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51
Lakes, reservoirs and ponds, vol. 10(1):51-70, 2016
©Romanian Limnogeographical Association
INDICATION OF TEMPERATURE INVERTED
MICROBIAL ASSIMILATIVE CAPACITIES
(EXTRACELLULAR ENZYMES ACTIVITIES) IN THE
PELAGIC OF LAKE SEVAN (ARMENIA)
Arevik MINASYAN1,2, Bernhard KARRASCH2
1UNESCO Chair in Life Sciences, International Postgraduate Educational Center, Acharian
31, Yerevan 0040, Armenia, Tel: +37410 624170, Fax: +37410 612461, E-mail:
arevik_m@inbox.ru
2UFZ - Helmholtz Center for Environmental Research, Brückstrasse 3a, 39114 Magdeburg,
Germany, Tel: +49 391 810 9620, Fax: + 49 391 810 9150 E-mail:
bernhard.karrasch@ufz.de
Abstract
Pioneering records of extracellular enzymes activities (EEA) in Lake Sevan waters highlight
dependence of heterotrophic functioning on physicochemical characteristics and bacterial
assemblage. Values of EEA, ranged 0.11-30.39 μg C/P L-1h-1, were higher in upper layers
compared to the omission in deeper parts. Particles associated (ecto-) enzymes mainly
predominated over free dissolved (exo-) enzymes. In June activities of all studied enzymes
followed similar pattern, particularly, decreasing at thermocline and increasing twice/more in
cold deeper waters. Regardless higher bacterial density and temperature in June, with no
similar records up to now, EEA revealed reverse relationship to temperature and bacteria
data and were significantly lesser than in March. Our finding might be suggested as
temperature inverted impact to heterotrophic activities in eutrophic conditions. We assume
that observed, with temperature raise, declined EEA was due to blocked enzymatic active
center from colloids and DOM components interaction, which, in overall, may suppress
organic substrate utilization and result in weakening of first and rate limiting step of
biological self-purification in Lake Sevan waters. Therefore, since temperature is co-
regulator of assimilative/carrying capacity of aquatic ecosystems, climate warming might
have unexpected negative feedbacks also through lowering assimilative capacities of water
bodies, jeopardizing their quality and ecology.
Keywords: freshwater alpine lakes, Lake Sevan, climate change, eutrophication,
temperature, bacteria, extracellular enzymatic activity
52
1 INTRODUCTION
In all aquatic ecosystems autochtonous and allochtonous inputs of
organic matter are the essential link in the energy and nutrients cycles,
driven by microbial decomposers (Wetzel, 1992). Organic matter of aquatic
ecosystems, mostly possesses heterogeneous nature based on molecular
weight, chemical composition, its availability and origin, turnover, as well
as significance to the microorganisms (Münster&Chròst, 1990), and is only
partly (< 5 %) assimilable (Chróst, 1991). The initial degradation of those
complex molecules into for bacteria easily utilizable oligo- to monomeric
compounds via depolymerization is induced by extracellular enzymes
(Hoppe, 1991; Hoppe et al., 1988; Meyer-Reil, 1991; Karrasch et al., 2003a,
b). This is the first step of microbial self-purification processes, which is the
key mean to regulate the turnover of in- and organic compounds in aquatic
environments (Hoppe, 1983). The activity of a certain microbial
extracellular enzymes can provide useful insights into rates of nutrient
mineralization and organic matter processing (Sinsabaugh el al., 2008).
Microbial extracellular enzyme activities in aquatic environments represent
the microbial physiological processes that have a direct influence on
ecosystem level transformations of carbon and nutrients (Jackson et al.,
2013). Moreover, the capability of microorganisms in aquatic environments
to uptake a certain compound can depend indirectly from quantities and
functioning of extracellular enzymes serving to liberate the compound from
POM and polymeric DOM and lift the monomers into the cells (Chróst,
1991; Hoppe, 1991; Karrasch et al., 2003a, b).
