1. INTRODUCTION

Rubber Industry is a major thrust industry in Kerala, which makes a significant contribution to national economy. Kerala accounts for 85 per cent of area under rubber cultivation and 92 per cent of the rubber processing industries are located and scattered all over the state. The state has 44 rivers and 32 river basins and most of the natural rubber processing industries are established in river basins (Central Environmental Authority, 1990). The regional concentration is mainly due to the availability of raw material, technical compulsions for timely processing and other locational advantages including adequate availability of water for processing and conventional facilities for disposal of the effluent. However the intensity of rubber production is very high in Kottayam district (Survey of Rubber based Industries, 1995-1996). Also it generates many employment opportunities to rural population having lower level of education. The technology used by most of raw rubber manufactures is very old and this results in low productivity and high environmental damage which people do not tolerate any longer. A closer look reveals that rubber industry consumes large volumes of water, uses tones of chemicals and other utilities and discharges enormous amounts of wastes and effluents. Water pollution, through industrial discharges which is mainly in the form of effluent or wastewater, is one of the biggest problems.

Bioremediation is a pollution control technology that uses biological systems to catalyze the degradation or transformation of various toxic chemicals to less harmful forms. Bioremediation could be employed for the treatment of various industrial effluents. The general approaches to bioremediation are to enhance natural biodegradation by native organism (intrinsic bioremediation), or carry out environmental modification by applying nutrients or aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Unlike conventional technologies, bioremediation can be carried out on site.

2 It is well known that the essential conditions in the environment for the well being of aquatic populations are basic to understand the problems created by pollution and evaluation of its detrimental effects. Since the effluents are rich in nutrients due to the loading of organic wastes, they afford ideal habitats for different microorganisms including bacteria, fungi and algae. The study on the floristic pattern and ecology of waste water algae and other microbes is of prime importance to understand the basic problem created on account of pollution, there are numerous reports dealing with the floristic and ecology of lentic and lotic alagae (Ganapati, 1940; Desikachary, 1959; Lakshminarayana, 1965; Vyas and Kumar, 1968) but the algal flora of waste water system have been little investigated (Kamat, 1982; Trivedi et al., 1982; Vijayakumar et al., 2005; Boominathan et al., 2007; and Senthil et al., 2012a). In comparison with freshwater systems, algae in waste water are exposed to different environmental stress and a study on the biological parameter of such water bodies certainly paves the way for future waste treatment programmes, using indicator species and hence the present investigation on the biodiversity of microbes in rubber effluent, such as bacteria, fungi and cyanobacteria in order to understand their utility in waste water treatment.

The utilization of cyanobacteria in effluent treatment is a recent phenomenon. Since 1980, momentum of using cyanobacteria in waste water treatment has increased (Manoharan and Subramanian, 1992a, b and 1993a; Dash and Mishra, 1999a; Vijayakumar et al., 2005; Ganapathy Selvam et al., 2011 and Sanjay et al., 2011) cyanobacteria could be a good candidate for treatment of industrial effluents because of the following reasons. i) Cyanobacterial growth does not require energy rich compounds like other non-photosynthetic microorganisms. ii) They have simple growth requirements which use wastes as a source of reductant. This gives them an edge over other photosynthetic bacteria. 3 iii) Many cyanobacteria combine photosynthesis and nitrogen fixation. This is another advantage over other eukaryotic photosynthetic organisms. iv) Separation of cyanobacterial biomass is much easier than other microbial biomass due to their size.

By keeping the above facts in mind, the present study was carried out by using two cyanobacterial species (filamentous and unicellular) isolated from the natural habitat for bioremediation.

Suspended cultivation of microalgae has been employed to remove various nutrients and inorganic chemicals, some difficulties limit the practical application of suspended microalgae which include (a) monospecificity and good operation conditions are hard to be maintained, and (b) microalgae are difficult to be separated from effluent before discharge. Recently the use of immobilization to entrap microalgae for the removal of nutrients from waste waters show the potential to solve the above problems (Patnaik et al., 2001, Boominathan, 2005 and Vijayakumar and Manoharan, 2012). Immobilized living cells have some advantages over suspended cells as they provide simple treatment for liquid, final separation of cells not required, and metabolic activities remain constant for longer periods (Vijayakumar, 2012).

Entrapment in porous gels and foams is one of the popular techniques for the immobilization of whole cells as they generally do not use toxic reagents. The most commonly used matrices for photosynthetic cells are agar and alginate gels and polyurethane and polyvinyl foams. A disadvantage of the use of agar and alginate is their low mechanical stability for long term use in bioreactors; moreover, calcium alginate gels are disrupted by phosphate ions. Polyvinyl and polyurethane foams offer better mechanical properties and are neutral to most commonly used ions (Brouers and Hall, 1986) and hence in the present study polyurethane foam was used for entrapment of cyanobacterial cells. 4 To develop suitable and efficient treatment systems, it is obligatory to understand the mutual influence and interactions between the effluents and organisms, so that manipulations to improve the treatment systems become feasible. Therefore, in the present study, cyanobacterial cells, capable of growing in waste water have been chosen and their role on the physicochemical properties of the effluent was studied. In turn, the influence of the effluent on the biochemistry of the cyanobacteria to an extent possible was also investigated.

By keeping all the above in mind, the present investigation is aimed: (i) To isolate and identify microbes such as bacteria, fungi and cyanobacteria from rubber effluent. (ii) To find out the indicator species among cyanobacteria to treat rubber effluent. (iii) To study treatment of rubber effluent with both free and immobilized cyanobacteria particularly removal of various chemicals. (iv) To treat the effluent with polyurethane foam immobilized cyanobacteria. (v) To find out the influence of rubber effluent on the biochemical characteristics of cyanobacteria and (vi) To find out the effect of treated effluent on crop plants. 2. REVIEW OF LITERATURE

2.1. Biodiversity of microbes Algae in general, and cyanobacteria in particular, are assuming an increasing importance in biotechnology. Therefore it is necessary that a detailed survey of different habitats are made, to know what cyanobacterial species are available and subsequently to try to isolate, purify and establish a collection which could be used for a variety of purpose. There are numerous reports dealing with the floristic and ecology of lentic and lotic algae but the algal flora of waste water system have not been investigated much (Singh et al., 1969; Singh and Saxena, 1969; Rai and Kumar, 1976a, b, 1977, 1979; Trivedi et al., 1982; Gunale, 1991; Kanhere and Gunale, 1997; Tarar et al., 1998). In comparison to freshwater system, algae in waste waters are exposed to different environmental stress and a study on the biological parameters of such water bodies certainly paves the way for future waste treatment programmes using the indicator species. Palmer (1969) listed the algae tolerating different kinds of pollution and compared them with clean water algae.

The algal flora and physico-chemical characteristics of effluents of the Indian Oil Refinery, Barauni, the Sindri Fertilizer Factory, Sindri; and the Mohan Meakin Brewery, Ghaziabad were studied by Kumar et al. (1974). The studies indicate that algae can tolerate and grow in highly polluted waters. The blue-green algae, flagellates and euglenoids are mostly associated with organically rich effluents and low in dissolved oxygen.

Agrawal and Kumar (1978) made physico-chemical and biological analyses of the mercury containing effluents discharged by the Kanoria Chemical Factory Renukoot and the Rothas Paper Industry. They were highly toxic and did not harbour any algal populations. Subsidiary factors possibly responsible for the lack of algae in the effluents include the presence of some 6 amounts of zinc, copper, chlorides and organic matter and the deficiency of such major nutrients as phosphate and nitrate.

The changes in algal flora in the Cauvery river due to industrial and domestic pollution were studied by Paramasivam and Sreenivasan (1981). It was found that in clear water zones, Chlorophyceae and Bacillariophyceae dominated. Below the outfalls of distillery and sewage wastes, Cyanophyceae dominated. Oscillatoria sub-brevis was found dominant in pulpmill wastes.

Ecological study (Ramaswamy et al., 1982) of algae in waste water from a rubber tyre factory near Mysore, Karnataka showed the occurrence of blue green alga Microcoleus for the first time in the waste water of this type of industry. Inspite of the absence of nitrates and phosphates, diatoms were abundantly present in the effluent stream.

Blue green algae were dominant in the industrially polluted eutrophic Hussain Sagar lake, Hyderabad and were favoured by high orthophosphate levels. The percentage of total phytoplankton declined gradually from surface to bottom. Dense blue-green algal population was observed during summer in surface and middle strata and during winter in the bottom water. Low concentration of oxygen and high concentration of orthophosphate were associated with the building up of the blue green algal population. Accumulated surplus phosphorus revealed that they had developed in the bottom stratum (Khan and Seenayya, 1982).

Results of a two year ecological study on the algae inhabiting the effluent stream of a paper factory near Mysore, Karnataka and algae in freshwater lentic and lotic systems have been compared by Somashekar and Ramaswamy (1983). Seasonal variation in the algal flora has also been studied by them. The possibility of using indicator species in controlled waste treatment ponds for monitoring pollution has been stressed. 7 Chemical and biological assessment of the water pollution have been made at five sites in Rangpo stream of Sikkim Himalayas by Venu et al. (1984). The stream receives effluents from Sikkim distilleries. The list of algal species in the stream has been compiled and grouped under Cyanophyceae, Chlorophyceae, Bacillariophyceae and Euglinophyceae. Chemical data of the effluent and the stream water were correlated with biological data. Laboratory experiments have also been conducted with regard to the growth of Chlorella vulgaris, the dominant species in the stream.

Sahai et al. (1985) surveyed the algal flora of effluents from fertilizer factory, sugar factory, distillery and township sewage. They correlated the distributional pattern with the physico-chemical characteristics of the effluents. In all the polluted habitats cyanophycean members dominated. Only Oscillatoria was the most dominant in fertilizer factory effluent and township sewage, Oscillatoria, Microcystis, Chlorella, Closterium and diatoms were dominant in sugar factory and distillery effluents.

The seasonal occurrence and distribution of aquatic fungi in relation to  2 2+  BOD, dissolved CO2, NO3 , SO4 , Ca , Cl temperature and pH of 3 lakes of Jabalpur were investigated by Hasija and Khan (1987). Aquatic fungi were found in maximum number and diversity during winter season, while they could 2  not be found during summer. High SO4 and Cl content in one lake totally suppressed the occurrence of fungi. Reasons for restricted distribution of aquatic fungi in other lakes could not be ascertained.

