Investigation of specific microorganisms in the salt lakes of Southern Russia

1 Department of Biology, Laboratory of Biotechnology, Federal State Institution of Higher Education, Astrakhan State University, Astrakhan, Russia. 2 Department of General Ecology and Hydrobiology, State Institution of Higher Education, Astrakhan State Technical University, Astrakhan, Russia. 3 Department of Botany, Biology, Ecosystems and Land Resources, Federal State Institution of Higher Education, Astrakhan State University, Russia. 4 Department of Physiology, Morphology, Genetics and Biomedical, Federal State Institution of Higher Education, Astrakhan State University, Russia. 5 Department of Biotechnology, Federal State Institution of Higher Education, Astrakhan State University, Russia.


INTRODUCTION
Fish-processing industry is one of the priorities in the territory of seaport towns.Waste waters, which are difficult to recycle, such as brines and bitterns (salt solutions), with total dissolved solids of 170-190 g l-1, are generated while producing salted fish.Fish-processing enterprises of the Astrakhan and other regions produce *Corresponding author.E-mail: aveatab@mail.ru.Tel: +7 903 349 6228.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License about 400 m 3 of brines per month.Waste water contains different organic substances (proteins, peptides, amino acids, fats, etc.) and mineral substances (mostly sodium chloride).Existing physical and chemical methods of brines treatment are costly and ineffective as they do not completely remove proteins from the medium, thus making brine reutilization complicated.Dumping of used salt solutions into sewerage or onto the ground is especially damaging to the environment.The search of brine waste treatment techniques to remove organic substances and to return salt solution into the working cycle remains an urgent and complicated task.
The greatest efficiency of purification of media from different contaminants is shown by biological techniques based on the same principles of the processes of natural ecosystems purification (Laliberte et al., 1994).Microorganisms play the leading role in purification and are capable of decomposing any organic substance of natural origin due to their having great variety of enzyme systems and great metabolism ability (Hoffmann, 1998;Rapper and Miller, 2005;Munoz and Guieysse, 2006;Vijaya et al., 2010;Cheunbarn and Peerapornpisal, 2010).A specific group of microorganisms should be used for brine neutralization, as high salinity decreases the degradation speed of organic substances, which the brine contains.
Halophilic microorganisms' vitality processes are not inhibited by media with high salt contents.They are widely spread among bacteria, achaea, fungi, yeast, as well as algae and protozoa.Extremely halophilic archaea (haloarchaea) and cyanobacteria that can grow in saturated NaCl solution (32%) are the most typical representatives of this group.Their characteristic feature is that the minimal NaCl content limit for their growth is 12-15%, while a number of other organisms, capable of growing in the saturated NaCl solution, need considerably lower concentration (6-12%), than extreme halophiles.
In natural conditions halophiles can be found in saline lakes, some seas and thrive in large quantities in salterns, where water is intensively evaporated (Bauld, 1981;Guererro et al., 1994;Kondratyeva, 1996;Stal, 2000;Zavarzin and Kolotilova, 2001;Thajuddin et al., 2002;Vorobieva, 2007).Sea lagoons and brine and natron lakes are one of the ecotopes of extreme halophilic microorganisms, where evaporation significantly increases salinity of sea water (Stal, 2000).Cyanobacterial mats form vigorously there, with phototrophic bacteria being the main link (D'Antoni D'Amelio et al., 1989).Cyanobacteria are basic producers of organic substance among phototrophic bacteria, and they form the mat structure.There is also a degradation activity, carried out by different bacteria (Fourcans et al., 2004;Zakharyuk et al., 2010).Cyanobacteria of Microcoleus genus are typical shapegenerating element in hypersaline reservoirs.Microcoleus is an organism, generating trichome bundle inside a general mucous sheath (Gerasimenko et al., 2003).Microcoleus forms thick skin which performs the functions of aquifuge where extracellular sheathes are seen at the bottom of saline sea lagoons with salinity of 17%.
Physiological feature of halophilic microorganisms' growth in highly mineralized media is determined by osmotic resistance of their cells, which can change intracellular conditions to decrease significantly the inhibitory action of salt.Such features of halophilic microorganisms, especially heterotrophs, participating in decomposition of organic substances, can be used to develop methods of brines treatment after fish salting.Cyanobacteria are not only known to be resistant to extreme saline conditions, but are also applied in wastewater treatment (Maclean et al., 1972;Uma and Subramaninan, 1990;Subramaninan and Uma, 1996;Shashirekha et al., 1997;Karna et al., 1999;Malliga et al., 1996;Kalavathi et al., 2001;Sharma et al., 2012).
This work aimed to detect the community of halophilic microorganisms in highly mineralized water ecosystems of the Lower Volga and to research the capacity of detected microorganisms to degrade organic constituent of brines.

