- Email: [email protected]
Effect of halothane on the growth of microbial species isolated from a peat biofilter
• 7•
,~
,~5~
ECOLOGICAL ENGINEERING ELSEVIER
Ecological Engineering 7 (1996) 151- 155
Short Communication
Effect of halothane on the growth of microbial species isolated from a peat biofilter Antonio M. Martin *, Kantha D. Arunachalam, Edward Whelan Department of Biochemistr3', Memorial University of Ne~Jbundland, St. John's. New?[bundland. Canada A IB 3X9
Received 11 October 1995; accepted 22 March 1996
Abstract Growth studies were conducted on microbial cultures isolated from a peat biofilter through which halothane gas, an anaesthetic used for animals, had been circulated. The microorganisms were further exposed to various concentrations of halothane in the growth medium. At concentrations of halothane up to 2.7% (v/v), the microbial populations, although initially affected, recovered to levels similar to those not exposed to halothane. However, a higher concentration of halothane (5.3%, v / v ) was lethal to the microorganisms. Kevwords: Anaesthetic gas; Halothane; Microorganisms; Mixed culture; Microbial adaptation
1. Introduction Halothane (1,1,1-trifluoro-2,2-chlorobromoethane) is an inhalation anaesthetic used by veterinarians. Leakage o f anaesthetic gases into the air of surgical operating rooms has been studied by Piziali et al. (1976), who reported their deleterious effects on human health. The installation o f scavenger systems in operating theaters to reduce the level of anaesthetic gas pollution was recommended by the Association of Anaesthetists of Great Britain and Ireland (Vickers, 1975), and later endorsed by the British Department of Health and Social Security (1976). Studies on anaesthetic waste scavenging systems for the reduction of halothane concentrations in a chemical warfare-proof operating theater have been reported (Yoganathan e t al., 1991). Halothane is a chlorofluorocarbon (CFC). It has been acknowledged that volatile CFCs have a destructive effect upon the ozone layer, and laws and guidelines now exist
* Corresponding author. Tel.: (1-709) 737-4324; Fax: (I-709) 737-4000; e-mail: [email protected] 0925-8574/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0925- 8574(96)00006-7
152
A.M. Martinet al./ EcologicalEngineering 7 (1996) 151-155
for the use of CFC gases. Halothane has been included in the Montreal protocol as one of the agents breaking down the ozone layer, although the amount of halothane used is small in comparison with other CFCs employed in industrial activities, such as those of the 'freon' type (N0rreslet et al., 1989). Therefore, the reduction of its emissions is an objective that should be pursued. Methods for the treatment and purification of waste gases recently have gained importance, including the use of biological methods for exhaust gas purification (Ottengraf, 1986; Ottengraf et al., 1986). Biofiltration is a technique having potential for the treatment of waste gases (Bohn, 1992). It is based on the use of microorganisms attached to appropriate support materials to degrade pollutants. To make biofiltration applicable on a larger scale, it is necessary to study the effects of xenobiotic compounds on microbial populations. Martin (1991) presented a comprehensive study on the mechanisms of removal of waste gases using peat biofilters. The present work reports the effects of halothane on microbial populations isolated from a peat biofilter through which this gas had been circulated.
2. Materials and methods
2.1. Isolation of microorganisms from the biofilter Sphagnum peat of a low degree of decomposition from an area close to St. John's, Newfoundland, was used as the microbial support material in a biofilter. Peat samples (10 g) were taken from the biofilter at five-day intervals over 20 days. H 2 0 (90 ml) was added to each sample, and each was homogenized (Janke and Kunkel Ultra-Turrax T25, IKA-Works, Cincinnati, OH, USA) for 5 min at 8000 rpm, then allowed to settle for 30 rain and decanted. Afterwards, serial dilutions were prepared in duplicate by adding 1 ml of supernatant to 9 ml H20 and vortex-mixed for 30 s. Each dilution was plated by pipetting 0.1 ml of the suspension onto Bacto nutrient agar (Difco Laboratories, Detroit, MI, USA) in Petri dishes, spreading it evenly with a glass L-rod, and incubating at 30°C for 48 h. Individual colonies on the plates could be differentiated at a dilution of 10 5. Each colony type was cultured by successively streak-plating until colonies of similar appearance were isolated on each plate. Each culture was maintained by inoculating onto a nutrient agar slant, followed by incubation at 30°C for 24 h and then storage at I°C. Each was also tested for the ability to grow anaerobically by the agar stab method. 2.2. Study of the growth patterns of the microorganisms The microorganisms from the colonies that grew most abundantly in the peat biofilter, which were found to be anaerobic, were selected for the studies of the effects on them of halothane at different concentrations. These studies were conducted using Gyrotory shaker baths (Models G76 and G76D, New Brunswick Scientific, Edison, NJ, USA) at 120 rpm and 30°C. In separate experiments in covered 125 ml erlenmeyer flasks, each culture was inoculated into 75 ml of Bacto nutrient broth to which halothane
A.M. Martin et al. / Ecological Engineering 7 (1996) 151-155
153
had been added to give concentrations of 0.7, 1.3, 2.7 and 5.3% (all v / v ) . Control experiments were conducted with no halothane added.
