microbial extracellular enzyme activities in humex lake skjervatjern

Environment International, Vol. 18, p. 637-647, 1992 0160-4120/92 $5.00 +.00 Printed in the U.S.A. All rights reserved. Copyright ©1992 Pergamon Press Ltd.

M I C R O B I A L E X T R A C E L L U L A R ENZYME ACTIVITIES IN HUMEX LAKE SKJERVATJERN

U. MOnster Max-Planck-lnstitute for Limnology, D-2320 PI0n, F.R.G.

El 9207-168 M (Received 31 July 1992; accepted 3 September 1992)

Two microbial ¢xtracellular enzyme activities (MEEA) were studied in HUMEX Lake Skjcrvat- jcrn: acid phosphatase (APHA) and leucine aminopeptidase (LeuAMPA). Both enzyme activities varied in the vertical and horizontal scale in both lake sites. APHA varied in the acidfied Basin A between 945-1706 nmol L -x h -x and LeuAMPA between 3.7-25 nmol L "t h'L Both MEEA reached maxima in 0.5 m depth. In the control site (Basin B), APHA was lower by a factor of two, and varied between 156-669 nmol L t h x. LouAMPA reached similar values as in Basin A and varied between 7.8-34.8 nmol L -x h-L Maxima of APHA were found in the upper layer (0-2 m), while LeuAMPA had only one distinct maxima at 2-2.5 m depth. The number of bacteria (AFDC) varied between 4.4-8.8 10 ~ cells mL -x and was not significantly different in either side, but both had maxima in the thermocline. Highest specific LeuAMPA activities were found in the thermocline (3.2-4.5 fmol L a h a cell "l) in both sides and varied between 0.4-4.5 fmol L 1 h "l cell "t in both water columns. The main contributor (60-70%) to LeuAMPA was found in the microplankton fraction, retained on Nuclepor¢ filters with pore sizes between 2.0-0.2 ~tm. APHA was retained less even on a filter with pore size smaller than 0.2 Ixm. About 50-70% of APHA passed through 0.2 Ixm-0.1 Ixm Nuclepore filters and could be found in the dissolved organic matter (DOM) fraction. APHA and bacteria counts (AFDC) showed a distinct gradient from the littoral zone to the pelagial in the surface water samples (0.2 m depth). APHA and LeuAMPA are regarded as important regulators for nutrient availabilty to microplankton. However, all data from vertical and horizontal samples showed that Lake Skjervatjern is a strongly gradient-dominated aquatic ecosystem. Watershed-littoral effects arc more pronounced in the shallow, acidified Basin A than in the control side, Basin B.

INTRODUCTION

Dissolved organic matter (DOM) is a main carbon and energy source in many aquatic environments (Wetzel 1983; MUnster and Chr6st 1990). Carbon of DOM may exceed that of particulate organic matter (POM) by a factor of 10-100 (Thurman 1985). This ratio is much more pronounced in humic than in clear water systems. In such environments, DOM com- prises the main substrate pool for microheterotrophs. Among them, bacteria are the most efficient utilizer of this DOM (Azam and Cho 1987). They play a

Present address: Univv,'sity of Hclsinki, Lammi Biological Station, SF-16900 Lammi, Finland.

central role in the microbial food web (Pomeroy and Wiebe 1988; Azam etal. 1983). In clear water systems, bacterial activity and biomass are closely coupled to phytoplankton development and grazing activities by phagotrophs (Azam and Cho 1987, Azam et al. 1983; Riemann and S~ndergaard 1984; Chr6st etal. 1989). However, in humic waters, the autochthonous production of DOM by autotrophs may not cover the carbon and energy requirements of bacteria (Hessen 1992a; Hessen etal. 1990; Salonen 1981; Salonen et al. 1992; Tranvik 1989). Humic waters may have a higher carrying capacity for bacteria compared to clear water systems (Tranvik 1992). External allochthonous carbon, energy, and nutrient sources are related to this higher carrying

637

638 U. Mtinster

capacity (Hessen 1985; Salonen and Hammar 1986; Tranvik 1992). However, about 80-90% of this allochthonous material consists of DOM and is recalcitrant to microbial utilization (Wetzel 1984, 1990). High molecular weight organic compounds are the dominant solutes in this DOM (MOnster 1985; Wet- zel 1990). There is a lack of basic knowledge about the mechanisms supporting this higher bacteria-carrying capacity in humic waters (MUnster 1991). Infor- mation is also needed on the effects of acidification on food web structures and interactions in humic brown- water lakes.

