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The measurement of electron transport system activity in river biofilms
Wat. Res. Vol. 24, No. 4, pp. 441--445, 1990 Printed in Great Britain.All rights reserved
0043-1354/90 $3.00+ 0.00 Copyright © 1990 Pergamon Press plc
THE MEASUREMENT OF ELECTRON TRANSPORT SYSTEM ACTIVITY IN RIVER BIOFILMS S. A. BLENKINSOPP*,t and M. A. LOCK School of Biological Sciences, University College of North Wales, Bangor, Gwynedd LL57 2UW, Wales (First received January 1989; accepted in revised form October 1989)
Abstract--The factors affecting the measurement of electron transport system (ETS) activity in river biofilm through the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan) have been studied. Methanol extracts INTformazan more effectively than either propanol or ethanol. A concentration of 0.02% INT was chosen and samples should be incubated for less than 8 h. ETS activity is optimal at a circumneutral pH. ETS stimulators (NADH, NADPH and succinate) added as a check of the assay produced an increase in INT-formazan indicating that ETS activity was being measured. This assay is quick, easy to use and ideal for field studies. Key words--method, river biofilms, electron transport system activity, INT
INTRODUCTION
River biofilm (epilithon, periphyton) is a complex community of microorganisms ranging from strict autotrophs to strict heterotrophs, depending on environmental conditions present at the time. This mixed microbial consortium exists within a polysaccharide matrix, which is thought to play a role in nutrient trapping, transport between cells and extracellular enzyme activity (Lock et al., 1984; Costerton et al., 1987; Jones and Lock, 1989). Thus, in order to obtain realistic measurements of activity within a biofilm, it is necessary to keep the biofilm intact so as not to disturb the intracellular and extracellular processes which occur within it. All respiring microorganisms possess an active electron transport system (ETS), and ETS activity is a measure of the ability of microorganisms to reduce an indicator under defined conditions. Tetrazolium salts such as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5phenyl tetrazolium chloride (INT) are commonly used as ETS indicators. INT is water-soluble in its oxidized form and, when reduced, forms a water insoluble deposit, iodonitrotetrazolium formazan (INT-formazan), within the cell. These red formazan deposits have been used to distinguish between respiring and non-respiring aquatic bacteria using light microscopy (Zimmermann et al., 1978). Extracted formazan can be measured spectrophotometrically to quantify ETS activity in marine (Vosjan, 1982), estuarine (Jeffrey and Paul, 1986), freshwater planktonic and benthic (Jones and Simon, 1979) and soil environments (Trevors et al., 1982). This paper deals *N~e Dither. tPresent address: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. 441
with the spectrophotometric approach and its application to fiver biofilm activity. In his review article, Trevors (1984a) notes that the INT assay is (i) operable over a wide range of environmental tempertures, (ii) measures both aerobic and anaerobic respiration and (iii) does not need the addition of substrates such as glucose or yeast extract to stimulate ETS activity. These features make the INT assay an attractive field method, particularly when dealing with thick biofilms in which anaerobic respiration may occur in the deeper layers. Also, not having to add an external substrate to demonstrate activity is most desirable, because of difficulties involved in determining a suitable substrate type and concentration for all the microbes in a mixed microbial consortium. There are currently two main spectrophotometric methods used to measure ETS activity. The first, developed for marine plankton work, involves incubating an enzyme extract prepared from the cells with an INT solution and a substrate solution containing ETS stimulators (NADH, N A D P H and succinate). This method gives the maximum potential ETS activity of the community. The second method, described for soil work, involves incubating the "intact" microbial community in an INT solution without ETS stimulators. This will give lower values than the first method because it measures the in situ ETS activity. The first method was not suitable for our needs because it would have meant disrupting the biofilm, and therefore its functioning (Ladd et al., 1979; Murray et al., 1987), before measuring its ETS activity. Recent literature on antibiotic resistance (Costerton et al., 1987) and biocide effectiveness (Costerton et al., 1987; LeChevallier et al., 1988) has highlighted problems with the extrapolation of techniques
442
S.A. BLENKINSOPPand M. A. LOCK
b e t w e e n different m i c r o b i a l c o m m u n i t i e s , in particular p l a n k t o n i c a n d a t t a c h e d c o m m u n i t i e s (biofilms), a n d highlights the need to evaluate m e t h o d s i n t e n d e d for biofilm work. In this p a p e r we i n v e s t i g a t e d the effect o f e x t r a c t i o n solvent, I N T c o n c e n t r a t i o n , length o f i n c u b a t i o n , w a t e r p H a n d E T S s t i m u l a t o r s o n E T S activity m e a s u r e d in intact river biofilm using a m o d i f i c a t i o n o f a m e t h o d used o n soil by T r e v o r s et al. (1982). MATERIALS AND METHODS
Biofilm studies Biofilm was grown on acid-washed, pre-combusted glass beads (1.5 mm dia) strung on nylon monofilament line. Bead strings were 32 cm long (15.27 cm 2 surface area). With the exception of the beads used in the solvent trial, which were colonized in a freshwater aquarium, the bead strings were tied onto perspex plates and inserted into plastic tubes (10.2era dia, 1.5m long). The tubes were fastened to a specially designed frame on the river bed (Blenkinsopp, unpublished Ph.D. thesis) to facilitate sampling, especially during high water periods. Bead strings were either light-incubated in tubes with perspex windows to allow autotrophic/heterotrophic growth, or dark-incubated to allow primarily heterotrophic growth in the fourth order Clywedog river, North Wales. Site details have been published (Lock and Ford, 1985). All bead strings were colonized for a minimum of 6 weeks before use. Standard assay followed in the trials INT does not dissolve readily in water, thus a 30-min sonication period (Dawe Sonicleaner) was used routinely, pausing