For free-living microbes utilizing DOM, the ambient substrate field
is radically different (Traving et al., 2015). Enzyme significant activities
have been measured on particles (Grossart et al., 2007; Ziervogel&Arnosti,
2008) as due to high nutrient concentrations on particles (Vetter et al., 1998)
the free-enzyme strategy is profitable for particle-associated microbes. In
contrast, surface-attached enzymes represent the most cost-efficient
strategy, as free enzymes should not be profitable due to the dilute nature of
DOM (Chróst, 1990; Somville&Billen, 1983). The enzymatic hydrolyzation
processes in aquatic ecosystems and the rate of them are influenced by a
complex of environmental factors (e.g. physicochemical parameter,
including temperature, colloid sorption, surface charge of particles, etc.),
that affect the activity and availability of extracellular enzymes, and by that
mean, may directly impact the ecological stability of entire aquatic
ecosystems (Arnosti, 2003). Although, investigations in various aquatic
environments have characterized enzymes to give different responses to
water temperature changes, the majority of scientists agreed for enzymes to
53
follow the “Q10” rule for temperature dependent enhanced catalytic rate.
Thus, Christian&Karl (1995) have found activities of leucine-
aminopeptidase and β-glucosidase to significantly vary in positive response
to temperature changes in subtropical North Pacific, the equatorial Pacific
and the Southern Ocean regions with very pronounced differences among
latitudinal and climatic zones. This supports similar results of latitudinal
gradient in the relative activities of proteases and polysaccharases in marine
microbiota from Kriss et al. (1963). Study in Antarctic has showed activity
of extracellular chitinases, regardless their high barotolerance at neutral pH,
to reduce with low temperature (Helmke&Weyland, 1986), meanwhile
Hoppe and Gocke (1993) that demonstrated for N-S Atlantic a clear
dependency of heterotrophic activity on temperature, suggested probability
of compensatory adaptation of enzymes to low temperature over nutrients
supply for bacterial growth and functioning to a specific extent.
Schweitzer&Simon (1994) have defined low temperature being one of the
limiting factors to bacterial heterotrophic functioning for Lake Constance.
Also Meyer-Reil&Köster (1992) marked activities of enzymes to depend
positively on the temperatures. Study from Sinsabaugh&Linkins (1988),
Chappel&Goulder (1994) determined temperature, as well as light
(Sinsabaugh&Linkins, 1988; Sabater et al., 1998) to control enzymatic
degradation rate in the streams. In the study from Kirschner et al. (1999) of
the Alte Donau (Danube), temperature and carbon supply were suggested as
the main limiting factors for bacterial functioning during the cold seasons
and the highest values of EEA recorded for hot season (especially during
phytoplankton bloom) and consequently lower EEA at the cold weather.
Other data demonstrate substrate availability being the major factor for
extracellular enzymatic activities (Meyer-Reil, 1987) and bacterial
activities; concentration and discharge of bioavailable nutrients
(Romaní&Sabater, 1999), and the humic material (Freeman et al., 1990).
Chappel&Goulder (1994), as well as Sabater&Romaní (1996), as a
biological indicator for heterotrophic processes in aquatic ecosystems, have
outlined microbial activities being positively aligned to environmental traits.
In light of altogether, the main aims of the present work were to
study the dependence of activities of certain extracellular enzymes in
correlation to water physicochemistry upon the quantitative characteristics
and distribution of bacterioplankton. The goal of this investigation was to
examine whether extracellular enzymes display characteristics of local
adaptation on latitude level on Lake Sevan example. This task is very
important because of the adaptation of certain microbial enzymes to a
particular temperature regime that may or not sustain microbial decomposer
activity under global warming conditions in freshwater lakes. For that
54
purpose EEA rates as an indicator of waters assimilative/carrying capacities
have been quantified in Lake Sevan waters to indicate OM load limits
depending on physicochemical traits to outline standing stocks of the
baseline structure. We hypothesized that microbial EEA in deep alpine lakes
(such as Lake Sevan) would be more sensitive to temperature seasonal
changes, and that EEA changes would show temperature dependence
sensitivity with increasing water temperature. The pioneering results derived
from this study suppose to improve our ability to model the effects of global
warming on decomposition of DOM in alpine freshwater lakes, to serve for
development of appropriate management strategies in further monitoring
and for better elucidation of the remediation program of the freshwater
reservoirs conservation worldwide, and for Lake Sevan, particularly.