The objectives of this project were to study the distribution and variation of microbial extracellular enzyme activities (MEEA) in two different basins of the HUMEX Lake Skjervatjern and to examine whether MEEA plays an important role in heterotrophic processes in Lake Skjervatjern.

MATERIAL AND METHODS

Samples were taken from HUMEX Lake Skjervat- jern in southwestern Norway. This lake was divided into two parts with a plastic curtain. Basin A is artifi- cially acidified by irrigating the catchment area with sulfuric and nitric acid. Basin B is used as a control side. A more detailed description of the lake is given by Gjessing (1992). Water samples were taken in vertical and horizontal profiles with a Ruttner sampler. After sampling, lake water was immediately filtered through a 100 I.tm plankton net into 1L plastic bot- tles washed with acid and distilled water. Samples were kept in coolers during sampling and until analyses started. Data about hydrological, physical, chemical, and biological parameters can be found in other contributions.

Microbial extracellular enzyme assays were performed as described by Hoppe (1983) and modified according to Miinster et al. (1989). Two fluorogenic substrates from Sigma were used to study the Leucine- aminopeptidase activity (LeuAMPA) and the acid phosphatase activitiy (APHA): L-leucine-7-amido- 4-methyl-coumarin (Leu-AMC) and 4-methylum- belliferylphosphate (MUF-PO4). For stock solutions, MUF-PO4 was dissolved in Millipore-Q water and Leu-AMC in ethanol to get a final concentrat ion of 10 mmol. Both stock solutions were stored deep- frozen until used. Then, 30 IxL of these stock solutions were added to 2.970 mL of lake water (< 100 l~m size fraction) to get a final substrate concentration of 100 lxmol in each enzyme assay. Enzyme assays were prepared and performed at 25°C in 3.5 mL quarz cuvettes (Hellma), which were kept at constant tempera- ture in the cuvette holder of the fluorimeter by an

external thermostat (Haake). The enzyme act ivi t ies were measured as the inc rease of f luorescence in the cuvettes with a Shimadzu spectrofluorimeter (Model RF-500). The excitation wavelengths for all enzyme assays were 330 nm for MUF-PO4 and 365 nm for Leu-AMC. The emission wavelength was set at 450 nm in both assays. The incubation time during the fluorescence reading was 10 min in both assays. Lake water was preincubated at 25°C for 2 h before enzyme assays started. In pretests, saturation levels of enzyme assays were examined by enzyme kinetics. In 80-90% of measurements, enzymes were saturated at a substrate level of 80-100 ~tmol.

For fract ionation experiments , lake water was passed through a prewashed 100 Ixm plankton net and Nuclepore filters ( 0 45 mm) by gravitation only. The nominal pore sizes of the Nuclepore fil ters were 10 txm, 2 ~tm, 1 lxm, and 0.2 lxm. After each filtration step, both enzyme assays were performed. As a result of this method, MEEA could be measured in five different size fractions: (FI: 100-10 I.tm; F2: 10-2 ~tm; F3:2-1 Ixm; F4:1.0-0.2 Ixm; F5" <0.2 I.tm). Their contributions to the total community activity were calculated in relation to the value of the <100 Ixm size fraction.

For bacteria counts, 25 mL subsamples were taken from each depth and fixed in 1-2% formalin. Bacteria were stained with acriflavine and counted according to Bergstr0m et al. (1986).

RESULTS AND DISCUSSION

In marine and clear water systems, measurements of MEEA are now regarded as an important parameter to study processing and utilization of natural biopolymers by bacteria (Chr6st 1989; Hoppe 1991; Bill6n 1991; Wetzel 1991). Close relationships have been found between MEEA and bacteria biomass (Bill6n 1991), substrate uptake (Hoppe et al. 1988; Chr6st et al. 1989), and bacteria production (Chr6st 1989, 1991) in clear waters with different trophy. Therefore, MEEA are considered to be important system regulators for whole lake metabolism (Wetzel 1991). Similar measurements of MEEA in humic waters are rather scarce, but Mtinster (1991) has emphasized the importance of MEEA in humic waters. However, MEEA in humic environments are more exposed to inhibition and translocation by dissolved humic matter (DHM). DHM is therefore regarded as a functional modulator of MEEA (Wetzel 1991). Thus, data about distribution and activities of MEEA are needed to understand better the role of microbial activities and their interactions in the food webs of humic waters.

Microbial extracellular enzymes in the HUMEX Lake 639

DISTRIBUTION AND VARIATION OF APHA AND LeuAMPA

The vertical distributions of APHA and LeuAMPA in the acidified (Basin A) and control side (Basin B)

are shown in Fig. 1. APHA varied in the Basin A within the water column between 945-1706 nmol L "l h "l

(mean 1246+263 nmol L -1 h l I and LeuAMPA between 3.7-24.9 nmol L 1 (mean 9.1+7.2 nmol L 1 h-l). Maxima of both MEEA were found at

2000'

1750'

.-. 1500'

5 • ~ 1250.

