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The measurement of electron transport system (ETS) activity in freshwater sediment
Water Res. Vol. 18. No. 5, pp 581-584. 1984 Printed in Great Britain
0043-135484 $3.00+0.00 Pergamon Press Ltd
THE M E A S U R E M E N T OF ELECTRON TRANSPORT SYSTEM (ETS) ACTIVITY IN FRESHWATER SEDIMENT JACK T. TREVORS Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada NIG 2W1 ( Recei~'ed August 1983)
Abstract--Electron transport system (ETS) activity was measured in freshwater sediment at 4, 10 and 20°C by following the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium (INT-formazan). The rate of INT-formazan formation was dependent upon the incubation temperature and nutrient amendments. Electron transport system activity also correlated very highly with O_, consumption rates in the same sediment. Key words--activity, electron transport, microbial, sediment
INTRODUCTION The characterization of microbial bioactivity in sediment is an aspect important to the understanding of the aquatic environment (Liu and Strachan, 1981). The numbers, types and activities of microorgarrisms in sediments have been investigated using a variety of methods. Microorganisms have been enumerated and isolated using plating techniques or have been examined by direct microscopic observation (Polonenko et al., 1979). Also, sediment activities such as 02 consumption, CO_, evolution and radioactive glucose uptake have been used as measures of bioactivity. Enzyme assays such as phosphatase and dehydrogenase assays can measure the specific activity of a large part of the microbial population in sediment. Liu and Strachan (1981) described a field method for determining the biological and chemical activity of sediments using resazurin reduction. Using this method, it was reported that microbial activity accounted for 2-40% of the resazurin reduced in sediment from freshwater lakes. Tetrazolium salts have been used as electron acceptors to measure microbial electron transport system (ETS) activity in both soil (Casida, 1977) and aquatic environments (Jones and Simon, 1979; Maki and Remsen, 1981; Peele and Colwell, 1981). Zimmerman et aL (1978) described the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to determine respiring bacteria in the aquatic environment. Electron transport system activity is a characteristic of respiring cells, and can be measured by observing dense, stable, formazan granules with light microscopy (Maki and Remsen, 1981; Peele and Colwell, 1981). Trevors et al. (1982) have described a method for measuring ETS activity in soil by extracting the I N T - f o r m a z a n from soil and measuring the concentration by spectrophotometric analysis. This paper describes the effect of temperature and sediment amendments on
ETS activity and its relationship to 02 consumption rates in sediment. MATERIALS AND METHODS Sediment samples were taken with plexiglass cores from the top 5 cm of a freshwater stream near Floradale, Ontario, Canada and transported at 4°C in the dark. Various characteristics of the sediment were measured by techniques previously described by Tam and Trevors (1981). The enumeration of aerobic heterotrophs was made using the standard spread plate technique. Serial decimal dilutions of the sediment were prepared in sterile distilled water, and 0. I-ml amounts were spread on the surface of sediment extract agar. This medium was prepared using sediment extract obtained by autoclaving I kg of fresh sediment in 21. of distilled water for 30 min at 121~C. The sediment extract was collected by centrifugation at 3000g for 15rain. The sediment extract agar was prepared as follows: glucose, 2.5 g; yeast extract (Difco), 0.5 g; peptone (Difco), 5.0g; FePO4, 0.01 g; agar (Difco), 15.0 g; sediment extract, 100 ml; distilled water, 900 ml. The medium was sterilized by autoclaving at 121°C for lJmin. Triplicate plates of each dilution were incubated aerobically in the dark at 4, 10 and 20°C for 21 days. The development of colonies was recorded and reported as numbers g-t dry wt of sediment. Total microscopic counts of bacteria were carried out using acridine orange and epifluoreseent microscopy. A 10-g sample (wet wt) of sediment was placed in a sterile 50-ml Erlenmeyer flask. Sterile controls were prepared by autoclaving flasks containing sediment for 1.5 h at 121°C on two consecutive days. Flasks amended with either glucose or yeast extract (Difco) received l ml of a 1% (w/v) sterile solution. Non-amended sediment received l ml of sterile distilled water. Each flask received 1.5 m[ of a 0.4% (w/v) filter sterilized aqueous solution of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-pheny[ terazolium chloride (INT) (Sigma Chemical Co., St Louis, MO). All solutions were thoroughly mixed into the sediment with a sterile glass rod. Flasks were capped with sterile serum stoppers and incubated for the desired length of time at 4, 10 and 20°C. All trials were carried out in triplicate. Sediment samples prepared in the same manner were also used to determine O 2 consumption rates by gas chromatography as previously described (Trevors et al., 1982). Aerobic gas phase conditions were maintained by injecting pure 02 to replace that consumed in respiration. All data are reported as the average of triplicate flasks.
