- Email: [email protected]
Effect of tylosin and tylosin fermentation waste on microbial activity of soil
EFFECT OF TYLOSIN AND TYLOSIN FERMENTATION WASTE ON MICROBIAL ACTIVITY OF SOIL M. W. M. BEWKK Department
of Applied
Biology, University of Cambridge. Cambridge. CB2 3DX. England
(Arcepred 7 Fthm-y
Pembroke
Street.
1978)
Summary-The
effect of tylosin fermentation waste and pure tylosin on microbial activity in soil was investigated. The addition of tylosin fermentation waste at concentrations equivalent to 0.4, I.2 and 2 tonnes ha-’ in the field stimulated microbial activity as measured by Oz uptake, CO* release and N mineralization. After 10 weeks incubation half of the C in the waste had been released as CO2 while mineralization of N ranged between 31 and 38%. Tyiosin was detected in the leachates, and at IO weeks between 20 and 32% of the tylosin present in the waste had been leached from the soil. Addition of high concentrations of pure tylosin. equivalent to those which would be found in 4. 20 and 40 tonnes fermentation waste ha -’ in the held. resulted in a decrease in microbial respiration for S--7 weeks after the addition of the antibiotic. N mineralization was reduced throughout the experiment, compared with the control. A higher con~ntration of tylosin was detected in the leachates in this experiment and after 10 weeks the quantity of tylosin detected represented between 16 and 2.4:); of the tylosin added. It is suggested that the full fertthzer potential of large additions of antibiotic waste to soil could be reduced to some extent due to the inhibitory activity of the residual antibiotic.
INTRODUCTION
The subject of antibiotics in soils has been extensively investigated in relation to their production in soil, disappearance of antibiotics added to soil, and the effects of antibiotics on microbial processes in soil (Bewick, 1977). There have been few reports on the use of antibiotic fermentation wastes as fertilizers. An investigation by Uhhar and Bucko (1974) suggested that penicillin and streptomycin fermentation wastes could increase the growth and yield of lucerne, while newspaper reports indicate that Pfizer Ltd of Groton. CT, U.S.A. had increased yields of potatoes using antibiotic fermentation waste as a fertilizer (Brayne. 1973; Warner, 1973). None of these reports mentioned the possible effects of the residual antibiotic, or the effect of the waste on microbial activity in soil. 1 have investigated the effect of different additions of pure tylosin, and residue from a tylosin fermentation. on microbial activity of soil. Aspects of microbial activity examined were 0, uptake, CO, release and N mineraIization. together with a determination of the amounts of extractable tylosin in the soil.
MATERIALS AND METHODS
Microbial activity was studied in a modified John Innes potting compost consisting of 3 parts peat, 7 parts loam and 8 parts sand. pH 6.1, using the apparatus described by Tribe and Bewick (1978). This artificial soil mixture produced a light, free-draining soil. enabling periodic leachings to be made over 10 weeks. and permitted reproducibility of results in different runs of the same experiment. O2 uptake, COz, NO,. NH:, NW3 and tylosin release were measured. The tylosin fermentation waste used (kindly supplied by Dista Products Ltd. Speke, Liverpool) was the material remaining after the broth containing the 403
antibiotic had been removed. The antibiotic itself is a broad-spectrum macrohde widely used in veterinary medicine. To ease handling the material was dried at 105’C for 24 h and then ground to a powder. This produced a material of known, constant, nutrient composition. the tylosin present being unaffected by the treatment. Pure tylosin was suppiied by Dista Products. Dried tylosin waste was added to the soil in amounts equivalent to 0.4, 1.2 and 2 tonnes ha-‘; the quantities added to each tube (2Scm dia) were 125. 