1000.

750.

500

250'

j\\ .J \

0 0.5 1

B A S I N A] 50

45

40

~ . _ _ ~, 35

\ 25 ,~ "1P--- -"~"

20 ~ 15 ~ 10

~ " - ~ = I - - ' ~ ~ ' 5

0 1.5 2 2.5 3 3.5 4

Depth ( m )

I --m-- APHA ~ AMPA I

[BAS IN B] 1000 50

900 45

800 40

60o 3o

500 / 25

20 ~ 4 0 0 / ' " 15 <~

200 -" . . . . J I 10

100 5

0 0 0 1 2 3 4 5 6 7 8

0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 Depth ( m )

I - - m - A P H A - - - k - A M P A

Fig. I. Vertical distribution of acid phosphatase (APHA) and aminopeptidase (AMPA) in two sites of HUMEX Lake Skjervatjem.

640 U. Mllnstcr

0.5 m depth. The MEEAin the control side (Basin B) were lower by a factor of 2 for APHA than in Basin A and varied between 156.3-668.9 nmol L "1 h "1 (mean 363.9+210.9 nmol L 1 h 1) for APHA and 7.8- 34.8 nmol L "l h "1 (mean 15.2+7.7 nmol L "1 h "1) for LeuAMPA. Maxima of APHA activities were found in the upper water layer (0-2.5 m); LeuAMPAmax- ima were found in a sharp peak at 2.0-2.5 m depth. Phosphate concentrat ions in Skjervatjern were 1-2 lxg P-PO4 L "1 (Gjessing 1992) in the epilimnion and increased slightly in the thermocline and the hypo- limnion. The availability of phosphate for microbial utilization was therefore lowest in the trophogenic zone (0-2 m), where the highest primary production was measured (Brettum 1992). The metabolic demand for phosphorus is therefore expected to be higher in this layer, which might be seen then in the APHA distribution (Fig. 1). The reason for this higher APHA in the acidified Basin A compared to Basin B is not clear. However, the data on primary production (Brettum 1992) gave similar relationships and supported the observation on APHA distribution. Primary production was higher by a factor of two in Basin A despite the similar phosphate concentrations in both basins. Therefore, higher demand and turnover of phosphorus in Basin A were probably regulated by higher APHA activity. This is supported by twofold higher values of APHA in Basin A (Fig. 1). Phosphorus seemed to be the limiting nutrient in the nutrition of the microplankton. This was supported by recent nutrient enrichment experiments in this lake in which addition of P-PO4 (100 gg P L "l) decreased the APHA slightly. Further increase of P-PO4 to 500 gg P-PO4 L t caused a 50% decrease in the APHA. Kinetics with APHA also gave significant inhibition patterns after addition of P-PO4 (100-500 I.tg P-PO4 L'I). A detailed analysis of the kinetic data, combined with the results from the size fractionation experiments, revealed that the degree of inhibition by P-PO4 was different in different size fractions. The dissolved free APHA (<0.2 gm size fraction) seemed to be less sensitive to P-PO4 inhibition compared to the particle bound fractions (<100-1.0 gm size fractions). Further, the inhibition in the Basin A was more pronounced. Similar results have been reported by Miinster (1991) for the polyhumic lake Mekkojarvi (southern Finland). Data in the literature indicate that phosphatase is synthesized in higher amounts when phosphate becomes a limiting nutrient for microbial growth (Chr6st 1991). Thus, APHA seems to be an important regulator of phosphorus regeneration and nutrition of microplankton in both sides of Skjervatjern.

LeuAMPA showed a different distribution pattern in both sides (Fig. 1). However, as a difference from the APHA results, the range of activities was similar in both sides of the lake. According to Gjessing (1992), nitric acid is added as an acidifying agent on the catchment area, which means that nitrogen may be of less importace as a limiting element for the nutrition of the microplankton. This is also consistent with nutrient enrichment experiments, where addition of organic nitrogen sources (e.g., amino acids) gave rise to lower microbial activities compared to P-PO4-addi- tions.

According to MUnster (1991), LeuAMPA may not specifically cleave only leucine from the peptide chain, but cleave more unspecifically many amino acids from the N-terminal polipeptide chain in an exo-cleaving mechanism. LeuAMPA is fur ther regarded as a good measurement for heterotrophic utilization of biopolymers by bacteria (Hoppe 1983; Chr6st 1991) and may be closely related to the distribution of bacterial biomass (Bill6n 1984, 1991). The distribution pattern of LeuAMPA will be therefore further discussed in connection with patterns of bacteria distribution in the water columns.