581
JACK T. TREvoRs
582
2o0
Table 1. Characteristics of the sediment Dry weight (% fresh wt) 68.6 Sand (0o) 51 Silt (°o) 36 Clay (%) 13 pH 7.0 Total bacterial count 3.2 x 10L° No. of heterotrophs (4:C) 2.0 x l06 No. of heterotrophs (10:C) 9.9 x 106 No. of heterotrophs (20=C) 3.1 x 107 Values are expressed on a per gram dry wt of sediment basis where applicable.
160
a
t20
80 >
40 I--la..I 0
At the desired time, a subsample o f sediment (approx. 0.5 g on a dry wt basis) was removed from each flask. The sediment was placed in a test tube and 10 ml o f methanol was added. The tube was mixed with a vortex mixer for 1 min, and the methanol extract filtered through a W h a t m a n No, 5 filter paper (W. & R. Balston Ltd, England). If additional methanol was required to extract the I N T - f o r m a z a n . the total volume of methanol was recorded and the proper correction made. The filter and extracted sediment were dried at 105°C for 24h, the filter and sediment weighed, and the dry weight of sediment determined. The I N T - f o r m a z a n concentration in the methanol extract was measured spectrophotometrically at 480 n m against a blank o f a methanol extract o f sediment containing no INT. A standard curve o f I N T - f o r m a z a n (Sigma) in methanol was used to quantify the concentration of INTzfo&mazan in the extract. The correlation coefficient f o r - t h e standard curve was 0.99 at the 9 5 ~ level. All data are reported on a per gram dry weight o f sediment basis. RESULTS
Some.characteristics of the sediment are presented in Table 1. Viable bacterial counts were less than the total bacterial count. It is also important to note that the number of heterotrophs enumerated at each incubation temperature, increased with the temperature. All incubations were carried out aerobically, since the ETS and 02 consumption rates were later determined in sediment incubated with an aerobic gas phase.
"7
L
"--
I
2
5
4
.5
6
Days
Fig. 2. ETS activity in non-amended and amended sediment at 10°C. Legends and symbols same as Fig. 1.
Electron transport system activity (Figs 1, 2 and 3) was stimulated by both glucose and yeast extract at 4, I0 and 20°C. Non-amended sediment displayed relatively low ETS activity at all three incubation temperatures. The presence of glucose or yeast extract brought about a 100~ increase in ETS activity at 4°C after 6 days. The same amendments at 10°C increased ETS activity about 327~ in the same period of time. When the incubation temperature was 20°C (Fig. 3), the sediment demonstrated as much ETS activity in 2-3 days, as the same sediment did at 10°C in 4 days. Oxygen consumption rates, measured in the same sediment displayed trends that were closely related to ETS activity (Figs 4, 5 and 6). Oxygen uptake rates were dependent on the incubation temperature and the type of amendment added to the sediment. In general, yeast extract initially served as a better substrate for increasing ETS activity and O2 consumption. However, as the length of incubation increased to 3 days at 20°C or 6 days at 10 and 4°C, the ETS activity was essentially the same in the glucose and yeast extract amended sediment. Yeast extract initially stimulated 02 uptake only slightly more than glucose. At the end of the incubation
20o~
o*
20O 5"
160
g
,oo
~ 120 E
~
~
120
~
80
80
~ 40 m kUA I
2
3
4
5
6
Days
Fig. 1. ETS activity in non-amended sediment at 4°C. Non-amended (O), amended with glucose ( e ) , amended with yeast extract (A), sterile controls o f all the above treatments demonstrated no activity (A).
pw
o
I
2
3
Days
Fig. 3. ETS activity in non-amended and amended sediment at 20°C. Legends and symbols same as Fig. 1.