frequently to shake the vessel vigorously and break up the powdery clumps. The pauses also served to prevent the solution from over-heating. In earlier trials gentle heating ( 6 0 ° 0 followed by sonication resulted in loss of activity when compared with sonication alone (unpublished data). Since INT is light-sensitive, it was kept in foil-covered vessels throughout the assay. Bead strings were placed in sterile wide-mouthed laboratory jars (500ml) containing 100ml of the appropriate concentration of INT (Aldrich) in river or aquarium water, where appropriate. Controls were prepared by adding bead strings to 2% formaldehyde in water. The controls were prepared a minimum of 30 rain prior to start of the assay and then rinsed briefly in water before being placed in INT solution. (All water was filter-sterilized using 0.2/am Whatman filters.) Incubations were performed at a known temperature for a known time period. After the incubation period each string was rinsed in water to remove any unbound INT-formazan and the beads slipped off the string into a vial containing 3-10 ml of chilled methanol. Each vial was sonicated, with pauses, for 5 min to disrupt the polysaccharide matrix and allow easy solvent access. Vials were then placed in a freezer for I h for extraction. Checks were made periodically to ensure that the solvent did not become saturated. If the colour of the extract approached that of the highest standard, a known volume of solvent was added to dilute the colour accordingly. The solvent containing the extracted INT-formazan was then briefly vortexed and filtered through a Whatman G F / F filter. At this point the solvent extract is stable for several days if refrigerated. The INT-formazan extract was measured spectrophotometrically at 480 nm against a solvent blank. A stock solution of 30#g/ml INT-formazan (Sigma) in solvent was used to prepare a standard curve of 0-30 # g/ml (corr. coeff. = 0.99). The final ETS activity results were calculated by subtracting the killed controls from the sample values and expressed per cm 2 for all trials excluding the ETS stimulation trial results, which were expressed per cell.
Effect o f type of extraction solvent on biofilm 1NT-formazan recovery
Fifteen bead strings, including 3 killed controls, were incubated at room temperature (17.6°C) for 1 h in 0.02% INT at pH 7.2. Four replicates and one killed control were extracted with each solvent and read against a standard curve of INT-formazan prepared with the appropriate solvent. The solvents compared were ethanol, methanol and propanol (GPR).
Effect o f I N T concentration on dark-grown biofilm ETS actiriO' A concentration of 0.02% INT was used by Zimmermann et al. (1978) to determine the proportion of bacterial cells actively respiring based on counts of cells with red formazan deposits. This concentration was used as a base-line value for this trial. Dark-grown beads usually had a less visible biofilm than the light-grown material, thus we were concerned with obtaining a measurable activity signal from the dark community. A concentration trial was therefore performed on the dark community to optimize assay conditions and the concentration chosen was used for both communities in later trials. Three replicates and I killed control per INT concentration (0.2, 0.02. 0.002%) were incubated at 1OC for 15 h at pH 7.2. Effect o[ incubation length on light-grown bio[ilm E T S activit)' Three replicates and l killed control per time period (1,4, 8, 12 and 16h) were incubated in 0.02% INT, pH 7.2, at 10C. Effect of pH on light-grown biofilm ETS activity The buffering capacity of the biofilm may have an effect on ETS activity. Fiebig (unpublished Ph.D. thesis) noted that river biofilm can buffer pH-adjusted river water in the direction of the unadjusted pH. A preliminary pH trial of bead strings in pH-adjusted fiver water showed that with the surface area of biofilm used per ml of water (15.27cm2/100ml) in these trials, the water pH was not altered when compared with the control of adjusted water alone (unpublished data) over an 18 h period. With this confirmed, the pH trial was performed. Three replicates and one killed control per pH level (4.0, 5.7, 7.0 and 9.0) were incubated in 0.02% INT for 15h at 10C. The 0.02% INT solution was adjusted to the appropriate pH with 0.05 N sodium hydroxide or 0.05 N sulphuric acid. Effect of E T S stimulators on biofilm ETS activity The purpose of this trial was to confirm that the assay was measuring ETS activity, through the use of known ETS stimulators. This trial was performed on both light-grown and dark-grown biofilms incubated at 10°C, for 13h at pH 7.2. Beads were incubated in 0.02% INT solution only (non-amended treatment) and 0.02% INT solution with the following additions (amended treatment): N A D P H 0.08mg/ml, NADH 0.5mg/ml and sodium succinate 4.0 mg/ml (Quinn, 1984). There were 8 replicates per treatment and 4 killed controls per treatment. To increase the number of replicates, the bead strings were unstrung and the beads well mixed to avoid any string effects. A mean of 36 randomly chosen beads were used per replicate. Cell counts were performed on light and dark-grown biofilm samples, collected from each treatment at the end of the incubation period, to ensure that any trends noted were not simply due to differences in cell numbers per cm 2. Cells were counted using the method of Hobbie et al. (1977), following a 90 s sonication in 2% formaldehyde in filter-sterilized river water to remove cells from the beads. The ETS activity values per cell are only a relative measure because measured ETS activity is due to both intracellular and extracellular enzyme activity (Trevors, 1984a). Also, enzymes, including those involved in electron transport, may be released from dead cells and may survive for a limited time in soil, sediment
ETS activity in river biofilms
443
(Trevors, 1984a) and river biofilms (Lock et al., 1984). However, because the enzymes responsible for intracellular and extracellular ETS activity arise from cells within the biofilm, and the acridine orange direct counts method includes all intact cells, without distinguishing between those that are viable and non-viable, it is reasonable to express ETS activity relative to cell numbers.
such as the 0.2% concentration, may be toxic to fiver biofilm and should not be used in its study. A concentration of 0.02% was used for the remainder of the assays.
