2006

Physico-chemical monitoring and mathematical modelling  (made at IBP SB RAS and ICS SB RAS):
The mathematical model of vertical stratification of sulphur cycle components in Lake Shira was improved. In particular, for modelling of microstratification of phototrophic bacteria observed in chemocline zones, the constant of hydrogen sulphide concentration limit below which the growth stops was introduced into the equation of specific growth rate of green sulphur bacteria. The biological meaning of such modification is that obligate anaerobic microorganisms are unable to grow in a microaerophilic zone, which in model calculations corresponds to the depth of zero concentration of hydrogen sulphide at the upper border of hydrosulphuric zone. As a result, in model calculations the microstratification of phototrophic bacteria in the chemocline zone was obtained, i.e. a more appropriate description of the microstratification of bacterial population, which increases the predictive capacity of the model.
The one-dimensional (in vertical direction) mathematical model was modified for the investigation of the annual temperature and salt regimes of the water bodies taking into account spring snow melting both at the lower and upper surfaces of the ice cover. Calculations were made on the basis of the developed one-dimensional model using field data. The results of calculations of the thermal and salt regimes of lakes Shira and Shunet correspond to the field data.

Microbiological investigations (made at IBP SB RAS, IMB RAS and IBIW RAS):
Using a thin-layer multi-syringe sampler, the structure of species diversity and spatial distribution of dominant bacterial forms was analyzed in the chemocline zones of lakes Shira and Shunet. The analysis of 16S rRNA fragments was made by PCR method with universal bacterial primers and subsequent analysis using DGGE (Fig. 1). In the chemocline of Lake Shunet, 7 unindentified dominant bacterial forms were revealed, which we symbolically A,B,C,D,E,F,G. It was discovered that there is a clear-cut vertical spatial separation of dominant bacterial forms along the gradient of redox potential, as well as that the species composition in the chemocline of Lake Shunet suffered a qualitative change during the investigated period - from May to September. It became clear that the bacterial comminuty of the chemocline of Lake Shira differs from the same of Lake Shunet. In particular, only two dominant species, E and G, were discovered in Shira, these two species being among the dominant ones in Shunet, too. 

Fig. 1:  PCR/DGGE - analysis of 16S rRNA fragments of bacterial communities of the chemocline of lakes Shira and Shunet. The meaning of symbols above the tracks: М - microaerophilic zone (traces of oxygen are present), R - redox zone (zero concentrations of oxygen and hydrogen sulphide), S₁, S₂, S₃ - hydrosulphuric zone, with the increase of number the depth increases by 5 cm. For all series of Shunet, except May, the depth interval between the adjacent tracks is 5 cm. In the "Shunet 25.05.2005" series the difference between M and R is 15 cm, between the rest - 5 cm. The depth in all series increases from right to left. The track for Shira was obtained from the sample corresponding to the middle of "pink" layer with traces of hydrogen sulphide. Below the tracks the numbers of sampler syringes are indicated (they are necessary for exact identification of samples in different analyses).

Protozoological investigations (made at IBP SB RAS):
The monitoring of the seasonal dynamics of the protozoan community was continued in the chemocline of Lake Shunet. The number, biomass and production of the main components of protozooplankton of the chemocline zone were calculated for the current year.
The laboratory cultures of the most numerous (in the chemocline of Lake Shunet) species of phytoflagellates and two species of infusoria were obtained. The laboratory experiments aimed at cultivation of these species on artificial media and various substrates were conducted.
It was found out that the laboratory cultures of hydrocoles isolated from the chemocline zone of Lake Shunet are able to grow and develop in aerobic conditions in daylight, which is much more intense than the illumination of their natural habitat. Thus, these organisms cannot be ascribed to anaerobic ones, - on the contrary, thay apparently possess a high degree of tolerance and are able to exist in the wide range of concentration of hydrogen sulphide, oxygen and illumination. 
It was shown that the specific growth rates of Cryptomonas salina population in natural environment varied from 1,15 to 3,15 h-1, the growth rates of the laboratory culture - 2,31-3,82 h-1. Such high growth rates of Cryptomonas population in the lake under light limitation conditions are possibly caused by the additional nutrition of these organisms by way of mixotrophy.
The specific growth rates of the laboratory cultures of infusoria Cyclidium spp. and  Euplotes spp. on the investigated substrates are considerably higher than in the water body environment.

