Mierob Eeol (1986) 12:193-203

MICROBIAL ECOLOGY

| Springer-VerlagNew York Inc. 1986

Relationship Between Microbial Activity of Stream Sediments, Determined by T h r e e D i f f e r e n t M e t h o d s , a n d A b i o t i c V a r i a b l e s J. H. Baker F.B.A. River Laboratory, East Stoke, Wareham, Dorset BH20 6BB, England

Abstract.

Microbial activity of stream sediments has been determined by three distinct methods: phosphatase levels, maximum uptake velocity of radiolabeled glucose, and carbon dioxide production rates. These methods have been applied to different types o f sediment (mud, sand, gravel) from the same stream and to 5 samples from two different streams for comparison. Temperature, discharge, and 8 other abiotic variables for each sample were also determined. The 3 activity methods correlated closely with each other and were measured with a similar precision. Phosphatase activity could be predicted for all sites from bulk density. The largest proportion of the variance associated with carbon dioxide production was explained by variations in percent o f organic matter, but the relationship did not hold for all streams. Maximum uptake velocity, compared with the other 2 activity measurements, was poorly explained by any of the abiotic variables.

Introduction Many techniques for the determination of microbial activity in sediments have been suggested, but few studies have compared several of these techniques on a range of different sediments. Consequently, in this work, three techniques have been used on the same samples of mud, sand, and gravel sediments from a small stream in central southern England. The techniques were measurement of alkaline phosphomonoesterase activity, heterotrophic potential, and carbon dioxide evolution. Phosphatase activity has been suggested as an index of microbial activity in both sediments [20] and soils [5, 1 1]. Methods for its measurement have been critically reviewed by Malcolm [ 17]. It is recognized that several enzymes are involved in the release of organically bound phosphate, but for convenience those enzymes are hereafter simply referred to as phosphatase. Moreover, this work is concerned with total phosphatase activity regardless of the many locations within the system where the enzymes might be found [2]. Heterotrophic potential may be expressed as the maximum uptake velocity ( V ~ ) of an added substrate, in this case glucose. Its application to sediments was pioneered by Harrison et al. [12], and it has subsequently been used extensively as a relative measure of microbial activity [1, 8]. Carbon dioxide production rates for stream sediments have seldom been reported [9], although

194

J.H. Baker

t h e t e c h n i q u e is f r e q u e n t l y u s e d f o r soil [ 19, 23]. I t suffers f r o m t h e d i s a d v a n tages t h a t all l i v i n g o r g a n i s m s , n o t j u s t m i c r o b e s , p r o d u c e CO2 a n d t h a t far less is p r o d u c e d u n d e r a n a e r o b i c c o n d i t i o n s t h a n u n d e r a e r o b i c c o n d i t i o n s . N e v e r t h e l e s s it is a d i r e c t m e t h o d , c o m p a r e d w i t h t h e o t h e r t w o i n d i r e c t m e t h ods. I n a d d i t i o n to t h e s e 3 m e a s u r e m e n t s o f m i c r o b i a l a c t i v i t y , 8 a b i o t i c v a r i a b l e s were d e t e r m i n e d for each sample. T h e abiotic variables h a v e b e e n d i v i d e d into 2 g r o u p s , e n v i r o n m e n t a l v a r i a b l e s - - t e m p e r a t u r e a n d d i s c h a r g e (flow r a t e ) and s e d i m e n t v a r i a b l e s - - b u l k density (dry weight/total v o l u m e including natural v o i d s ) a n d t h e p e r c e n t a g e s o f silt, s a n d , grit, g r a v e l , a n d o r g a n i c m a t t e r . T h e a i m s o f t h e w o r k w e r e t o see h o w w e l l t h e d e t e r m i n a t i o n s o f a c t i v i t y a g r e e d with each other and w h e t h e r they could be statistically explained and predicted by any o f the abiotic variables.