Importance of the killed control
INT-formazan was produced asymptotically over the 16 h incubation period at 10°C (Fig. 1); its rate of production decreased at about 8 h of incubation.
The control chosen for these assays was a killed control whereby the control sample of biofilm was killed with formaldehyde prior to incubation under identical conditions as the samples. It is very important to have a killed control because (i) chemical INT reduction may occur in the sample environment, e.g. in systems containing humic acids, nonenzymatic formazan formation may occur by their activity (Schindler et al., 1976), (ii) in light-grown material, algal pigments are extracted with organic solvents and absorb at the wavelength used for formazan detection and (iii) ETS stimulator such as succinate, NADH and NADPH are known to reduce certain brands of INT chemically (Packard and Williams, 1981).
Effect of incubation length on biofilm ETS activity
Effect of p H on biofilm ETS activity Water pH was shown to significantly affect biofilm ETS activity (ANOVA, P <0.05). INT-formazan values were barely detectable at pH 4 and increased to pH 7 (Fig. 2). There was a slight but not significant increase at pH 9. Thus, it appears that a circumneutral pH is optimal for INT-formazan production in fiver biofilms.
Statistical analysis Standard curve regressions and statistical analyses were performed using Statgraphics Version 2.1 (STSC Inc., Rockville, MD 20852, U.S.A.). RESULTS
Effect of extraction solvent on biofilm INT-formazan recovery Methanol extracted significantly more INTformazan than propanol and ethanol (ANOVA, P < 0.05). The INT-formazan levels were 4.88 + 0.41, 3.45___ 0.16 and 2 . 8 8 _ 0.21 pg/cm 2, respectively ( m e a n _ SE, n = 4). Propanol and ethanol had approximately the same extraction capability (multiple range analysis). HPLC-grade methanol (May & Baker) was therefore used in the remainder of the assays.
Effect of I N T concentration on biofilm ETS activity The three concentrations of I N T yielded INTformazan values which were not equal to each other (ANOVA, P < 0.05) and were all significantly different from each other (multiple range analysis). 0.02% I N T yielded the most INT-formazan (6.05 _ 0.46 #g/ cm z) followed by 0.002% I N T (3.47 + 0.39/~g/cm2); 0.2% I N T had the lowest values (1.57 _ 0.22 #g/cm 2) ( m e a n _ SE, n = 3). High concentrations of INT, 12
Effect of ETS stimulators on biofilm E T S activity INT-formazan values were 2.77 _ 0.08 and 1.81 -t-0.05 ( x 1044-13g/cell), for the light and darkgrown biofilms, respectively, in the "non-amended treatment" (INT only) and were 6.82 _+ 0.50 and 3.64 _+ 0.12 ( x l0 -~3 g/cell) for the light and darkgrown biofilms, respectively, in the "amended treatment" (INT plus ETS stimulators) (mean _+ SE, n = 8 ) . INT-formazan values were significantly greater in the amended treatments thus firmly supporting that the assay is measuring ETS activity in fiver biofilms (two-way ANOVA, P < 0.05). Also, the INT-formazan values were significantly greater in the light-grown biofilm than in the dark-grown biofilm (two-way ANOVA, P < 0.05). Cell counts were very similar in the light and dark-grown biofilms for both non-amended and amended treatments (4.7-6.3 x 10 7 cells/cm2).
FIELD
DATA
Representative field data from the Clywedog fiver are shown in Table I. Mean INT-formazan values ranged from 0.58/~g/cmE/h in a dark-grown early spring biofilm to 2.06/zg/cm:/h in a light-grown late spring biofilm. 6
[j+j+ ,o
2
3
I
I
i
I
4
I
t
J
I
i
i
8 Incubotion
J
I
12
I
I
t
I
16
(h)
Fig. 1. Effect of incubation length on fiver biofilm ETS activity (mean _ SE, n = 3).
0 3
J*l+lll 4
III 5
Irlll
IIJ 6
IlilJ 7
IIII 8
II 9
pH
Fig. 2. Effect of pH on river biofilm ETS activity (mean + SE, n = 3).