2 MATERIAL AND METHODS
2.1 Study site
Lake Sevan is one of the largest freshwater alpine lake in the world
at 1900 m above sea level situated approximately 60 km north of Armenia
capital Yerevan, possesses a surface area of 1270 km2 and a maximum
depth of 80 m. Lake Sevan, the largest freshwater lake in the South
Caucasus region and nearby territories (Iran, Turkey). Hrazdan River is the
only outflowing river of Lake Sevan that becomes Lake Yerevan (an
artificial water body in the southwestern part of the city) on the territory of
c. Yerevan (the capital of Armenia). Lake Sevan - River Hrazdan - Lake
Yerevan water cascade continues again as River Hrazdan joining River Arax
in the Ararat valley, along with Armenian-Turkish borders, then flowing
along the Armenian-Iranian and Iranian-Azerbaijan borders to meet with
the Kura River and inflow into the Caspian Sea. It has soft water with
mineralization = 700 mg L-1. Salinity is about 0.36 - 0.37 ppt, total hardness
of its water is 4.65 - 4.90 meq L-1, conductivity 598 - 634 µS cm-1. Lake
Sevan with its huge socioeconomic importance for Armenia is very unique
itself also due to its endemic fishes: e.g. Sevan trout (Salmo ischchan
gegarkuni), Sevan koghak (Capoeta capoeta sevangi) and Sevan beghlou
(Barbus geokschaicus). Due to artificial decline of its water level (down to
20.2 m), started in 1933, the morphometric, physicochemical and biological
parameters of the lake were heavily impacted. Hypolimnion total volume
decreased for up to 40 %, and basin water average temperature increased for
2-3˚C (Lind&Taslakyan, 2005). Decomposition of loading organic matter
decreased the oxygen content in water column in general with oxygen
saturation tending in the lake floor zone to low values, especially for hot
55
seasons during the lake stratification. The Lake started its evolution toward
from oligo- to meso-eutrophic stage. Altogether collapsed entire balance of
the lake through affecting the natural limnological properties of it.
Moreover, the changing climatic conditions and new strategy on water
management started since 2002, contributed to a quick rerise of the lake’s
water level up to 3.6 meters that caused problems of flooding numerous
settlements and forestlands, which can threaten water quality of the Lake.
2.2 Material collection
The geographical coordinates of the sampling points are 40° 29'
35.50' N and 45° 11' 31.34" E (Figure 1). Water sampling of the Lake north-
western sector (Minor Sevan) along vertical profile (0; 5; 10; 15; 20; 25; 30;
35; 40; 45; 50; 55; 60m) was done with a Ruttner sampler.
Figure 1. Map of Lake Sevan catchment basin (“X” is the illustration of the
position of the sampled vertical profile)
For the microscopic analyses 40 ml of the samples were immediately
fixed with 0.2 µm filtered formalin (final concentration 2 % v/v) and stored
at 4ºC for further determination of the abundance and biomass of bacteria.
The non-preserved samples were collected for the quantification of
56
the EEA. As no negative effects on the quantification of EEA after several
days of storing the samples in the dark at 4ºC were observed by the authors,
we had followed the same procedure. All samples were transported (in cold
and dark) to the host institute in Germany (UFZ -Helmholtz Centre for
Environmental Research, Department of River Ecology) for proceeding
(started on 15-th March and 15-th June for samples collected on 12th March
and 13th June respectively).
2.3 Quantification of bacterial abundance and biomass
Total abundance and biomass of bacteria were determined by
fluorescence microscopy (acridine orange (AO) method after Hobbie et al.
(1977) on 0.2 μm Irgalan black stained polycarbonate nuclepore filters).