Blue green algae play an important role in all types of water bodies whether being heterocystous or non-heterocystous. Mildly polluted ponds of Kanpur recorded the highest population of algae during summer, while lowest during winter months. Anabaena beckii, A. flos-aquae and A. orientalis were the only heterocystous members recorded from the polluted ponds (Pandey and Tripathi, 1988).

8 Fifteen pathogenic fungi were isolated from the effluents of a Gelatin Factory. Six of them were pathogenic to man, one to crops, and eight to both. Most of them occurred only in winter and originated from the first lagoon of the waste treatment system (Saxena et al., 1990).

The changes in phytoplankton size and species composition in the Nile water near the Egyptian Starch and Glucose factory were studied by Kobbia et al. (1993). The number of species found in the polluted section was low but always higher than in the unpolluted section.

Water samples from river were collected at polluted and unpolluted sites and analysed for physico-chemical properties such as temperature, electrical conductivity, pH, DO, BOD, COD, chloride, phosphate and nitrate by Srivastava and Singh (1995). They also collected algal samples from those sites and correlated their distribution with physico-chemical parameters.

Abo-Shehada and Sallal (1996) reported the occurrence of various types of heterotrophic Gram negative bacteria, in raw sewage, such as Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogens, Aeromonas hydrophila and Pseudomonas aeruginosa.

A total of 703 soil samples were collected from different rice fields of Manipur comprising of 8 districts. Altogether 110 blue-green algal forms belonging to 34 genera were identified. Nostoc punctiforme was found as the most dominant form. The maximum number (21) of BGA were recorded from soil sample of Khongiom (Thoubal district) having pH 7.0 and water holding capacity 38.97 per cent, whereas the minimum number (5) was observed from Phubala (Bishnupur district) having pH 4.1 and water holding capacity 42.1 per cent. pH of the soil proved to be an important factor affecting the growth and distribution of BGA (Devi et al., 1999).

9 Kousar et al. (2000) isolated 13 fungal species from dye effluent amended soils and these fungi were used for the decolourization studies of three textile dyes viz., scarlet direct red, fast greenish blue and brilliant direct violet.

Four different habitats namely, stream, campus soil, rice field soil and plant surfaces of Dargakona area were studied by Nandi and Rout (2000) for algal component during July to October 1999. A total of 66 algal species belonging to 41 genera were identified. It was noted that the number of algal species were more in the stream compared to other habitats. In the stream, green algae and diatoms dominated. Blue-greens proliferated more in the soil. Nitrogen fixers like Oscillatoria, Scytonema, Nostoc, Anabaena were also detected from the soil.

Colonization of structures of archaeological importance at various regions of the globe by cyanobacteria and algae and biodeterioration of monuments by these microorganisms has been reviewed (Adhikary, 2000). The possible measures for controlling the biological growth on the structures of cultural property has also been presented. Certain cyanobacterial species forming blackish-brown crust/tuft on the exposed rock surface of temples and monuments of India were also reported. Eleven different species of cyanobacteria belonging to Gloeocapsopsis, Lyngbya, Phormidium, Plectonema and Tolypothrix were the major components of the crusts/tufts collected from different locations of the country. These organisms grew slowly, possessed a well defined sheath around their cells/trichome and survived the extreme climatic conditions during summer months prevailing on the rock surfaces.

Shannon – Weaver index of diversity and other components of diversity were applied to surface plankton population by Manna et al. (2000) to study the water quality of a lotic sewage-fed freshwater ecosystem. Severe organic loadings caused low diversity by reducing the number of species (species richness) but did not increase the evenness (equitability).

10 Cyanobacteria are common in eutrophic natural waters. Being favoured by warm, stable and nutrient-enriched waters, they may constitute an important part of the phytoplankton community in Wastewater Treatment Plants (WWTP). The phytoplankton communities of two ponds (facultative and maturation) of the WWTP of Esmoriz (North Portugal) were studied by Vasconcelos and Pereira (2001) with special reference to cyanobacteria. During the study period (January-July, 1999) cyanobacteria were frequently dominant in the ponds ranging from 15.2 to 99.8 per cent of the total phytoplankton density. The main species were Planktothrix mougeotii, Microcystis aeruginosa and Pseudoanabaena mucicola.

Jain et al. (2001) isolated bacteria such as Xanthomonas fragariae, Bacillus megaterium and B. cereus from the activated sludge of a distillery waste water treatment plant and used for effluent treatment. They reported the removal of COD and colour from anaerobically digested distillery waste water from 55 to 68 per cent and 38 to 58 per cent respectively by these bacteria.

An investigation was carried out by Sulaiman et al. (2002) to assess the impact of dye factory effluent on the dynamics of microbial population viz., bacteria, fungi, actinomycetes and dinitrogen fixing free living organisms in horizon wise soil samples drawn at the discharge point and at 10 and 20 m lateral distances. Comparison was made with an unpolluted soil. At the effluent discharge site, in the 0-15 cm soil layer, there was a suppression of bacterial, fungal and actinomycetous population to the extent of 63, 100 and 59 per cent respectively. A further progressive reduction in their population was evident in deeper soil layers upto 45 cm. At 10 m and 20 m lateral distances, in general, the microbial population progressively decreased as the distance increased. The fungal population was almost nil in the polluted habitat which was moderately sodic. The free living nitrogen fixing bacterium, Azotobacter was totally absent in the dye effluent polluted soils. But other free living nitrogen fixing bacteria like Beijerinckia and Derxia, almost doubled in surface soil of the polluted site, 11 which further increased progressively with increasing soil depth and its lateral distances.

Abed et al. (2002) studied the microbial diversity of benthic cyanobacterial mats inhabiting a heavily polluted site in a coastal stream (Wadi Gaza) and monitored the microbial community response induced by exposure to degradation of four model petroleum compounds in the laboratory. Phormidium and Oscillatoria-like cyanobacterial morphotypes were dominant in the field. Bacteria belonging to different groups, mainly the Cytophaga-Flavobacterium- Bacteriodes group, the  and  subclasses of the class Proteobacteria, and the green nonsulfur bacteria, were also detected.

Fifteen different strains of blue green algae (including one green alga) collected from the waste waters of fruit processing industrial areas were screened for production of algal biomass from mango processing waste by Sunita and Rao (2003).

Cyanobacterial survey of dye industry effluent has been carried out by Vijayakumar et al. (2005). They isolated 24 species of cyanobacteria distributed in 9 genera falling under 5 different families. Among cyanobacteria, Oscillatoria with nine species was found to be the dominant genus in that habitat.

Role of cyanobacteria in distilleries effluent was studied in Kanchipuram, Tamil Nadu, India. Totally 12 species of cyanobacteria belonging to 6 genera falling under 4 families were identified by Ganapathy Selvam et al. (2011). Among the cyanobacteria isolated, Nostoc muscorum was selected to treat the effluent. Distilleries effluent was the potential source of cyanobacteria. Nostoc muscorum was found to be the most dominated genus in this effluent. The inoculation of Nostoc muscorum resulted in removal of various chemicals such as nitrogen, ammonia, phosphorus from the effluent. It is concluded that N. muscorum could be potentially employed for the treatment of distilleries effluent. 12 Biodiversity of cyanobacteria in industrial effluents such as dye, paper mill, pharmaceutical and sugar were selected by Vijayakumar et al. (2007). The physico-chemical characteristics of all the effluents studied were more or less similar. Totally 59 species of cyanobacteria distributed in four different effluents. Among the effluents, sugar mill recorded the maximum number of species (55) followed by dye (54), paper mill (45) and pharmaceutical (30). Except pharmaceutical effluent, others recorded heterocystous cyanobacteria. In total 26 species of cyanobacteria were recorded in common to all the effluents analysed. Of them, Oscillatoria with 13 species was the dominant genus which was followed by Phormidium (8), Lyngbya (2), Microcystis (2) and Synechococcus with single species each. The abundance of cyanobacteria in these effluents was due to favourable contents of nutrients.

An investigation was carried out by Boominathan et al. (2007) to assess the impact of dairy effluent on the microbial diversity viz., bacteria, fungi and cyanobacteria. Results of one year ecological study revealed that together 9 species of bacteria, 11 species fungi and 20 species of cyanobacteria were isolated from the effluent stream. Among bacteria, Pseudomonas with two species and others with single each were recorded. Aspergillus was dominant group of algae, inhabint all kinds of water (effluents), recorded 20 species. Oscillatoria with 11 species was the dominant genus followed by Phormidium (5), Plectonema (2), Aphanocapsa and Chlorogloea with single species each. Higher amounts of phosphates and nitrates, with sufficient amount of oxidizable organic matter, limited DO content and slightly alkaline. pH were probably the factors favouring the growth of microbes especially cyanobacteria.

Impact of rubber effluent on the microbial diversity viz., bacteria, fungi and cyanobacteria were analysed by Senthil et al. (2012a). Results of one year ecological study revealed that altogether 10 species of bacteria, 15 species of fungi and 42 species cyanobacteria were isolated from the effluent stream. Among the bacteria, Pseudomonas with two species and others with single species each were recorded. Aspergillus was dominant among fungi with 7 13 species followed by Penicillium with two cyanobacteria, one of the dominant group of algae, inhabiting all kinds of water one of the dominant genus followed by Lynbgya (8), Phormidium (4), Chroococcus and Microcystis with two species each. Nutrients were probably the factors favouring the growth of microbes.

Cyanobacterial populations from three different industrial effluents such as chemical, distillery and oil refinary have been isolated and identified by Vijayakumar et al. (2012). Their diversity has been correlated with physico- chemical characteristics of the effluents. Altogether 63 species of cyanobacteria were recorded from these effluents. Among the effluents, distilleries contained the maximum number of species (63) followed by chemical (52) and oil refinary (43). Except oil refinary effluent, others recorded heterocystous cyanobacteria. Totally 34 common species were observed in all the effluents. Of them, Oscillatoria with 14 species was the dominant genus followed by Lyngbya (7), Phormidium (6), Chroococcus, Aphanocapsa, Aphanotheca, Synechocystis and Plectonema with single species each.