S e d i m e n t activity
583
3o
25-
24
c~ 2 0 - o
E :k
15
8
8 ~. E
~o
Q
g
5
o
12
E 6
A
o
2
i
3
4
5
6
o
Fig. 4. 02 c o n s u m p t i o n in n o n - a m e n d e d a n d a m e n d e d s e d i m e n t at 4~C. L e g e n d s a n d s y m b o l s s a m e as Fig. 1.
25 'o~ 2 0 ::L 15
8 E 5
C 2
3
A
2
l
i
3
Days
Days
t
•
i
4
5
6
Days Fig 5 02 consumption in nonamended and amended sediment at IO~C Legends and symbols same as Fig I
Fig. 6. 02 c o n s u m p t i o n in n o n - a m e n d e d a n d a m e n d e d s e d i m e n t at 20:C. L e g e n d s a n d s y m b o l s s a m e as Fig. 1.
comparison to be made with the non-amended measurements. Table 2 shows the lines of best fit and the correlation coefficients obtained when ETS activity was plotted against O2 consumption and analyzed using regression analysis. The correlation coefficients were high, indicating the relationships between the two measurements of sediment activity. The use of INT for measuring ETS activity was dependent upon the presence of respiring cells. Also, ETS system activity has been shown to be well correlated with O 2 consumption in soil samples (Trevors et al., 1982) suggesting that the reduction of INT was related to respiration.
DISCUSSION
period 02 uptake was always higher in the sediment amended with yeast extract. The effect of sediment amendments was not the main aspect of this study, where only one relatively low concentration of two nutrients were added. The important point was the relatively low ETS activity and O2 uptake measured in the non-amended sediment. However, the addition of nutrients allowed a
Since microbial activity in the natural environment is under less than optimal conditions, it is important to obtain accurate rates of ETS activity and O2 consumption in non-amended sediment over a range of temperatures that are common in the natural environment. It is clear that a relationship does exist between INT reduction and 02 consumption, with O2 being the normal preferred acceptor. It is possible
Table 2. Correlations between ETS activity (Y) and O: consumption (x) in non-amended and amended sediment Incubation conditions 4C Non-amended Glucose amended Yeast extract amended 10:C Non-amended Glucose am~ra~ed Yeast extract amended 20=C Non-amended Glucose amended Yeast extract amended
Line of best fit
Correlation coefficient
Y = 22.84 + 1.89x Y = 63.24 + 2.33x Y = 55.23 + 2.26x
0.82 0.74 0.86
Y = 27.81 + 1.99x Y = 61.73 + 5.70x Y = 93.92 + 3.02x
0.90 0.94 0.87
Y = 8.31 + 2.79x Y = 27.39 + 6.53x Y = 41.88 + 5.0Ix
0.91 0.95 0.86
Lines of best fit and correlation coefficients (at the 95% level) were calculated from the data presented in Figs 1-6 using a regression analysis program.