374 and 624mg respectively. The amounts of pure tyiosin added to each tube were 2.6, 12.9 and 25.8 mg. As the concentration of tylosin in the waste was 2.1 mg g-t these amounts of pure tylosin represented additions to the leaching tubes equivalent to 4, 20 and 40 tonnes fermentation waste ha-’ respectively. The tylosin and tylosin fermentation waste were added to 70g samples of air-dried soil in leaching tubes, with 70g samples of unamended soil acting as controls. Four replicates were made of each treatment and the control. To measure the initial amounts of NO;, NH; and tylosin the soil was brought to saturation and leached (Tribe and Bewick, 1978). The moisture content of the soil was then adjusted by suction to 13% of the wet weight. Incubation was carried out at 23% Estimations of O2 uptake were made daily until uptake had fallen to 2-3 ml day-‘, when readings were taken every 34 days. CO1 and gaseous NH, release were estimated weekly, while the concentrations of NO;, NH: and the tylosin were measured every fortnight. COz release was measured using Shaw’s (1959) apparatus and the weight of CO2 obtained was converted to voiume for comparison with 0, uptake. WI;, NO; and NH, release were determined using a modified Markham Stitl method (Tribe, 1961). Tylosin determinations involved the extraction of a portion of the leachate with CHCI,
M. W. M. BEWICK
404
and ether into 0.1 N HCI. Absorption of the tylosin in HCI was determined at 290nm using a Hi&r Watts Uvispec spectrophotometer. Interference from the soil was estimated by extracting a portion of the leachatc from the control. RESllLTS E‘cwlloltatiorl
wustr
The results of the effect of different rates of fermentation waste on respiration (Fig. 1) show O2 uptake and COz release produced from the breakdown of the waste alone. with allowance made for control respiration. Increases in respiration due to the addition of the fermentation waste occurred for approximately 8 10 weeks after the beginning of the experiment. Release of N from the waste. measured as NH:. NO; and NH,. followed a similar pattern (Fig. Za), with maximum release of N recorded where the greatest amount of waste was added (2 tonnes ha-‘). After 6 weeks there was little increase in the N mineralized either from the waste itself. or from the control (Fig. la). Release of volatile NH, was only detected in the first fortnight. Maximum NH, volatilization appeared in the control with 5 pg g- ’ being released after 2 weeks. while the addition of fermentation waste appeared to inhibit NH3 release (Table 1). With all additions of fermentation waste low con~ntratjons of tylosin were released from the soil throughout the experiment (Fig. 3a). At 0.4 tonnes ha-’ the lowest amounts of tylosin release were recorded. At the end of the experiment. in the treatments of 0.4, 1.2 and 2 tonnes ha- ‘. of the initial tylosin concentrations of 0.26. 0.77 and 1.29 mg respectively. 22, 32 and 20”; of the tylosin present had been recovered. Analysis of the waste showed it to be 43.7:; C and 6.34”, N: from this the proportions of C and N released from the waste during the experiment were determined. After 10 weeks half of the C had been released as CO2 in all treatments, while the mineralization of N ranged from 31-38”V After 1 week the
Time,
ratios of CO, release: O2 uptake (RQ) in soil amended with 0.4, 1.2 and 2 tonnes ha-’ were 0.78, 0.94 and 0.96 respectively. After 10 weeks, using cumulative 0, uptake and CO, release data. they ranged from 0.7-0.8. Purr tylosin An initial inhibition in 0, uptake and COz release occurred at all concentrations of added tylosin (Fig. 4). In terms of CO2 release this inhibition was maintained for 7 weeks when an increase in the CO2 released from the treatments, compared to the control, occurred. Inhibition of O2 uptake appeared to be in two stages, an initial inhibition which lasted for 2 weeks followed by a second inhibitory period between week 4 and half way through week 5. At the highest concentration of tylosin although a decrease in inhibition occurred after 2 weeks, there was still some degree of inhibition until 5$ weeks. In terms 80
300 r
0
(0)
2
4
Time,
6
weeks
weeks
Fig. I. Cumulative 0, uptake and CO2 release resulting from the breakdown of tylosin fermentation waste in soil. _ Oz uptake, ----- CO, release 04.4, A-l.2 and m-2 tonnes ha-‘.