2.2. Bioremediation The treatment of effluents may broadly be termed as physical, chemical and biological (Bhaskaran, 1977). By physical methods it is possible to remove about 80-90 per cent suspended solids and 10-15 per cent of BOD from wastes (Corning, 1976). Effluents can be treated by chemical coagulatns like alum, carbon dioxide from flue gas, sulphuric acid, ferric chloride and lime (Kbziorowski and Kuchaski, 1972) and biological treatments such as anaerobic digestion, trickling filter (Chakrabarty and Trivedy, 1965; Madhavakrishna et al., 1967) activated sludge process (Chakrabarty et al., 1967; Nallathamby, 1977) and oxidation ditch (Chakrabarty, 1972) are available. Reports on the treatment of domestic sewage by stabilization ponds (Ludwig et al., 1951; Oswald et al., 1953; Meron et al., 1965; Arceivala et al., 1970; Gloyna, 1971; Patil et al., 1975), septic tank followed by absorption trenches (Cooper and Rezek, 1977) and activated sludge process (Humenik and Hanna, 1969) are also available. 14 2.2.1. Suspended cultivation Many studies have demonstrated the success of using the algal cultures to remove nutrients from waste water rich in nitrogenous and phosphorus compounds (Neos and Varma, 1966; Kalisz, 1974; Saxena et al., 1974; Matusiak et al., 1976; Oswald et al., 1978; Chan et al., 1979; Rodrigues and Oliveria, 1987) and hence, have been used extensively in stabilization ponds (Fitzgerald and Rohlich, 1958; Witt and Borchardts, 1960; Gloyna, 1971), lagoons (Neel et al., 1961) and in tertiary treatment of sewage (Gates and Borchadts, 1964; Hemens and Stander, 1969; Knapp, 1971) for the removal of pollutants from the waste water.

The BOD and COD are widely recognized as important parameters for the measurement of the organic strength of waste waters. By using acclimatized algal cultures, considerable reduction of BOD and COD in sago mill waste water, dairy waste water and tannery waste water has been reported (Govindan, 1983, 1984, 1985).

Suspended cultivation of microalgae is one of the biological processes for the removal of nitrogenous compounds from wastewaters. Several species of microalgae have been studied including the green algae – Chlorella (Przytocka et al., 1984; Tam and Wong, 1989; de la Noe and Basseres, 1989), Scenedesmus (Martin et al., 1985; Tam and Wong, 1989; de la Noe and Basseres, 1989), Chlamydomonas (Taylor et al., 1988) and the blue green algae – Spirulina (Kosaric et al., 1974), Phormidium (de la Noe and Basseres, 1989; Pouliot et al., 1989), Oscillatoria (Fogg and Thake, 1987; Hashimoto and Furukawa, 1989; Manoharan and Subramanian, 1992a, b and 1993a), Anabaena (Taylor et al., 1988; Lee et al., 1995). These studies concluded that microalgae efficiently take up nitrogenous compounds, phosphorus and heavy metals from the effluents.

Ayala and Vagas (1987) did experiments on Spirulina culture in waste effluent media. They found that massive cultivation of Spirulina in waste 15 effluent media could improve the prospects for individual production of this biomass and the nutrient elements available in waste effluent were used by micro algae. The biomass obtained from intensive cultivation of Spirulina in these waste water media could be used as pigment-protein supplement in animal feed and as raw material for certain chemicals.

A general survey of various polluted environs was made and the effluents discharged by different factories were analysed for algal growth by Ahluwalia et al. (1989). The effluents from an electroplating plant was found highly toxic for algal growth. The final effluent of ghee factory was neutral and supported algal growth with all the concentrations employed. However, effluent discharged after treatment with H2SO4 had severe effect on algal growth even at 2 per cent concentration. The effluents of steel, automobile and fertilizer factories were also inhibitory, at relatively higher concentrations. However, relatively lower concentrations of some effluents supported the algal growth.

Tadros and Phillips (1992) studied the growth, nutrient removal and quality of Spirulina maxima on waste effluent media of different sources. The removal rate of N and P was rapid during the first week of growth. At the end of the second week, more than 90 per cent of the total N and P was removed. The mass of algae was high and they suggested that Spirulina may be integrated into the effluent treatment system.

Red mud, a waste material obtained from aluminium factory in the processing of bauxite ore, has been used as flocculant in the treatment of dairy-waste water by Namasivayam and Ranganathan (1992). Its efficiency was compared with the conventional flocculant, alum. Red mud removed 77, 65, 73 and 95 per cent of turbidity, BOD, COD, oil and grease respectively at a dosage of 1304 mg per litre of effluent compared to 94, 80, 86 and 93 per cent respectively for alum treatment at a dosage of 476 mg per litre of effluent.

16 Manoharan and Subramanian (1992a, b and 1993a) analysed the physico-chemical characteristics of domestic sewage, paper mill and ossein effluents under laboratory conditions by inoculating a cyanobacterium Oscillatoria pseudogeminata var. unigranulata. They observed a significant reduction of BOD and COD and the removal of various nutrients such as nitrates, ammonia and phosphorus from the effluents.

A cyanobacterium (Phormidium bohneri) was used to remove nutrients from municipal wastewater by Lessard et al. (1994). Field experiments suggested that the use of cyanobacteria was a viable alternative for small communities. Satisfactory reductions in ammonium, nitrate, nitrite and phosphates were achieved.

The nutrient removal and growth capacity of Phormidium bohneri were studied on anaerobic dairy (cheese factory) effluent. Among the 3 concentrations of effluent used (30, 40 and 50 mg NH3-N/litre), the highest growth rate and algal biomass were found in the more diluted effluent. The removal rate of ammonium nitrogen during the first 6 days was similar for all treatments (3.1 mg NH3-N/litre per day). In contrast, the rate of removal of phosphorus was proportional to the amount of phosphate present in the medium, 3 with a maximum value of 4.9 mg P-PO4 /litre per day (Blier et al., 1995).

Results on the growth response of the cyanobacterium, Westiellopsis prolifica in paper mill waste-water showed that the alga can grow well in the wastewater with basal nutrient medium. A significant reduction in the level of sodium (68%), potassium (50%), calcium (71.23%), chloride (23%), sulphate (74%), phosphate (90%) and chemical oxygen demand (78%) was recorded when Westiellopsis prolifica was grown in the paper mill wastewater (Dash and Mishra, 1999b).

Prakasham and Ramakrishna (1998) reviewed the work carried out by different authors using cyanobacteria for the removal of metal iron, nitrogenous 17 compounds and phosphates from the industrial wastewaters. From their review, they suggested that cyanobacteria are ideal tool for the treatment of industrial effluents.

The treatment of dairy industry effluent by biological methods, producing high value for the alternative costly method, has been discussed by Panesar et al. (1999).

Jain et al. (2001) reported that the removal of COD and colour from anaerobically digested distillery waste water ranges from 55 to 68 and 38 to 58 per cent respectively due to the growth of Xanthomonas fragariae, Bacillus megaterium and B. cereus.

A laboratory scale experiment was conducted in tannery effluent using cowdung as the seed material for aerobic digestion. The BOD removal of 95.8 per cent was obtained at an optimum organic load of 0.6 kg BOD/m1d. Biokinetic coefficients were calculated for the data obtained to study the metabolic performance of the microorganisms (Prakash, 2001).

Sharma et al. (2002) used a mixed culture of cyanobacteria to study the decolourization and COD reduction of digested distillery spent wash. On supplementing the diluted effluent with 1 per cent single super phosphate, about 63 per cent decolourization and 72 per cent COD reduction were achieved after 20d of incubation at 30-35oC.

Murugesan (2003) reported that the white-rot fungi such as Phanerochaete chrysosporium, Corius versicolor, Trametes versicolor etc., are efficient in decolourizing paper and pulp mill effluents. He also found that Gliocladium virens, a saprophytic soil fungus decolourised paper and pulp mill effluents by 42 per cent due to the production of hemicellulase, lignin peroxidase, manganese peroxidase and laccase.

18 Vijayakumar et al. (2005) investigated the role of cyanobacterium, Oscillatoria brevis in the treatment of dye industry effluent. They reported that within 30 days, more than 60 per cent of colour has been reduced. Nutrients such as nitrates and phosphates have been completely removed. An increase in DO content and reduction in BOD and COD upto 90 per cent have also been reported.

Iyagba et al. (2008) observed treating and disposing of the effluent of an 3- + indigenous rubber company rich in PO4 and NH4 and also determine the effect of the effluent on soil fertility. The basic method used for the biological treatment was aerobic digestion with glucose and magnesium amendments. Most naturally occurring aerobic heterotrophic bacteria in the rubber effluent were found to be capable of utilizing prominent among these bacteria were the genera of Micrococcus, Bacillus, Staphylococcus, Aerobacter, Proteus, Corynebacterium, Streptococcus, Aeromonas and Pseudomonas. The possibility of using the effluent as soil supplement was established.

Malaysia is the third largest rubber producer in the world, whereby the rubber industry is an economically and socially significant industry. Rubber industry consumes large volumes of water, uses chemicals and other utilizes and produces enormous amounts of wastes and effluent. Discharge of untreated rubber effluent to waterways resulted in water pollution that affected the human health, with a new global trends towards, sustainable development, the industry needs to focus on cleaner production technology, waste minimization, utilization of waste, resource recovery and recycling of water. It also adhres to the future trends of rubber effluent in Malaysia by reviewing various treatment technologies for natural rubber industry implemented by Thailand (Mitra et al., 2010).

Indira et al. (2011) reported that two sources of pollution, a sewage effluent and a tannery effluent and a comparison made with the ground water Guindy. Microalgae Lyngbya sp. and Oscillatoria sp. capable of surviving toxic 19 effects of the pollutants were used to degrade the pollutants rendering the effluents fit for further use. Water samples analysed from effluents revealed that sweage was less polluting than tannery effluent. The sewage showed a tolerable range particularly for cyanophycean algae.

Many industrial establishments due to scarcity of water – the natural resource and high water bills are prompted to recycle or reuse treated water for the process that may require freshwater. The rising demand for cleaner environmental requires cost effective and ecofriendly methods for control of pollution from industrial discharges, sewage etc. Bioremediation is one such method that offers a more suitable alternative to highly expensive physical and hazardous chemical methods of cleaning sites contaminated by discharge of raw effluents and waste water. Phycoremediation, which employs algae in clean-up process in a novel technique for bioremediation which is non-hazardous, less expensive and an environment friendly process (Kamaleswari and Sivasubramanian, 2011).