Investigations of phyto- and zooplankton (made at IBP SB RAS and KSU):
The monitoring of C, N, P and PUFA (polyunsaturated fatty acids) of the seston and zooplankton of Lake Shira was conducted in the epilimnion and in the oxic and anoxic zones of the hypolimnion. For the estimation of the flow of biogenic elements and PUFA, that are lost in hydrosulphuric zone, sediment traps were used. It was found out that the average flow of biogenic elements being part of dead zooplankton into the anoxic zone was 3.5 mg С/m2/day or 3.2% of zooplankton daily production. The flow of nitrogen and phosphorus was 0.5 mg N and 0.07 mg Р /m2/day, correspondingly. The treatment of samples that are necessary fot the calculation of lost biogenic elements being part of the dying phytoplankton is not finished yet. However, according to literature data, about 20-25% of phytoplankton primary production settles in lakes with temporary hydrosulphuric zones (Veronesi et al., 2002), the loss of P being disproportionately lower. Thus, stratified lakes show an extremely high degree of closing of carbon and biogenic elements on the level of pelagic oxic zone. About 75-80% of phytoplankton primary production and up to 96% of zooplankton production mineralizes in the oxic zone. It was revealed that the plankton community retains not only biogenic elements, but also a part of PUFA. Acids that are indispensable for animals - a18:3w3 (0.36 mcg/l), 18:3w6 (0.12 mcg/l), and hardly synthesizable 20:5w3 (0.61 mcg/l), 22:6w3 (0.07 mcg/l) were observed in the oxic hypolimnion and were absent in the hydrosulphuric zone. The essential acid 18:2w6 was an exception, but its concentration, with the transition to the hydrosulphuric zone, became almost 2 times as lower  (0.26 mcg/l - oxic hypolimnion and 0.16 mcg/l hydrosulphuric zone). The proportion of PUFA in the seston and bodies of crustaceans A.salinus is represented in the table.

Table.
The proportion (%) of essential polyunsaturated acids (PUFA) in the seston and zooplankton of lake Shira on different horizons.

It is evident that the synthesis of essential acids is limited by the hypolimnion oxic zone, while in zooplankton they are present on all horizons. Hence, the retention (accumulation) and spatial redistribution of PUFA in the lake is realized through zooplankton. 
Thus, it was revealed that in a stratified water body with a hydrosulphuric zone the cycle of not only biogenic elements, but also of essential biochemical compounds (PUFA) becomes more complete within the bounds of the pelagic oxic zone due to the specific organization of plankton community functioning.

Bioluminescent testing of lake water (made at IBP SB RAS):
The sensitivity of the following bioluminescent test-systems was investigated: 1) bioluminescent system with quinone, 2) enzymes of bioluminescent bienzymatic system immobilized into starch gel, 3) jointly immobilized enzymes and substrates of bienzymatic system - with a view to the effect of the water of Lake Shira. It was shown that the water samples taken along the shoreline of the lake inhibited the intensity of luminescence of the soluble bienzymatic system NADH:FMN -oxidoreductase-luciferase to the extent of 20% on average, while they didn't affect the immobilized bienzymatic system. The intensity of luminescence of the bienzymatic system NADH:FMN - oxidoreductase-luciferase in solution and in immobilized state in the presence of the water samples taken from the depth of the lake decreased with the depth in the range of 4-6 metres. The difference in sensitivity between the soluble and immobilized bienzymatic systems is explained by the fact that the polymer carrier used for immobilization prevents toxic substances of water samples from penetration into enzymes. 
As a result of investigations, a modification of the bioluminescent system with quinone was made, and the qualitative and quantitative composition of reaction mixture components was selected. With the aim of detection of sensitivity of the modified system to redox active compounds by the example of model compound solutions, the effect of sulphur compounds and phenols on the kinetics of bioluminescent system with quinone was studied. The dependencies of changes in the kinetic parameters of bioluminescence from the redox characteristics of model solutions were established. It was shown that the most sensitive parameters of the bienzymatic system with quinone affected by a reducing agent are the parameters of luminescence detention. The efficiency of diminishing of the induction period and the time of reaching of bioluminescence maximum in the presence of a reducing agent is determined by the concentration and reducing properties of exogenous compounds. The comparison showed that the bioluminescent bienzymatic system with quinone is specific to the effect of reducing agents, its sensitivity being higher than the same of the bienzymatic system without quinone. 
A new system was adapted to the conditions of water testing of natural water body. The water samples taken along the shoreline and from the depth of lakes Shira and Shunet were studied. It was demonstrated that the bioluminescence of the bienzymatic system with an oxidant is highly sensitive to the redox characteristics of the water from natural water bodies. High correlation coefficients were noticed between the changes in the kinetic parameters of bioluminescence and the characteristics of the lake water (redox potential, oxygen and hydrogen sulphide content). The obtained results allowed to make conclusions about the possibilities and advantages of using the new bioluminescent system with an oxidant for ecological monitoring of natural water ecosystems with distinct redox characteristics.