584
JACK T. TREVORS
that not all species of bacteria can reduce the INT. This may cause an underestimation of the sediment ETS activity. Also. INT may inhibit or suppress respiratory activity in some species of bacteria. However, many of these same problems also exist with the 2.3,5-triphenyl tetrazolium chloride assay and other sediment enzyme assays. This does not detract from the usefulness of the technique as a measure of sediment bioactivity. In the past, INT and T T C have been used to obtain measures of bioactivity in sediments without fully understanding the mechanisms. This report has correlated the relationship between INT reduction and O,. consumption. Electron transport system activity can also be measured in anaerobically incubated sediment or soil (Trevors et al., 1982). However, in the present study only aerobic incubation conditions were used, to allow correlations with O, consumption to be made. It has been recommended by Casida (1977) that short incubation periods of 6-h at 37°C with yeast extract or glucose as the substrate may be useful in measuring microbial dehydrogenase activity in soil. In the present study, temperatures of 4, 10 and 20°C were used as they approximate a range of temperatures that are found in the natural environment. Regardless of the choice of temperature and the-concentration and type of substrate added, the use of INT allows a rapid quantitative determination of ETS activity in sediment.
Acknowledgement--This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. REFERENCES
Casida L. E. (1977) Microbial activity in soil as measured by dehydrogenase determinations. Appl. ent'ir. Microbiol. 34, 630-636. Jones J. G. and Simon B. M. (1979) The measurement of electron transport system activity in freshwater benthic and planktonic samples. J. appl. Bact. 46, 305--315. Liu D. and Strachan W. M. (1981) A field method for determining the chemical and biological activity of sediments, Water Res. 15, 353-359. Maki J. S. and Remsen C. C. (1981) Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl. envir. Microbiol. 41, 1t32-1138. Peele E. R. and Colwell R. R. (1981) Application of a direct microscopic method for enumeration of substrateresponsive marine bacteria. Can. J. Microbiol. 27, 1071-1075. Polonenko D. R., Mayfield C. I. and Inniss W. E. (1979) A direct observation technique for the study of microorganisms in sediment samples. Wat. Air Soil Pollut. I 1, 237-245. Tam T. Y. and Trevors J. T. (1981) Effects of pentachlorophenol on asymbiotic nitrogen fixation in soil. War. Air Soil Pollut. 16, 409--414. Trevors J. T., Mayfield C. I. and Inniss W. E. (1982) Measurement of electron transport system (ETS) activity in soil. Microbial Ecol. 8, 163-168. Zimmerman R., Iturriaga R. and Becker-Birck J. (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. envir. Microbiol. 36, 926-935.
0043-135484 $3.00+0.00 Pergamon Press Ltd
THE M E A S U R E M E N T OF ELECTRON TRANSPORT SYSTEM (ETS) ACTIVITY IN FRESHWATER SEDIMENT JACK T. TREVORS Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada NIG 2W1 ( Recei~'ed August 1983)
Abstract--Electron transport system (ETS) activity was measured in freshwater sediment at 4, 10 and 20°C by following the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium (INT-formazan). The rate of INT-formazan formation was dependent upon the incubation temperature and nutrient amendments. Electron transport system activity also correlated very highly with O_, consumption rates in the same sediment. Key words--activity, electron transport, microbial, sediment
INTRODUCTION The characterization of microbial bioactivity in sediment is an aspect important to the understanding of the aquatic environment (Liu and Strachan, 1981). The numbers, types and activities of microorgarrisms in sediments have been investigated using a variety of methods. Microorganisms have been enumerated and isolated using plating techniques or have been examined by direct microscopic observation (Polonenko et al., 1979). Also, sediment activities such as 02 consumption, CO_, evolution and radioactive glucose uptake have been used as measures of bioactivity. Enzyme assays such as phosphatase and dehydrogenase assays can measure the specific activity of a large part of the microbial population in sediment. Liu and Strachan (1981) described a field method for determining the biological and chemical activity of sediments using resazurin reduction. Using this method, it was reported that microbial activity accounted for 2-40% of the resazurin reduced in sediment from freshwater lakes. Tetrazolium salts have been used as electron acceptors to measure microbial electron transport system (ETS) activity in both soil (Casida, 1977) and aquatic environments (Jones and Simon, 1979; Maki and Remsen, 1981; Peele and Colwell, 1981). Zimmerman et aL (1978) described the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to determine respiring bacteria in the aquatic environment. Electron transport system activity is a characteristic of respiring cells, and can be measured by observing dense, stable, formazan granules with light microscopy (Maki and Remsen, 1981; Peele and Colwell, 1981). Trevors et al. (1982) have described a method for measuring ETS activity in soil by extracting the I N T - f o r m a z a n from soil and measuring the concentration by spectrophotometric analysis. This paper describes the effect of temperature and sediment amendments on