(b)
60
8
IO
0
I
I
I
I
1
2
4
6
8
10
Time,
weeks
Fig. 2. Cumulative N uptake or release from soil amended with tylosin fermentation waste and pure tylosin. (a) fermentation waste in amounts equivalent to +---0, +--0.4, A-l.2 and D-2 tonnes ha-’ (b) tylosin in amounts equivalent to those contained in fermentation waste added at. +-O, O-3. a-20 and 040 tonnes ha.-‘.
405
Tylosin and microbiai activity (a)
(b)
‘^P----60
t
Time,
weeks
Time,
weeks
Fig. 3. Cumulative release of tylosin from soil amended with tylosin fermentation tylosin. (a) fermentation waste in amounts equivalent to, W.4, A-l.2 and m-2 pure tylosin in amounts equivalent to those contained in fermentation waste added and O-40 tonnes ha-‘.
of both O2 uptake and CO* release the high concentration of tylosin, equivalent to tbat present in 40 tonnes fermentation waste ha-‘, produced the maximum inhibition of microbial activity. In all treatments the amount of NH,+, NO; and NH, leached from the soil was lower than in the control (Fig. 2b). This apparent inhibition of N mineralization was maintained throughout the experiment. As in the first experiment NH, was only detected in the first 2-3 weeks of incubation and the highest concentrations of 5 pg g-’ soil were detected in the control. As would be expected the concentrations of tylosin found in the leachates were much greater than those found when fermentation waste was added to the soil (Fig. 3b). Maximum tylosin release occurred in the first ieaching of the soil and represented 13, 18 and 19% of the tylosin added in amounts equivalent to those contained in additions of fermentation waste at 4, 20 and 40 tonnes ha-‘. Subsequent concentrations detected in the leachates were much lower. At the end of the experiment the total amount of tylosin which had been detected in the Ieachates represented 16, 24 and 21% of the tylosin added at the three concentrations. Tylosin contains 59X0/;, C (Hamil et al.. 1960) and determinations of the C released from tylosin as CO, showed that after 10 weeks 29, 14 and 4% of the C present in tylosin added to the soil in amounts equivalent to 4, 20 and 40 tonnes fermentation waste ha- ’ respectively was
waste and pure tonnes ha-’ (b) at. 04, n-20
released as COZ. Tylosin contains 1.6% N (Hamil a/., 1960) but the proportion of N released from tylosin could not be determined as the amount N detected in the leachates was always less than the control.
et the of in
DISCUSSION I found that the addition of fermentation waste to soil produced an increase in microbial activity as measured by O2 uptake, CO2 release and N mineralization. Microbial activity was increased for 10 weeks after the addition of the waste when half of the C in the waste had been released as CO,, and about 35% of the N had been released as NH: and NO;. The total release of tylosin represented between 20 and 32% of the tylosin present in the waste, with a maximum cumulative release of 4 pg g- ’ soil occurring with the addition of 2 tonnes fermentation waste ha-‘. The higher amounts of tylosin added to the soil were equivalent to those present in applications of 4, 20 and 40 tonnes fermentation waste ha-’ in the field, and with these amounts microbial activity in terms of O2 uptake, CO2 release and N mineralization was inhibited. The degree of inhibition was relatively similar, in terms of microbial respiration and N mineralization (Figs. 2b and 4). and did not appear to be proportiona to the amount of tylosin added.
Table 1. NH, volatilization from soil amended with tylosin fermentation waste and pure tylosin NH, Fermentation
Time (weeks) 1 2 3
Total release
Control
0.4
4.5 0.5 0
0 0.4 0
5.0
0.4
volatilization
waste treatments
(tonnes ha- ‘) 1.2
(fig N g-’ soil week-‘) Addition of pure tylosin equivalent to that present in fermentation waste