Sanjay et al. (2011) reported that cyanobacterial species isolated from the pharmaceutical and textile industries were analysed. Isolation and utilization of the locally generated cyanobacterial biomass for remediation of private industrial activities will generate a source of revenue of some potential cyanobacterial species: Oscillatoria sp., Synechococcus sp., Nodularia sp., Nostoc sp. and Cyanothece sp. dominated the effluents and mixed cultures showed varying sensitivity. Contaminant was removed by all the species, either as individuals or mixtures, at both concentrations. The abundance of cyanobacteria in this effluent was due to favourable content of organic matter, rich calcium and nutrients as nitrates and phosphates with less DO content.

Biodiversity and its application of cyanobacteria for the treatment of domestic and industrial effluents have received more attention during the recent years. Cyanobacteria have the capacity to utilize nitrogenous compounds, ammonia and phosphates. In addition, they accumulate metal ions such as Cr, 20 Co, Cu and Zn very effectively. It has been observed that immobilized cyanobacteria have great potential than its counterparts, i.e., free cells. Immobilization of success Vijayakumar et al. (2012) reported that the application of cyanobacteria for the removal of metal ions, nutrients, pesticides from the waste water to different effluents.

Dye industry effluent was treated with cyanobacteria for removing colour and other nutrients. Oscillatoria brevis and Westiellopsis prolifica were selected for the study based on their dominant occurrence in the effluent. Organisms were used in both free and immobilized conditions. These organisms not only removed the organic and inorganic chemicals but also reduced the intensity of the colour from the effluent. The result revealed that within 30 days, more than 75% colour has been removed. Nutrients such as nitrites, phosphates and ammonia were completely removed. Increase in DO content and reduction of BOD, COD upto 95% have been reported (Vijayakumar and Manoharan, 2012). Among the two conditions, immobilized cyanobacteria were more effective than that of free cells. It is concluded that Oscillatoria had a little edge over than Westiellopsis can successfully be used not only to reduce pollution load but also for colour removal purposes.

2.2.2. Immobilization Suspended cultivation of microalgae is one of the biological processes for the removal of nutrients from the wastewaters. However, some difficulties limit the practical application of suspended microalgae which include (a) monospecificity and good operation conditions are hard to be maintained and (b) microalgae are difficult to be separated from the effluent before discharge. Therefore, only few processes such as stabilization pond (Li et al., 1991) and high rate algal pond (Svoboda and Fellowfield, 1989) have been developed. Recently, the use of immobilization to entrap microalgae for removal of nutrients from wastewaters shows potential to solve the above problems (Chavalier and de la Noe, 1985; de la Noe and Proulx, 1988; Robinson et al., 1988; Lee et al., 1995). Several matrices such as agarose (Wickstrom et al., 21 1982), Carrageenan (Chavalier and de la Noe, 1985), Chitosan (de la Noe and Proulx, 1988) and alginate (Robinson et al., 1988; Lee et al., 1995) have been used for the immobilization of microalgae. Process involving immobilized cells have been attempted in the treatment of effluents containing materials such as phenols (Wisecarver and Fan, 1989), paper mill sludge (Gijzen et al., 1988), distillery waters (Subramanian et al., 1992), rubber press wastes (Jayachandran et al., 1994), olive oil mill wastes (Vassilev et al., 1997) and heavy metals (Stoll and Duncan, 1997).

The production of polysaccharide by immobilized cells of Porphyridium cruentum in a polyurethane prepolymer has been studied by Thepenier and Gudin (1985). The oxygen evolution rate has been evaluated. Cells divide in the polyurethane foam, colonized it and produced large quantities of polysaccharide for more than 8 weeks.

Gel-immobilized cells of Zymomonas mobilis grown on high glucose media were examined by Grote et al. (1986) by a freeze etching technique using transmission electron microscopy, and in a scanning electron microscope equipped with a Robinson detector. Both methods were found to be suitable for electron microscopy of high water gels. The study revealed that immobilized cells of Z. mobilis, which are facultative anaerobes, form microcolonies throughout the gel.

Yang et al. (1993) used an entrapment of mixed microbial cells in polymeric cellulose triacetate to remove the pesticide Ethylene Dibromide (EDB), Trichlopropane (TCP) and nitrate contaminated in the groundwater. The system was able to remove (aerobically) more than 90 per cent of EDB (influent concentration of 300 g/l) at more than 30 minutes of hydraulic retention time (HRT). TCP (influent concentration of 2.81 g/l) could not be detected in the effluent at the same HRT. The system was also able to remove (anaerobically) more than 99 per cent of nitrate (influent concentration of NO3-N ranging from 50 to 850 mg/l) at an HRT of more than 2 hours. They suggested that this 22 system had shown very promising results in respect of the removal of trace pesticide and nitrate contaminated groundwater and could also be considered as an alternative for direct treatment of nitrate-rich water.

Anabaena doliolum and Chlorella vulgaris immobilized on chitosan   3 2 were more efficient at removing NO3 , NO2 , PO4 and Cr2O7 from waste waters than free cells or cells immobilized on agar, alginate and carrageenan (Mallick and Rai, 1994). Carrageenan-immobilized cells, however, were better 3 in removing NH4 and NO2. The PO4 uptake capacity was significantly 3 increased in cells starved in PO4 for 24 h. Agar-immobilized cells had good metal and nutrient uptake efficiency but had a slow growth rate.

The removal of nitrogenous compounds from wastewaters using calcium alginate entrapped cyanobacterium, Anabaena CH3 was studied by Lee et al. (1995) in batch as well as semicontinuous cultures. Results of the batch cultivation showed that the removal rates of nitrate and ammonium nitrogen were between 15-23 and 7-30 N / litre per d.g. Anabaena CH3 respectively. The observed specific growth rates of Anabaena CH3 for different initial nitrate and ammonium concentration were between 0.35–0.65 and 0.2–0.56 /d, respectively. Results of the semicontinuous cultivation showed that the average growth rate of

Anabaena CH3 and ammonium removal rate were 83/mg per d and 86 mg

N/litre per d.g. Anabaena CH3 respectively. The optimum growth conditions for o immobilized Anabaena CH3 were pH 7-9 and temperature 30-40 C.

The immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam and their efficiency in the production of citric acid and extracellular enzymes were investigated by Sanroman et al. (1996). The different morphology of the obtained bioparticles seriously modify the productivity of citric acid and extracellular peroxidases by A. niger and P. chrysosporium respectively. The best results are obtained in both cases, when fungi developed inside the cube foam.

23 Alkaline protease production by mycelium of Aspergillus on agar, sodium alginate and polyacrylamide gel matrices was studied by Nehra et al. (1998). Immobilized mycelia performed better over a wide range of temperature and pH compared to free mycelia. Among all matrices, mycelia entrapped in agar gave better results upto 4th cycle of reuse. For longer reuse sodium alginate proved better and performed well upto 7th cycle of reuse. The maximum protease activity was in 7th cycle of reuse by mycelia entrapped in sodium alginate and reduced by only 15 per cent of the initial value.

Cultures of Anabaena azollae AS-DS-SK, A. variabilis – SAo, Nostoc muscorum DOH, N. muscorum – Kew-SK, Oscillatoria – Kew-SK, and Oscillatoria – DB-SK-1 were used for immobilization on polyurethane foam, sugarcane and paper wastes by Balachandar and Kannaiyan (1998). Growth, chlorophyll-a content and ammonia excretion were investigated by them. In general, immobilization of N2 fixing cyanobacteria on solid matrices stimulated growth and ammonia excretion when compared to free-living condition. Among the solid matrices used, polyurethane foam proved to be the best facilitating better colonization on the surface and in the pores, which increased the growth and ammonia production by the cyanobacteria. Nostoc muscorum – DOH showed maximum growth, chlorophyll-a content and higher ammonia excretion.

Growth, heterocyst differentiation, nitrogenase activity, ammonia production, ammonia uptake, glutamine synthetase (GS) activity, CO2-fixation and hill activity have been studied in the wild-type Anabaena variabilis and its NaCl-resistant (NaClr) mutant strain immobilized in calcium alginate gel by Chauhan et al. (1999). Immobilization of the cells in calcium alginate gel increased heterocyst differentiation and nitrogenase activity both in wild-type and its NaClr strain.

Patnaik et al. (2001) reported that the immobilization of cyanobacterium Spirulina platensis in 1.5 per cent alginate gave the best quality of bead and 15- 16 beads were formed per ml of aqueous solution of alginate. The immobilized 24 cells were used in a batch process for treatment of diluted sewage. They found that, after 8 days, 95 per cent of BOD, 77 per cent of COD, 90 per cent of ammonia, and 94 per cent of TSS were removed from the effluent.

For the effective treatment of tannery effluent, immobilized Flavobacterium sp. was used by Elangovan et al. (2002). They compared the efficiency of immobilized cells with that of free cells and found that the immobilized cells were efficient in the removal of various nutrients, BOD and COD as compared to free cells.

The cyanobacteria Anabaena torulosa was immobilized onto an oxygen electrode using a poly hydroxyl ethyl methaoxylate matrix. The behaviour of the organism towards some toxicants was investigated via inhibition of its photosynthetic activity; which could be monitored by the changes of photosynthetic oxygen release (Tay Chia et al., 2009). Using lead and 2,4- dichlorophenoxy acetic acid (2, 4-D) as the toxicants, it was shown that the cyanobacteria response was not affected by cell age or phase of cell growth. The results showed that the immobilized organism can be used as a toxicity biosensor for the assessment of Pb toxicity in river water samples.

Mohamed and Ola (2007) reported that, to evaluate the different uses of immobilized algae. Details of the techniques of immobilization and the effects of immobilization and the effects of immobilization on cell function are included special concern to the use of immobilized algae for waste water treatment and heavy metals removal has been taken into consideration. The use of immobilized algae in these processes is efficient and offers significant advantages in bioreactors.

2.3. Biochemical studies 2.3.1. Biomass and pigments The recent interest in algal and more specifically cyanobacterial biomass production using waste waters has necessitated a thorough understanding of the 25 influence of these waters on the physiology and biochemistry of these organisms. No serious attempt has yet been made in this direction.