2.2.1, Counting of colony-forming units The microorganisms in the broth cultures were subjected to serial dilution and spread plating to find the viable cell count for each sample. At seven intervals of 12 h, 1 ml of the broth culture from each flask was pipetted into sterile H 2 0 in a test tube and vortex-mixed for 30 s. Successive dilutions were conducted as previously indicated. Then, 0.2 ml of each dilution was pipetted onto nutrient agar in a petri dish, and incubated for 36 h at 30°C.
2.2.2. Determination of total dry biomass Decanted culture broth was centrifuged at ~ 16000 × g for 10 min. The supernatant was discarded and the cell pellet was removed and vortex-mixed with distilled H~O for 10 s. The cell solution was dried overnight at 110°C and weighed. All the experiments were conducted in duplicate, and aseptic techniques were observed throughout for all microbiological procedures. Reported values represent means of the two microbial populations found to abundantly grow in the peat biofilter. The graphical representations and the calculation of means and standard deviations were conducted using Microsoft Excel 5.0c, Microsoft (USA).
3. Results and discussion The effects on the microorganisms of adding halothane to the culture media are presented in Fig. 1. For halothane concentrations of 0.7 to 2.7% ( v / v ) , the number of colony-forming units was lower than for the control (with no added halothane) in the
1 X 1010
:~ l x 1 0 6
.~_
:K .£o
lx104
o (..) 1 x 1 0 2
1 0
12
24
36
48
60
72
Time (h)
Fig. 1. Colony-formingunits at various halothane concentrations, in % (0, 0; 0.7, It; 1.3, ©; 2.7, [~; 5.3, zx). Means and standard deviations. Only the upper limit of each standard deviation is indicated. For clusters of experimental points, only the largest standard deviation is shown.
A.M. Martin et al. / Ecological Engineering 7 (1996) 151-155
154
1.4 1.2
1 1
i
~" 1.0 ~/
/o//
0.8 E .o 0.6 .0 n
0.4
0.2 0.0
I
I
I
I
I
I
I
0
12
24
36
48
60
72
Time (h) Fig. 2. Microbial dry biomass at various halothane concentrations, in % (0, 0 ; 0.7, I ; 1.3, C)" 2.7, E]; 5.3, A ). Means and standard deviations. Only the upper limit of each standard deviation is indicated. For clusters of experimental points, only the largest standard deviation is shown.
initial stages of the experiments. However, over the remaining time, the populations showed a recovery, and at 72 h the counts were similar to that of the control. The exception was observed in the culture to which 5.3% ( v / v ) halothane was added. In this case the microbial concentration decreased practically to extinction in 12 h, and did not recover. A similar pattern was found in the total biomass concentrations of the cultures (Fig. 2). The biomass increased in the control, but it did not increase when 5.3% ( v / v ) of halothane was added. With intermediate amounts of halothane (0.7, 1.3 and 2.7%, v / v ) , the increase in the total biomass was less than the control in the initial stages of the experiments. However, there was eventual recovery in these cases over the time of the experiments. The initial inhibition of the microorganisms, evidenced by a delay in the increase of the number of colony-forming units and the total biomass, indicates that the halothane was harmful to the microorganisms. At the lower concentrations of halothane, the microorganisms apparently adapted to it. When the concentration of halothane in the medium was 5.3% ( v / v ) , no adaptation was observed.