DISTRIBUTION OF BACTERIAL NUMBERS

The distribution of numbers of bacteria in both studied sides are shown in Fig. 2. Numbers of bacteria varied between 4.5-8.8x106 cells mL "t (mean 6.7+1.4 lx106 cells mL "1) in Basin A and 4.4-8.5x106 cells mL -1 (mean 5.5+1.3x106 cells mL -1) in Basin B. There were now significant differences in the numbers of both sides; these numbers were also insignificantly different from data from other humic (Salonen et al. 1992; Bergstr0m et al. 1986; Tranvik 1989), and eutrophic lakes (Overbeck 1979), but slightly higher than in oligotrophic lakes (Wetzel 1983) and marine waters (Bratbak 1988). A similar distribution pattern was found by Hessen (1992b) in September 1991 in Skjervatjern. Maxima of cell numbers (8.5-8.8x106 cells mL "1) were found between 1.0 and 2.5 m in both sides. The most obvious gradients of physical and chemical parameters were also observed at these depths. Variation and dynamics of organic nutrients, like dissolved free (DFAA) and dissolved combined amino acids (DCAA), were also more pronounced in these depths than in others.

Further analysis of mean diameters and biovolumes of bacteria revealed that, in the thermocline, the proportinal contribution of larger bacteria increased from 10-20% to 40-50%. In surface water, the majority (40-60%) of bacteria had a biovolume of 0.008- - ; 3 0 . 0 0 9 Ixm cell -1 compared to 0.011-0.035 gm 3 cell -I


IBASIN A

g . I II H La H H H ll H La La

0.2 0.5 1.0 1.5 2.0 2.5 3.0

Depth ( m )

I 1 ~ AFDC ]

BASIN B 1

_• 8.0

... 7.0 6.0 5.0- 4.0-

<~ 3.0- 2.0- 1.0- 0.0 '3.0' '4.0' 'so' 'so' 0.2 1.0 2.0 . . 7.0

0.5 1.5 2.5 3.5 4.5 5.5 6.5 Depth ( rn )

I ~ AFDC I

Fig. 2. Vertical distribution of bacteria numbers (AcriFlavine Direct Counts:AFDC) in two sites of HUMEX Lake Skjervatjern.

in the thermocline. The mean diameter remained relatively constant at all depths and sampling sides

and varied only between 0.22-0.25 gm in all water

samples. The reason for this shift in bacteria size is not clear but may be related to grazing pressure and nutrient availability.

642 U. MOnster

MICROBIAL CONTRIBUTORS TO MEEA

In most clear-water systems (marine and fresh water), the major contributors of MEEA can be found in the bacterioplankton size fraction retained on 1-0.2 lxm pore size filters (Hoppe 1991; Bill6n 1991; Chr6st 1991). However, there are less data available on humic environments. According to M(Jnster (1992), MEEA in humic lakes may have a different size class distribution compared to clear water systems. Humic waters contain larger amounts of detritus with the dominance of dissolved humic matter (DHM). This DHM may act as a modifier at different levels of whole lake metabolism (Wetzel 1991). Analyzing the size class distribution of APHA and LeuAMPA in Skjer- vatjern (Fig. 3) showed that there is a large amount of dissolved free APHA in both sides. According to the applied fractionation techniques, about 60% of total APHA activity (<100 ~tm size fraction) was not associated with any particles (dead or alive), whereas LeuAMPA was mostly found in particle-associated size fractions. The reason for the high amount of dissolved free APHA is not clear. In surface water samples, the main contributors of LeuAMPA (40-

50%) were found in the bacterioplankton size fractions, F4 (retained on 1.0-0.2 ~tm pore size filter). In water from metalimnion, the main contributors (35-40%) were found in larger size fractions, F3 (retained on 2.0-1.0 lxm pore size filter), which may contain phago-trophic flagellates and other algae. Such a distribution pattern has also been found in polyhumic Mekkojllrvi (MUnster 1992). There is no clear explanation for these size class distributions of MEEA in humic waters. Further improvements of such size fraction techniques by passing lake water through a 0.1 lxm filter cartridge (Nuclepore No. 720010, pressure <5 kPa) confirmed the former results. Again, 40-55% of APHA was found in the dissolved free fraction, passing a 0.1 lxm pore size filter cartridge. Therefore, it has to be questioned whether filtration techniques are suitable approaches to differentiate MEEA according to their association to particles of different sizes. MEAA synthesizing and metabolic active bacteria may be attached to larger dead particles or even living organisms which can mimic MEEA contribution from larger organisms. Further methodological studies are needed to solve these problems.