444
S. A. BLENKINSOPP and M. A. LOCK Table 1. Representative electron transport system (ETS) activity results from Clywedog river (North Wales) biofilms. Sample statistics are the mean ± SE, with sample size in parentheses
Date
pH
Incubation temp. ('C)
1/2:88 30/3/88 12/5/88
7.2 7.2 7.2
10 10 12
,ug INT-formazan/cm2/h
Incubation time (h)
Artificial substratum
Light-grown biofilm
Dark-grown biofilm
1 13 3
Glass beads Glass beads* Spurr resint
0.94 ± 0. I 0 (3) ND 2.06 ± 0.08 (4)
ND 0.58 ± 0.02 (7) t.87 ± 0.19 (4)
ND, not determined. *The beads were removed from the string for this incubation. +Transmission electron microscope epoxy resin (Spurt, 1969). DISCUSSION
The factors affecting the measurement of ETS activity in river biofilm have been investigated. Methanol was the superior solvent and extracted INT-formazan from the biofilm more effectively than either ethanol or propanol. This finding is in contrast with Burton and Lanza (1986) who used another commonly used tetrazolium salt, triphenyltetrazolium chloride (TTC), to measure ETS activity in freshwater lake sediments. The extraction performance of solvents used for TTC-formazan recovery was tetrachloroethylene-acetone > propanol > ethanol > methanol. Tetrachloroethylene-acetone was not used in this study, because tetrachloroethylene is a suspected carcinogen (Aldrich Chemical Co. Ltd Catalogue 1987-1988). Whether the difference in solvent extraction ability is due to the type of tetrazolium salt or the system in which it was converted to formazan is unclear. However, it does emphasize the need for preliminary trials prior to setting assay procedures. Concentrations of up to 0.4% INT (maximum solubility in water) have been reported in an estuarine study (Jeffrey and Paul, 1986). However, this concentration (0.4%) was based on a soil study by Trevors (1984b) which revealed that dehydrogenase (ETS) activity is positively correlated with the initial concentration of INT. Our results show that in a river biofilm, INT-formazan does not increase with the initial concentration of INT. The lowest concentration used in the study (0.002% INT) yielded more formazan than the highest concentration (0.2%) suggesting that 0.2% may be toxic. The middle concentration, 0.02% INT, yielded the greatest amount of extracted formazan and was chosen for the remainder of the study. Ideally, to avoid changes within the biofilm community over the course of the assay, the incubation time chosen should be kept to a minimum. Casida (1977) used triphenyltetrazolium chloride to measure ETS activity in soils, and noted population shifts for both aerobic and anaerobic bacteria during 24 h incubations at 37~'C with yeast substrate. The dehydrogenase curves showed a change at about 9-12 h and increased substrate concentrations did not produce linear activity with time. A change at about 8 h was noted in this study (Fig. 1). The biofilm population was not monitored over the time course; however, it is possible that a population shift could
have occurred. Based on the time course, incubation should be less than 8 h. However, under low growth or low temperature conditions, for example, it may be necessary to incubate samples longer until measurable INT-formazan formation occurs. Incubations in this study were performed at 10°C. In theory, the curve (Fig. 1) should shift to the right at lower temperatures. The majority of the trials in this study were performed over a 15 h period due to the distance from the laboratory to the field site (3 h round-trip plus sampling time). It was felt that even though an overnight incubation meant a 15 h incubation time, this was preferable to the changes which could occur in the biofilm by holding the samples 24 h prior to running a shorter assay. The importance of the killed control was shown by the light controls which were typically pale green in colour and had higher values than their dark counterparts, due to absorption at 480 nm by algal pigments. Higher control values than normal were noted in the ETS stimulated (amended) controls, particularly in the light-grown samples, but they were still well below the values measured in the ETS stimulated samples. The higher control values in the ETS stimulated controls were probably due to the ETS stimulators reducing the INT chemically (Packard and Williams, 1981). Although the effect of environmental toxicants on ETS activity has not been attempted in this study, work by Trevors et al. (1983) has shown that ETS activity (determined using direct microscopic counts of formazan deposits) of A n k i s t r o d e s m u s braunii decreased as exposure time to HgC12 increased. It is therefore quite possible that the amount of INT-formazan extracted from river biofilms will decrease on exposure to toxicants, allowing this assay to be used as an environmental monitor. Further work is needed in this area. We therefore conclude that experimental parameters must be carefully tested prior to performing the ETS assay. At our study site, (i) methanol is the best solvent, (ii) 0.02% INT is the suggested concentration, (iii) the incubation time should be kept to a reasonable minimum (e.g. less than 8 h) to avoid community changes within the biofilm, (iv) the pH of the INT solutions must be kept constant and (v) it is not necessary to add ETS stimulators to get a measurable response. We have found the method sensitive, easy and quick. Seasonal field data will be presented in a future publication.
ETS activity in river biofdms Acknowledgements --We thank Dr J. T. Trevors for suggesting the INT assay as an appropriate method for river biofilm work. We also thank S. E. Jones, D. Fiebig and M. Croy for useful suggestions. S. Blenkinsopp is supported by an NSERC Canada post-graduate scholarship and an Overseas Research Students Award for which she is most grateful. We are also thankful for the funding provided by the University College of North Wales for this work. REFERENCES