Reservoirs with extreme conditions
The research was carried out on the selected saline lakes, located in the territory of the Lower Volga, which are water bodies of natural (Baskunchak, Selitrennoe, Tinaki) and anthropogenic origin (Mramornoe).
Lake Baskunchak is connected with one of the greatest deposits of high-quality common salt.It contains 98% sodium chloride and is considered to be one of the best.The length of the longest axis of the lake is 18 km; the width is 6-9 km, with the total area of 106 km 2 .The lake is fed by underground waters, located close to the ground, with rock salt as waterproof stratum, deposited 240 million years ago at the bottom of shallow Lower Permian basin.Underground waters dissolve salt, transform into brine and flow on the surface of the lake as springs.
Lake Selitrennoe is located in the Northern-eastern part of the region, where the ruins of the capital of the Golden Hordethe City of Sarai-Batu, founded by Batu Khan on the bank of the Achtuba river, were found.In that place a small lake Selitrennoe is located in a natural relief low.The length of the lake is approximately 100 m, the width is 50 m and maximal depth is about 1 m.The water has pink coloring and viscous consistency.The bottom is covered with white salt layer.The silt is black creamlike mud of high dissolved solids, saturated with hydrogen sulfide.Lake Tinaki is situated 4.5 km to the West of the Volga and 12 km to the North-West of Astrakhan and belongs to South-western group of Astrakhan saline lakes.The lake origin is connected with ancient recession of the Caspian Sea.The lake leach is water solution of different salts.The leach has pink or orange-red coloring in frost-free season.There is mud under leach along the full area that is black, plastic, sticky and easily soiled mass.It has a sensible smell of hydrogen sulfide.The silts are generally considered to have medical properties.Lake Mramornoe appeared in the place of Baskunchak gypsum deposit development.Gypsum extraction is carried out in the area of subterranean salt massif from the opencast mine with the depth of 40-42 m.Water-bearing non-subartesian floor or level was uncovered in the base of the opencast mine, which resulted in forming the lake with total area of 5000 m 2 and maximal depth of 10 m

Sampling
Water samples were drawn according to the microbiology rules twice a year -springtime and autumn; five samples from the inshore water of each reservoir to isolate halophilic organisms.Investigations were carried out in two years.For lakes the sampling point was selected after taking into consideration such factors as geography, whether there are freshwater (rivers or streams) or wastewater inflows, depth, tides, currents etc. Containers were washed 3-4 times with water from the exact site of sampling prior to taking the sample.The samples were carefully and gently poured into the containers without making bubbles (Dworkin et al., 2006).Also sampled waste water was taken after salting the fish Abramis brama of the genus, bream and family of Cyprinidae.The content of sodium chloride in the brine taken after fish salting was 67 g/l.

Isolation and purification of the species
Phototrophic and heterotrophic bacteria were isolated by enrichment culture methods considering the biochemical properties (Netrusov et al., 2005;Starr et al., 1992;Horikoshi et al., 2011).Isolation of heterotrophic microorganisms was carried out with the fish-and-peptone broth using the water from the reservoirs.
Bacterial community isolation was carried out in double replication, with temperature of 22-25°С.