Counting was performed with a Zeiss Axioplan epifluorescence microscope
(365nm excitation filter, 395nm beam splitter, 397nm emission filter). A
minimum of 20 fields per filter were counted by means of a new Porton grid
(Graticules, Ltd.) to obtain confidence level of 95%. The cell volume (V)
was calculated as follows:
cell volume = π/4 x W2 x (l - W/3) (with W: width in μm and L: length in
μm) Bacterial biomass in carbon was determined using the non-linear
equation derived from Simon&Azam (1989):
cell carbon / fg = (cell volume/μm3) 0.59 x 88.6 x 1.04878
2.4 Quantification of extracellular enzymes activities
The EEA were determined as a measure in accordance with the
method published by Hoppe (1983) modified by Karrasch (2005). A
technique based on 4-methylumbelliferyl (MUF)α- and β-D-
glucopyranoside, phosphate, β-D-galactopyranoside, N-acetyl-β-D-
glucosaminide) and L-leucine7-amino-4-methyl-cumarin (AMC) labeled
enzyme substrates was used to quantify rates of extracellular breakdown of
dissolved and particulate organic macromolecules (Karrasch et al., 2003a,
b). The rates of EEA are expressed in terms of maximum velocity of
hydrolysis (µg C L-1h-1) or (µg P L-1h-1in the case of MUF-phosphate). 10 µl
of substrate analogue (final concentration 500 µM, MUF-phosphatase 125
µM L-1) was supplied to the samples. To divide the total EEA into
ectoenzymes (enzymes associated with bacteria and particles) and free in the
water dissolved enzymes activities, a filtration was performed using 0.2 μm
57
nuclepore polycarbonate filters whereby the <0.2 μm fraction defines free
dissolved enzymes. 200 µl subsamples (0.2 μm filtered and non-filtered
sample) were added into each well of microplate (4 replicates) and
supplemented with previously added AMC and MUF-substrates. Incubation
time was 24h at ambient sample temperature in the dark. Standard of AMC
and MUF solutions were used for calibration (10 µl of 0.5 µmol L-1; internal
standard addition method). Fluorescence quantification was done using a
microplate fluorescence reader (Labsystems, Ascent software) at λ=364 nm
(excitation) and λ=445 nm (emission).
3 RESULTS
In March transparency of Lake Sevan water reached up to 13 m and
dropped down to 8 m in June. Temperature fluctuated from +3.5˚C - +18˚C.
Dissolved oxygen was decreasing from surface (14.2 mg L-1) up to the
upper edge of thermocline at 20 m (12.4 mg L-1) with further lowering at
bottom. The detection limit for nitrite ion that quickly converts into nitrate
in natural waters, was below <0.023 mg L-1 for over the water column.
Concentrations of nitrate ion were in the range of 22 µg N L-1 up to 277 µg
N L-1, ammonium was of 34 µg N L-1 - 152 µg N L-1 (data of March).
Phosphate was accounted for 58.6 µg P L-1 up to 82.0 µg P L-1.
In summer total phosphorus content had increased up to values typical for
eutrophic waters (Minasyan, 2010).
Figure 2. Vertical distribution of the total bacterial abundance (*109 cells L-1)
and biomass (µg C L-1) in Lake Sevan waters
58
In March bacteria were counted from 2.4*109 cells L-1 up to
5.3*109 cells L-1 (see Fig.2), with minimal numbers for the surface waters.
At 50 m depth was registered the highest value for spring followed with
gradual drop down at bottom. Reverse situation of higher bacterial density
starting with the highest values for surface (6.9*109 cells L-1) and gradual
decrease for deeper waters was counted in June. Insignificant oscillations in
vertical distribution were observed with decrease started at the thermocline
= 5.3*109 cells L-1 (25 m) and continued up to the lowest for the bottom
waters. Bacterioplankton dominated by small-size single cells with mean
volume in March equaled to 0.08 µm3. Comparatively medium-size
bacterial cells were observed in June (cell mean volume 0.20 µm3) with
biomass values accounted for 68.4 - 108.54 µg C L-1 (Figure 2).
Figure 3. Vertical distribution of hydrolysis rates of EEA in Lake Sevan in 2010
March/June.