Materials and M e t h o d s

Sample Sites Most of the samples were obtained from Bere Stream (National Grid reference SY 858925), a relatively unpolluted small stream in central southern England. Bere Stream is spring-fed from a chalk aquifer and consequently has a pH averaging 8. I and an alkalinity between 4.0 and 4.6 meq 1-1. The sediment of Bere Stream is composed of small juxtaposed areas of mud, sand, and gravel [ 15]. Grab samples were obtained from all types of sediment at different times over a 2 year period, except that samples containing large pieces of recognizable plant material were discarded. For comparison, 4 samples were also obtained from Ober Water (SU 225035), a soft water stream with a pH range from 6.1-7.2 and an alkalinity of 0.2-0.6 meq 1-1 [18], and I from Slepe Stream (SY 962856), which drains an acid heath and has a pH of 5.3-5.9 (alkalinity 0.15 meq 1-1). Activity measurements were always started within 1 hour of sample collection after transport to the laboratory in 500 ml screw top glass jars.

Heterotrophic Potential The technique used has been described by Griffiths et al. [8]. Briefly, the sediments were diluted 1:1000 v/v with sterilized stream water and 10 ml aliquots were dispensed into 50 ml serum bottles. Uniformly labeled 14C-glucose (Amersham) was added to duplicate bottles to give final concentrations of 12.5, 25, 50, and 100 gg l -~ . The bottles were incubated at 15"{2for periods that varied according to the expected activity of the sample, but typically 5 hours. The activity was terminated with 5 N sulphuric acid (0.2 ml), and respired carbon dioxide was trapped with 2-phenylethylamine (0.15 ml) while the bottles were being shaken in the cold for at least l hour. Controls were prepared at each glucose concentration by adding the sulphuric acid at the beginning of the incubation. Labeled bacteria (and other particulates) were harvested on 0.2 #m porosity membrane filters, and both cells and respired 14CO2 were assayed on a Packard 2425 liquid scintillation counter using Koch-Light cocktail no. 502. Maximum uptake rate for glucose ( V ~ ) was determined by the LeeWilson modification of the Lineweaver-Burk plot [7].

Alkaline Phosphatase Assay Phosphatase was released from the sediment by ultrasonic treatment of a sediment slurry in 0.1 M Tris (pH 8.0) at 20 kHz and 50 W [20]. The optimum time for sonication was found to be 45

Microbial Activity of Stream Sediments

195

s. Phosphatase activity was determined using p-nitrophenyl phosphate (PNP) as a substrate and assaying the colored nitrophenol produced spectrophotometrically at 418 nm. The final concentration of PNP prior to incubation was 0.001 M, and 6 replicate tubes were incubated for 1 hour at 25*(2. The pH used (pH 8.0) was similar to that o f the natural water and did not vary during the incubation period. The reaction was terminated with sodium hydroxide (2 N), and calcium chloride solution (0.5 M) was added to prevent interference by humic compounds [24]. Six control tubes were prepared by adding the hydroxide at the beginning of the incubation. The mean reading of the control tubes was subtracted from each of the active tubes.

Carbon Dioxide Production The production rates of CO2 (under aerobic and anaerobic conditions) were determined by gas chromatography as described by Baker et al. [1]. A Packard 438 chromatograph was used with a 2m Porapak N column connected to a flame ionization detector. The oven was operated isothermally at 40"C, and the injection port and detector were at 150* and 250"C, respectively. For detection purposes the CO2 was reduced to methane by a hot nickel catalyst placed between the end of the column and the detector. Sediment subsamples (2.5 ml) were placed in Bijou bottles and sealed With serum closures. They were incubated at 15*(2 in the dark, and headspace samples (0.3 ml) were removed daily for analysis over a 72 hour period. Anaerobic bottles were gassed with helium for 1 rain before incubation. Integration of the CO2 peaks was carried out by a Spectra-Physics 4100 integrator. Four replicate subsamples were incubated aerobically and 4 anaerobically. The CO2 production rate was calculated by regression analysis.

Sediment Properties After oxidation of the organic matter with peroxide overnight, the mineral component of the sediments was fractionated by wet sieving through sieves with apertures of 10, 2, 1, and 0.125 ram. The fractions retained by these sieves were arbitrarily designated as pebbles, gravel, grit, and sand, respectively. The fraction passing and 0.125 m m sieve was obtained by difference and designated silt, although it also included all of the clay. The percentage organic matter was estimated by loss-on-ignition at 550"C in a muffle furnace of a sample of known weight previously dried at 105"C for 24 hours. It is recognized that this method will overestimate organic matter slightly due to the decomposition of carbonates and clay minerals, but this error is not thought to be serious.