Photosynthetic conversion of domestic and industrial wastes into algal biomass has been successfully developed in California, USA (Oswald, 1973) and Israel (Shelef et al., 1976). This system has been considered much more promising to operate with cyanobacteria because of their filamentous nature which allows mechanical harvesting. It is difficult with most micro algal species because of their small size (Benemann et al., 1977).

The toxic effects of Hindustan Petroleum (Caltex) Ltd., Gnanapuram, oil refinery effluent on green alga Scenedesmus incrassatulus and the blue-green alga Synechococcus aeruginosus were observed by Reddy et al. (1983). The growth decreased with increasing concentrations of effluent in both the algae but blue green alga S. aeruginosus showed a higher tolerance. Log phase was not expressed in any concentration of the effluent in both the algae. The effluent inhibits the growth and also reduces the synthesis of biochemical products like proteins, pigments and activity of acid phosphatase. Photosynthesis and respiration processes were also inhibited in both algae.

Mass culture of the economic important algae in synthetic medium is very costly. Taking into consideration, high organic content of domestic sewage of Berhampur and paper-mill effluent of Rayagada (Orissa) were investigated to utilize them as cheap and efficient media for algal biomass production as well as its implication in pollution abatement programme. Four local isolates of N2 fixing blue-green algae. Scytonema schmidlei, Anabaena cylindrica, Calothrix marchica, Gloeotrichia echinulata and one highly protein containing form Spirulina platensis were used as the test organism (Patnaik et al., 1995).

The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations by Marquez et al. (1995). At high DO concentration (1.25 mM) the growth rate decreased upto 36 per cent 26 compared with that of low concentration (0.063 mM). The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O2 evolution. They also reported that the photosynthetic pigments, such as phycocyanin, carotenoid and chlorophyll-a decreased distinctly.

Laboratory cultivation of Chlorella and Spirulina using an effluent from a fertilizer company was carried out by Anaga and Abu (1996). Approximately 6.1 mg ml1 was obtained for Chlorella in the effluent (pH 7.4). While 2.7 mg ml1 was obtained for Spirulina in a 50:50 mixture of the effluent and filtered sea water (pH 8.3). It was concluded that this non-sewage effluent could be used for the production of micro algal biomass and value-added biochemicals.

Ganesh (1996), investigated the feasibility of using whey and dairy effluent as substrates for microbial biomass protein (MBP) production by cultures of the algae Spirulina maxima and S. platensis. Biomass production was greater in 50 per cent than in 25 per cent whey. The inocula of S. maxima and S. platensis respectively yielded 0.56 and 2.33 gl1 biomass, which were correlated with moisture content and organic matter.

The nutrient removal and growth capacity of Phormidium bohneri were studied on anaerobic dairy effluent by Blier et al. (1996). Among the 3 concentrations of effluent used (30, 40 and 50 mg NH4-N/l), the highest growth rate and algal biomass were found in the more dilute effluent. A reduction in chlorophyll and phycobiliproteins were noted in all concentrations studied. It was concluded that the growth of P. bohneri in anaerobic dairy effluent is feasible for the production of useful amount of biomass in parallel with tertiary treatment.

Anand and Hopper (1987) reported that different salinity concentration such as 10, 40, 80 and 100 per cent (NaCl) influenced the pigments, 27 photosynthesis, protein content and phycobilin leaching in the cyanobacterium, Oscillatoria sancta.

The total content of phycobiliproteins was estimated at 28.4 per cent of dry weight of Spirulina subsalsa being higher than that of other blue-green algae. S. subsalsa was shown to be an ideal source for edible protein (Qifang et al., 1988).

Wenzhou et al. (1991) isolated and purified two types of biliproteins, C-phycocyanin and allophycocyanin from blue-green alga Spirulina platensis cultured in seawater. Low light intensity induced an increase in biliprotein content. The biliprotein content was decreased in nitrogen starvation and recovered by addition of nitrogen source. These results showed that biliproteins can serve as a “nitrogen pool” in S. platensis cultured in sea water.

The qualitative and quantitative carotenoid composition for (i) a red and a green strain of Oscillatoria limnetica and a green strain of Spirulina platensis cultivated under identical conditions and (ii) a red and a green strain of S. subsalsa grown under identical conditions have been reported (Aakermann et al., 1992). No correlation between colour and carotenoid content was obtained. However differences in carotenoid composition between Oscillatoria and Spirulina strains were observed.

Structure, composition and extraction of phycobiliproteins such as C-phycocyanin and allophycocyanin from Spirulina platensis have been reported by a number of people (Brejc et al., 1995; Guangce et al., 1996; Naidu et al., 1999; Ming and Feng, 1999).

Isolation of cyanobacteria was attempted from herbicide applied rice soils. The predominant genera was Westiellopsis followed by Anabaena, Nostoc and Oscillatoria. The herbicide tolerance was further tested by growing the cyanobacterial cultures in BG-11 medium supplemented with varying 28 concentrations of the commonly used rice herbicide, viz. butachlor under in vitro condition. The chlorophyll-a, phycobiliproteins and ammonia excretion were assessed at periodic intervals. Westiellopsis showed the maximum tolerance followed by Anabaena, Nostoc and Oscillatoria (Selvakumar et al., 2002).

The effect of light irradiance and temperature on growth rate, biomass composition and pigment production of Spirulina platensis were studied in axenic batch culture (Manoj Kumar et al., 2011). Growth kinetics of cultures showed a wide range of temperature tolerance from 20 to 40°C. Maximum growth rate, cell production with maximum accumulation of chlorophyll and phycobilli proteins were found at temperature 35°C and 2,000 lux light intensity, carotenoid content was found maximum at 3500 lux. Improvement in the carotenoid content with increase in light intensity is an adaptive mechanisms of cyanobacterium S. platensis for photoprotection could be a good basis for the exploitation of microalgae as a source of biopigments.

The cyanobacterium Spirulina platensis is an attractive alternative source of the pigment chlorophyll, which is used as a natural colour in food, cosmetic and pharmaceutical products. The influence of light intensity and pH for Spirulina platensis growth, protein and chlorophyll ‘a’ content were examined by Pandey et al. (2010). The production of Spirulina platensis was optimized in terms of biomass and metabolites. The dry weight of Spirulina platensis was 0.91 g/500 ml and protein and chlorophyll ‘a’ content were 64.3 per cent and 13.2 mg/g respectively at pH 9.

2.3.2. Macromolecules The influence of environment on the physiology of an organism also results in profound changes in the biochemical composition of the organism. Changes in protein and amino acid profiles due to variety of stress are well known. Environmental-stress-induced modifications of protein synthesis have observed in microbes, plants and animals (Schlessinger et al., 1982; Kimpel and 29 Key, 1985; Berg et al., 1987). However, the mechanisms which govern gene expression during stress and the biological significance of the stress induced proteins are not well understood. A few cases are known in which exposure to a certain stress has been found to induce protein responses typical of another stress (Berg et al., 1987: Edelman et al., 1988) or even tolerance to another stress (Harrington and Alm, 1988).

Many cyanobacteria express a marked tolerance to various stresses such as water stress and desiccation (Brock, 1975; Potts and Friedmann, 1981; Potts et al., 1983; Potts and Bowman, 1985), salt and osmatic stress (Fogg et al., 1973), heat shock and salinity (Bhagwat and Apte, 1989) resulting in considerable alternations in their protein synthesis patterns. The cyanobacterial response to these stresses varied with time. Two prominent types of modification were noted; the synthesis of certain proteins was significantly enhanced; and synthesis of specific set of proteins was induced de novo (Apte et al., 1987; Bhagwat and Apte, 1989).

In Spirulina platensis, elevated temperature determine changes in filament morphology, growth rates, macro molecular composition, lipid and fatty acid composition and phycobiliproteins – chlorophyll ‘a’ ratio. At 42oC a significant decrease in protein content (22%), a marked increase of lipid (43%) and of carbohydrates (30%) were observed (Tomaselli et al., 1988).

When cells of Synechococcus PCC 7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of Mr 92000, 48000-50000 and 35000 appeared.

Specific iron-limited outer membrane proteins (IRMPs) of Mr 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC 6308. Under magnesium limitation polypeptides of Mr 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. Phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of Mr 32000 30 and one of over 100000 being induced, whereas calcium limitation had no apparent affect (Scanlan et al., 1989).

The effect of different concentrations of nitrate-nitrogen, phosphate, bicarbonate and sodium chloride in the growth medium on the growth rate and yield of Spirulina maxima was investigated by by Tadros (1991). The growth rate and the yield coincided with increasing the concentration of the nutrients upto a certain concentration and these leveled off. The total carbohydrate increased significantly when the nitrate-nitrogen phosphate and bicarbonate were in low concentrations compared to the control while the total protein decreased. On the other hand, increasing the concentration of sodium chloride in the growth medium led to increasing the carbohydrate percentage.

Influence of papermill effluent on the growth and biochemical characteristics of Oscillatoria pseudogeminata var. unigranulata was investigated by Manoharan and Subramanian (1992a). They found that except carbohydrate, all other biochemical components have been drastically reduced with 100 per cent effluent. Biomass content was also low when compared to control.

Effect of different nitrogen levels and light quality on growth, protein and pigment synthesis in Spirulina fusiformis was investigated by Subramanian and Jeeji Bai (1992). They stated that protein synthesis was enhanced in blue, yellow and red light in nitrogen deficient cultures and in highest nitrate levels. In white and green light, however, protein content progressively increased from deficient to sub-maximal levels and decreased again at the maximum levels (2.5 and 5.0 g/l). In absolute terms blue light yielded highest protein content followed by yellow, white and green light. Pigment synthesis in white and green lights seemed to be negatively affected. Except for the maximum nitrogen level in red light all other chromatic regions yielded higher absorption peaks with 0.625 and 1.25 g/l of nitrate.

31 Influence of ossein effluent on the biochemical composition of Oscillatoria pseudogeminata var. unigranulata was investigated with respect to carbohydrate, free amino acids, total organic nitrogen, protein and lipid. Except carbohydrate and free amino acids, other biochemical components showed significant reduction in their content (Manoharan and Subramaniyan, 1996).