Acknowledgements This work was partially funded by a grant from the Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Mr. Paul Bemister, Department of Biochemistry, and Dr. Lenka Husa, Director of Animal Care Services, for their assistance.
A.M. Martin et al. / Ecological Engineering 7 (1996) 151 -155
155
References Bohn, H., 1992. Consider biofiltration for decontaminating gases. Chem. Eng. Prog., 88: 34-40. Department of Health and Social Security, 1976. Health Services Development. Pollution of Operating Departments etc. by Anaesthetic Gases. Health Circular HC(76)38. HMSO, London. Martin, A.M., 1991. Peat as an agent in biological degradation: Peat biofilters. In: A.M. Martin (Ed.), Biological Degradation of Wastes. Elsevier Applied Science, London, pp. 341-362. Norreslet, J., Friberg, S., Nielson, T.M. and R#mer, U., 1989. Halothane anaesthetic and the ozone layer. Lancet, i: 719. Ottengraf, S.P.P., 1986. Exhaust gas purification. In: H.-J. Rehm and G. Reed (Eds.), Biotechnology, Vol. 8. VCH, Weinheim, pp. 425-452. Ottengraf, S.P.P., Meesters, J.J.P., van den Oever, A.H.C. and Rozema, H.R., 1986. Biological elimination of volatile xenobiotic compounds in biofilters. Bioprocess Eng., 1: 61-69. Piziali, R.L.. Whitcher, C., Sher, R. and Moffat, R.J., 1976. Distribution of waste anesthetic gases in the operating room air. Anesthesiology, 45: 487-494. Vickers, M.D., 1975. Pollution of the atmosphere of operating theatres. Advice to members from the Council of the Association of Anaesthetists of Great Britain and Ireland, Anaesthesia, 30: 697-699. Yoganathan, S., Johnston, I.G., Parnell, C.J., Houghton, I.T. and Restall, J., 1991. Determination of contamination of a chemical warfare-proof operating theatre with volatile anaesthetic agents and assessment of anaesthetic gas scavenging systems. Br. J. Anaesth., 67: 614-617.
,~
,~5~
ECOLOGICAL ENGINEERING ELSEVIER
Ecological Engineering 7 (1996) 151- 155
Short Communication
Effect of halothane on the growth of microbial species isolated from a peat biofilter Antonio M. Martin *, Kantha D. Arunachalam, Edward Whelan Department of Biochemistr3', Memorial University of Ne~Jbundland, St. John's. New?[bundland. Canada A IB 3X9
Received 11 October 1995; accepted 22 March 1996
Abstract Growth studies were conducted on microbial cultures isolated from a peat biofilter through which halothane gas, an anaesthetic used for animals, had been circulated. The microorganisms were further exposed to various concentrations of halothane in the growth medium. At concentrations of halothane up to 2.7% (v/v), the microbial populations, although initially affected, recovered to levels similar to those not exposed to halothane. However, a higher concentration of halothane (5.3%, v / v ) was lethal to the microorganisms. Kevwords: Anaesthetic gas; Halothane; Microorganisms; Mixed culture; Microbial adaptation
1. Introduction Halothane (1,1,1-trifluoro-2,2-chlorobromoethane) is an inhalation anaesthetic used by veterinarians. Leakage o f anaesthetic gases into the air of surgical operating rooms has been studied by Piziali et al. (1976), who reported their deleterious effects on human health. The installation o f scavenger systems in operating theaters to reduce the level of anaesthetic gas pollution was recommended by the Association of Anaesthetists of Great Britain and Ireland (Vickers, 1975), and later endorsed by the British Department of Health and Social Security (1976). Studies on anaesthetic waste scavenging systems for the reduction of halothane concentrations in a chemical warfare-proof operating theater have been reported (Yoganathan e t al., 1991). Halothane is a chlorofluorocarbon (CFC). It has been acknowledged that volatile CFCs have a destructive effect upon the ozone layer, and laws and guidelines now exist
* Corresponding author. Tel.: (1-709) 737-4324; Fax: (I-709) 737-4000; e-mail: [email protected] 0925-8574/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0925- 8574(96)00006-7
152
A.M. Martinet al./ EcologicalEngineering 7 (1996) 151-155
for the use of CFC gases. Halothane has been included in the Montreal protocol as one of the agents breaking down the ozone layer, although the amount of halothane used is small in comparison with other CFCs employed in industrial activities, such as those of the 'freon' type (N0rreslet et al., 1989). Therefore, the reduction of its emissions is an objective that should be pursued. Methods for the treatment and purification of waste gases recently have gained importance, including the use of biological methods for exhaust gas purification (Ottengraf, 1986; Ottengraf et al., 1986). Biofiltration is a technique having potential for the treatment of waste gases (Bohn, 1992). It is based on the use of microorganisms attached to appropriate support materials to degrade pollutants. To make biofiltration applicable on a larger scale, it is necessary to study the effects of xenobiotic compounds on microbial populations. Martin (1991) presented a comprehensive study on the mechanisms of removal of waste gases using peat biofilters. The present work reports the effects of halothane on microbial populations isolated from a peat biofilter through which this gas had been circulated.
