentrations. This difference in effect can be explained by considering the nature of the soil surface charge and that of the respective substrates for Nitrosomonas and Nitrobatter and further by assuming that either potassium ethyl xanthate, CS, or both are adsorbed to the soil surface. Soil particles have a net negative charge and adsorb NH: ions while NO; remains in solution. It might be expected therefore that NH: oxidation will proceed faster in the presence of soil, which might concentrate the substrate, than in liquid culture. Soil would provide no such advantage for NO;-oxidizers and indeed its presence may be a disadvantage if the bacteria became adsorbed to the soil surface, from
I:-:--,_ I-
Ll
-
0.04
z = 51
-
0.03
5 *
-
0.02
P 2
001
5
I
1
8
4
1
eooo
.\”
Tams Ih)
Fig. 5. Carbon disulphide evolved from soil (0) and liquid (0) over a 5 h period as a percentage of that applied. Standard errors for evolution from soil are illustrated. Standard errors for evolution from liquid were in all cases less than 7%.
233
Inhibition of nit&c :ation by xanthate
which their substrate, NO;, would be repelled. This may explain the increased oxidation rate for Nitrosomonas in soil as opposed to liquid culture in the absence of xanthate (Table 1). If potassium ethyl xanthate or its breakdown products are adsorbed to the soil surface then Nitrosomonas is likely to be inhibited to a greater extent than Nitrobacter in soil. This is in fact the case with NO; oxidation unaffected even at very high concentrations. The greater sensitivity of Nitrobacter in liquid culture is less easily explained but Nitrosomonas may be adapted to growth on surfaces and may avoid xanthate in solution, e.g. by wall growth. The only inhibitory effect is on the length of the lag phase, with no decrease in oxidation rate and, in fact, some evidence of stimulation at a xanthate concentration of 0.8 pg ml-’ (Table 1). Rates of CS2 evolved over 5 h periods from soil and solution did not vary significantly during the period of incubations and release from lag was not due to complete breakdown of xanthate. Ashworth et al. (1979) measured cumulative CS2 levels and found that approximately 25% of CSr applied to soil as potassium ethyl xanthate was released within 10 days with little further release up to 40 days. They also found breakdown to be strongly temperature dependent and reported that, under the alkaline conditions prevailing in this study, xanthate breaks down to give CS, as a major product but also H,S and another unidentified minor product. These other compounds may also be inhibitory to nitrifiers and may interfere with the C’S, assay in liquid, but not in soil which binds HIS strongly. This may be an additional reason for the higher rates of evolution from solution observed in this study. However, the release from lag was not associated with reduction in CS, concentration in either liquid or soil studies. Rodgers et Ql. (1982) found that 100 pg potassium ethyl xanthate ml-’ was bacteriostatic for NitrosomonaS over 48 h. This is in agreement with our results at lower xanthate concentrations and during much longer incubations, and similar in scale to observed lag times. Our results also demonstrate the care required when using the MPN method for enumeration of nitrifying bacteria. Matulewich et al. (1975) drew attention to the importance of choosing the correct incubation period. Growth of NH:-oxidizers in their system was not detected until 2&55 days and in some cases maximum numbers of NO;-oxidizers had not been reached by 113 days. This problem has also been found here with non-xanthate treated cells giving counts of 2.43 x 104cells ml-’ after 4 weeks and weeks. More 6.36 x lo6 cells ml-’ after 7
significantly, all samples of xanthate-treated cells exhibited lag periods greater than 4 weeks, though growing in xanthate-free medium. Thus, although standardization of incubation times and conditions may allow comparison of counts from similar sources, this may not apply following any treatment likely to induce a lag period. Acknowledgements-S.
E. Underhill would like to acknowledge receipt of a Science and Engineering Research Council Studentship and we would like to thank J. W. Parsons for his assistance in preparing the soil. REFERENCES
Alexander M. (1965) Most-probable-number method for microbial populations. In Methodr of Soil Analysis (C. A. Black et al., Eds), pp. 1467-1472. American Society of Agronomy, Madison, Wisconsin. Ashworth J., Briars G. G.. Evans A. A. and Matula J. (1977) Inhibitive of nit&cation by nitrapyrin, carbon disulphide and trithiocarbonate. Journal of Food Science and Agriculture 28, 673683.