Gordillo et al. (1999) studied the effect of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of cyanobacterium Spirulina platensis (Arthrospira). They found that increasing

CO2 levels did not cause any change in maximum growth rate while it decreased maximum biomass yield. Protein and pigments were decreased and carbohydrate increased by high CO2, but the capability to store carbohydrates was saturated.

CO2 affected the pigment content; phycocyanin, chlorophyll and carotenoids were reduced in around 50 per cent but the photosynthetic parameters were slightly changed.

Dairy effluent and cyanobacteria cultured in effluent for 3, 5, 7, 9 and 11 days were studied by X-ray diffraction to understand the interaction between them. Physico-chemical properties, total protein and carbohydrate content of effluent before and after culture of cyanobacteria, change in biomass, total proteins, carbohydrates, chlorophyll,  and  carotene contents of cyanobacteria were analysed. X-ray spectra showed an increase in the crystalline nature of the biomass and decrease in crystalline nature of effluent. Results suggest that organic and inorganic substances present in effluent are absorbed and metabolized by cyanobacteria (Sharma et al., 2003).

Renuga (2005) observed a significant growth of nitrogen-fixing blue-green algae and an increase chlorophyll content, protein and carbohydrate accumulation in the cells grown with diluted tannery effluent.

Karanth and Madaiah (2011) studied the biochemical constituents of seven species of cyanobacteria namely, Calothrix fusca, Gloeocapsa livida, 32 Lyngbya limnetica and Scytonama bohneri isolated from parekal sulfur spring. The species namely Oscillatoria acuminata from petrochemicals refinery, O. calcuttensis from dairy effluent and O. foreiaui from sewage drain located in the Western Ghats of S. Indian water labortatory culture conditions. The biochemical constituents were analysed in term of total carbohydrate, total protein, total free amino acids, total lipids, fatty acid and mineral contents. The analysis showed that maximu amount of total carbohydrate in S. bohneri (28.4% dry weight) and minimum in O. foreami (8.0% of dry weight). Maximum amount of total protein and total free amino acids were in O. foreain (7% dry weight), O. calcuttensis showed higher amount of total lipids (20% dry weight). A total of 12 types of fatty acids were detected among which lauric acid was a highest quantity in all the seven species.

Total protein, lipid and carbohydrate content of seston in four seasonal sampling campaigns in a tropical hypereutrophic reservoir, physical and water chemical variables and taxonomic composition of phytoplankton were measured in parallel. Seston lipid and carbohydrate contents exhibited highest values during the day, while protein content was highest at night. Carbohydrate content was negatively correlated with nitrate and nitrite concentrations. Lipid content was negatively correlated with temperature and positively with soluble reactive phosphorus (SRP) concentrations. Protein content was positively correlated with temperature and negatively with SRP concentrations. In all sampling campaigns are correlated to the biovolume of phytoplankton (Iola and Alessandra, 2008).

2.3.3. Fatty acids Among eukaryotes, -linolenic acid (18:3) and certain related polyunsaturated fatty acids occur as major fatty acids only in photosynthetic organisms, where they are concentrated in chloroplasts as components of the acyl lipids (Benson, 1964). Blue-green algae are the only prokaryotes which photosynthesize as do green plants, and their fatty acid composition is thus of particular interest from the evolutionary stand point. Many filamentous blue green algae have been shown to contain polyunsaturated fatty acids (Nichols and 33 Wood, 1968). With respect to their content of polyunsaturated fatty acids, the strains fall into two readily distinguishable groups. Some have a high content of these fatty acids, and others either do not contain these compounds, or contain very little of them. All filamentous strains examined belong to high polyunsaturated fatty acid group (Kenyon and Stanier, 1970).

Lipid content of various microalgae grown under different environmental conditions and different types of water ranging from fresh, brackish to sea water and hyper saline conditions varied considerably. The lipids of blue-green algae have close affinity to bacteria than eukaryotic algae. Considerable differences in lipid components of blue-green algae with those of eukaryotic algae and higher plants are known (Nichols, 1970).

The bacterial type of fatty acid composition is relatively common among unicellular blue-green algae, whereas the presence of large quantities of polyenoic fatty acids is characteristic of most filamentous blue-green algae (Kenyon, 1972). The analyses so far conducted have shown that the members of blue-green algae are uniquely diverse with respect to fatty acid composition, some have a fatty acid composition of the bacterial type, some are chloroplast type, and some types hitherto not described in bacteria or in chloroplast (Holton and Blecker, 1972).

The fatty acids of 32 strains of filamentous blue-green algae have been analysed by Kenyon et al. (1972). All filamentous strains except two of the Spirulina types contain relatively large amount of polyunsaturated fatty acids, whereas the Spirulina type contain large amount of -linolenic acid. Oscillatoria group is characterized by the presence of both octadecatetraenoic acid and trienoic fatty acids (predominantly -linolenic acid) as the major fatty acid with highest degree of unsaturation.

There seems to be a wide variation in the composition of fatty acids between individual classes of marine algae, marine and freshwater algae and 34 also between algae and terrestrial plants. Algae synthesize generally straight chain saturated and unsaturated fatty acids with even numbered carbon atoms (Pohl and Zurheids, 1979).

Effect of temperature (Holton et al., 1964) and light (Dohler and Datz, 1980) on the fatty acid composition of Anacystis nidulans was studied. Palmitic and palmitoleic acid totaled approximately to 90 per cent at all the temperatures. However, the levels of palmitoleic to palmitic acid decreased as the temperature was raised; the ratio of total unsaturated to saturated acid remained approximately 10 at temperatures between 26 to 35o but at 41o the saturated acids predominated.

A number of cyanobacteria showing a high degree of adaptation to life under reduced oxygen tensions as witnessed by their potency of facultative anoxygenic CO2 photoassimilation with sulfide as electron donor were found to lack polyunsaturated fatty acids in their lipids. Lack of polyunsaturated fatty acids was found in representatives of different taxonomic groups. One of the strains lacking polyenoic acids was Oscillatoria limnetica, which can alternatively grow aerobically or anaerobically with sulfide as electron donor. This organism was found to synthesize monounsaturated fatty acids by desaturation of their saturated counterparts, in the presence as well as in the absence of molecular oxygen (Oren et al., 1985).

Some unusual fatty acids such as cyclopropanic, acetylenic, hydroxyl, epoxy, oxo acids which are common in bacteria, fungi and some terrestrial plants were not found in many groups of algae. Arachidic acid (20:0), behenic acid (22:0) and fatty acids with still longer chains are rarely present. Small quantities of odd chain fatty acids such as 13:0, 15:0, 17:0 and 19:0 have also been reported (Pohl and Zurheids, 1979; Orcutt et al., 1986; Jahnke et al., 1989).

The lipid biosynthesis in green algae is influenced by the physiological state of the cell and availability of nutrients, especially nitrogen. At low nitrogen 35 concentrations all of the green algae contained large amounts of total lipids. These amounts decreased significantly with increasing nitrogen concentrations. However, in blue-green algae the total lipid content was not usually influenced by culture conditions (Piorreck et al., 1984; Piorreck and Pohl, 1984). Effect of light and dark incubation on the lipid and fatty acid composition of five cyanobacteria were studied (Al-Hasan et al., 1989). All light grown cyanobacteria contained in their lipid extracts the four major lipid classes characteristics of chloroplasts. However, in their fatty acid composition, they differ markedly from those of chloroplast and responded differently to dark inoculation.

Fatty acid types from anaerobically grown Oscillatoria limnetica have been reported by Jahnke et al. (1989). The principle fatty acids detected were, 14:0, 16:0, 16:1, 18:0 and 18:1. In addition, small amounts of both 17:0 and 17:1 fatty acids were also detected.

Changes in growth and fatty acid content in Spirulina platensis were examined after transferring cells into media containing various concentrations of ammonium chloride by Manabe et al. (1992). They found that photosynthetic O2 evolution rate decreased, with increasing ammonium chloride concentration. On the other hand, total fatty acid content markedly increased after addition of ammonium chloride to a concentration of 15-50 mM. The increases in palmitic acid and oleic acid content were especially remarkable. 1.5 to 2 fold increase in -linolenic acid content was observed compared to untreated cells.

Qualitative and quantitative estimations of fatty acids from a cyanobacterium Oscillatoria pseudogeminata var. unigranulata influenced by four different effluents (domestic, ossein, paper mill and tannery) were studied by Manoharan and Subramanian (1993b). They reported the occurrence of 17 different fatty acids including two unidentified and six short chain along with linolenic acid (C18.3).

36 The fatty acid composition of the lipids of Spirulina was studied by Pascaud (1993). He reported that the fatty acid composition of the lipids of Spirulina is characterized by huge levels of the long-chain polyunsaturated fatty acids (PUFAs) belonging to the omega 6 series, including the essential fatty acid linolenic 18:2 omega 6. The fatty acids composition depends on the strain of Spirulina and the culture conditions. Most strains are rich in gamma-linolenic acid 20:3 omega 6 belonging to the omega 6 series, and poor in linolenic acid 18:3 omega 3, some are poor in omega 6 PUFA but rich in linoleic acid.

The total lipid content was reduced to half both in the halosensitive Calothrix marchica ARM 659 and in the halotolerant Calothrix bharadwajae ARM 558 when grown at 50 and 350 mM NaCl respectively. Common fatty acids detected in the extracted lipids of both the strains were from C8 to C18. In response to NaCl the quantity of caprylic acid, nonanoic acid and trimyristolein were substantially decreased whereas, capric, undecanoic, lauric, palmitoleic, heptadeconoic and stearic acids increased in the halotolerant strain. The halosensitive Calothrix marchica differed from that of the halotolerant Calothrix bharadwajae in the absence of nonanoic acid and palmitoleic acid (Senthil et al., 1993).

Effect of effluent such as domestic, ossein, paper mill and tannery on lipid content of Oscillatoria pseudogeminata var. unigranulata was studied by Manoharan and Subramanian (1995). All the effluents brought down the total lipid level considerably as compared to control.

Lipids have a vital role in tolerance to several physiological stresses in a variety of organisms including cyanobacteria. The mechanism of desiccation tolerance relies on phospholipids bilayers which are stabilized during water stress by sugars, especially by trehalose. Unsaturation of fatty acids also counteracts water or salt stress. Their role in stress tolerance in cyanobacteria have been reviewed by Singh et al. (2002).

