Cyanobacteria in wetlands of the industrialized Sambalpur District of India

Background Cyanobacteria are common components of phytoplankton communities in most freshwater ecosystems. Proliferations of cyanobacteria are often caused by high nutrient loading, and as such can serve as indicators of declining water quality. Massive industrialization in developing countries, like India, has polluted fresh water bodies, including wetlands. Many industries directly discard their effluents to nearby water sources without treatment. In the Sambalpur District of India effluents reach the reservoir of the worlds largest earthen dam i.e Hirakud Dam. This study examines cyanobacteria communities in the wetlands of Sambalpur District, Odisha, India, including areas subjected to industrial pollution. Result & Discussion The genera Anabaena, Oscillatoria, Chroococcus, Phormidium were dominant genera of polluted wetlands of Sambalpur districts. A positive correlation was found between total cyanobacterial species and dissolved oxygen levels, but cyanobacterial diversity was inversely related to BOD, COD, TSS, and TDS. High dissolved oxygen content was also associated with regions of lower cyanobacteria biomass. Conclusion Cyanobacterial abundance was positively correlated to content of oxidisable organic matter, but negatively correlated to species diversity. Lower dissolved oxygen was correlated to decreased diversity and increased dominance by Anabaena, Oscillatoria, Chroococcus, Phormidium species, observed in regions characterized by deteriorated water quality.


Background
Wetlands support a wide array of flora and fauna and deliver many ecological, climatic and societal functions. Scientists often refer to wetlands as the "kidneys" of the earth. However, many wetlands are subjected to urban and industrial pollution which disturb the aquatic ecosystem. Sambalpur is one of the main cultural and business centres of Odisha, India. It lies between 20°30′-22°30′N latitude and 83°E-85°1′longitudes with a total geographical area of 6,698 km 2 Figure 1. The Hirakud reservoir in Sambalpur was built primarily for hydropower generation, but is also used for irrigation, fisheries and drinking water. Sambalpur contains various freshwater ecosystem types, including lakes, reservoirs, ponds and wetlands. Cyanobacteria have been shown to be key primary producers at the base of the food web of many of these types of systems, i.e. freshwater (Muthukumar et al., [1], paddy fields (Bhattacharyya et al., [2], soils (Adhikary [3], desert (Bhatnagar et al., [4], temple (Deepa et al., [5] estuaries (Palleyi,[6], hotspring and marine hypersaline (Aharon et al., [7], Komarek et al., [8]. However, large proliferations of cyanobacteria are often caused by high nutrient loading, and therefore can be indicators of declining water quality (Rivas et al., [9]; Garcia et al., [10], which can be associated with a range of problems, such as low oxygen levels and the production of algal toxins (Komarek, [11]. Anthropogenically-driven increase in eutrophication and pollution have led to increases in the frequency and intensity of cyanobacteria blooms in ecosystems worldwide (Hecky [12]; Rivas et al., [9]; Garcia et al., [10].
This investigation focused on determining cyanobacterial composition and diversity in wetland ecosystems under the influence of industrial pollution in Sambalpur, India. Little information is available on cyanobacteria in wetlands of this region. In Orissa many researchers (Ghadai et al., [13]; Dey et al., [14,15]; Prasanna and Nayak [16]; Sahu [17]; Adhikary [3] have focused on cyanobacteria in rice fields. Dey and Bastia [15]; Dash et al., [18] have studied algal flora of Simlipal biosphere. In the last five years, about 40 iron factories have been established in Sambalpur and its neighboring Jharsuguda district. In addition, a Super-thermal power plant, a paper mill and several cement factories are located in these two districts. Open cast coal mines of the Mahanadi coal fields are located in Western Orissa which includes Sambalpur, Jharsuguda and Sundargarh districts. Many of these industries discharge untreated water into local channels which drain into Hirakud reservoir. The municipal wastes of cities of this region also mix into the Mahanadi, the main river of this region. This study was carried out to provide important missing data needed to define algal flora within impacted wetland ecosystems of this region Das [19].