MEEA in Different Size Fractions I

t . .

LL E

o

= o

100'

90'

80'

70'

60"

50"

40"

30"

20'

10"

0" F1 F 2 F 3 F 4

Size Fractions

N |

F5

[ ~ APHA In BASIN A ~ APHA in BASIN B ~ AMPA in BASIN B ~ AMPA in BASIN A ]

Fig. 3. Size distribution of two microbial extracellular enzyme activities (MEEA) in two sites of HUMEX Lake Skjervatjern: AMPA (aminopeptidase activity); APHA (acid phosphatase activity). Definit ion of size fractions: FI : 100-20 ~m; F2 :10-2 .0 ~m;

F3:2.0-1.0 tim; F4:1.0-0.2 l.tm; F5:<0.2 ~sn.


A

;! BASIN A

.2O00

" " m / \

m l - t _

i m ~ m ~ m | 1 N N N N l N N N N

0.2 o.s 1.o 1.s zo 2.s Depth ( rn )

m

t- I 3.0

--1750

-1500 ~,, ..c:

• 1250 ~ 0

.750

'500

'250

I m~I~IAFDC-"-APHA I

B BASIN A

10

9

8

7

6

5

4

3

2

1

0

ol 1 m t f l m m mML t 0.2 0.5 1.0

la m m

~ N m 1.5 2.0 2.5

Depth ( m )

5O

45

40

30

J - - - z 5 E= v

20 <

15 ~;

-10

0 3.0

I mAFDO-*-AMPA

Fig. 4. (A) Relation between acid phosphatase activity (APHA) and bacteria numbers, AcriFlavine direct counts (AFDC); and (B) relation between aminopeptidase activity (AMPA) and AFDC in vertical profiles in Basin A of Humex Lake Skjervatjern.

RELATION BETWEEN MEEA AND AFDC

It is assumed that the main contributors of MEEA are bacteria, especially for LeuAMPA. Therefore, the distribution of MEEA together with AFDC for both sites is plotted in Figs. 4 and 5. It can be clearly seen that API-IA and LeuAMPA in Basin A did not follow

the distribution pattern of bacterial numbers, but in Basin B, LeuAMPA covaried slightly with AFDC (Fig. 5).

Highest LeuAMPA was found at 0.5 m depth in Basin A, whereas in Basin B, LeuAMPA maxima were found in 2.0-2.5 m depth. Consequently, the

644 U. Mflnster

A BASIN B I

800 10

z 800 B ~" "~ 500

¢E 400 5

I t . 2 <

i i = = | l = = = i

0.2 .0 2.0 3.0 4.0 5.0 6.0 7.0 0.5 1.5 2.5 3.5 4.5 5.5 6.5

Depth ( rn )

I ~ AFDC ~ APHA I

B BASIN, B I

50 10

40 8 F:cP ~ ' 3 5 I 7 "~ ~3o " '~'~1 " ' I ra i . 18

o= t ~ VI~H~ ~ la._l_m_~J. 5

.~2o 4 g lii ii i -3

H1HIHIHIHIH I OI ~ ~ ,, = , , , : =,, ,= ,, =, ,, ,o, :0 0.2 1.0 2.0 3.0 4.0 5.0 6.0 7.0

0.5 1.5 2.5 3.5 4.5 5.5 6.5 Depth ( m )

I ~ AFDC ~ AMPA I

Fig. 5. (A) Relation between acid phosphatase activity (APHA) and bacteria numbers, AcriFlavine direct counts (AFDC); and (B) relation between aminopeptidase activity (AMPA) and AFDC in vertical profiles in Basin B of HUMEX Lake Skjervatjern.

specific LouAMPA activity (fmol L "I h "I cell "l) differs with depth in both sides. Highest specific LeuAMPA (4.56 fmol L "I h "I cell "I) was found at LeuAMPA maxima at 0.5 m in the Basin A compared to lower specific LeuAMPA (1.74 fmol L I h "l cell l) at 0.5 m in Basin B. However, at LeuAMPA maxima in

Basin B (2.0-2.5 m depth), the specific LeuAMPA was in a similar range (3.65-4.24 fmol L "I h "1 cell "1) as in Basin A. Concerning the maxima of proteolytic activities and their relation to the bacterioplankton, there are no significant differences in the two sides. The reason for these distribution patterns is not clear,