ETS activity and its relationship to 02 consumption rates in sediment. MATERIALS AND METHODS Sediment samples were taken with plexiglass cores from the top 5 cm of a freshwater stream near Floradale, Ontario, Canada and transported at 4°C in the dark. Various characteristics of the sediment were measured by techniques previously described by Tam and Trevors (1981). The enumeration of aerobic heterotrophs was made using the standard spread plate technique. Serial decimal dilutions of the sediment were prepared in sterile distilled water, and 0. I-ml amounts were spread on the surface of sediment extract agar. This medium was prepared using sediment extract obtained by autoclaving I kg of fresh sediment in 21. of distilled water for 30 min at 121~C. The sediment extract was collected by centrifugation at 3000g for 15rain. The sediment extract agar was prepared as follows: glucose, 2.5 g; yeast extract (Difco), 0.5 g; peptone (Difco), 5.0g; FePO4, 0.01 g; agar (Difco), 15.0 g; sediment extract, 100 ml; distilled water, 900 ml. The medium was sterilized by autoclaving at 121°C for lJmin. Triplicate plates of each dilution were incubated aerobically in the dark at 4, 10 and 20°C for 21 days. The development of colonies was recorded and reported as numbers g-t dry wt of sediment. Total microscopic counts of bacteria were carried out using acridine orange and epifluoreseent microscopy. A 10-g sample (wet wt) of sediment was placed in a sterile 50-ml Erlenmeyer flask. Sterile controls were prepared by autoclaving flasks containing sediment for 1.5 h at 121°C on two consecutive days. Flasks amended with either glucose or yeast extract (Difco) received l ml of a 1% (w/v) sterile solution. Non-amended sediment received l ml of sterile distilled water. Each flask received 1.5 m[ of a 0.4% (w/v) filter sterilized aqueous solution of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-pheny[ terazolium chloride (INT) (Sigma Chemical Co., St Louis, MO). All solutions were thoroughly mixed into the sediment with a sterile glass rod. Flasks were capped with sterile serum stoppers and incubated for the desired length of time at 4, 10 and 20°C. All trials were carried out in triplicate. Sediment samples prepared in the same manner were also used to determine O 2 consumption rates by gas chromatography as previously described (Trevors et al., 1982). Aerobic gas phase conditions were maintained by injecting pure 02 to replace that consumed in respiration. All data are reported as the average of triplicate flasks.
581
JACK T. TREvoRs
582
2o0
Table 1. Characteristics of the sediment Dry weight (% fresh wt) 68.6 Sand (0o) 51 Silt (°o) 36 Clay (%) 13 pH 7.0 Total bacterial count 3.2 x 10L° No. of heterotrophs (4:C) 2.0 x l06 No. of heterotrophs (10:C) 9.9 x 106 No. of heterotrophs (20=C) 3.1 x 107 Values are expressed on a per gram dry wt of sediment basis where applicable.
160
a
t20
80 >
40 I--la..I 0
At the desired time, a subsample o f sediment (approx. 0.5 g on a dry wt basis) was removed from each flask. The sediment was placed in a test tube and 10 ml o f methanol was added. The tube was mixed with a vortex mixer for 1 min, and the methanol extract filtered through a W h a t m a n No, 5 filter paper (W. & R. Balston Ltd, England). If additional methanol was required to extract the I N T - f o r m a z a n . the total volume of methanol was recorded and the proper correction made. The filter and extracted sediment were dried at 105°C for 24h, the filter and sediment weighed, and the dry weight of sediment determined. The I N T - f o r m a z a n concentration in the methanol extract was measured spectrophotometrically at 480 n m against a blank o f a methanol extract o f sediment containing no INT. A standard curve o f I N T - f o r m a z a n (Sigma) in methanol was used to quantify the concentration of INTzfo&mazan in the extract. The correlation coefficient f o r - t h e standard curve was 0.99 at the 9 5 ~ level. All data are reported on a per gram dry weight o f sediment basis. RESULTS
Some.characteristics of the sediment are presented in Table 1. Viable bacterial counts were less than the total bacterial count. It is also important to note that the number of heterotrophs enumerated at each incubation temperature, increased with the temperature. All incubations were carried out aerobically, since the ETS and 02 consumption rates were later determined in sediment incubated with an aerobic gas phase.