2. Materials and methods
2.1. Isolation of microorganisms from the biofilter Sphagnum peat of a low degree of decomposition from an area close to St. John's, Newfoundland, was used as the microbial support material in a biofilter. Peat samples (10 g) were taken from the biofilter at five-day intervals over 20 days. H 2 0 (90 ml) was added to each sample, and each was homogenized (Janke and Kunkel Ultra-Turrax T25, IKA-Works, Cincinnati, OH, USA) for 5 min at 8000 rpm, then allowed to settle for 30 rain and decanted. Afterwards, serial dilutions were prepared in duplicate by adding 1 ml of supernatant to 9 ml H20 and vortex-mixed for 30 s. Each dilution was plated by pipetting 0.1 ml of the suspension onto Bacto nutrient agar (Difco Laboratories, Detroit, MI, USA) in Petri dishes, spreading it evenly with a glass L-rod, and incubating at 30°C for 48 h. Individual colonies on the plates could be differentiated at a dilution of 10 5. Each colony type was cultured by successively streak-plating until colonies of similar appearance were isolated on each plate. Each culture was maintained by inoculating onto a nutrient agar slant, followed by incubation at 30°C for 24 h and then storage at I°C. Each was also tested for the ability to grow anaerobically by the agar stab method. 2.2. Study of the growth patterns of the microorganisms The microorganisms from the colonies that grew most abundantly in the peat biofilter, which were found to be anaerobic, were selected for the studies of the effects on them of halothane at different concentrations. These studies were conducted using Gyrotory shaker baths (Models G76 and G76D, New Brunswick Scientific, Edison, NJ, USA) at 120 rpm and 30°C. In separate experiments in covered 125 ml erlenmeyer flasks, each culture was inoculated into 75 ml of Bacto nutrient broth to which halothane
A.M. Martin et al. / Ecological Engineering 7 (1996) 151-155
153
had been added to give concentrations of 0.7, 1.3, 2.7 and 5.3% (all v / v ) . Control experiments were conducted with no halothane added.
2.2.1, Counting of colony-forming units The microorganisms in the broth cultures were subjected to serial dilution and spread plating to find the viable cell count for each sample. At seven intervals of 12 h, 1 ml of the broth culture from each flask was pipetted into sterile H 2 0 in a test tube and vortex-mixed for 30 s. Successive dilutions were conducted as previously indicated. Then, 0.2 ml of each dilution was pipetted onto nutrient agar in a petri dish, and incubated for 36 h at 30°C.
2.2.2. Determination of total dry biomass Decanted culture broth was centrifuged at ~ 16000 × g for 10 min. The supernatant was discarded and the cell pellet was removed and vortex-mixed with distilled H~O for 10 s. The cell solution was dried overnight at 110°C and weighed. All the experiments were conducted in duplicate, and aseptic techniques were observed throughout for all microbiological procedures. Reported values represent means of the two microbial populations found to abundantly grow in the peat biofilter. The graphical representations and the calculation of means and standard deviations were conducted using Microsoft Excel 5.0c, Microsoft (USA).