Ashworth J., Rodgers G. A. and Briggs G. G. (IY7Y) Xanthates as inhibitors of fertilizer nitrogen transformation in soil. Chemistry and Industry (London) 3, 9G92.
Ashworth J., Penny A., Widdowson F. V. and Briggs G. G. (1980) The effects of injecting nitrapyrin (‘N-serve’), carbon disulphide or trithiocarbonates, with aqueous ammonia, on yield and %N of grass. Journal of the Science of Food and Agriculture 31, 229-237.
Campbell N. E. R. and Aleem M. I. H. (1965) The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria. 1. Ammonia and hydroxylamine oxidation by Nitrosomonas. Antonie van Leeuwenhoek 31, 124136 Goring C. A. (1962) Control of nitrification by 2-chloro-6-(trichloromethyl)pyridine. Soil Science 93, 211-218. Matulewich V. A., Strom P. F. and Finstein M. S. (1975) Length of incubation for enumerating nitrifying bacteria present in various environments. Applied Microbiology 29, 265-268.
Parr J. F. (1973) Chemical and biochemical considerations for maximizing the efficiency of fertilizer nitrogen. Journal of Environmental Quality 2, 75-84.
Ratnayake M. and Audus L. J. (1978) Studies on the effects of herbicides on soil nitrification, II. Pesticide Biochemistry and Physiology 8, 17&185. Rodgers G. A. and Ashworth J. (1982) Bacteriostatic action of nitrification inhibitors. Canadian Journal of Microbiology 28, 1093-l 100. Skinner F. A. and Walker N. (1961) Growth of Nitrosomonas europaea in batch and continuous culture. Archiv fur Mikrobiologie 38, 339-349.
Smith A. J. and Hoare D. S. (1968) Acetate assimilation by Nitrobacter in relation to its ‘obligate autotrophy’. Journal of Bacteriology 95, 844855.
0038-0717/85 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
INHIBITION OF NITRIFICATION BY POTASSIUM ETHYL XANTHATE IN SOIL AND IN LIQUID CULTURE SUSAN
Department
of Microbiology,
E. UNDERHILL*
Marischal
College,
and J. I.
University
PROSSER
of Aberdeen,
Aberdeen,
Scotland
AB9 1AS
(Accepted 30 October 1984) Summary-Inhibition of nitrification by potassium ethyl xanthate has been investigated in liquid batch culture and in soil, using axenic cultures of Nitrosomonus and Nitrobacter. In all cases the major effect was induction of a prolonged lag rather than a reduction in specific oxidation rates, although this was also found for Nitrosomonas at high xanthate concentrations. Inhibition of both ammonium and nitrite oxidation was reduced in soil, because of immobilization of xanthate or its breakdown products at the soil surface. However, in liquid culture nitrite oxidation was more sensitive while ammonium oxidation was more sensitive in soil. It is proposed that this also results from accumulation of xanthate, or its breakdown products at the soil surface. Xanthate was shown to have a bacteriostatic, rather than bactericidal effect, but xanthate-treated cells exhibited a significantly longer lag when transferred to xanthate-free medium, emphasizing the need for caution when using the most probable number method in such studies.
INTRODUCTION
but with some variation between different herbicides. None of these studies involved axenic cultures and interpretation of results is therefore complicated by the possibility of microbial breakdown of added compounds, selection of different nitrifier strains in different situations and by undefined interactions with other organisms. These problems have been eliminated in the present study by the use of axenic cultures of nitrifying bacteria inoculated into sterile soil and liquid medium.