Environmental Variables Discharge (mas - l) of Bere Stream was calculated from a continuous record of water depth using calibrations obtained previously with a current meter. Water temperature at Bere Stream was continuously recorded with a mercury-in-steel thermometer. Spot sediment temperatures were obtained from each site when samples were collected.

Statistics Correlation coefficients were determined nonparametrically by Spearman's rank method [21 ]. Also the coefficient of variation (CV) for each activity was determined from the appropriate formula. Thus for phosphatase SD • 100 CVt - - .g•

Results for V~,~ refer to mineralized 14CO2 only

(ng glucose g-' h-') Aerobic CO2 production (nmol g-' h-')

Vm~

Phosphatase (~mol PNP g-' h-')

Range

12.9 10.7

7.25-236

100

12.8

5.14-23.6

4.62-34.2

2.48-43.2

Range

Coefficient of variation (%) Mean

7.03-366

0.0609-5.49

Activity value

94.5

1.78

Mean

Table 1. The mean, range, and precision of microbial activity values from all sediment types in Bere Stream

Microbial Activity of Stream Sediments

197

Where SD = standard deviation, .~ = mean and n = no. of subsamples. For carbon dioxide production CV2

SE(bt) • 100 bt

where bi = slope of the regression of CO2 concentration on time and SE(bt) was the standard error of b~. V~.~ is the reciprocal of the slope (b2) of a Lineweaver-Burk plot. To a first order approximation, the variance of 1/b = variance (b)/b4[ 13]. Hence the coefficientof variation of Vm,~(CV3)= SE(1/b2) x 100 l/b2

SEC02)x 100 b22 x l/b2

SEC02)x 100 b2

9Linearregression analyses have been used to determine the proportion of the variation in microbial activity explained by sediment variables. This proportion is called the coefficientof determination (r2). It is acknowledgedthat regression analysis assumes that the independent variable is measured Without error; although this assumption is not true in the current analyses, the effect is not thought to be significant.

Results The total n u m b e r o f sediment samples from Bere Stream studied over a 2 year Period was 34, o f which 17 were " m u d , " i.e., in which the silt fraction exceeded 6 5%. The means, ranges, and levels o f precision for the 3 estimates o f microbial activity are given in Table 1. The results are expressed per gram o v e n dry weight. M a x i m u m glucose uptake rates (Vmax) were d e t e r m i n e d on the first 15 samples f r o m both i n c o r p o r a t e d and mineralized r a d i o c a r b o n counts together, and from mineralized 14CO2 alone. As there was a very strong correlation (P < 0.001) between these 2 measures o f V . . . . subsequent determinations ~ were calculated f r o m mineralized ~4CO2 alone. All o f the glucose uptake experiments resulted in significant regressions, a n d no negative slopes were encountered as reported by others [6, 16]. Total carbon dioxide p r o d u c t i o n rates were d e t e r m i n e d on only 14 o f the samples. A n a e r o b i c CO2 p r o d u c t i o n rates were 49.1% +__ 4.2% (95% confidence limits) o f aerobic CO2 p r o d u c t i o n rates. High correlations were f o u n d between each possible pair o f the 3 activity estimates (Table 2), although Vm~x a n d c a r b o n dioxide p r o d u c t i o n rates were not correlated at the 0.1% level. The extent to which each activity estimate Was correlated with the e n v i r o n m e n t a l a n d sediment variables was also calCulated. N o n e o f the activity m e a s u r e m e n t s was correlated with either o f the environmental variables, but all 3 activity m e a s u r e m e n t s were significantly correlated with all 6 sediment variables (Table 3). T o determine the extent to Which each activity estimate can be explained by the sediment variables, linear regressions were carried out and the coefficients o f d e t e r m i n a t i o n (r 2 values) are given in Table 4. The models which describe the data best were obtained after logarithmic t r a n s f o r m a t i o n o f phosphatase a n d Vmax, but no transformation was necessary for the aerobic CO2 p r o d u c t i o n rates. The equations for the best fitted regressions are given below:

J. H. Baker

198 Table 2. activity

Levels o f correlation between the 3 estimates o f microbial

Activities correlated

Spearman rank correlation coefficient (rs)

No. o f pairs (n)

Probability (P)

Phosphatase and Vma~ Phosphatase and COz CO2 and Vma~

0.724 0.811 0.783

28 14 12