Blenkinsopp S. A. (1989) The ultra-structure, storage products and electron transport system activity of river biofilms. Ph.D. thesis, University of Wales, Bangor, Wales. Burton G. A. Jr and Lanza G. R. (1986) Variables affecting two electron transport system assays. AppL envir. Microbiol. 51, 931-937. Casida L. E. Jr (1977) Microbiol metabolic activity in soil as measured by dehydrogenase determinations. Appl. envir. Microbiol. 34, 630~36. Costerton J. W., Cheng K.-J., Geesey G. G., Ladd T. I., Nickel J. C., Dasgupta M. and Marrie T. J. (1987) Bacterial biofilms in nature and disease. A. Rev. Microbiol. 41, 435-464. Fiebig D. M. (1988) Riparian zone and stream water chemistries, and organic matter immobilization at the stream-bed interface. Ph.D. thesis, University of Wales, Bangor, Wales. Hobbie J. E., Daley R. J. and Jasper S. (1977) Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. envir. Microbiol. 33, 1225-1228. Jeffrey W. H. and Paul J. H. (1986) Activity measurements of planktonic microbial and microfouling communities in a eutrophic estuary. Appl. envir. Microbiol. 51, 157-162. 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. Jones S. E. and Lock M. A. (1989) Hydrolytic enzyme activity in the biofilms from two contrasted streams. Freshwat. Biol. 22, 289-296. Ladd T. I., Costerton J. W. and Geesey G. G. (1979) Determination of the heterotrophic activity of epilithic microbial populations. In Native Aquatic Bacteria: Enumeration, Activity and Ecology (Edited by Costerton J. W. and Colwell R. R.). American Society for Testing and Materials, Philadelphia, Pa. LeChevallier M. W., Cawthon C. D. and Lee R. G. (1988)
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Inactivation of biofilm bacteria. Appl. envir. Microbiol. 54, 2492-2499. Lock M. A. and Ford T. E. (1985) Microcalorimetric approach to determine relationships between energy supply and metabolism in river epilithon. Appl. envir. Microbiol. 49, 408--412. Lock M. A., Wallace R. R., Costerton J. W., Ventullo R. M. and Charlton S. E. (1984) River epilithon: toward a structural-functional model. Oikos 42, 10-22. Murray R. E., Cooksey K. E. and Pfiscu J. C. (1987) Influence of physical disruption on growth of attached bacteria. Appl. envir. Microbiol. 53, 2997-2999. Packard T. T. and Williams P. J. leB. (1981) Rates of respiratory oxygen consumption and electron transport in surface seawater from the northwest Atlantic. OceanoL Acta 4, 351-358. Quinn J. P. (1984) The modification and evaluation of some cytochemical techniques for the enumeration of metabolically active heterotrophic bacteria in the aquatic environment. J. appl. Bact. 57, 51-57. Schindler J. E., Williams D. J. and Zimmerman A. P. (1976) Investigation of extracellular electron transport by humic acids. In Environmental Biogeochemistry (Edited by Nriagu J. O.). Ann Arbor Science, Ann Arbor, Mich. Spun" A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31--43. Trevors J. T. (1984a) Electron transport system activity in soil, sediment and pure cultures. CRC Crit. Rev. Microbiol. 11, 83-100. Trevors J. T. (1984b) Effect of substrate concentration, inorganic nitrogen, O 2 concentration, temperature and pH on dehydrogenase activity in soil. Pl. Soil 77, 285-293. Trevors J. T., Mayfield C. I. and Inniss W. E. (1982) Measurement of electron transport system (ETS) activity in soil. Microb. Ecol. 8, 163-168. Trevors J. T., Mayfield C. I. and Inniss W. E. (1983) The use of electron transport system activity for assessing toxicant effects on algae. Wat. Air Soil Pollut. 19, 361-367. Vosjan J. H. (1982) Respiratory electron transport system activities in marine environments. Hydrobiol. Bull. 16, 61-68. Zimmermann 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.
0043-1354/90 $3.00+ 0.00 Copyright © 1990 Pergamon Press plc
THE MEASUREMENT OF ELECTRON TRANSPORT SYSTEM ACTIVITY IN RIVER BIOFILMS S. A. BLENKINSOPP*,t and M. A. LOCK School of Biological Sciences, University College of North Wales, Bangor, Gwynedd LL57 2UW, Wales (First received January 1989; accepted in revised form October 1989)
Abstract--The factors affecting the measurement of electron transport system (ETS) activity in river biofilm through the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan) have been studied. Methanol extracts INTformazan more effectively than either propanol or ethanol. A concentration of 0.02% INT was chosen and samples should be incubated for less than 8 h. ETS activity is optimal at a circumneutral pH. ETS stimulators (NADH, NADPH and succinate) added as a check of the assay produced an increase in INT-formazan indicating that ETS activity was being measured. This assay is quick, easy to use and ideal for field studies. Key words--method, river biofilms, electron transport system activity, INT
INTRODUCTION
River biofilm (epilithon, periphyton) is a complex community of microorganisms ranging from strict autotrophs to strict heterotrophs, depending on environmental conditions present at the time. This mixed microbial consortium exists within a polysaccharide matrix, which is thought to play a role in nutrient trapping, transport between cells and extracellular enzyme activity (Lock et al., 1984; Costerton et al., 1987; Jones and Lock, 1989). Thus, in order to obtain realistic measurements of activity within a biofilm, it is necessary to keep the biofilm intact so as not to disturb the intracellular and extracellular processes which occur within it. All respiring microorganisms possess an active electron transport system (ETS), and ETS activity is a measure of the ability of microorganisms to reduce an indicator under defined conditions. Tetrazolium salts such as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5phenyl tetrazolium chloride (INT) are commonly used as ETS indicators. INT is water-soluble in its oxidized form and, when reduced, forms a water insoluble deposit, iodonitrotetrazolium formazan (INT-formazan), within the cell. These red formazan deposits have been used to distinguish between respiring and non-respiring aquatic bacteria using light microscopy (Zimmermann et al., 1978). Extracted formazan can be measured spectrophotometrically to quantify ETS activity in marine (Vosjan, 1982), estuarine (Jeffrey and Paul, 1986), freshwater planktonic and benthic (Jones and Simon, 1979) and soil environments (Trevors et al., 1982). This paper deals *N~e Dither. tPresent address: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. 441