Under certain conditions, application of ammoniumbased fertilizers is followed by significant oxidation of ammonium to nitrate. As well as increasing rates of loss of N from the soil, through leaching or denitrification of nitrate, unacceptable levels of nitrate may be reached in run-off waters. To prevent this, nitrification inhibitors may be applied along with such fertilizers (Parr, 1973). The most commonly used of such inhibitors is N-Serve (nitrapyrin) (Goring, 1962), which inhibits the cytochrome oxidase component involved in ammonium oxidation (Campbell and Aleem, 1965), but the use of carbon disulphide based inhibitors has recently been investigated (Ashworth et al., 1977,1979, 1980). The most efficient of these is potassium ethyl xanthate which is oxidized chemically in soil, releasing carbon disulphide which is a strong inhibitor of nitrification at levels which have little effect on other microbial processes. Rodgers and Ashworth (1982) distinguished between bactericidal and bacteriostatic actions of several inhibitors including potassium ethyl xanthate which they found to be bacteriostatic. They observed complete inhibition of nitrification by potassium ethyl xanthate at concentrations of 10 mgl-‘, in aqueous culture, but no evidence of bactericidal effects. At 10 mg l-‘, nitrification was inhibited for 14 days. In soil, Ashworth et al. (1979) reported inhibition for 6 weeks at a concentration of 315 mg kg-’ soil. Ratnayake and Audus (1978) studied the effects of a range of herbicides on several measures of activity and growth of nitrifying bacteria in liquid culture and in soil reperfusion systems. They also report reduced sensitivity to inhibition in soil,
MATERIALS AND METHODS
Soil isolates of Nitrosomonas europaea and Nitrobatter sp. were kindly supplied by Dr R. M. MacDonald, Rothamsted Experimental Station, Harpenden, England. Nitrosomonas was routinely subcultured in 100ml inorganic medium (Skinner and Walker, 1961) contained in 250 ml Erlenmeyer flasks fitted with side-arms half-filled with aqueous sodium carbonate solution (5% w/v). This facilitated aseptic neutralization of acid formed during ammonium oxidation. Nitrobacter was routinely cultured in Skinner and Walker’s medium with (NH,),SO, replaced by NaNO, (1.42 g l-‘, equivalent to 410 pg NOT-N ml-‘). Both were incubated at 27°C on a New Brunswick Scientific Rotary Incubator. Growth was assessed by spot tests for NH: (Nesslers reagent) and NO; and NO; (Griess llosvay’s reagents I and II). Cultures were checked regularly for contamination by microscopic examination and by inoculation onto nutrient agar plates which were then incubated at 27°C for 3-4 weeks. Liquid culture Nitrosomonas and Nitrobacter were grown at 27°C in 250 ml Erlenmeyer flasks containing 150 ml Smith and Hoare’s (1968) medium with 50 pg NH:-N ml-’ as (NH,)$Od and 50 pg NOT-N ml-’ as NaNO,
*Present address: Department of Chemical Engineering, The Polytechnic of Wales, Pontypridd, Mid Glamorgan, CS37 IDL, U.K. 229
230
SUSAN
E.
UNDERHILL
respectively. Potassium ethyl xanthate was added aseptically as 1 ml of a filter sterilized solution following autoclaving of the medium at 120°C for 15 min to give final concentrations in the range O-8 pgml-‘. Flasks were inoculated with 1 ml of fully grown cultures of either Nitrosomonas or Nitrobacter, previously subcultured at least twice in Smith and Hoare’s medium. Samples were removed daily (Nitro somonas) or twice daily (Nitrobacter) and were analyzed for NH:, NO, and NO; using a Technicon Autoanalyser II. Samples were also plated onto nutrient agar and incubated at 27°C to check for contamination by heterotrophs. Contaminated flasks were discarded. Viable cell numbers of Nitrosomonas were estimated by the most probable number (MPN) method (Alexander, 1965). A l/l0 dilution series was constructed and five 100~1 samples from each dilution were mixed with 1.50~1 Skinner and Walker’s (1961) medium in the wells of microtitre plates. Plates were incubated for up to 7 weeks at 27°C with water lost by evaporation replaced where necessary. Growth was assessed periodically by change of the medium indicator from red to yellow, as a result of acid production accompanying NH: oxidation. After 7 weeks, spot tests for NO; were carried out on all wells. Soil incubation studies Air-dried soil was adjusted to pH 7.6 by addition of Ca(OH), and 80 g portions were autoclaved for 30 min at 120°C in 2.50 ml Erlenmeyer flasks. Each flask was then supplied, aseptically, with 20ml of a sterile solution containing 50,ug NH:-N ml-’ as (NH,),SO, sulphate or 5Opg NOT-N ml-’ as NaNO,, 1 ml of a fully grown culture of Nitrosomonas or Nitrobacter, prepared as described above, and 1 ml of the appropriate filter sterilized potassium ethyl xanthate solution, giving final concentrations in the range O-80 pg ml-‘. The soil was then mixed to ensure even distribution of solutions and cells. Three flasks were prepared for each treatment and each flask was weighed regularly with water lost through evaporation being replaced. Soil samples of approximately 2.5 g were removed daily and twice daily for Nitrosomonas and Nitrobacter respectively. Each sample was then weighed accurately and NO; and NO; concentrations estimated on a Technicon Autoanalyser following extraction by shaking for 30 min in 1 M KC1 (2.5 g: 12.5 ml) followed by filtering. When necessary, samples were stored at - 15°C. Samples were also checked for contamination by heterotrophs as described above and contaminated flasks discarded. Rate of carbon disulphide production CS, production from potassium ethyl xanthate in soil and in liquid medium was measured using Conway units as described by Ashworth et al. (1979). Evolution from liquid culture was determined in 1 ml samples removed regularly from Smith and Hoare’s (1968) medium containing potassium ethyl xanthate and shaking at 27°C. The sample was placed in the inner compartment of a Conway unit and 2 ml KOH in ethanol (10% w/v) was added to the outer compartment. The units were sealed, incubated for 5 h, when
and J. I.