2.0
4
(tonnes ha- ‘) 20
40
0 0.3 0
1.3 0. t 0
0 0.9 0
0 0.6 0.1
1.4 0.3 0
0.3
I .4
0.9
0.7
1.7
406
M. W. M.
BEW~CK
Fig. 4. Cumulative OZ uptake and CO2 release resulting from the breakdown of pure tylosin in soil. ___ O2 uptake. ----- CO, release tylosin added in amounts equivalent to those contained in fermentation waste added at. ct--4, n-20 and c)-_jO tonnes ha-*. (The figures presented here show the effect on microbial respiration due to the tvlosin alone. with allowance made for the basal resuiration of the soil.) When increasing amounts of fermentation waste were added to the soil (Figs 1 and 2a) changes in microbial respiration and N mineralization were directly proportional to the amount of waste added. Tylosin is a basic antibiotic and Nissen (1954) found that basic antibiotics. at rates 16 times greater than those I used, caused an inhibition in CO2 evofution for at least 14 days after addition to the soil. Extending the experiments to 70 days I found that although the amounts of antibiotic were lower than those used by Nissen. inhibition of microbial respiration occurred for up to 7 weeks after addition of the antibiotic. The influence of tylosin in the soil will be especially important in terms of the release of NH; and NO; as plant nutrients. A reduction in available N was seen when amounts of antibiotic. equivalent to those present in applications of 4. 20 and 40 tonnes fermentation waste ha -I in a field situation, were added to the soil. and this would naturally have an effect on crop growth and yield. My results suggest that in the experiments of Pfizer Ltd. in the U.S.A. (Brayne. 1973: Warner, 1973). when mycelium was added to soil in amounts up to 104 tonnes ha-‘. equivalent to 31.2 dry tonnes ha-‘. the concentrations of residual antibiotic in these wastes could have reduced the potential microbial activity in the soil. and therefore N mineralization. Recent investigations in this laboratory have shown that mycelium from an oxytetracycline fermentation, an antibiotic produced by Pfizer, contains initially 4O.OCKl pg oxytetracyc~in~ g- ‘. approximately 20 times
the level of antibiotic in tylosin fermentation waste. Although this concentration decreases with time due to the instability of oxytetracycline it is possible that the fertilizer potential of the waste used in the Pfizer experiments was counteracted to some extent by the presence of high levels of antibiotic. With the present knowledge concerning the action on the soil microRora of the addition of organic matter in combination with antibiotics this reduction in fertilizer potential is speculative. But the deleterious effect of antibiotics on the soil microflora shown here should not be ignored. especially in the case of tylosin, an antibiotic more resistant to breakdown that oxytetracycline. AcknoM,/edyu,,lenrs-This research was carried out with the aid of the Leaf Fellowship from Trinity College, Cambridge. My thanks also go to Dr H. T. Tribe for his comments and suggestions during the course of this investigation. REFEREbCES BKAYNE M.
F. (1973) Pfizer feeds my~iium-grown baked potatoes to employees. Norwicli Bull. (Oct. i 2). BEWICK M. W. M. (1977) Considerations of the use of antibiotic fermentation wastes as fertilizers. A review of their past use and potential effects on the soil ecosystem. Com~}?~}?~~u~f~ Bureau of Soits. special publication No. 4. Commonwealth Agricultural Bureaux. Slough. England. HAMIL R. L., HARVEY M. E.. STAMPER M. and WILEY P. F. (1960) Tylosin. a new antibiotic: II Isolation, properties and preparation of desmycosin. a microbiologically active degradation product. A~1tihiotics ~~~~~~~tb~r. It. 328.-334.
Tylosin
and microbial
NISSEN T. V. (1954) Effects of antibiotics on carbon dioxide production in soil. Nature. Lond. 174, 226227. SHAW K. (1959) Determination of organic carbon in soil and plant material. J. Soil Sci. 10. 316326. TRIRF. H. T. (I 961) Microbiology of cellulose decomposition in soil. Soil Sci. 92, 61-77. TRIBE H. T. and BEWICI( M. W. M. (1978) A simple electro-
lytic
407
activity respirometer-leaching
tube
combination
metabolism studies. J. Soil. Sci. 29. In press. UHLIAR J. and BUCKO M. (1974) Moznost vyuzitia lovych odpadov z vyroby St&l. Akad. rrm?d. VZd. WARNER N. J. (1973) Once product fattens potatoes.
for
sotI
pricms-
antibiotik v vast1inne.j vyrob 20. 9233930. dumped at sea. antibiotic byPror.idmw d. (Oct. 22).
of Applied
Biology, University of Cambridge. Cambridge. CB2 3DX. England
(Arcepred 7 Fthm-y
Pembroke
Street.