BASIN A I

II[ZL I 1500 7

= oll [ ] • . . ,250

511 N I B I B I _ I - - - I - ' I - I lib /,ooo

! - <

a~' 4 -750 "i- n

<~ "500 <

"250

C, , , , , , , , , , , , ,0

A-1 A-3 A-5 A-7 A-9 A-11 A-2 A-4 A-@ A-8 A-tO A-12

Depth ( m )

I AFDC--A--APHA I Fig. 6. Horizontal distribution of acid phosphatase activity (APHA) and bacteria numbers (AcriFlavine direct counts: AFDC) in surface water (0.2 m) of HUMEX Lake Skjervatjern in Basin A. Sampling station AI is in the vicinity of floating moss mats and peat layers, whereas station A12 is in the longest distance in the opposite direction in the vicinity of a rocky shoreline with less vegetation

and water plants. The others (A2-Al l ) are between these two littoral stations.

but may be related to the quality of available substrates. According to Brettum (1992), primary production was highest at 0.5-1 m depth in Basin A and Basin B. It can be assumed that photosynthesis of phytoplankton and the release of utilizable dissolved organic matter (UDOM) by algae are closely coupled to bacteria activities (Azam and Cho 1987; Mtlnster and Chr6st 1990). The higher availability of UDOM at depths with high photosynthetic activities may be a reasonable indication of lower specific LeuAMPA in epilimnion. According to Hcsson (1992b), respiration in the thcrmocline (2-2.5 m) may exceed that of carbon fixation, which means that hctcrotrophic processes are more pronounced than autotrophic in the thermocline. This observation is consistent with LeuAMPA and AFDCA distributions in Basin B, but lesser so in Basin A. It can be assumed that, in the more shallow Basin A, mixing processes and external nutrient loadings are more rapidly changed compared to Basin B. The autotrophic-heterotrophic interactions as shown in Basin B are less stabilized in Basin A. External events have stronger effects to the lake metabolism in the Basin A than in Basin B. However, these LeuAMPA and APHA data support the hypothesis that MEEA arc reliable parameters to

measure microbial activities and subsequent substrate utilization in humic waters as has boon found in previous studies (MUnster 1991, 1992; MUnster ¢tal . 1992). At this stage of studies on bacterioplankton in Skjorvatjern, no uptake measurements with 14C-labelled substrates were carried out. These measurements have been scheduled for the next experimental season in 1992 and will provide more information on DOM processing and utilization in Lake Skjervatjern.

HORIZONTAL DISTRIBUTION OF MEEA

Wetzel (1990) emphasized that a large number of lakes have pelagial/littoral ratio of 0.001-0.1. This means that land water interfaces may be of great importance to the water chemistry and the metabolism of the biota in those lakes. Depending on the hydrological conditions, the productivity and biomass at these interfaces can exceed that of the pelagial. Inorganic and organic nutrients can be loaded to the recipient water bodies and can affect their whole trophic structures (Wetzel 1984). A main constituent of the organic loadings is dissolved organic matter (DOM) which is mostly recalcitrant to microbial

646 U. MOnster

utilization. Such compounds are derived from ligno- ce l lu loses and humic material. The chemical characterization of DOM pools, especially the humic fractions from Skjervatjern, support this assumption (Malcolm 1992). According to Wetzel (1991, 1992), especially humic matter can act as a vehicle for nutrient transport from the wetlands to the recipient water bodies. Also, MEEA can be immobilized by DHM in the littoral zones and translocated and reactiviated in the open water areas (Wetzel 1991, 1992). Therefore, the horizontal distribution of APHA and AFDC was measured in the shallow Basin A at 0.2 m depth samples within the longest distance of both littoral sites in Basin A (for sampling locations in Skjervatjern, see Gjessing 1992). Water samples were taken at the land-water interface from the shal- lowest part (A1) to the opposite site (A12). The results are shown in Fig. 6. There is a clear gradient from sample station A1 to the opposite site in A12. Highest APHA was found in A1 (1668.4 nmol L "l h") at the land-water interface and lowest at A8 (1202.9 nmol L "l h "1) in the pelagial (mean 1301.7+136.8 nmol L 1 h-l). APHA showed a similar range of activities in surface water as previously found in the vertical profile (Fig. 1). Bacteria counts (AFDC) also gave a clear gradient from sample stations A1 to A12. Again, highest bacteria counts were found at Sta- tion A1 (6 .7×106 cel ls mL 1 ) and l o w e s t at Station A8 (4.7×106 cel ls mL l ) in the pe lag ia l (mean 5.5+0.7×106 cel ls mL'l) . There seems to be a higher impact of the watershed at station A1 compared to the opposite site at station A12. This may be due to the higher water inflow at A1 than at A12 and the larger wetland and littoral zone at A1 compared to A12. Obviously, Skjervatjern is a gradient-dominated aquatic ecosystem where littoral processes may partially translocate nutrients and microbial activities to the recipient water bodies as already described by Wetzel (1992). But there are still open questions related to the sinks or sources for micro-heterotrophs in those interfaces. Further studies are needed to address those issues.