PROSSER
excess KOH was neutralized with acetic acid and the solution titrated with 20mM iodine in potassium iodide, with sodium starch glycollate as indicator. Potassium ethyl xanthate solution was removed from the inner well between titrations. Evolution from soil was measured by placing 5 g of air dried soil, with pH and moisture content adjusted as described above, in the inner compartment. This was then mixed with 1 ml of a 32% w/v potassium ethyl xanthate solution in water. KOH in ethanol was placed in the outer well and carbon disulphide evolution measured as described above, except that 5 mM iodine was used to increase sensitivity. The solution in the outer compartment was renewed after each measurement while the same xanthate-soil matrix was used for each determination. RESULTS
In liquid or soil culture, growth was assessed by NO; (Nitrosomonas) or NO; (Nitrobacter) production, following a lag period. For Nitrosomonas, growing in liquid culture, the lag period was followed by an exponential increase in NO; concentration and a deceleration phase, due to a reduction in pH which prevented full conversion of NH: (Fig. 1). The reduction in pH was noted as a change in medium indicator (phenol red) from red to yellow at pH 7.
I 203
I
I
I
400
600
600
I loo0
Time (h)
Fig. I. Effect of potassium ethyl xanthate concentrations of Opgml-’ (01, 1pgml-’ (01, 2pgml-’ (Cl), 3figrnl-’ (a) and 8 pgml-’ (A) on growth of Nitrosomonasin liquid culture.
f ‘9 r
30-
E ?t
2.0 -
3
lo-
g 5 "c
oo-
8 I
-1.0 -
Time (h)
Fig. 2. Effect of potassium ethyl xanthate concentrations of Opg ml-’ (0) and 8 pgml-’ (0) on growth of NitroSOM~~~S in soil.
231
Inhibition of nitrification by xanthate
Time
trations, is due to evaporation. In soil (Fig. 2), the greater buffering capacity increased the duration of the exponential phase and allowed complete oxidation of added NH: to NO;. Although NHr could have been lost by volatilization, the initial concentration was such that it was in excess, until depleted by growth of Nitrosomonas, and any volatilization would not affect either the duration of the lag period or the specific oxidation rate. For Nitrobacter the exponential phase continued until NO; was exhausted in both soil and liquid culture (Figs 3 and 4). Xanthate concentrations are expressed as pg ml-’ of soil solution. This allows a more meaningful comparison with concentrations in liquid culture although it was not possible to quantify precisely the extent of absorption and immobilization of xanthate by the soil and the consequent effect on its concentration in the soil solution. A concentration of 1 pgml-’ is equivalent to a concentration of 0.26 pg g soil-‘. The effect of xanthate was therefore assessed by its effect on the duration of the lag and the specific rate of product formation, termed the specific oxidation rate. The latter was calculated from the slope of a semi-logarithmic plot of product concentration vs time during the exponential phase. The duration of the lag was calculated as the time when the gradient during the exponential phase intercepted the initial product concentration. Three replicates were used for each treatment. Where possible results were calculated from plots of means of these replicates. Variability where lag periods were prolonged, however, necessitated calculation from plots of single replicate points. The effects of a range of xanthate concentrations on lag periods and specific oxidation rate are summarized in Table 1. In soil the specific rate of oxidation by Nitrosomorws in the absence of xanthate was greater than that in liquid culture, although the length of the lag was increased. The reverse was found for Nitrobacter with soil oxidation rates and lag times 71 and 19% respectively of those found in liquid culture. In liquid culture xanthate greatly increased the duration of the
(h)
Fig. 3. Effect of potassium ethyl xanthate concentrations of Opgml-’ (0) 2pgml-’ (0) and 8 pgml-’ (IJ) on growth of Nitrobacter in liquid culture. f ‘d z
15 r
2,o -0 5 E m I?
