The use of chloramphenicol in the study of the denitrification process: Some side-effects

Soil Biol. B&hem. Vol. 26. No. I. DD.925-921. 1994

Pergamon

Copyright 0’ 1994i%vier Sc&e Ltd Prinled in Great Britain. All rights reserved 0038-07I7/94 $7.00+ 0.00

003%0717(93)EOO16-F

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COMMUNICATION

THE USE OF CHLORAMPHENICOL THE DENITRIFICATION PROCESS:

IN THE STUDY OF SOME SIDE-EFFECTS

L. DENDOOVEN,P. SPLATT and J. M. ANDERSON Department of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, England (Accepted 24 Seprember 1993)

Chloramphenicol is routinely used to determine the denitrification enzyme activity (DEA) of a soil (Martin et al., 1988; Groffman and Tiedje, 1989; Parsons et a/., 1991). As an antibiotic it inhibits de nouo protein synthesis by bacteria but it is assumed that it does not affect the functioning of existing enzymes (Franklin and Snow, 1989). We are investigating the controls over N,O: N, gas ratios during brief denitrification events and hypothesize that the persistence and derepression of nitrate, nitrite and nitrous oxide reduction enzymes are key factors in these processes. Chloramphenicol is a useful tool in such studies as it can be used to determine the initial concentrations of the reduction enzymes and their dynamics when anaerobiosis is triggered. During the course of these experiments it was found that chloramphenicol was an active reagent in the denitrification process. It indirectly enhanced CO, and N,O production and nitrite was formed as a degradation product of chloramphenicol (Fig. 1); a combination of effects which could lead to a misinterpretation of the dynamics of the denitrification process. The experiments were carried out with soil under permanent pasture at the Agriculture and Food Research Council Institute for Grassland and Environmental Research at North Wyke, Devon. The soil, a clayey pelostagnogley has an inorganic fraction of 36.6% clay, 47.7% silt, 13.9% fine and 1.8% coarse sand (Armstrong and Garwood, 1991). Soil samples were taken in November 1992 from the 0 to 1Ocm layer of an experimental plot that had received no fertilizer for at least 10 yr. The organic C content of the upper 10 Cm was 5.29% and the organic N content 0.62%. At collectlon, the sol1 pH,,,o, was 6.0 and the gravimetric water content was 36.9%. The moist soil was sieved (5 mm) and sub-samples of 7.5 g were added to 100 ml Erlenmeyer flasks. The flasks were air-sealed tightly with a silicone rubber stopper and incubated aerobically for 5 days at 25°C. After 5 days, lOm1 of a degassed KNO, solution (3.39 mM) was added together with different concentrations of chloramphenicol (0, 0.22, 0.44, 0.88, 1.84 or 3.68 mM). This resulted in a concentration of ca 100 mg NO; N kg-’ soil providing a sufficient amount of electron acceptor for an anaerobic incubation of 48 h at 25°C. The effective concentrations of chloramphenicol were 0, 150, 300, 600, 1250 and 2500 mg kg-’ soil. The samples were shaken for 15 min to eliminate diffusion constraints on nitrate. After shaking, two sub-samples of each treatment were selected at random for assays of NO; and NO;. Each sub-sample was extracted for 30 min with 50 ml of H,O,,,,, . The extracts were filtered through a GFC Whatman filter paper and samples retained for analysis of nitrate and nitrite. The Erlenmeyers were resealed, vacuum sucked for 15 min and flushed with He for 15 min. After purging of ail 0,, 10 ml of C,H, (10% v/v) was added to half of the sub-samples applied with KNO, and to half of those applied with KNO, + chloramphenicol. All sub-samples were then 925

kept at 25°C for 49 h. After 2, 5, 14, 29 and 49 h, two sub-samples were selected at random from each treatment. We routinely measured nitrate and nitrite on an autoanalyser (Burkhard Instruments, U.K.) by the sulphanilamide and sulphanilamide-copper hydrazine methods respectively. Nitrate concentrations were found to be lower in the presence of chloramphenicol possibly as a consequence of interference with the reduction of NO; to NO; (Brooks et al., 1992). We also noted that chloramphenicol increased measured values for NH: determined by the alkaline phenate-sodium nitroprusside method on the same automatic analyser. Samples were therefore analysed for NO; on an ion chromatograph (Dionex, U.K.). The headspace of each Erlenmeyer was sampled and analysed for N,O and CO, using a Pye Unicam 46OOg.c. fitted with a thermal conductivity detector at 30°C. The CTR column from Alltech (a Porapack Q column of 2 m in line with a Molecular sieve + Porous Polymer of I .82 m) with the carrier gas He flowing rate of flow 35 ml min-’ was maintained at 30°C. The concentrations of N,O and CO, were corrected for gas dissolved in the water (Moraghan and Buresh, 1977). After measurement of N,O and CO,, samples were analysed for NO; and NO;. Chloramphenicol was found to increase the CO, production when nitrate and nitrous oxide were used as electron acceptors. The increase was also found not to be proportional to the chloramphenicol concentrations in the soil slurry (Fig. 2). The addition of 100 mg glucose C kg-’ did not change the pattern of increase. The sharpest proportional increase in C mineralization was found at concentrations of 15Omg chloramphenicol kg-’ soil and was presumably related to the diversion of C from protein synthesis to respiratory processes (Smith and Tiedje, 1979). This increase in CO, production was accompanied by an enhanced N,O production (Fig. 3). Further increases in the CO, production with higher application rates of chloramphenicol were much smaller (Fig. 2) and not accompanied by an increase in N,O production (Fig. 3). These smaller increases were presumably due to a biological decompo-

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600

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Fig. 2. The CO, production rate (mg CO, C kg-’ h-‘) of the Rowden soil amended with 3.390 mM KNO, and different concentrations of chloramphenicol (mg kg-’ soil) anaerobically incubated for 48 h at 25°C. (0) Control and (0) 100 mg glucose C kg-‘. (I) plus and minus standard error of the estimates.