A c k n o w l e d g m e n t ~ These studies are part of the HUMOR/ HUMEX Project which were supported by the CEC (Commission of the European Communities) within the STEP program, contract No. STEP-CT90-0112 and by the Max-Planck-Gesellschaft, F.R.G. Further, I would like to acknowledge the University of Helsinki for technical support during my stay at Lammi Biological Station. Thanks are also directed to Riitta Ilola and Jaakko Vainionpiii for counting the bacteria and nutrient measurements at Lammi Biological Station. Valuable discussions and suggestions from K. Salonen and Lauri Arvola are also greatly appreciated.

REFERENCES

Azam, F.; Cho, B.C. Bacterial utilization of organic matter in the sea. In: Fletcher, M.; Gray, I". R. G.; Jones, J. G., eds. Ecology of Microbial Communities. New York, NY: Cambridge Univer- sity Press; 1987: 261-281.

Azam, F.; Fenchel, T.; Field, J.G.; Gray, LS.; Meyer-Reil, L.-A.; Thingstad, F. The ecological role of water column microbes in the sea. Mar. Ecol. Prog. Ser. 10: 257-263; 1983.

Bergstr6m, I.; Heininen, A.; Salonen, K. Comparison of acridine orange, acriflavine, and bisbenzimide stains for enumeration of bacteria in clear and humic waters. Appl. Environ. Microbiol. 51: 664-667; 1986.

Bill6n, G. Heterotrophic utilization and regeneration of nitrogen. In: Hobble, J.E.; leB Williams, P. J., eds. Hetrotrophic activity in the sea. NATO SAD. New York: Plenum Press; 1984: 313- 355.

Bill6n, G. Protein degradation in aquatic environments. In: Chr6st, R. J., ed. Microbial enzymes in aquatic environments. New York: Brock/Springer Series; 1991: 123-143.

Bratbak, G. Production, amount and fate of bacterial biomass in marine waters. Ph. D. Thesis. University of Bergen; 1988.

Brettum, P. Further analysis on the totale volume and composition of the phytoplankton in Lake Skjervatjern. HUMOR/ HUMEX Newsletter (Norwegian Institute for Water Research, Oslo) 2: 24-28; 1992.

Chr6st, R.J. Characterization and significance of ~-glucosidase activity in lake water. Limnol. Oceanogr. 34: 660-672; 1989.

Chr6st, R. J. Microbial ectoenzymes in aquatic environments. In: Overbeck, J.; Chr6st, R. J., eds.. Aquatic microbial ecology: Biochemical and molecular approaches. New York: Brock/ Springer Series; 1990: 47-78.

Chr6st, R. J. Environmental control of the synthesis and activity of aquatic microbial ectoenzymes. In: R. J. Chr6st, ed. Microbial enzymes in aquatic environments. New York: Brock/Springer Series; 1991: 29-59.

Chr6st, R. J.; Miinster, U.; Rai, H.; Albrecht, D.; Witzel, P.K.; Overbeck, J. Photosynthetic production and exoenzymatic degradation of organic matter in euphotic zone of an eutrophic lake. J. Plankt. Res. l h 223-242; 1989.

Gjessing, E. The HUMEX Project: Experimental acidification of a catchment and its humic lake. Environ. Int. 18: 535-543; 1992.

Hessen, D.O. The relation between bacterial carbon and dissolved humic compounds in oligotrophic lakes. FEMS Microb. Ecol. 31: 215-223; 1985.

Hessen, D.O. Dissolved organic carbon in a humic lake: Effects on bacterial production and respiration. Hydrobiologia 229: 115-123; 1992a.

Hessen, D.O. Acidification of the HUMEX Lake; Effects on epilimnetic pools and fluxes of carbon. Environ. Int. 18: 649- 657; 1992b.

Hessen, D.O.; Andersen, T.; Lyche, A. Carbon metabolism in a humic lake: pool sizes and cycling through zooplankton. Lim- nol. Oceanogr. 35: 84-99; 1990.

Hoppe, H.-G. Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl-substrates. Mar. Ecol. Progr. Ser. II: 299-308; 1983.

Hoppe, H.-G. Microbial extracellular enzyme activity: A new key parameter in aquatic ecology. In: R.J. Chr6st, ed. Microbial enzymes in aquatic environments. New York: Brock/Springer Series; 1991: 60-83.