-1.0 0
20
40
60
80
100
120
Time I h) Fig. 4. Effect of potassium ethyl xanthate concentrations of 0 pg ml-’ (0) and 80 pg ml-’ (0) on growth of Nitrobacter
in soil.
Final pH was 6.5. At a xanthate concentration of 8 p g ml-’ NH: oxidation was completely inhibited. The gradual increase in NO; levels at this concentration, and following growth at lower concen-
Table 1. Specific oxidation rates and lag periods for Nifrowmonas and Nitrobacfer in soil and liquid culture. Each value is the mean of three replicates. Standard errors are less than 10 and 5% of the mean for Nitrosomonas and Nirrobocter respectively. *Represents a significant decrease andt a significant increase in comparison to the control value at the 5Y level. NG indicates no erowth Nitrosomonas
Xanthate concentration (fig ml_‘) 0 0.2 0.8 1.0 2.0 3.0 6.0 8.0
Liquid culture Specific oxidation rate Lag period W’) (h) 0.030 0.026 0.020’ 0.017’ 0.019: 0.014; NG NC
Soil incubation Specific oxidation rate Lag period (h-l) (h)
6.4 6.1 9.3 10.8’ 270* 502; NG NG
0.038 0.037 0.044 0.038 0.030 0.035
29 20 51. 31* 82* 85*
0.038
92’
0.023
16
0.023 0.022 0 021
17 14 1x
Nitrobacter 0.0 0.2 0.8 1.0 8.0 10.0 80.0
0.032 0.032 0.045t NC
85 134’ 5078 NG
SUSAN E. UNDERHILL and J. I. PROWR
232
lag. Above a concentration
of 3 ,ug ml-’ no growth of during the sampling period (7 weeks). No growth of Nitrobacter was observed above a concentration of 0.8 pg ml-‘. Thus, Nitrobacter is more sensitive than Nitrosomonas to xanthate in liquid culture with respect to its effect on the lag period. The specific oxidation rate of Nitrosomonas was significantly reduced above a xanthate concentration of 0.2pgmll’, with inhibition increasing at higher concentrations until the effect on lag period prevented further assessment. The effect on specific oxidation rate of Nitrobacter could only be studied up to 0.8 pg ml-’ but at this concentration specific oxidation rate increased significantly over the control, suggesting a stimulation of NO; oxidation and growth despite an increase in the lag period. In soil the effect of xanthate on both organisms was less marked. There was no effect on specific oxidation rate of Nitrosomonas up to a concentration 8 pg ml-’ while that of Nitrobacter was unaffected at concentrations of 80pgmll’. The duration of the lag for Nitrosomonas increased with increasing xanthate concentration but the effect was less marked than in liquid culture such that growth was observed at all concentrations applied. Xanthate concentrations up to 80 pgml-’ had no effect on the lag period of Nitrobacter. In soil, therefore, Nitrosomonas was more sensitive to xanthate than Nitrobacter and again the major effect was on the lag period rather than the specific rate of oxidation. Rates of evolution of CS, from potassium ethyl xanthate added to both liquid medium and to soil were measured, to dete-mine whether the extent of the lag period merely represented the time when carbon disulphide release ceased (Fig. 5). Evolution from liquid was followed for a longer period because this medium resulted in greatly extended lags. In both cases the rate of evolution of CS2 varied little over the incubation periods described above. Rates of evolution in soil are one order of magnitude less than those in liquid culture. This is thought to be due to immobilization of xanthate or CS, at the soil surface and also to increased resistance to diffusion of CS, by the soil matrix during assay. In no case does this reduced rate of evolution affect the qualitative Nitrosomonas was observed