A = α-glucosidase; B = ß-glucosidase; C= phosphatase; D = leucin aminepeptidase;
E = ß-galactosidase; F = N-acet;
EEA <0.2 = external enzymes; EEA-Ecto = ectoenzymes; M = March; J = June
59
The EEA, except for leucine-aminopeptidase, ranged from 0.11 to
10.41 μg C/P L-1 h-1, with the lowest values for β-D-galactosidase of the
maximal up to 2 % share (Figure 3). Activity of leucine-aminopeptidase was
one magnitude higher (from 5.93 up to 30.39 μg C L-1 h-1), with up to 74 %
proportional share in the total EEA. In March started from 15 m depth up to
the bottom increasing bacterial density was in concordance with increasing
enzymatic activities (relatively high for β-D-glucosidase (2.36 - 3.26 μg C
L-1 h-1) and N-acetyl-β-D-glucoseaminidase (3.00 - 3.73 μg C L-1 h-1) up to
6.37 μg P L-1 h-1 for phosphatase and the highest of 30.39 μg C L-1 h-1 for
leucine-aminopeptidase). In the upper layers, particularly for 0-5 m linked to
relatively low bacterial densities nearly for all enzymes were registered
higher activities and relative decrease at 10 m (euphotic zone with water
lower temperature) and furthermore.
In June both values of bacterial densities and EEA were higher for
upper layers up to 10 m (euphotic zone with water higher temperature). The
values of activity of phosphatase, β-D-galactosidase were higher at surface
and α- and β-D-glucosidase and N-acetyl-β-D-glucoseaminidase at 5 m
depth. The rates of activities for all 6 studied enzymes followed similar
pattern. Started from thermocline all enzymes, except of leucine-
aminopeptidase, had demonstrated decreased activities up to the range of β-
D-galactosidase measured in March. Their activities decreased at
thermocline (25 m) and tend to increase approximately twice or more in
hypolimnion. The hydrolysis rates of α-D-glucosidase, β-D-glucosidase and
phosphatase at the bottom layer were up to 2.5 times higher than at the
thermocline. Activity of N-acetylglucoseaminidase, registered “above” -
“at” - “below” thermocline, differs approximately three times.
4 DISCUSSION
The concentration of nitrogen and phosphorus in Lake Sevan waters
was in range typical for mesotrophic lakes stepping into the first stage of
eutrophy (Wetzel, 2000). Low oxygen content at bottom characterized Lake
Sevan being at meso- toward first stage of eutrophy too. The relatively equal
distribution of bacteria in March was with low values at the upper layers and
increase in hypolimnion. Windy weather and typical spring overturn that
equalized also water temperature could be the main reasons for
homogeneous distribution of bacteria in Lake Sevan. In June in the
epilimnion of Lake Sevan the water higher temperatures were registered.
Together with more organic matter approaching from enhanced human
60
summer activities in the Lake basin, such as recreation, agricultural, sewage
discharge, etc., higher concentrations of available nutrients of autochtonous
and allochtonous origin, increase mainly N and P inputs (Hovhannisyan,
2010). Additionally, a solid amount of organic matter entering the Lake
Sevan ecosystem from the flooded territories (reports of the Ministry of
Nature Protection of Republic of Armenia) can be essential for development
of microbial community.
According to Lind&Taslakyan (2005) algal “blooming” in Lake
Sevan during the hot seasons causes TN/TP ratio increase toward P-
limitation in the upper layers and P-trapping in the Lake floor, and a lower
nutrient availability in the epilimnion clearly enhance the “bottom-up”
control reflected in the relatively increased bacterial density in epilimnion
and decreased in hypolimnion. Our past studies (Kosolapov et al., 2010;
Hahn et al., 2012) revealed similar tendency of increasing quantitative
characteristics (abundance and biomass) of bacterial community, especially
in hot seasons together with higher algal numbers (Minasyan&Karrasch,
2015), demonstrate continuing evolution of Lake Sevan from meso- toward
eutrophic stage. Altogether confirm high oxygen demand and could be one
of the reasons of its depletion in Lake Sevan hypolimnion.
Extracellular enzymes activities as a first step of biological self-
purification capacity of aquatic ecosystems undergo the influence of a
multitude of physicochemical, as well as biological processes and
interactions existing therein (e.g. Karrasch et al., 2003a, b). The majority
studies indicate temperature (the “Q10 rule”) as one of the environmental
trait to regulate cell metabolism, which was attributed also for EEA
(Münster et al., 1992), with seasonal character of extracellular enzymes
sensitivity over temperature (Fenner et al., 2005; Koch et al., 2007; Trasar-
Cepeda et al., 2007). The affinity of enzyme systems decreases at low
temperatures, therefore, their interaction has an indirect effect on enzymatic
activity (Zweifel, 1999; Pomeroy&Wiebe, 2001; Reyes et al., 2008) of
certain aquatic ecosystem.