1978)
Summary-The
effect of tylosin fermentation waste and pure tylosin on microbial activity in soil was investigated. The addition of tylosin fermentation waste at concentrations equivalent to 0.4, I.2 and 2 tonnes ha-’ in the field stimulated microbial activity as measured by Oz uptake, CO* release and N mineralization. After 10 weeks incubation half of the C in the waste had been released as CO2 while mineralization of N ranged between 31 and 38%. Tyiosin was detected in the leachates, and at IO weeks between 20 and 32% of the tylosin present in the waste had been leached from the soil. Addition of high concentrations of pure tylosin. equivalent to those which would be found in 4. 20 and 40 tonnes fermentation waste ha -’ in the held. resulted in a decrease in microbial respiration for S--7 weeks after the addition of the antibiotic. N mineralization was reduced throughout the experiment, compared with the control. A higher con~ntration of tylosin was detected in the leachates in this experiment and after 10 weeks the quantity of tylosin detected represented between 16 and 2.4:); of the tylosin added. It is suggested that the full fertthzer potential of large additions of antibiotic waste to soil could be reduced to some extent due to the inhibitory activity of the residual antibiotic.
INTRODUCTION
The subject of antibiotics in soils has been extensively investigated in relation to their production in soil, disappearance of antibiotics added to soil, and the effects of antibiotics on microbial processes in soil (Bewick, 1977). There have been few reports on the use of antibiotic fermentation wastes as fertilizers. An investigation by Uhhar and Bucko (1974) suggested that penicillin and streptomycin fermentation wastes could increase the growth and yield of lucerne, while newspaper reports indicate that Pfizer Ltd of Groton. CT, U.S.A. had increased yields of potatoes using antibiotic fermentation waste as a fertilizer (Brayne. 1973; Warner, 1973). None of these reports mentioned the possible effects of the residual antibiotic, or the effect of the waste on microbial activity in soil. 1 have investigated the effect of different additions of pure tylosin, and residue from a tylosin fermentation. on microbial activity of soil. Aspects of microbial activity examined were 0, uptake, CO, release and N mineraIization. together with a determination of the amounts of extractable tylosin in the soil.
MATERIALS AND METHODS
Microbial activity was studied in a modified John Innes potting compost consisting of 3 parts peat, 7 parts loam and 8 parts sand. pH 6.1, using the apparatus described by Tribe and Bewick (1978). This artificial soil mixture produced a light, free-draining soil. enabling periodic leachings to be made over 10 weeks. and permitted reproducibility of results in different runs of the same experiment. O2 uptake, COz, NO,. NH:, NW3 and tylosin release were measured. The tylosin fermentation waste used (kindly supplied by Dista Products Ltd. Speke, Liverpool) was the material remaining after the broth containing the 403
antibiotic had been removed. The antibiotic itself is a broad-spectrum macrohde widely used in veterinary medicine. To ease handling the material was dried at 105’C for 24 h and then ground to a powder. This produced a material of known, constant, nutrient composition. the tylosin present being unaffected by the treatment. Pure tylosin was suppiied by Dista Products. Dried tylosin waste was added to the soil in amounts equivalent to 0.4, 1.2 and 2 tonnes ha-‘; the quantities added to each tube (2Scm dia) were 125. 