37 Senthil et al. (2012b) reported that the impact of rubber industry effluent on the amino acid and fatty acid contents of two cyanobacteria, Oscillatoria salina and Microcystis aeruginosa were chosen as the test organism. Altogether 16 different amoni acids and 24 different fatty acids were detected in both test organisms. Some of the amino acids and fatty acids found in control were not detected from the effluent grown cyanobacteria. Effluent grown cyanobacteria recorded in higher quantity of total amino acids and fatty acids when compared to control.

2.4. Effect of industrial effluent on crop plants Reuse of treated waste waters is not being practiced on a large scale in many of the developing countries like India and also in many of underdeveloped countries. In a few situations where sewage waters are treated, they are used for raising crops, mostly fodder crops in sewage farms maintained by the local administration. However many of these countries are moving into the same situation as that of the developed countries with respect to the disposal of waste waters. Background information, problems, past experiences in this regard in India and other developing countries are limited and can be considered as scanty (Behera and Mishra, 1982; Pervez, 1986; Singh and Mishra, 1987; Kannabiran and Pragasam, 1993; Vijayaranjan and Lakshmanachary, 1993). However information and experiences are available in developed countries on the reuse of treated waste waters and these can be taken as guidance for planning though they have been generated in entirely different environmental conditions.

The results of three years of experiments concerning the crop yields, crop quality (both chemical and microbiological) on three crops such as cereal, a forage crop and an oil-bearing crop have been compiled and reported by Monte and Sousa (1992). Slightly better crop yield obtained in the plots irrigated with effluent lead to the conclusion that the nitrogen content of the facultative pond effluent can replace the nitrogen, the nitrogen from commercial fertilizers.

38 Rajula and Padmadevi (2000) investigated the effect of two different forms of effluent samples i.e., before recycling (sample I) and after recycling (Sample II) from an automotive industry on seed germination, growth and biochemical contents of Helianthus annus. They found that seedlings grown in Sample I showed a gradual decrease in germination percentage and growth with increase in effluent concentration. On contrary, seedlings grown in Sample II showed an increase in germination percentage and growth when the concentration of the effluent was increased. Similarly, with increasing effluent concentration there was a decrease in the biochemical contents (protein, carbohydrate and amino acids) in the seedlings grown in sample I and an increasing amount of biochemical contents in the seedlings grown in sample II was recorded.

An attempt has been made to irrigate two selected green leaf vegetables, such as species of Amaranthus and Trigonella sp. with dairy effluent (Maruthi et al., 2003). The results revealed that effluent with highly concentration increased the rate of seed germination and there was no adverse effect on nitrogen, carbohydrate and fat contents of seeds suggested that the dairy effluent may be beneficially utilized after proper dilutions for agricultural purposes, with respect to eco-friendly management of industrial effluent.

The effect of different concentrations of the sewage waste on the germination of Rabi crops (Triticum aestivum var. WH-147, Brassica campestris var. RH-30 and Hordeum vulgare var. BH-75) and Kharif crops (Sorghum vulgare var. Ramganj, Penniseum typhoides var. Nandi-3 and Zea mays var. KH-101) was studied by Khatrt et al. (2003). Germination percentage was found to be maximum in 50 per cent concentration of the sewage waste water while differential response was observed for seedling growth. Wheat, barley and yellow sarson showed similar trend of seedling growth on application of 50 per cent sewage waste water, while pearl millet and maize showed maximum growth under 100 per cent concentration of sewage 39 waste water. In case of Sorghum, both 50 and 100 per cent concentration of sewage waste water showed adverse effects on radicle as well as plumule length.

A green house studies was conducted at the University of Benin, Nigeria to evaluate the effect of brewery effluent on some soil chemical properties and growth of maize. The experiment, which was organized in a completely randomized design, had three replications with 0, 25, 50, 75 and 100% effluent concentration in a 2 kg soil. Results showed that organic carbon, N, P, Na and Mg concentration in the soil were reduced while K, Ca, C/N ratio, soil pH were increased. There were no changes observed in the soil textural class. The growth of maize plant as well as chlorophyll content was enhanced with brewery effluent treatments when compard with the control (Orhue et al., 2005a).

Sharma et al. (2011) reported that, to evaluate the impact of Amul dairy effluent on certain physico-chemical properties of soil and on growth, and quality of Lady’s finger (Abelmoschus esculentus) and Guar (Cymopsis tetragonoloba). The effluent used in different concentrations 20, 40, 60, 80 and 100%. The pH of the waste water was near about neutral but it contained an enough amount of nitrogen, phosphate, chloride, calcium, carbonates, bicarbonates and suspended dissolved solids when compared with freshwater. Soil receiving the wastewater showed no significant changes in water soluble salts, electrical conductivity, cation exchange capacity, pH, total organic carbon etc. Moreover waste water irrigation resulted in increased growth and nutrients of the both crops. 3. MATERIALS AND METHODS

3.1. Source of effluent To study the biodiversity and bioremediation of rubber effluent, samples were collected from Nijavalli latex, situated at Cochin, Kerala, India for a period of one year (November 2010 to October 2011). Samples were collected in large sterilized container and brought to the laboratory. Physico-chemical characteristics were done on the same day when the samples were brought to the laboratory. The effluent samples were filtered through cotton to remove suspended coarse particles before use.

Glassware used were of Borosil make, well cleaned with cleaning solution followed by the detergent and washed repeatedly with distilled water. Chemicals used were of AR grade.

3.2. Physico-chemical analysis of dairy effluent Physico-chemical characteristics such as Colour pH Temperature Total suspended solids Total dissolved solids Free carbon-di-oxide Carbonate Bicarbonate BOD COD Dissolved oxygen Nitrate Nitrite Ammonia 41 Total phosphorus Inorganic phosphate Organic phosphate Calcium Magnesium Chloride and biochemical characteristics such as carbohydrate and protein were analysed.

3.2.1. Methods of effluent analysis pH was recorded at the collection site with BDH indicator papers. In the laboratory the pH was checked again with Elico-india pH meter.

Free carbon-di-oxide; carbonate and bicarbonate alkalinities were determined titrimetrically (Mackereth, 1963) and expressed as mg l1.

Dissolved oxygen (DO) was estimasted by the Winkler’s method modified by Strickland and Parsons (1972) following necessary precautions. The 1 result was expressed as mg O2 l .

TSS, TDS, BOD and COD were estimated as per the Standard Methods (APHA, 2000).

Nitrates and nitrites were determined according to the method of 1 Strickland and Parsons (1972) and expressed as mg NO2-N l and mg NO3-N l1 respectively.

Ammonia nitrogen was estimated following the method of Solorzano 1 (1969) and expressed as mg NH3-N l .

Total phosphorus was measured by the method of Menzel and Corwin (1965) and expressed as mg P l1. Inorganic phosphate was determined by the 1 method of Murphy and Riley (1962) and expressed as mg PO4-P l .

Calcium and magnesium were estimated titrimetrically (Mackereth, 1963) and expressed as mg l1. 42 Chloride was determined titrimetrically (Strickland and Parsons, 1972) and expressed as mg l1.

Carbohydrate from the water sample was estimated following the methods of Dubois et al. (1956).

Protein was estimated according to the method of Lowry et al. (1951).

3.3. Biodiversity of microbes in rubber effluent The effluent samples were aseptically collected in a pre-sterilized bottle for the isolation of bacteria and fungi. For cyanobacterial survey, 10 places were selected along the effluent stream.

3.3.1. Isolation of bacteria Ten ml of the dairy effluent sample was taken in a 250 ml conical flask containing 90 ml sterile distilled water. The flask was shaken on an electric shaker to get a homogenous suspension and different dilutions viz., 101, 102, 103, 104 and 105 were made by transferring serially 10 ml of the effluent suspension to 90 ml of sterile distilled water. One ml each of 104 and 105 dilutions were plated separately in petridishes containing nutrient agar medium.

Composition of nutrient agar medium Peptone - 5 g Beef extract - 3 g NaCl - 5 g Agar - 15 g Distilled water - 1000 ml The pH of the medium was adjusted to 7. The inoculated plates were incubated at 25  2oC for one or two days and bacteria appearing on the medium were identified based on colony characteristics, Gram staining and by various biochemical methods as given by Bergey’s (1984) Manual of Determinative Bacteriology.

43 3.3.2. Isolation of fungi One ml of 103 dilution made in the above study (isolation of bacteria) was plated in petridishes containing Potato Dextrose Agar medium (PDA).

Composition of PDA medium Potato - 250 g Dextrose - 20 g Agar - 15 g Distilled water - 1000 ml The pH of the medium was adjusted to 5.6. Streptomycin sulphate (100 mg l1) was added to the medium to prevent the bacterial growth. The plates were incubated at 25  2oC for five days and fungi appearing on the medium were mounted over a clean slide, stained with lactophenol and cotton blue and observed under the microscope.

The fungi were identified by using standard manuals, such as Manual of soil fungi (Gillman, 1957), Dematiaceous Hyphomycetes (Ellis, 1971), Hyphomycetes (Subramanian, 1971) and More Dematiaceous Hyphomycetes (Ellis, 1976).

3.3.3. Isolation of cyanobacteria Cyanobacterial samples were collected from various collection spots (10 places) along with effluent in polythene bags. Standard microbiological methods were followed for the isolation of cyanobacteria. Algal samples were microscopically examined and plated on solid agar medium (BG11, Rippka et al., 1979). For the isolation of unicellular cyanobacteria, effluent samples were used.

Composition of BG11 medium

NaNO3 - 1.500 g

MgSO4.7H2O - 0.075 g

K2HPO4.3H2O - 0.040 g 44

CaCl2.2H2O - 0.036 g Ferric ammonium citrate - 0.006 g Citric acid - 0.006 g EDTA (disodium salt) - 0.001 g Solution with Trace elements - 1 ml Distilled water - 1000 ml pH - 7.1

Trace elements

H3BO3 - 2.860 g

ZnSO4.7H2O - 0.222 g

MnCl2 .4H2O - 1.810 g

Na2MoO3. 2H2O - 0.390 g

CuSO4.5H2O - 0.079 g

CO(NO3)2.6H2O - 0.049 g Distilled water - 1000 ml For solid agar medium, agar powder at 1.2 per cent was added to the above medium.