Results and discussion
During the present investigation water samples were collected from 10 sites in wetland and stagnant water bodies associated with different industrial applications. The physicochemical properties of water of all site are described in Table 1 including pH, total suspended solids (TSS), total dissolved dolids (TDS), dissolved oxygen (DO), biological oxygen demand (BOD), chemical oxygen demand (COD),  Cyanobacterial strains were observed under a light microscope and further described with the aid of Camera Lucida, which helped to define taxonomically important characteristics, such as shape of vegetative cell, presence of heterocysts and akinetes (Figures 2, 3, 4, and 5). Results showed that Nostocales, and Chroococcales are major groups in the wetlands of this region. Over the study period, a total of 55 species and 20 different cyanobacteria genera were isolated and documented (Table 2) of which 30 were heterocystous forms and 24 were non-heterocystous. Relative abundance percentages of individual species are shown in Table 2.
Oscillatoriales, Nostocales and Chroococcales species showed the highest relative abundance values. Chroococcus turgidus and Nostoc punctiforme were found in almost all sites except Site 3 and Site 5 respectively. Two species of Haplosiphon were only found in samples from the guest house pond (control), although it is one of the major species observed in rice paddy fields by Das [19]. In terms of frequency of occurrence the genera Anabaena, Chroococcus, Gloeocapsa, Nostoc, Oscillatoria and Phormidium were observed at all sites, while at the other end of the spectrum Coelospherium and Fischerella were only observed in 33% ( Table 3). Presence of Anabaena, Oscillatoria and Chroococcus indicate the polluted values of wetlands.
Diversity index of cyanobacterial populations at the study sites were calculated using the Shannon-Wienner Method (1949) ( Table 4). Sites 3, 4, 6, 8 and 10 had indices over 3 (Table 4). Sites 1, 2, 7 and 9 had indices between, 2 and 3, while Site 5 had the lowest value, i.e. 1.24. The two sites with the lowest diversity, Sites 1 and 5, also exhibited the lowest mean DO values and the highest mean TSS, TDS and COD values.
Correlation analysis (Table 5) of physicochemical properties of water samples and total cyanobacterial species (TCS) revealed a positive correlation ( Figure 6) between dissolved oxygen and TCS (r=0.9385 p<0.01), which supports the findings of Muthukumar et al., [1]. In addition, the negative correlations between TCS and TSS, TDS, BOD, and COD, although not significant at the 0.01 level, further suggest that reduced water quality is associated with lower cyanobacterial diversity. By contrast, cyanobacterial abundance or biomass can increase in polluted systems associated with heavy nutrient loads. Kim et al., [22] found a positive correlation between prevalence of cyanobacteria and the levels of pollution in reservoir water, including the following species Anabaena azollae, Anabaena oscillarioides, Aphanothece microscopic, Chroococcus limneticus, Chroococcus turgidus, Chroococcus tenax, Gloeocapsa, Lyngbya, Oscillatoria, Phormidium. The crucial role in physicochemical parameters in defining algal community composition and abundance in agricultural and wetland ecosystems of tropical and temperate regions has been examined by a number of researchers (Kohler, [23]; Chellappa et al., [24]; Prasanna et al., [25].
Rapid cyanobacterial growth in the micro aerophilic condition has been observed by Stewart and Parsons [26]. Rai and Kumar [27] did not find heterocystous cyanobacteria in polluted water, although in our study various heterocystous species, including the genera Anabaena, and Nostoc, were commonly present. Presence of Anabaena, and some other blooming cyanobacteria have been linked to low DO content in eutrophic waterbodies (Moss, [28]; Mbonde et al., [29]. In terms of non-heterocystous taxa, Oscillatoria has been found to be tolerant to polluted water (Singh and Saxena [30]. Our observation of the widespread presence of the non-heterocystous genera Oscillatoria, Phormidium, Gloeocapsa, and Chroococcus corresponds to

Conclusions
Cyanobacteria are important primary producers at the base of the microbial food web in many aquatic environment. The composition and diversity of cyanobacterial communities can provide insights into changes in water quality. The present study provides a baseline of information on cyanobacterial composition associated with tropical wetland habitats under the influence of significant industrial development. This information can also be used to identify candidate species for use in bioremediation of industrial waste, since the species found in these systems are adapted to the stresses imposed by the components of the waste.

Study site
The study was conducted in different wetlands of Sambalpur districts, Odisha. Nine sampling sites (Figures 1  and 7) were selected from different industrial regions. A Guest house pond at Sambalpur University was selected as a site not directly influenced by industrial development. The sites are as follows: Site1, Paddy field water near  Surface grab water samples were collected in bottles from each site for microscopic analysis and isolation of cyanobacterial strains.

Collection of water sample
Water sample are collected in 250 ml air tight plastic jars. A few rocks from wetlands were collected to examine attached cyanobacteria.

Physicochemical properties of water
The physicochemical analyses of the water samples were carried out by using standard methods (APHA, [33] and Trivedy and Goel [34].

Isolation and enumeration of cyanobacteria
One ml water samples were added to agar plates made with 25 ml of sterilized BG ± 11 medium in petri dishes     Figure 6 Correlation between Cyanobacterial diversity index and physicochemical parameter of water.
Deep et al. Aquatic Biosystems 2013, 9:14 Page 9 of 12 http://www.aquaticbiosystems.org/content/9/1/14 (Rippka et al., [35] The dishes were kept under 7.5 W/ m 2 light intensity at 25±0.5 o C in a culture room. After 10-12 days of incubation, algal colonies appeared on the agar plates. The number of colonies of each species were recorded (CFU), after microscopic observation. Colonies were then isolated and spread on to fresh agar plates. After development, colonies appearing in agar plates were examined microscopically and transferred to agar slants. This process was repeated until individual pure colonies were obtained free from any contamination.

Microscopic analysis
Isolated cyanobacterial strains were observed under microscope MLX-TR. For morphometric analysis Camera Lucida Drawings was done and data related to trichome shape, filament colour, akinete and heterocyst shape, size, position, number recorded. Identification of cyanobacteria was done using the keys given by Desikachary [36] and Komarek and Anagnostidis [37,38].  S-the Number of Species in the Sample Pi-relative abundance of i th species or Kind measures,n i /N N-total no of individuals of all kinds n i -no of individual of i th species f. Correlation coefficient were calculated as per [40], using Microsoft Excel 2007 Package and analysed for their significant using Pearson's table