Registered in Lake Sevan waters reduced EEA with increased
temperatures was quite contrary with no similar records in the existing
literature. In June water higher temperature and higher bacterial density
were coupled with the mean values of activities of total enzymes being
lower, than those in March (an exceptions of N-acetylglucosaminidase)
(Figure 3). Reduced activities of 6 studied enzymes in both fractions (ecto-
and exoenzymes) was quantified especially right at the thermocline level
(20-25 m) - “above” - “at” - “below”, differing approximately three times.
Meanwhile, the abundance of bacteria of those depths did not differ so
dramatically (5.91-4.47*106 cells L-1), with less difference in
61
picocyanobacterial density (0.18-0.16*106 cells L-1) (Minasyan&Karrasch,
2015). Activity of leucine-aminopeptidase that had the highest share in EEA
in Lake Sevan also comparatively declined in summer. This observation is
in contrast to the statement of Hoppe (1983) of observed typical EEA to
reach its maximums during the hot periods, when water temperature,
bacterial density and uptake of leucine are also the highest, together with
increased nutrients input from human activities. Leucine-aminopeptidase
(due to their high values activity of leucine-aminopeptidase was not
included in the total calculations), with the higher proportional share in cold
season (in average 2.4 times higher in March) are similar to data from
Münster (1991) of the higher activity of ecto- fraction of leucine-
aminopeptidase to be in oligotrophic waters.
Observed in Lake Sevan dominance of aminopeptidase versus
glucosidase is common for other aquatic ecosystems (Stursova et al., 2006;
Karrasch et al., 2011) too. Lancelot&Billen (1984) showed tight coupling of
aminopeptidase activity to primary production, similar to data of Lake
Sevan. Hoppe (1986) also showed extracellular glucosaminidase activities,
being with the highest values during the spring phytoplankton bloom mainly
in March with higher dependency of EEA on total bacterial density. Rath et
al. (1993) demonstrated activity of α-D-glucosidase (associated with
phytoplankton biomass and dissolved monomeric carbohydrates achieved
from forestlands) and N-acetyl-P-D-glucosaminidase (chitobiase) to
decrease from eutrophic to oligotrophic conditions, which is in contrast to
our observation. In Lake Sevan high activity of N-acetylglucosaminidase
was accompanied with high activity of β-glucosidase (hydrolyze cellulose)
and phosphatase, more likely associated with deciduous leaf litter
decomposing, approaching Lake Sevan from flooded forestland areas
(March).
Chrόst et al. (1989) offered potential close correlation between
ectoenzymatic activity of α-glucosidase and algal community, which is
similar to what we seen in Lake Sevan in June. This indicates the particle
associated self-purification capabilities in Lake Sevan waters to be higher in
March (mesotrophic stage). If to couple these observations and finding from
Cunha et al. (2010) about ectoenzymes being responsible for the hydrolysis
of the major components of DOM consisting of labile oligo- to monomeric
molecules originating mainly from autochthonous processes like viral lysis
(Fuhrman 1999) exudations from phytoplankton (Fogg, 1977, Baines&Pace,
1991) and zooplankton excretions (Lampert, 1978; Jurmars et al. 1989), that
are easily accessible for extracellular enzymes, linked with data on high
algal and viral densities in Lake Sevan (Minasyan&Karrasch, 2015), we can
assume tendency of increasing concentration of DOM, originating from
62
algal exudation of increased grazing, especially in hot seasons.
General biochemical considerations upon the role of temperature on
reaction rates and physiology within the zone of biokinetic temperatures
(e.g. Arrhenius, 1989, the empiric Q10, derived from the vant’ Hoff
equation) stimulates the cell growth (including bacterial and algal cells) and
their metabolic rates. With increased primary production the presence of
high DOM concentrations (e.g. exudation, sloppy feeding, etc.) is very
likely raises the probability of the occurrence of enzyme inhibiting
compounds that might block the enzymatic active centers and/or the
significantly changed tertiary structures of them (Tietjen&Wetzel, 2003).