with the spectrophotometric approach and its application to fiver biofilm activity. In his review article, Trevors (1984a) notes that the INT assay is (i) operable over a wide range of environmental tempertures, (ii) measures both aerobic and anaerobic respiration and (iii) does not need the addition of substrates such as glucose or yeast extract to stimulate ETS activity. These features make the INT assay an attractive field method, particularly when dealing with thick biofilms in which anaerobic respiration may occur in the deeper layers. Also, not having to add an external substrate to demonstrate activity is most desirable, because of difficulties involved in determining a suitable substrate type and concentration for all the microbes in a mixed microbial consortium. There are currently two main spectrophotometric methods used to measure ETS activity. The first, developed for marine plankton work, involves incubating an enzyme extract prepared from the cells with an INT solution and a substrate solution containing ETS stimulators (NADH, N A D P H and succinate). This method gives the maximum potential ETS activity of the community. The second method, described for soil work, involves incubating the "intact" microbial community in an INT solution without ETS stimulators. This will give lower values than the first method because it measures the in situ ETS activity. The first method was not suitable for our needs because it would have meant disrupting the biofilm, and therefore its functioning (Ladd et al., 1979; Murray et al., 1987), before measuring its ETS activity. Recent literature on antibiotic resistance (Costerton et al., 1987) and biocide effectiveness (Costerton et al., 1987; LeChevallier et al., 1988) has highlighted problems with the extrapolation of techniques
442
S.A. BLENKINSOPPand M. A. LOCK
b e t w e e n different m i c r o b i a l c o m m u n i t i e s , in particular p l a n k t o n i c a n d a t t a c h e d c o m m u n i t i e s (biofilms), a n d highlights the need to evaluate m e t h o d s i n t e n d e d for biofilm work. In this p a p e r we i n v e s t i g a t e d the effect o f e x t r a c t i o n solvent, I N T c o n c e n t r a t i o n , length o f i n c u b a t i o n , w a t e r p H a n d E T S s t i m u l a t o r s o n E T S activity m e a s u r e d in intact river biofilm using a m o d i f i c a t i o n o f a m e t h o d used o n soil by T r e v o r s et al. (1982). MATERIALS AND METHODS
Biofilm studies Biofilm was grown on acid-washed, pre-combusted glass beads (1.5 mm dia) strung on nylon monofilament line. Bead strings were 32 cm long (15.27 cm 2 surface area). With the exception of the beads used in the solvent trial, which were colonized in a freshwater aquarium, the bead strings were tied onto perspex plates and inserted into plastic tubes (10.2era dia, 1.5m long). The tubes were fastened to a specially designed frame on the river bed (Blenkinsopp, unpublished Ph.D. thesis) to facilitate sampling, especially during high water periods. Bead strings were either light-incubated in tubes with perspex windows to allow autotrophic/heterotrophic growth, or dark-incubated to allow primarily heterotrophic growth in the fourth order Clywedog river, North Wales. Site details have been published (Lock and Ford, 1985). All bead strings were colonized for a minimum of 6 weeks before use. Standard assay followed in the trials INT does not dissolve readily in water, thus a 30-min sonication period (Dawe Sonicleaner) was used routinely, pausing frequently to shake the vessel vigorously and break up the powdery clumps. The pauses also served to prevent the solution from over-heating. In earlier trials gentle heating ( 6 0 ° 0 followed by sonication resulted in loss of activity when compared with sonication alone (unpublished data). Since INT is light-sensitive, it was kept in foil-covered vessels throughout the assay. Bead strings were placed in sterile wide-mouthed laboratory jars (500ml) containing 100ml of the appropriate concentration of INT (Aldrich) in river or aquarium water, where appropriate. Controls were prepared by adding bead strings to 2% formaldehyde in water. The controls were prepared a minimum of 30 rain prior to start of the assay and then rinsed briefly in water before being placed in INT solution. (All water was filter-sterilized using 0.2/am Whatman filters.) Incubations were performed at a known temperature for a known time period. After the incubation period each string was rinsed in water to remove any unbound INT-formazan and the beads slipped off the string into a vial containing 3-10 ml of chilled methanol. Each vial was sonicated, with pauses, for 5 min to disrupt the polysaccharide matrix and allow easy solvent access. Vials were then placed in a freezer for I h for extraction. Checks were made periodically to ensure that the solvent did not become saturated. If the colour of the extract approached that of the highest standard, a known volume of solvent was added to dilute the colour accordingly. The solvent containing the extracted INT-formazan was then briefly vortexed and filtered through a Whatman G F / F filter. At this point the solvent extract is stable for several days if refrigerated. The INT-formazan extract was measured spectrophotometrically at 480 nm against a solvent blank. A stock solution of 30#g/ml INT-formazan (Sigma) in solvent was used to prepare a standard curve of 0-30 # g/ml (corr. coeff. = 0.99). The final ETS activity results were calculated by subtracting the killed controls from the sample values and expressed per cm 2 for all trials excluding the ETS stimulation trial results, which were expressed per cell.
Effect o f type of extraction solvent on biofilm 1NT-formazan recovery
Fifteen bead strings, including 3 killed controls, were incubated at room temperature (17.6°C) for 1 h in 0.02% INT at pH 7.2. Four replicates and one killed control were extracted with each solvent and read against a standard curve of INT-formazan prepared with the appropriate solvent. The solvents compared were ethanol, methanol and propanol (GPR).