374 and 624mg respectively. The amounts of pure tyiosin added to each tube were 2.6, 12.9 and 25.8 mg. As the concentration of tylosin in the waste was 2.1 mg g-t these amounts of pure tylosin represented additions to the leaching tubes equivalent to 4, 20 and 40 tonnes fermentation waste ha-’ respectively. The tylosin and tylosin fermentation waste were added to 70g samples of air-dried soil in leaching tubes, with 70g samples of unamended soil acting as controls. Four replicates were made of each treatment and the control. To measure the initial amounts of NO;, NH; and tylosin the soil was brought to saturation and leached (Tribe and Bewick, 1978). The moisture content of the soil was then adjusted by suction to 13% of the wet weight. Incubation was carried out at 23% Estimations of O2 uptake were made daily until uptake had fallen to 2-3 ml day-‘, when readings were taken every 34 days. CO1 and gaseous NH, release were estimated weekly, while the concentrations of NO;, NH: and the tylosin were measured every fortnight. COz release was measured using Shaw’s (1959) apparatus and the weight of CO2 obtained was converted to voiume for comparison with 0, uptake. WI;, NO; and NH, release were determined using a modified Markham Stitl method (Tribe, 1961). Tylosin determinations involved the extraction of a portion of the leachate with CHCI,
M. W. M. BEWICK
404
and ether into 0.1 N HCI. Absorption of the tylosin in HCI was determined at 290nm using a Hi&r Watts Uvispec spectrophotometer. Interference from the soil was estimated by extracting a portion of the leachatc from the control. RESllLTS E‘cwlloltatiorl
wustr
The results of the effect of different rates of fermentation waste on respiration (Fig. 1) show O2 uptake and COz release produced from the breakdown of the waste alone. with allowance made for control respiration. Increases in respiration due to the addition of the fermentation waste occurred for approximately 8 10 weeks after the beginning of the experiment. Release of N from the waste. measured as NH:. NO; and NH,. followed a similar pattern (Fig. Za), with maximum release of N recorded where the greatest amount of waste was added (2 tonnes ha-‘). After 6 weeks there was little increase in the N mineralized either from the waste itself. or from the control (Fig. la). Release of volatile NH, was only detected in the first fortnight. Maximum NH, volatilization appeared in the control with 5 pg g- ’ being released after 2 weeks. while the addition of fermentation waste appeared to inhibit NH3 release (Table 1). With all additions of fermentation waste low con~ntratjons of tylosin were released from the soil throughout the experiment (Fig. 3a). At 0.4 tonnes ha-’ the lowest amounts of tylosin release were recorded. At the end of the experiment. in the treatments of 0.4, 1.2 and 2 tonnes ha- ‘. of the initial tylosin concentrations of 0.26. 0.77 and 1.29 mg respectively. 22, 32 and 20”; of the tylosin present had been recovered. Analysis of the waste showed it to be 43.7:; C and 6.34”, N: from this the proportions of C and N released from the waste during the experiment were determined. After 10 weeks half of the C had been released as CO2 in all treatments, while the mineralization of N ranged from 31-38”V After 1 week the
Time,
ratios of CO, release: O2 uptake (RQ) in soil amended with 0.4, 1.2 and 2 tonnes ha-’ were 0.78, 0.94 and 0.96 respectively. After 10 weeks, using cumulative 0, uptake and CO, release data. they ranged from 0.7-0.8. Purr tylosin An initial inhibition in 0, uptake and COz release occurred at all concentrations of added tylosin (Fig. 4). In terms of CO2 release this inhibition was maintained for 7 weeks when an increase in the CO2 released from the treatments, compared to the control, occurred. Inhibition of O2 uptake appeared to be in two stages, an initial inhibition which lasted for 2 weeks followed by a second inhibitory period between week 4 and half way through week 5. At the highest concentration of tylosin although a decrease in inhibition occurred after 2 weeks, there was still some degree of inhibition until 5$ weeks. In terms 80
300 r
0
(0)
2
4
Time,
6
weeks
weeks
Fig. I. Cumulative 0, uptake and CO2 release resulting from the breakdown of tylosin fermentation waste in soil. _ Oz uptake, ----- CO, release 04.4, A-l.2 and m-2 tonnes ha-‘.
(b)
60
8
IO
0
I
I
I
I
1
2
4
6
8
10
Time,
weeks
Fig. 2. Cumulative N uptake or release from soil amended with tylosin fermentation waste and pure tylosin. (a) fermentation waste in amounts equivalent to +---0, +--0.4, A-l.2 and D-2 tonnes ha-’ (b) tylosin in amounts equivalent to those contained in fermentation waste added at. +-O, O-3. a-20 and 040 tonnes ha.-‘.