Finding of Karrasch (2005), Wetzel (1991) of the increased occurrence of
charged particle surfaces (e.g. clay and POM), an intensified of colloids and
further DOM components interaction with the extracellular enzymes, e.g.
creating van der Waals dipole linkages, covalent and hydrogen bonds, ionic
linkages, aggregation and precipitation, e.g. by organic acids and cross-
linking with reactive functional groups in proteins, nucleic acids and other
organic matter components, showed high potential to suppress enzymatic
activities.
In June higher abundances of bacteria, viruses and picocyanobacteria
(Minasyan&Karrasch, 2015) in Lake Sevan, were in concordance with the
similar tendency registered for primary productivity (up to 3 times higher)
and bacterial production (up to 8 times higher) quantifications (data not
shown). In a number of studies in deep lakes Chrόst&Siuda (2006);
Kisand&Tammert (2000) have demonstrated a close coupling between
enzyme activities and primary productivity. If to link these to the observed
bloom by cyanobacteria genera of Microcystis, Anabaena, and
Aphanizomenon (Minasyan et al., 2012) together with summer increased
standing stocks and applying temperature related physiological axiom, we
have to expect with increasing temperature raised EEA, which was the
opposite in Lake Sevan. Registered maximums of EEA (3.26; 6.37; 10.41;
30.39 μg P/C L-1 h-1; for β-glucosidase; phosphatase, N-
acetylglucosaminidase and leucine-aminopeptidase, respectively) that
demonstrates the upper limit of the self-purification capacities of Sevan
waters indicates also that increased discharge of substrates would impact the
ecosystem via accumulating and induce significant changes in the
community structure and by that threaten the ecology of Lake Sevan.
Presumably, the occurrence of organic matter equipped partially with high
reactive surface act/impacting inhibiting enzymes availabilities or activities
in the waters of Lake Sevan, coupled with water temperature and its water
mixing intensity are the most probable explanations of corresponding
fluctuations in enzymatic activity during our study, with displaying restrict
63
functionality of free dissolved enzymes versus their ecto- fraction. Since
EEA-rates are representing the maximum self-purification limits, our
findings of their considerable reduction in Lake Sevan observed at
thermocline, would probably encompass far-reaching consequences for the
ecology of freshwater lakes with typical thermal stratification (e.g. Jeppesen
et al., 2014) to demonstrate possible negative feedbacks of global warming
from reduced assimilative capacity. The world is warming as a result of
anthropogenic activities raising the atmospheric CO2 concentrations (IPCC,
2007). Discovering the inner processes that could determine water quality
through assessing assimilative/carrying capacities might help in defining
appropriate experimental sets for further monitoring of the ecological
situation and the trophic stage of aquatic ecosystems, especially in summer
period, when weakens the first and rate limiting step of the biological self-
purification leading the evolution of aquatic ecosystem to raised trophic
levels.
5 CONCLUSION
The occurrence especially of surface charged DOM could interact
with the extracellular enzymes either directly via blocking the zone around
the active center of enzymes or by allosteric inhibition thus disturbing the
access of substrate to it. Indirectly the autochthonously produced organic
matter with a high electrostatically potential could mediate with their
charged surfaces the adsorption of extracellular enzymes to colloids and
particles whereby, depending on the accessibility of the active center
(enzyme orioentation), the creation of an enzyme substrate complex could
be interfered. Both cases may decline EEA and suppress organic substrate
utilization and by that weaken the first and rate limiting step of biological
self-purification in Lake Sevan waters. Therefore, since temperature is co-
regulator of assimilative/carrying capacity of aquatic ecosystems, climate
warming might have unexpected negative feedbacks also through lowering
assimilative capacities of water bodies, jeopardizing their quality and
ecology.
ACKNOWLEDGMENT
The authors would like to express especial thanks the Otto Kinne
Foundation (OKF) and German Academic Exchange Service (DAAD) for
funding Arevik Minasyan’s research stay at the Helmholtz Centre for
Environmental Research - UFZ, Department of River Ecology. We are
grateful to Maren Mehrens (technician of UFZ - Helmholtz Centre for
64
Environmental Research) for technical assistance in performing the analyses
on quantifying extracellular enzymes activities rates.
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