Effect o f I N T concentration on dark-grown biofilm ETS actiriO' A concentration of 0.02% INT was used by Zimmermann et al. (1978) to determine the proportion of bacterial cells actively respiring based on counts of cells with red formazan deposits. This concentration was used as a base-line value for this trial. Dark-grown beads usually had a less visible biofilm than the light-grown material, thus we were concerned with obtaining a measurable activity signal from the dark community. A concentration trial was therefore performed on the dark community to optimize assay conditions and the concentration chosen was used for both communities in later trials. Three replicates and I killed control per INT concentration (0.2, 0.02. 0.002%) were incubated at 1OC for 15 h at pH 7.2. Effect o[ incubation length on light-grown bio[ilm E T S activit)' Three replicates and l killed control per time period (1,4, 8, 12 and 16h) were incubated in 0.02% INT, pH 7.2, at 10C. Effect of pH on light-grown biofilm ETS activity The buffering capacity of the biofilm may have an effect on ETS activity. Fiebig (unpublished Ph.D. thesis) noted that river biofilm can buffer pH-adjusted river water in the direction of the unadjusted pH. A preliminary pH trial of bead strings in pH-adjusted fiver water showed that with the surface area of biofilm used per ml of water (15.27cm2/100ml) in these trials, the water pH was not altered when compared with the control of adjusted water alone (unpublished data) over an 18 h period. With this confirmed, the pH trial was performed. Three replicates and one killed control per pH level (4.0, 5.7, 7.0 and 9.0) were incubated in 0.02% INT for 15h at 10C. The 0.02% INT solution was adjusted to the appropriate pH with 0.05 N sodium hydroxide or 0.05 N sulphuric acid. Effect of E T S stimulators on biofilm ETS activity The purpose of this trial was to confirm that the assay was measuring ETS activity, through the use of known ETS stimulators. This trial was performed on both light-grown and dark-grown biofilms incubated at 10°C, for 13h at pH 7.2. Beads were incubated in 0.02% INT solution only (non-amended treatment) and 0.02% INT solution with the following additions (amended treatment): N A D P H 0.08mg/ml, NADH 0.5mg/ml and sodium succinate 4.0 mg/ml (Quinn, 1984). There were 8 replicates per treatment and 4 killed controls per treatment. To increase the number of replicates, the bead strings were unstrung and the beads well mixed to avoid any string effects. A mean of 36 randomly chosen beads were used per replicate. Cell counts were performed on light and dark-grown biofilm samples, collected from each treatment at the end of the incubation period, to ensure that any trends noted were not simply due to differences in cell numbers per cm 2. Cells were counted using the method of Hobbie et al. (1977), following a 90 s sonication in 2% formaldehyde in filter-sterilized river water to remove cells from the beads. The ETS activity values per cell are only a relative measure because measured ETS activity is due to both intracellular and extracellular enzyme activity (Trevors, 1984a). Also, enzymes, including those involved in electron transport, may be released from dead cells and may survive for a limited time in soil, sediment
ETS activity in river biofilms
443
(Trevors, 1984a) and river biofilms (Lock et al., 1984). However, because the enzymes responsible for intracellular and extracellular ETS activity arise from cells within the biofilm, and the acridine orange direct counts method includes all intact cells, without distinguishing between those that are viable and non-viable, it is reasonable to express ETS activity relative to cell numbers.
such as the 0.2% concentration, may be toxic to fiver biofilm and should not be used in its study. A concentration of 0.02% was used for the remainder of the assays.
Importance of the killed control
INT-formazan was produced asymptotically over the 16 h incubation period at 10°C (Fig. 1); its rate of production decreased at about 8 h of incubation.
The control chosen for these assays was a killed control whereby the control sample of biofilm was killed with formaldehyde prior to incubation under identical conditions as the samples. It is very important to have a killed control because (i) chemical INT reduction may occur in the sample environment, e.g. in systems containing humic acids, nonenzymatic formazan formation may occur by their activity (Schindler et al., 1976), (ii) in light-grown material, algal pigments are extracted with organic solvents and absorb at the wavelength used for formazan detection and (iii) ETS stimulator such as succinate, NADH and NADPH are known to reduce certain brands of INT chemically (Packard and Williams, 1981).
Effect of incubation length on biofilm ETS activity
Effect of p H on biofilm ETS activity Water pH was shown to significantly affect biofilm ETS activity (ANOVA, P <0.05). INT-formazan values were barely detectable at pH 4 and increased to pH 7 (Fig. 2). There was a slight but not significant increase at pH 9. Thus, it appears that a circumneutral pH is optimal for INT-formazan production in fiver biofilms.
Statistical analysis Standard curve regressions and statistical analyses were performed using Statgraphics Version 2.1 (STSC Inc., Rockville, MD 20852, U.S.A.). RESULTS
Effect of extraction solvent on biofilm INT-formazan recovery Methanol extracted significantly more INTformazan than propanol and ethanol (ANOVA, P < 0.05). The INT-formazan levels were 4.88 + 0.41, 3.45___ 0.16 and 2 . 8 8 _ 0.21 pg/cm 2, respectively ( m e a n _ SE, n = 4). Propanol and ethanol had approximately the same extraction capability (multiple range analysis). HPLC-grade methanol (May & Baker) was therefore used in the remainder of the assays.
Effect of I N T concentration on biofilm ETS activity The three concentrations of I N T yielded INTformazan values which were not equal to each other (ANOVA, P < 0.05) and were all significantly different from each other (multiple range analysis). 0.02% I N T yielded the most INT-formazan (6.05 _ 0.46 #g/ cm z) followed by 0.002% I N T (3.47 + 0.39/~g/cm2); 0.2% I N T had the lowest values (1.57 _ 0.22 #g/cm 2) ( m e a n _ SE, n = 3). High concentrations of INT, 12
Effect of ETS stimulators on biofilm E T S activity INT-formazan values were 2.77 _ 0.08 and 1.81 -t-0.05 ( x 1044-13g/cell), for the light and darkgrown biofilms, respectively, in the "non-amended treatment" (INT only) and were 6.82 _+ 0.50 and 3.64 _+ 0.12 ( x l0 -~3 g/cell) for the light and darkgrown biofilms, respectively, in the "amended treatment" (INT plus ETS stimulators) (mean _+ SE, n = 8 ) . INT-formazan values were significantly greater in the amended treatments thus firmly supporting that the assay is measuring ETS activity in fiver biofilms (two-way ANOVA, P < 0.05). Also, the INT-formazan values were significantly greater in the light-grown biofilm than in the dark-grown biofilm (two-way ANOVA, P < 0.05). Cell counts were very similar in the light and dark-grown biofilms for both non-amended and amended treatments (4.7-6.3 x 10 7 cells/cm2).
FIELD
DATA
Representative field data from the Clywedog fiver are shown in Table I. Mean INT-formazan values ranged from 0.58/~g/cmE/h in a dark-grown early spring biofilm to 2.06/zg/cm:/h in a light-grown late spring biofilm. 6
[j+j+ ,o
2
3
I
I
i
I
4
I
t
J
I
i
i
8 Incubotion
J
I
12
I
I
t
I
16
(h)
Fig. 1. Effect of incubation length on fiver biofilm ETS activity (mean _ SE, n = 3).