"7
L
"--
I
2
5
4
.5
6
Days
Fig. 2. ETS activity in non-amended and amended sediment at 10°C. Legends and symbols same as Fig. 1.
Electron transport system activity (Figs 1, 2 and 3) was stimulated by both glucose and yeast extract at 4, I0 and 20°C. Non-amended sediment displayed relatively low ETS activity at all three incubation temperatures. The presence of glucose or yeast extract brought about a 100~ increase in ETS activity at 4°C after 6 days. The same amendments at 10°C increased ETS activity about 327~ in the same period of time. When the incubation temperature was 20°C (Fig. 3), the sediment demonstrated as much ETS activity in 2-3 days, as the same sediment did at 10°C in 4 days. Oxygen consumption rates, measured in the same sediment displayed trends that were closely related to ETS activity (Figs 4, 5 and 6). Oxygen uptake rates were dependent on the incubation temperature and the type of amendment added to the sediment. In general, yeast extract initially served as a better substrate for increasing ETS activity and O2 consumption. However, as the length of incubation increased to 3 days at 20°C or 6 days at 10 and 4°C, the ETS activity was essentially the same in the glucose and yeast extract amended sediment. Yeast extract initially stimulated 02 uptake only slightly more than glucose. At the end of the incubation
20o~
o*
20O 5"
160
g
,oo
~ 120 E
~
~
120
~
80
80
~ 40 m kUA I
2
3
4
5
6
Days
Fig. 1. ETS activity in non-amended sediment at 4°C. Non-amended (O), amended with glucose ( e ) , amended with yeast extract (A), sterile controls o f all the above treatments demonstrated no activity (A).
pw
o
I
2
3
Days
Fig. 3. ETS activity in non-amended and amended sediment at 20°C. Legends and symbols same as Fig. 1.
S e d i m e n t activity
583
3o
25-
24
c~ 2 0 - o
E :k
15
8
8 ~. E
~o
Q
g
5
o
12
E 6
A
o
2
i
3
4
5
6
o
Fig. 4. 02 c o n s u m p t i o n in n o n - a m e n d e d a n d a m e n d e d s e d i m e n t at 4~C. L e g e n d s a n d s y m b o l s s a m e as Fig. 1.
25 'o~ 2 0 ::L 15
8 E 5
C 2
3
A
2
l
i
3
Days
Days
t
•
i
4
5
6
Days Fig 5 02 consumption in nonamended and amended sediment at IO~C Legends and symbols same as Fig I
Fig. 6. 02 c o n s u m p t i o n in n o n - a m e n d e d a n d a m e n d e d s e d i m e n t at 20:C. L e g e n d s a n d s y m b o l s s a m e as Fig. 1.
comparison to be made with the non-amended measurements. Table 2 shows the lines of best fit and the correlation coefficients obtained when ETS activity was plotted against O2 consumption and analyzed using regression analysis. The correlation coefficients were high, indicating the relationships between the two measurements of sediment activity. The use of INT for measuring ETS activity was dependent upon the presence of respiring cells. Also, ETS system activity has been shown to be well correlated with O 2 consumption in soil samples (Trevors et al., 1982) suggesting that the reduction of INT was related to respiration.