405
Tylosin and microbiai activity (a)
(b)
‘^P----60
t
Time,
weeks
Time,
weeks
Fig. 3. Cumulative release of tylosin from soil amended with tylosin fermentation tylosin. (a) fermentation waste in amounts equivalent to, W.4, A-l.2 and m-2 pure tylosin in amounts equivalent to those contained in fermentation waste added and O-40 tonnes ha-‘.
of both O2 uptake and CO* release the high concentration of tylosin, equivalent to tbat present in 40 tonnes fermentation waste ha-‘, produced the maximum inhibition of microbial activity. In all treatments the amount of NH,+, NO; and NH, leached from the soil was lower than in the control (Fig. 2b). This apparent inhibition of N mineralization was maintained throughout the experiment. As in the first experiment NH, was only detected in the first 2-3 weeks of incubation and the highest concentrations of 5 pg g-’ soil were detected in the control. As would be expected the concentrations of tylosin found in the leachates were much greater than those found when fermentation waste was added to the soil (Fig. 3b). Maximum tylosin release occurred in the first ieaching of the soil and represented 13, 18 and 19% of the tylosin added in amounts equivalent to those contained in additions of fermentation waste at 4, 20 and 40 tonnes ha-‘. Subsequent concentrations detected in the leachates were much lower. At the end of the experiment the total amount of tylosin which had been detected in the Ieachates represented 16, 24 and 21% of the tylosin added at the three concentrations. Tylosin contains 59X0/;, C (Hamil et al.. 1960) and determinations of the C released from tylosin as CO, showed that after 10 weeks 29, 14 and 4% of the C present in tylosin added to the soil in amounts equivalent to 4, 20 and 40 tonnes fermentation waste ha- ’ respectively was
waste and pure tonnes ha-’ (b) at. 04, n-20
released as COZ. Tylosin contains 1.6% N (Hamil a/., 1960) but the proportion of N released from tylosin could not be determined as the amount N detected in the leachates was always less than the control.
et the of in
DISCUSSION I found that the addition of fermentation waste to soil produced an increase in microbial activity as measured by O2 uptake, CO2 release and N mineralization. Microbial activity was increased for 10 weeks after the addition of the waste when half of the C in the waste had been released as CO,, and about 35% of the N had been released as NH: and NO;. The total release of tylosin represented between 20 and 32% of the tylosin present in the waste, with a maximum cumulative release of 4 pg g- ’ soil occurring with the addition of 2 tonnes fermentation waste ha-‘. The higher amounts of tylosin added to the soil were equivalent to those present in applications of 4, 20 and 40 tonnes fermentation waste ha-’ in the field, and with these amounts microbial activity in terms of O2 uptake, CO2 release and N mineralization was inhibited. The degree of inhibition was relatively similar, in terms of microbial respiration and N mineralization (Figs. 2b and 4). and did not appear to be proportiona to the amount of tylosin added.
Table 1. NH, volatilization from soil amended with tylosin fermentation waste and pure tylosin NH, Fermentation
Time (weeks) 1 2 3
Total release
Control
0.4
4.5 0.5 0
0 0.4 0
5.0
0.4
volatilization
waste treatments
(tonnes ha- ‘) 1.2
(fig N g-’ soil week-‘) Addition of pure tylosin equivalent to that present in fermentation waste