0.05
differences described above between oxidation rates and lag periods of the different organisms in liquid and soil culture. To assess the effect of potassium ethyl xanthate on the viability of cells during the long lag periods observed, MPN counts were carried out on two cultures of Nitrosomonas incubated for 7 weeks. The first contained Smith and Hoare’s medium with 50 pg NH:-N ml-’ as (NH&SO, and 3 pg ml-’ potassium ethyl xanthate and the second contained Smith and Hoare’s medium with no NH: or xanthate. No growth occurred within 4 weeks in any of the samples taken from dilutions of the xanthate-treated cells while the non-xanthate treated cells gave a count of 2.53 x lo4 cells ml-’ at this time. After 7 weeks, however, xanthate inhibited cultures gave a count of 6.36 x lo6 cells ml-’ (SE 0.68 x 106) while nontreated cells gave 7.13 x 106cellsml(SE 0.41 x 106). These numbers are not significantly different at the 5% level. DISCUSSION
This study allows assessment of the effect of potassium ethyl xanthate on the growth and activity of both NH:- and NO;-oxidizing bacteria in the absence of interactions with other microorganisms. The major inhibitory effect of xanthate is the induction of long lag phases rather than a reduction in growth rate or specific oxidation rate, although this was also observed at some concentrations. Results show quantitative differences from those of other workers. Rodgers and Ashworth (1982) observed a lag of 14 days in liquid culture at a xanthate concentration 10 pg ml-‘, compared to 20 days at 3 pg ml-’ in our study. Ashworth et al. (1979) found a lag of 6 weeks after application of 315 mg kg-’ soil while a lag of 4 days was observed in our soil incubation studies at a concentration of 2.1 mg kg-’ soil. The shorter lags observed here probably result from differences in the experimental systems, in particular different incubation temperatures. Xanthate breakdown is reported by Ashworth et al. (1979) to be strongly temperature dependent. iions are less sensitive Both NH: and NO; o. to xanthate inhibition in son than in liquid culture presumably through immobilization of xanthate or CS, at the soil surface. However, within each of the two experimental systems the different processes are affected to different extents. In liquid culture, Nitrobccter is more sensitive than Nitrosomonas. In soil, Nitrosomonas is inhibited but Nitrobacter is unaffected even at very high xanthate concentrations. This difference in effect can be explained by considering the nature of the soil surface charge and that of the respective substrates for Nitrosomonas and Nitrobatter and further by assuming that either potassium ethyl xanthate, CS, or both are adsorbed to the soil surface. Soil particles have a net negative charge and adsorb NH: ions while NO; remains in solution. It might be expected therefore that NH: oxidation will proceed faster in the presence of soil, which might concentrate the substrate, than in liquid culture. Soil would provide no such advantage for NO;-oxidizers and indeed its presence may be a disadvantage if the bacteria became adsorbed to the soil surface, from
I:-:--,_ I-
Ll
-
0.04
z = 51
-
0.03
5 *
-
0.02
P 2
001
5
I
1
8
4
1
eooo
.\”
Tams Ih)
Fig. 5. Carbon disulphide evolved from soil (0) and liquid (0) over a 5 h period as a percentage of that applied. Standard errors for evolution from soil are illustrated. Standard errors for evolution from liquid were in all cases less than 7%.