3. Results and discussion The effects on the microorganisms of adding halothane to the culture media are presented in Fig. 1. For halothane concentrations of 0.7 to 2.7% ( v / v ) , the number of colony-forming units was lower than for the control (with no added halothane) in the
1 X 1010
:~ l x 1 0 6
.~_
:K .£o
lx104
o (..) 1 x 1 0 2
1 0
12
24
36
48
60
72
Time (h)
Fig. 1. Colony-formingunits at various halothane concentrations, in % (0, 0; 0.7, It; 1.3, ©; 2.7, [~; 5.3, zx). Means and standard deviations. Only the upper limit of each standard deviation is indicated. For clusters of experimental points, only the largest standard deviation is shown.
A.M. Martin et al. / Ecological Engineering 7 (1996) 151-155
154
1.4 1.2
1 1
i
~" 1.0 ~/
/o//
0.8 E .o 0.6 .0 n
0.4
0.2 0.0
I
I
I
I
I
I
I
0
12
24
36
48
60
72
Time (h) Fig. 2. Microbial dry biomass at various halothane concentrations, in % (0, 0 ; 0.7, I ; 1.3, C)" 2.7, E]; 5.3, A ). Means and standard deviations. Only the upper limit of each standard deviation is indicated. For clusters of experimental points, only the largest standard deviation is shown.
initial stages of the experiments. However, over the remaining time, the populations showed a recovery, and at 72 h the counts were similar to that of the control. The exception was observed in the culture to which 5.3% ( v / v ) halothane was added. In this case the microbial concentration decreased practically to extinction in 12 h, and did not recover. A similar pattern was found in the total biomass concentrations of the cultures (Fig. 2). The biomass increased in the control, but it did not increase when 5.3% ( v / v ) of halothane was added. With intermediate amounts of halothane (0.7, 1.3 and 2.7%, v / v ) , the increase in the total biomass was less than the control in the initial stages of the experiments. However, there was eventual recovery in these cases over the time of the experiments. The initial inhibition of the microorganisms, evidenced by a delay in the increase of the number of colony-forming units and the total biomass, indicates that the halothane was harmful to the microorganisms. At the lower concentrations of halothane, the microorganisms apparently adapted to it. When the concentration of halothane in the medium was 5.3% ( v / v ) , no adaptation was observed.
Acknowledgements This work was partially funded by a grant from the Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Mr. Paul Bemister, Department of Biochemistry, and Dr. Lenka Husa, Director of Animal Care Services, for their assistance.
A.M. Martin et al. / Ecological Engineering 7 (1996) 151 -155
155
References Bohn, H., 1992. Consider biofiltration for decontaminating gases. Chem. Eng. Prog., 88: 34-40. Department of Health and Social Security, 1976. Health Services Development. Pollution of Operating Departments etc. by Anaesthetic Gases. Health Circular HC(76)38. HMSO, London. Martin, A.M., 1991. Peat as an agent in biological degradation: Peat biofilters. In: A.M. Martin (Ed.), Biological Degradation of Wastes. Elsevier Applied Science, London, pp. 341-362. Norreslet, J., Friberg, S., Nielson, T.M. and R#mer, U., 1989. Halothane anaesthetic and the ozone layer. Lancet, i: 719. Ottengraf, S.P.P., 1986. Exhaust gas purification. In: H.-J. Rehm and G. Reed (Eds.), Biotechnology, Vol. 8. VCH, Weinheim, pp. 425-452. Ottengraf, S.P.P., Meesters, J.J.P., van den Oever, A.H.C. and Rozema, H.R., 1986. Biological elimination of volatile xenobiotic compounds in biofilters. Bioprocess Eng., 1: 61-69. Piziali, R.L.. Whitcher, C., Sher, R. and Moffat, R.J., 1976. Distribution of waste anesthetic gases in the operating room air. Anesthesiology, 45: 487-494. Vickers, M.D., 1975. Pollution of the atmosphere of operating theatres. Advice to members from the Council of the Association of Anaesthetists of Great Britain and Ireland, Anaesthesia, 30: 697-699. Yoganathan, S., Johnston, I.G., Parnell, C.J., Houghton, I.T. and Restall, J., 1991. Determination of contamination of a chemical warfare-proof operating theatre with volatile anaesthetic agents and assessment of anaesthetic gas scavenging systems. Br. J. Anaesth., 67: 614-617.