Growth conditions
To estimate degradation capacity of halophilic microorganisms against organic constituent of brines communities, an experiment was conducted to test the effectiveness of wastewater treatment after salting fish in heterotrophic and phototrophic community.The followings are the stages: 1) control waste water; 2) waste water with introducted heterotrophic microbial communities; 3) waste water with introducted phototrophic microbial communities (variant 3 -waste water, 75% of waste water and 25% of distilled water, 50% of waste water and 50% of distilled water).
Microorganisms were not introduced into the control containers with salt solution.Community of heterotrophic microorganisms was introduced into the waste water of 2 ml/100 ml.The titer of the cells was 10 9 cells /ml in fish-and-peptone broth.Introduction of biomass of phototrophic microorganisms was measured by the gravimetric method. 2 g of community was placed in 100 ml of test water.
weight to determine the dry weight of the biomass.Dry weight was 0.187 g.

Chemical analysis
The chemical analysis of water from the reservoirs under study included determination of the content of chlorine ions by means of the argentometric titration method (The Unified Methods of Research of Waters Quality, 1976;Clesceri et al., 1998).рН was evaluated with the pH-meter "Ekspert-001".Total dissolved solid was determined with high-precision conductometer.
Degradation ability of the microorganisms was estimated on the basis of change of the amount of solid residue minus the mass of sodium chloride in the salt solutions before the microorganisms were introduced and after a month's exposition.Dry residue was determined by methods of evaporation and calcination.10 ml of wastewater control was placed in a calcined, cooled and weighed in porcelain cup (m = 35,477 gr).The cup was placed in a water bath until complete evaporation of the liquid.The residue was dried to constant weight in thermostat at 105°C, cooled in a desiccator and weighed.It was calcined in a muffle furnace for one hour at 600°C.The residue was cooled in a desiccator and weighed.
Solid residue after deduction of the mass of sodium chloride is an indirect index of the content of some organic substance in the salt solution.Decrease of its mass proves the degradation of the organic component of the salt solution.

Analysis of the four lakes where halophilic bacteria exist
The experiment included testing of pH environment, Cl and Mg 2+ ions and total dissolved solids (Table 1) (Clesceri et al., 1998).Saline reservoirs under consideration belong to extremely high mineralized areas as per total dissolved solids rate.The maximal concentration of dissolved solids, Cl -and Mg 2+ ions content is observed in Selitrennoe and the minimal one is observed in Lake Tinaki.
The features of hydrochemical conditions of the lakes presuppose the presence of halophilic microorganisms.Phototrophic and heterotrophic microorganisms were detected in water bodies under consideration as a result of microbiological analysis.Phototrophs are presented by cyanobacteria, diatomaceous and green algae of Dunaliella and Asteromonas generation.Green algae and cyanobacteria prevailed in all the reservoirs under consideration.Purple thiobacteria were detected in the Tinaki.

Composition and degradation activity of heterotrophic bacteria
Halophilic heterotrophs are presented by gram-negative and gram-positive bacilli and cocci, and also by gram positive polymorphous and spore generating microorganisms in reservoirs under consideration.Gramnegative bacteria prevail in the lakes Selitrennoe and Baskunchak, while accumulative cultures are equally presented by gram-positive and gram-negative forms in the lakes Mramornoe and Tinaki.The least variety of morphotypes can be observed in Selitrennoe Lake communities.Detected halophilic microorganisms were capable of growing in different nutrient mediums, containing peptones, casein hydrolyzate, glycerine and glucose.
To estimate degradation capacity of halophilic microorganisms against organic constituent of brines communities, forming maximal biomass in liquid nutrient mediums, namely heterotrophic communities of the lakes Baskunchak, Tinaki and Mramornoe was used.The communities of microorganisms, detected in the Selitrennoe, were not used in the research because total dissolved solids of this reservoir and brines were significantly different, compared to the other water bodies.Bacteria of generation Pseudomonas, Bacillus, Micrococcus; archaea of generation Halobacterium were singled out in the brine microflora composition.
The chemical analysis revealed the reduction of organic substances in test samples without microorganisms, as well as in experimental samples with microorganisms; but, degradation is more intensive with halophilic microorganisms introduced to test samples, than with test samples, with organic decomposition observed in the conditions of natural microflora.Thus, after introducing communities of the Tinaki into brine samples, reduction of organic substances was 18.3%; that of Baskunchak was 20.6% and Mramornoe, 21.5%.The rest of the test samples showed 17,4% organic reduction (Figure 1).Hence, we observed insignificant intensification of natural degradation of brine organic substances from 1% to 4%.