233
Inhibition of nit&c :ation by xanthate
which their substrate, NO;, would be repelled. This may explain the increased oxidation rate for Nitrosomonas in soil as opposed to liquid culture in the absence of xanthate (Table 1). If potassium ethyl xanthate or its breakdown products are adsorbed to the soil surface then Nitrosomonas is likely to be inhibited to a greater extent than Nitrobacter in soil. This is in fact the case with NO; oxidation unaffected even at very high concentrations. The greater sensitivity of Nitrobacter in liquid culture is less easily explained but Nitrosomonas may be adapted to growth on surfaces and may avoid xanthate in solution, e.g. by wall growth. The only inhibitory effect is on the length of the lag phase, with no decrease in oxidation rate and, in fact, some evidence of stimulation at a xanthate concentration of 0.8 pg ml-’ (Table 1). Rates of CS2 evolved over 5 h periods from soil and solution did not vary significantly during the period of incubations and release from lag was not due to complete breakdown of xanthate. Ashworth et al. (1979) measured cumulative CS2 levels and found that approximately 25% of CSr applied to soil as potassium ethyl xanthate was released within 10 days with little further release up to 40 days. They also found breakdown to be strongly temperature dependent and reported that, under the alkaline conditions prevailing in this study, xanthate breaks down to give CS, as a major product but also H,S and another unidentified minor product. These other compounds may also be inhibitory to nitrifiers and may interfere with the C’S, assay in liquid, but not in soil which binds HIS strongly. This may be an additional reason for the higher rates of evolution from solution observed in this study. However, the release from lag was not associated with reduction in CS, concentration in either liquid or soil studies. Rodgers et Ql. (1982) found that 100 pg potassium ethyl xanthate ml-’ was bacteriostatic for NitrosomonaS over 48 h. This is in agreement with our results at lower xanthate concentrations and during much longer incubations, and similar in scale to observed lag times. Our results also demonstrate the care required when using the MPN method for enumeration of nitrifying bacteria. Matulewich et al. (1975) drew attention to the importance of choosing the correct incubation period. Growth of NH:-oxidizers in their system was not detected until 2&55 days and in some cases maximum numbers of NO;-oxidizers had not been reached by 113 days. This problem has also been found here with non-xanthate treated cells giving counts of 2.43 x 104cells ml-’ after 4 weeks and weeks. More 6.36 x lo6 cells ml-’ after 7
significantly, all samples of xanthate-treated cells exhibited lag periods greater than 4 weeks, though growing in xanthate-free medium. Thus, although standardization of incubation times and conditions may allow comparison of counts from similar sources, this may not apply following any treatment likely to induce a lag period. Acknowledgements-S.
E. Underhill would like to acknowledge receipt of a Science and Engineering Research Council Studentship and we would like to thank J. W. Parsons for his assistance in preparing the soil. REFERENCES
Alexander M. (1965) Most-probable-number method for microbial populations. In Methodr of Soil Analysis (C. A. Black et al., Eds), pp. 1467-1472. American Society of Agronomy, Madison, Wisconsin. Ashworth J., Briars G. G.. Evans A. A. and Matula J. (1977) Inhibitive of nit&cation by nitrapyrin, carbon disulphide and trithiocarbonate. Journal of Food Science and Agriculture 28, 673683.
Ashworth J., Rodgers G. A. and Briggs G. G. (IY7Y) Xanthates as inhibitors of fertilizer nitrogen transformation in soil. Chemistry and Industry (London) 3, 9G92.
Ashworth J., Penny A., Widdowson F. V. and Briggs G. G. (1980) The effects of injecting nitrapyrin (‘N-serve’), carbon disulphide or trithiocarbonates, with aqueous ammonia, on yield and %N of grass. Journal of the Science of Food and Agriculture 31, 229-237.
Campbell N. E. R. and Aleem M. I. H. (1965) The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria. 1. Ammonia and hydroxylamine oxidation by Nitrosomonas. Antonie van Leeuwenhoek 31, 124136 Goring C. A. (1962) Control of nitrification by 2-chloro-6-(trichloromethyl)pyridine. Soil Science 93, 211-218. Matulewich V. A., Strom P. F. and Finstein M. S. (1975) Length of incubation for enumerating nitrifying bacteria present in various environments. Applied Microbiology 29, 265-268.
Parr J. F. (1973) Chemical and biochemical considerations for maximizing the efficiency of fertilizer nitrogen. Journal of Environmental Quality 2, 75-84.
Ratnayake M. and Audus L. J. (1978) Studies on the effects of herbicides on soil nitrification, II. Pesticide Biochemistry and Physiology 8, 17&185. Rodgers G. A. and Ashworth J. (1982) Bacteriostatic action of nitrification inhibitors. Canadian Journal of Microbiology 28, 1093-l 100. Skinner F. A. and Walker N. (1961) Growth of Nitrosomonas europaea in batch and continuous culture. Archiv fur Mikrobiologie 38, 339-349.
Smith A. J. and Hoare D. S. (1968) Acetate assimilation by Nitrobacter in relation to its ‘obligate autotrophy’. Journal of Bacteriology 95, 844855.