Hoppe, H.-G.; Kim, S. J.; Gocke, K. Microbial decomposition in aquatic environments: Combined processes of extracellular enzyme activity and substrate uptake. Appl. Environ. Microbiol. 54: 784-790; 1988.


Malcolm, R.L. 13C-NMR spectra and contact time experiment for Skjervatjern fulvic and humic acids. Environ. Int. 18: 609-620; 1992.

Mflnster, U. Investigations about structure, distribution and dynamics of different organic substrates in the DOM of lake PluBsee. Arch. Hydrobiol. Suppl. 70: 429-480; 1985.

MUnster, U. Enzyme activity in eutrophic and polyhumic lakes. In: R.J. Chr6st, ed. Microbial enzymes in aquatic environments. New York: Brock/Springer Series; 1991: 96-122.

Mflnster, U. Microbial extraceUular enzyme activities and biopolymer processing in two acid polyhumic lakes. Arch. Hydrobiol. Ergebn. Limnol. 37; 1992. (In press)

Mfinster, U.; Eini6, P.; Nurminen, J. Evaluation of the measurements of extracellular enzyme activities in a polyhumic lake by means of studies with methylumbelliferyl-substrates. Arch. Hydrobiol. I15: 321-337; 1989.

M~Inster, U.; Chr6st, RJ. Origin, composition and microbial utilization of dissolved organic matter. In: Overbeck, J.; Chr6st, RJ., eds. Aquatic microbial ecology: Biochemical and molecular approaches. New York: Brock/Springer Series; 1990: 8-46.

MUnster, U.; Eini6, P.; Nurminen, J.; Overbeck, J. Microbial extracellular enzymes in a polyhumic lake: important regulators in detritus processing. Hydrobiologia 229: 225-238; 1992.

Overbeck, J. Studies on the heterotrophic function and glucose metabolism of microplankton in Plul3see. Arch. Hydrobiol. Beih. Ergebn. Limnol. 13: 56-76; 1979.

Pomeroy, L.R.; Wiebe, W. J. Energetics of microbial food webs. Hydrobiologia 156: 7-18; 1988.

Riemann, B.; Sendergaard, M. Bacterial growth in relation to phytoplankton primary production and extracellular release of organic carbon. In: Hobbie, J.E.; ieB Williams, P.J., eds. Heterotrophic activity of the sea, NATO SAD. New York: Plenum Press; 1984: 233-248.

Salonen, K. The ecosystem of the oligotrophic lake Pliijirvi. 2. Bacterioplankton. Verh. Intemat. Verein. Limnol. 21: 448-453; 1981.

Sslonen, K.; Hammar, T.On the importance of dissolved organic matter in the nutrition of zooplankton in some lake waters. Oecologia 68: 246-253; 1986.

Salonen, K.; Arvola, L.; Tulonen, T.; Hammar, T.; Metslli, T.-R.; Kankaala, P.; Miinster, U. Planktonic food chains of a highly humic lake. I. A mesocosm experiment during the spring primary production maximum. Hydrobiologia 229: 125-142; 1992.

Tranvik, L. Bacterioplankton growth, grazing mortality and quan- titative relatiosnship to primary production in a humic and a clearwater lake. J. Plankt. Res. 11: 985-1000; 1989.

Tranvik, L. Allochthonous dissolved organic matter as an energy source for pelagic bacteria and the concept of the microbial loop. Hydrobiologia 229: 107-114; 1992.

Thurman, M. Organic geochemistry of natural waters. Boston, MA: Nijhoff/Junk Publ.; 1985.

Wetzel, R.G. Limnology; 2nd edition. Philadelphia. PA: Saunders College; 1983.

Wetzel, R.G. Detrital dissolved and particulate organic carbon functions in aquatic ecosystems. Bull. Mar. Sci. 35: 503-509; 1984.

Wetzel, R.G. Land-water interfaces: Metabolic and limnological regulators. Verh. Int. Vet. Limnol. 24: 6-24; 1990.

Wetzel, R.G. ExtraceUular enzymatic interactions in aquatic ecosystems: storage, redistribution, and interspecffic communication. In: R.J. Chr6st, ed. Microbial enzymes in aquatic environments. New York: Brock/Springer Series; 1991: 6-28.

Wetzel, R.G. Gradient-dominated ecosystems: Sources and regulatory functions of dissolved organic matter in fresh- water ecosystems. Hydrobiology 229: 181-199; 1992.

microbial extracellular enzyme activities in humex lake skjervatjern

Documents