0 3
J*l+lll 4
III 5
Irlll
IIJ 6
IlilJ 7
IIII 8
II 9
pH
Fig. 2. Effect of pH on river biofilm ETS activity (mean + SE, n = 3).
444
S. A. BLENKINSOPP and M. A. LOCK Table 1. Representative electron transport system (ETS) activity results from Clywedog river (North Wales) biofilms. Sample statistics are the mean ± SE, with sample size in parentheses
Date
pH
Incubation temp. ('C)
1/2:88 30/3/88 12/5/88
7.2 7.2 7.2
10 10 12
,ug INT-formazan/cm2/h
Incubation time (h)
Artificial substratum
Light-grown biofilm
Dark-grown biofilm
1 13 3
Glass beads Glass beads* Spurr resint
0.94 ± 0. I 0 (3) ND 2.06 ± 0.08 (4)
ND 0.58 ± 0.02 (7) t.87 ± 0.19 (4)
ND, not determined. *The beads were removed from the string for this incubation. +Transmission electron microscope epoxy resin (Spurt, 1969). DISCUSSION
The factors affecting the measurement of ETS activity in river biofilm have been investigated. Methanol was the superior solvent and extracted INT-formazan from the biofilm more effectively than either ethanol or propanol. This finding is in contrast with Burton and Lanza (1986) who used another commonly used tetrazolium salt, triphenyltetrazolium chloride (TTC), to measure ETS activity in freshwater lake sediments. The extraction performance of solvents used for TTC-formazan recovery was tetrachloroethylene-acetone > propanol > ethanol > methanol. Tetrachloroethylene-acetone was not used in this study, because tetrachloroethylene is a suspected carcinogen (Aldrich Chemical Co. Ltd Catalogue 1987-1988). Whether the difference in solvent extraction ability is due to the type of tetrazolium salt or the system in which it was converted to formazan is unclear. However, it does emphasize the need for preliminary trials prior to setting assay procedures. Concentrations of up to 0.4% INT (maximum solubility in water) have been reported in an estuarine study (Jeffrey and Paul, 1986). However, this concentration (0.4%) was based on a soil study by Trevors (1984b) which revealed that dehydrogenase (ETS) activity is positively correlated with the initial concentration of INT. Our results show that in a river biofilm, INT-formazan does not increase with the initial concentration of INT. The lowest concentration used in the study (0.002% INT) yielded more formazan than the highest concentration (0.2%) suggesting that 0.2% may be toxic. The middle concentration, 0.02% INT, yielded the greatest amount of extracted formazan and was chosen for the remainder of the study. Ideally, to avoid changes within the biofilm community over the course of the assay, the incubation time chosen should be kept to a minimum. Casida (1977) used triphenyltetrazolium chloride to measure ETS activity in soils, and noted population shifts for both aerobic and anaerobic bacteria during 24 h incubations at 37~'C with yeast substrate. The dehydrogenase curves showed a change at about 9-12 h and increased substrate concentrations did not produce linear activity with time. A change at about 8 h was noted in this study (Fig. 1). The biofilm population was not monitored over the time course; however, it is possible that a population shift could
have occurred. Based on the time course, incubation should be less than 8 h. However, under low growth or low temperature conditions, for example, it may be necessary to incubate samples longer until measurable INT-formazan formation occurs. Incubations in this study were performed at 10°C. In theory, the curve (Fig. 1) should shift to the right at lower temperatures. The majority of the trials in this study were performed over a 15 h period due to the distance from the laboratory to the field site (3 h round-trip plus sampling time). It was felt that even though an overnight incubation meant a 15 h incubation time, this was preferable to the changes which could occur in the biofilm by holding the samples 24 h prior to running a shorter assay. The importance of the killed control was shown by the light controls which were typically pale green in colour and had higher values than their dark counterparts, due to absorption at 480 nm by algal pigments. Higher control values than normal were noted in the ETS stimulated (amended) controls, particularly in the light-grown samples, but they were still well below the values measured in the ETS stimulated samples. The higher control values in the ETS stimulated controls were probably due to the ETS stimulators reducing the INT chemically (Packard and Williams, 1981). Although the effect of environmental toxicants on ETS activity has not been attempted in this study, work by Trevors et al. (1983) has shown that ETS activity (determined using direct microscopic counts of formazan deposits) of A n k i s t r o d e s m u s braunii decreased as exposure time to HgC12 increased. It is therefore quite possible that the amount of INT-formazan extracted from river biofilms will decrease on exposure to toxicants, allowing this assay to be used as an environmental monitor. Further work is needed in this area. We therefore conclude that experimental parameters must be carefully tested prior to performing the ETS assay. At our study site, (i) methanol is the best solvent, (ii) 0.02% INT is the suggested concentration, (iii) the incubation time should be kept to a reasonable minimum (e.g. less than 8 h) to avoid community changes within the biofilm, (iv) the pH of the INT solutions must be kept constant and (v) it is not necessary to add ETS stimulators to get a measurable response. We have found the method sensitive, easy and quick. Seasonal field data will be presented in a future publication.
ETS activity in river biofdms Acknowledgements --We thank Dr J. T. Trevors for suggesting the INT assay as an appropriate method for river biofilm work. We also thank S. E. Jones, D. Fiebig and M. Croy for useful suggestions. S. Blenkinsopp is supported by an NSERC Canada post-graduate scholarship and an Overseas Research Students Award for which she is most grateful. We are also thankful for the funding provided by the University College of North Wales for this work. REFERENCES
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