Fig. 3. The N,O production rate (mg N,O N kg-’ h-‘) of the Rowden soil amended with 3.390 mM KNO, and different concentrations of chloramphenicol (mg kg-’ soil) anaerobically incubated for 48 h at 25°C. (0) Control and (0) 100 mg glucose C kg-‘. (I) plus and minus standard error of the estimates.

sition of chloramphenicol (Smith and Worrell, 1950). No evidence was found to indicate that a chemical decomposition of chloramphenicol resulted in the production of C02. This was demonstrated using soil samples sterilized on three successive days at 120°C for 45min and amended with different concentrations of chloramphenicol. No enhanced CO, production was found above amounts in controls after incubation under anaerobic conditions at 25°C for 48 h. The nitrate and nitrite concentrations were routinely measured in the stock solutions after 2 days and some nitrite was found in the solutions with 3.39 mM KNO, and chloramphenicol. As shown in Table 1, the nitrite concentration increased with increasing concentration of chloramphenicol. The nitrite concentration showed no significant changes with prolonged incubation and following the application of glucose. It became clear that a hydrolysis or oxidation of chloramphenicol, or an internal oxidation-reduction reaction formed nitrite as the absence of nitrate had no effect on the concentrations of nitrite (Table 1). The formation of nitrite in the solution could be due to different types of complicated reactions. Higuchi and Bias (1953) in a study on the kinetics of degradation of chloramphenicol suggested that due to the multiplicity of functional groups in chloram-

phenicol (Fig. I), the degradative mechanism could involve a number of mechanisms: hydrolysis of the amide, hydrolysis of the chloride, oxidation to the ketone or aldehyde, reduction of the nitro group etc. It is questionable whether buffering the solution to pH 7 with a phosphate buffer (Martin et al., 1988; Parsons et al., 1991) [solutions of the antibiotic in water are faintly acid with a pH of 5.5 (Bartz, 1948)] will stop the formation of nitrite as Higuchi et al. (1954) reported that the degradation of chloramphenicol in aqueous solution is largely independent of pH within the range 2-7. The timecourse of nitrite formed in the 3.39 mM KNO, stock solution with different concentrations of chloramphenicol and kept at 25°C in the dark, is given in Fig. 4. Nitrite was found within 10min in all solutions after the application of chloramphenicol. The nitrite concentration increased very rapidly in the solution with 3.68 mM chloramphenicol 1-l but remained low in the 0.22 mM and 0.44 mg chloramphenicol I-’ solution. The application of the 3.68 IIIM chloramphenicol solution to the sterilized soil samples gave results similar to these in the stock solution, indicating that the hydrolysis of chloramphenicol was not inhibited in soil. The possible formation of nitrite through

Table I. Nitrite concentrations (pg NO; N I- ‘) in H,O, in a solution of 3.39 IIIM KNO, and in a solution of 3.39 rn~ KNO, where 208 mg glucose C I-’ was added after I68 h and stored for 48 h amended with different concentrations of chloramphenicol and kept at 22°C ?r 2 Incubation 46 Chloramphenicol (mM)

H,O

0.00 0.22 0.44 0.88 1.84 3.68

ND 16 95 I57 103 302

48

time (h) 216

I68

+ KNO, + 208 mg glucose C I-’

+KNO, ND 46 (II) 61 (19) 169 (27) 172 (56) 391 (35)

ND 48 (2) 113 (8) 89 (28) 217 (65) 396 (35)

4Yp57) 108 (16) 90 (27) 247 (23) 391 (35)

Numbers in parentheses are standard deviation. ND = Not detectable: lower limit of detection was around

IO fig NO;

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and a continuous culture of denitrifying bacteria, was not observed in our study.

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Acknowledgements-We are grateful to Dr D. Scholefield for access to the Rowden plots and for comments on an early draft. The research was funded by the AFRC.

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REFERENCES

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(hours) Fig. 4. The NO; concentration (mg NO; N 1-l) in a stock solution of 3.390 mM KNOj amended with different concentrations of chloramphenicol (mg 1-l) kept for 48 h in the dark at 25°C. (m) Control; (0) 71.3 mg chloramphenicol 1-l; (0) 142.5 mg chloramphenicol I-‘; (0) 285 mg chloramphenicol 1-l; (A) 593.8mg chloramphenicol 1-l; and (A) 1187.5 mg chloramphenicol I-‘.

chemodenitrification was negligible as no NO; was detected in the soil amended with 3.39 mM KNO,. It was estimated that the increase in nitrite concentration due to the hydrolysis of chloramphenicol would be around 1 mg NO; N kg-’ soil after 48 h with a concentration of 2500 mg chloramphenicol. It is concluded that the use of chloramphenicol in studies on denitrification processes should be carried out with care. Storage of a stock solution before use should be avoided and the chloramphenicol concentration should be kept as low as possible. The application of 150mg kg-’ was sufficient to inhibit de novo synthesis of reduction enzymes in the Rowden soil without a substantial formation of nitrite during an incubation of 48 h at 25°C. Since part of the chloramphenico1 may be bound to soil surfaces (Firestone and Tiedje, 1979), effective concentrations may change with soil type and texture. Increases in CO, production due to a biological decomposition of chloramphenicol did not affect the N,O production but the diversion of C from protein synthesis to respiratory processes increased the N,O production irrespective of the amount of chloramphenicol applied. An inhibition of the activity of existing denitrification enzymes in acetylene-block incubations, as reported by Brooks et al. (1992) for sediments from a nitrate-contaminated aquifer

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