2.0
4
(tonnes ha- ‘) 20
40
0 0.3 0
1.3 0. t 0
0 0.9 0
0 0.6 0.1
1.4 0.3 0
0.3
I .4
0.9
0.7
1.7
406
M. W. M.
BEW~CK
Fig. 4. Cumulative OZ uptake and CO2 release resulting from the breakdown of pure tylosin in soil. ___ O2 uptake. ----- CO, release tylosin added in amounts equivalent to those contained in fermentation waste added at. ct--4, n-20 and c)-_jO tonnes ha-*. (The figures presented here show the effect on microbial respiration due to the tvlosin alone. with allowance made for the basal resuiration of the soil.) When increasing amounts of fermentation waste were added to the soil (Figs 1 and 2a) changes in microbial respiration and N mineralization were directly proportional to the amount of waste added. Tylosin is a basic antibiotic and Nissen (1954) found that basic antibiotics. at rates 16 times greater than those I used, caused an inhibition in CO2 evofution for at least 14 days after addition to the soil. Extending the experiments to 70 days I found that although the amounts of antibiotic were lower than those used by Nissen. inhibition of microbial respiration occurred for up to 7 weeks after addition of the antibiotic. The influence of tylosin in the soil will be especially important in terms of the release of NH; and NO; as plant nutrients. A reduction in available N was seen when amounts of antibiotic. equivalent to those present in applications of 4. 20 and 40 tonnes fermentation waste ha -I in a field situation, were added to the soil. and this would naturally have an effect on crop growth and yield. My results suggest that in the experiments of Pfizer Ltd. in the U.S.A. (Brayne. 1973: Warner, 1973). when mycelium was added to soil in amounts up to 104 tonnes ha-‘. equivalent to 31.2 dry tonnes ha-‘. the concentrations of residual antibiotic in these wastes could have reduced the potential microbial activity in the soil. and therefore N mineralization. Recent investigations in this laboratory have shown that mycelium from an oxytetracycline fermentation, an antibiotic produced by Pfizer, contains initially 4O.OCKl pg oxytetracyc~in~ g- ‘. approximately 20 times
the level of antibiotic in tylosin fermentation waste. Although this concentration decreases with time due to the instability of oxytetracycline it is possible that the fertilizer potential of the waste used in the Pfizer experiments was counteracted to some extent by the presence of high levels of antibiotic. With the present knowledge concerning the action on the soil microRora of the addition of organic matter in combination with antibiotics this reduction in fertilizer potential is speculative. But the deleterious effect of antibiotics on the soil microflora shown here should not be ignored. especially in the case of tylosin, an antibiotic more resistant to breakdown that oxytetracycline. AcknoM,/edyu,,lenrs-This research was carried out with the aid of the Leaf Fellowship from Trinity College, Cambridge. My thanks also go to Dr H. T. Tribe for his comments and suggestions during the course of this investigation. REFEREbCES BKAYNE M.
F. (1973) Pfizer feeds my~iium-grown baked potatoes to employees. Norwicli Bull. (Oct. i 2). BEWICK M. W. M. (1977) Considerations of the use of antibiotic fermentation wastes as fertilizers. A review of their past use and potential effects on the soil ecosystem. Com~}?~}?~~u~f~ Bureau of Soits. special publication No. 4. Commonwealth Agricultural Bureaux. Slough. England. HAMIL R. L., HARVEY M. E.. STAMPER M. and WILEY P. F. (1960) Tylosin. a new antibiotic: II Isolation, properties and preparation of desmycosin. a microbiologically active degradation product. A~1tihiotics ~~~~~~~tb~r. It. 328.-334.
Tylosin
and microbial
NISSEN T. V. (1954) Effects of antibiotics on carbon dioxide production in soil. Nature. Lond. 174, 226227. SHAW K. (1959) Determination of organic carbon in soil and plant material. J. Soil Sci. 10. 316326. TRIRF. H. T. (I 961) Microbiology of cellulose decomposition in soil. Soil Sci. 92, 61-77. TRIBE H. T. and BEWICI( M. W. M. (1978) A simple electro-
lytic
407
activity respirometer-leaching
tube
combination
metabolism studies. J. Soil. Sci. 29. In press. UHLIAR J. and BUCKO M. (1974) Moznost vyuzitia lovych odpadov z vyroby St&l. Akad. rrm?d. VZd. WARNER N. J. (1973) Once product fattens potatoes.
for
sotI
pricms-
antibiotik v vast1inne.j vyrob 20. 9233930. dumped at sea. antibiotic byPror.idmw d. (Oct. 22).