Composition and degradation activity of phototrophic cyano-bacterial communities
Phototrophic cyanobacterial communities in the forms of present blue-green films, flakes and structural communities were obtained after 6 months of accumulative culture exposition using water from Mramornoe and nutrient medium BG N -11.Dominating cyanobacteria were identified as Phormidium dimorphum and Ph.сalcareum.Experiments on defining vitality of the obtained cyanobacterial communities in different sodium chloride concentrations were carried out beforehand.The experiments revealed that development and maximal increase of biomass occur with NaCl content from 1% to 8%.The decrease of cyanobacteria growth was observed With NaCl content from 4 % to 8%.We consider it to be  caused by medium aggressively.Then cyanobacterial communities of the Mramornoe adapted to functioning in the brine under research (with sodium chloride concentration 67 g l-1), and the active growth of the mat was observed.21 days later, after the introduction of the community into brine samples, organic substances content reduced by 74-86%; introduction of cyanobacteria caused 74% reduction of organic substances content in 100% brine; 75% brine, 86% reduction; 50% brine, 80% reduction (Table 2).Mineral salts content in the brines did not change.
The experiments show that natural degradation of brine organic substances is intensified.And halophilic heterotrophic communities, detected in Baskunchak, Tinaki and Mramornoe gave insignificant 1%-4% intensification of natural degradation.The best results were achieved while treating organic substances with cyanobacterial communities of Mramornoe (by 56% -68% compared to test samples), which is consistent with the literature (Lee et al., 1995;Blier et al., 1995;Prakasham and Ramakrishna, 1998;Boominathan, 2005).
The result of microbiological research of accumulative cultures of cyanobacteria communities of Mramornoe showed that their structure and species composition makes them an integrated community, which consists of cyanobacteria and other microorganisms, namely bacteria and fungi.We singled out bacteria of We consider that the maximum effect of the removal of organic substances when cyanobacterial communities were introduced is connected with the fact that in natural conditions cyanobacteria always form cyanobacteria populations, it is difficult to divide them from bacteria satellites, and that the organisms have cooperated for thousands of years (Herbst and Overbeek, 1978;Jorgensen et al., 1983;Jungblut et al., 2005;Vijayakumar, 2005).This consolidation might play a great Bataeva et al. 2055 role in adaptation universality of both cyanobacteria and cyanobacterial communities.Possibility of symbiosis is predetermined by cyanobacteria creating a specific ecological nichemucous formationssheathes, where heterotrophs are immobilized (Carr and Whitton, 1982).Extracellular polymeric substances, secreted by cyanobacteria, play an important role, protecting associates from drying, toxic substances effect, performing functions of a shelter for them (Thajuddin and Subramanian, 2005).Providing heterotrophic microorganisms with different products of its metabolism (polypeptides, polysaccharides, auxins, antibiotics, vitamins and amino acids), which these microorganisms can utilize (Sirenko et al., 1997;Smith and Doan, 1999;Kondratyeva, 2001) is considered to be stimulating role of cyanobacteria in microorganism communities.In turn, microorganisms provide cyanobacteria with carbonic acid, vitamins, decompose organic compound substances, consume oxygen, disengaged in photosynthesis.

Conclusion
In this work, we study the communities of halophilic microorganisms of extreme reservoirs with total dissolved solids of 195.7 to 585.8 g/l in Southern Russia.Heterotrophic and phototrophic community of microorganisms was isolated.Isolated communities have degradation abilities against organic substances of salty wastes of a fish-processing plant.Phototrophic cyano-bacterial communities consisting of numerous microorganisms that perform different physiological functions and which are more resistant to physicochemical medium conditions appeared to be more active (degree of bio-degradation was 86%).Dedicated phototrophic communities are communities where edificator or formative components (builders) are cyanobacteria of Phormidium.Therefore, these organisms can be used for further research and development of fundamentally new methods of purification of highly mineralized sewage water.

Figure 1 .
Figure 1.Biodegradation of organic substances by heterotrophs communities.

Table 1 .
Some hydrochemical coefficients of lakes under consideration and their MPC for fish economical water bodies

Table 2 .
Biodegradation of organic substances by phototrophic communities.