DISCUSSION
period 02 uptake was always higher in the sediment amended with yeast extract. The effect of sediment amendments was not the main aspect of this study, where only one relatively low concentration of two nutrients were added. The important point was the relatively low ETS activity and O2 uptake measured in the non-amended sediment. However, the addition of nutrients allowed a
Since microbial activity in the natural environment is under less than optimal conditions, it is important to obtain accurate rates of ETS activity and O2 consumption in non-amended sediment over a range of temperatures that are common in the natural environment. It is clear that a relationship does exist between INT reduction and 02 consumption, with O2 being the normal preferred acceptor. It is possible
Table 2. Correlations between ETS activity (Y) and O: consumption (x) in non-amended and amended sediment Incubation conditions 4C Non-amended Glucose amended Yeast extract amended 10:C Non-amended Glucose am~ra~ed Yeast extract amended 20=C Non-amended Glucose amended Yeast extract amended
Line of best fit
Correlation coefficient
Y = 22.84 + 1.89x Y = 63.24 + 2.33x Y = 55.23 + 2.26x
0.82 0.74 0.86
Y = 27.81 + 1.99x Y = 61.73 + 5.70x Y = 93.92 + 3.02x
0.90 0.94 0.87
Y = 8.31 + 2.79x Y = 27.39 + 6.53x Y = 41.88 + 5.0Ix
0.91 0.95 0.86
Lines of best fit and correlation coefficients (at the 95% level) were calculated from the data presented in Figs 1-6 using a regression analysis program.
584
JACK T. TREVORS
that not all species of bacteria can reduce the INT. This may cause an underestimation of the sediment ETS activity. Also. INT may inhibit or suppress respiratory activity in some species of bacteria. However, many of these same problems also exist with the 2.3,5-triphenyl tetrazolium chloride assay and other sediment enzyme assays. This does not detract from the usefulness of the technique as a measure of sediment bioactivity. In the past, INT and T T C have been used to obtain measures of bioactivity in sediments without fully understanding the mechanisms. This report has correlated the relationship between INT reduction and O,. consumption. Electron transport system activity can also be measured in anaerobically incubated sediment or soil (Trevors et al., 1982). However, in the present study only aerobic incubation conditions were used, to allow correlations with O, consumption to be made. It has been recommended by Casida (1977) that short incubation periods of 6-h at 37°C with yeast extract or glucose as the substrate may be useful in measuring microbial dehydrogenase activity in soil. In the present study, temperatures of 4, 10 and 20°C were used as they approximate a range of temperatures that are found in the natural environment. Regardless of the choice of temperature and the-concentration and type of substrate added, the use of INT allows a rapid quantitative determination of ETS activity in sediment.
Acknowledgement--This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. REFERENCES
Casida L. E. (1977) Microbial activity in soil as measured by dehydrogenase determinations. Appl. ent'ir. Microbiol. 34, 630-636. Jones J. G. and Simon B. M. (1979) The measurement of electron transport system activity in freshwater benthic and planktonic samples. J. appl. Bact. 46, 305--315. Liu D. and Strachan W. M. (1981) A field method for determining the chemical and biological activity of sediments, Water Res. 15, 353-359. Maki J. S. and Remsen C. C. (1981) Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl. envir. Microbiol. 41, 1t32-1138. Peele E. R. and Colwell R. R. (1981) Application of a direct microscopic method for enumeration of substrateresponsive marine bacteria. Can. J. Microbiol. 27, 1071-1075. Polonenko D. R., Mayfield C. I. and Inniss W. E. (1979) A direct observation technique for the study of microorganisms in sediment samples. Wat. Air Soil Pollut. I 1, 237-245. Tam T. Y. and Trevors J. T. (1981) Effects of pentachlorophenol on asymbiotic nitrogen fixation in soil. War. Air Soil Pollut. 16, 409--414. Trevors J. T., Mayfield C. I. and Inniss W. E. (1982) Measurement of electron transport system (ETS) activity in soil. Microbial Ecol. 8, 163-168. Zimmerman R., Iturriaga R. and Becker-Birck J. (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. envir. Microbiol. 36, 926-935.