Plates were incubated at controlled conditions (temperature maintained at 28  2oC fitted with cool white fluorescent tube emitting 2500 lux for 18 hrs a day). Inoculated plates were regularly examined for the growth of cyanobacteria. Colonies appearing on solid medium were picked up and transferred to liquid medium. By repeated streaking, cultures were made unialgal and maintained in

BG11 liquid medium. Cyanobacteria were identified with the help of keys given by Desikachary (1959).

3.4. Bioremediation studies For bioremediation of rubber effluent, two cyanobacterial species such as Oscillatoria salina (filamentous) and Microcystis aeruginosa (unicellular) were selected based on their dominant occurrence throughout the study period as well 45 as by screening process. Both free and immobilized cells of O. salina and M. aeruginosa were used for the present study.

Oscillatoria salina Biswas Plant mass forming a deep blue-green, thin membrane extending over the muddy soil and finally after being separated floating on the surface of the water; filaments lying side by side in the stratum, straight, elongate, erect, scarcey curved, fragile, rapidly moving, not at all constricted at the joints 3-5  diam., apices of trichome straight, briefly tapering ending acuminately in a sharp point, hooked or twisted, not capitate; apical cell mucronate hyaline, calyptra absent; cells shorter than broad; 1.5 – 2  long, sometimes filament may be septa indistinct, not granulated, cell contents finely uniformly granular, almost homogenous blue green.

Microcystis aeruginosa Ktz Colonies when young round or slightly longer than broad, solid, when old becoming clathrate, with distinct hyaline colonial mucilage; cells 3-7  in diam., spherical, generally with gas-vacuoles.

3.4.1. Immobilization of cyanobacteria in polyurethane foam (PUF) cubes (Kannaiyan et al., 1994) (Fig.31) Procedure Polyurethane foam was cut into cubes of 10 mm; washed and soaked in distilled water. Two gram of PUF cubes were added into 250 ml conical flasks containing 150 ml of BG11 medium and sterilized. After sterilization, actively growing 15 days old cyanobacterial cultures were inoculated at 5 ml per flask. The flasks were then incubated for the period of 20 days under the light intensity of 2500 lux at 28  2oC.

46 3.4.1.1. Scanning electron microscopic study on PUF immobilized cyanobacteria (Rhodes et al., 1986) (Fig.32)

Polyurethane immobilized cells were fixed in 2.5 per cent glutaraldehyde for 2 hrs, washed in phosphate buffer and dried using graded (30 to 100%) ethanol. The dried specimens were treated with acetone; mounted on stubs and coated with platinum. Stub and specimen were then frozen by submerging in liquid nitrogen. The frozen preparation was examined in a SEM (ISI-100 A) equipped with Robinson backscatter electron detector. With this method, there is a separator and lower vacuum in the specimen chamber, providing about 30 min of viewing time with minimal distortion and image artifact.

3.4.2. Experimental conditions The following treatments were employed in order to study the interaction of cyanobacteria with the effluent (Fig.33 and 34). (i) Effluent inoculated with free cells of O. salina (FC1) (ii) Effluent inoculated with PUF immobilized cells of O. salina (PUF1)

(iii) BG11 medium inoculated with free cells of O. salina (CO) (iv) Effluent inoculated with free cells of M. aeruginosa (FC2) (v) Effluent inoculated with PUF immobilized cells of M. aeruginosa (PUF2)

(vi) BG11 medium inoculated with free cells of M. aeruginosa (CA) (vii) Uninoculated effluent– Control (C)

Uniform suspension of Oscillatoria and Microcystis (2 ml each) were inoculated separately as initial inoculum in each flask of the above treatments.

The experiment was conducted in batch cultures in duplicates for a total period of 20 days in 250 ml Erlenmeyer flasks under the same condition as described for the culture maintenance. Effluent samples (control and cyanobacteria treated) were periodically (every 5th day) analysed for various physico-chemical parameters.

47 3.4.2.1. Growth Growth of Oscillatoria and Microcystis were measured in terms of Chlorophyll ‘a’ (Mackinney, 1941) as the biomass component, at regular intervals.

3.5. Biochemical studies For biochemical studies, the following treatments were employed.

(i) BG11 medium inoculated with O. salina – Control (C1)

(ii) Effluent inoculated with O. salina – Treated (T1)

(iii) BG11 medium inoculated with M. aeruginosa – Control (C2)

(iv) Effluent inoculated with M. aeruginosa – Treated (T2)

Pigments such as chlorophyll ‘a’, carotenoids and phycobilins were estimated periodically (every 5th day). Carbohydrate, protein, amino acid, lipids and fatty acids were analysed on 20th day.

3.5.1. Estimation of Chlorophyll ‘a’ (Mackinney, 1941) Reagent 80 per cent methanol.

Procedure The cultures were centrifuged at 5,000 xg for 10 minutes. The pellets were washed with distilled water; suspended in 4 ml of 80 per cent methanol and vortexed thoroughly. Then the tubes were covered with aluminium foil to prevent solvent evaporation and incubated in a water bath set at 60oC for 1 hr, in dark with occasional shaking. After 1 hr, the contents were cooled and centrifuged at 5,000 xg for 5 minutes. The supernatant was saved and the above procedure was repeated twice with pellets to ensure complete extraction of the pigment. The pooled supernatant was made upto a known volume with 80 per cent methanol (To compensate the solvent loss during heating). The absorbancy was measured at 663 nm in Spectronic 20 against methanol as blank.

48 Calculation A663 x 12.63 x Volume of methanol Chl-a =  g ml1 Volume of algal sample where, A663 = Absorbance at 663 nm. 12.63 = Correction factor.

3.5.2. Estimation of Carotenoids (Davis, 1976) Reagent Acetone (85%)

Procedure The cultures were centrifuged at 5,000 xg for 10 minutes. The pellets were washed with distilled water; suspended in 3 ml of 85 per cent acetone and homogenized. The contents were centrifuged at 5,000 xg for 5 minutes and the supernatant was stored in refrigerator. The above procedure was repeated until the acetone colourless. The pooled supernatant was made upto a known volume with 85 per cent acetone. The absorbancy was measured at 450 nm in Spectronic 20 against acetone as blank.

Calculation D x V x f x 10 Carotenoids =  g ml1 2500 where, D = Absorbance at 450 nm. V = Volume of the sample. f = Dilution factor. 2500 = Extinction coefficient.

49 3.5.3. Estimation of Phycobilins (Phycobiliproteins) (Moreno et al., 1994) Reagent Phosphate buffer (0.05 M), pH 6.7.

Procedure The cultures were centrifuged at 5,000 xg for 10 minutes. The pellets were washed with distilled water; suspended in 3 ml of phosphate buffer (0.05 M) and homogenized. The contents were freeze thawed repeatedly and centrifuged at 5,000 xg for 5 minutes. The supernatant was stored in refrigerator. The above procedure was repeated to ensure complete extraction. The absorbancy of the pooled supernatant was measured at 565, 615 and 652 nm against phosphate buffer as blank.

Calculation A615 – 0.474 (A652) C-phycocyanin (PC) =  g ml1 5.34

A652 – 0.208 (A615) Allophycocyanin (APC) =  g ml1 5.09

A565 – 2.41 (PC) – 0.849 (APC) C-phycoerythrin (PE) =  g ml1 9.62

3.5.4. Estimation of Carbohydrate (Dubois et al., 1956) Reagents

A. Concentrated sulphuric acid (H2SO4) B. Phenol (5%)

Procedure The cultures were centrifuged at 5,000 xg for 10 minutes. From the pellet 100 mg was taken in a test tube and hydrolysed with 2 ml of concentrated o H2SO4 for 30 minutes at 100 C. To 0.5 ml of hydrolysate, 1 ml of 5 per cent 50 phenol and 5 ml of conc. H2SO4 were added and mixed thoroughly. The colour developed was measured at 490 nm in Spectronic 20 against the reagent blank. The amount of carbohydrate was calculated using a standard graph prepared from glucose and expressed as mg g1 dry weight.

3.5.5. Estimation of Total Protein (Lowry et al., 1951) Reagents A. 10 per cent TCA B. 1 N NaOH C. Alkaline sodium carbonate solution Prepared by dissolving 2 g sodium carbonate in 100 ml of 0.1 N NaOH. D. Copper sulphate – Sodium potassium tartrate solution

Prepared freshly every time by mixing CuSO4.5H2O (0.5%) and 1 per cent sodium potassium tartrate solutions in equal proportion. E. Alkaline copper reagent Prepared freshly by mixing 50 ml of reagent C and 1 ml of reagent D. F. Folin-Ciocalteu reagent Commercially available reagent was diluted with equal volume of water just prior to use.

Procedure The cultures were centrifuged at 5,000 xg for 10 minutes. From the pellet 100 mg was treated with reagent A and centrifuged at 10,000 xg for 10 minutes. The resulting pellet was resuspended in reagent B and boiled for 30 minutes; cooled and then recentrifuged to eliminate light scattering materials. The supernatant was made upto a known volume. To 0.1 ml of the supernatant 0.9 ml of distilled water and 5 ml of reagent E were added and allowed to stand for 10 minutes. Finally 0.5 ml of reagent F was added. The absorbancy was measured after 30 minutes at 750 nm in Spectronic 20 against the reagent blank. The amount of protein was calculated using a standard graph prepared from Bovine Serum Albumin (BSA) and expressed as mg g1 dry weight.

51 3.5.6. Extraction of protein for SDS-PAGE (Scanlan and Carr, 1988) Reagents (A) Sodium phosphate buffer - 0.1 M, pH 7.4 (B) Tris – HCl - 0.5 M, pH 6.8 The cultures were centrifuged at 10,000 xg for 10 min in refrigerated centrifuge. Equal amount of samples on dry weight basis was homogenized well with reagent A. The homogenate was centrifuged at 15,000 xg for 10 min at 4oC. From the supernatant, total protein was precipitated by the addition of powdered ammonium sulphate (47.2 g / 100 ml, yielding a 70 per cent saturated solution) at 4oC over 10-15 min with stirring, and was allowed to continue for a further 30 min before spinning in transparent tubes at 8,500 x g for 40 min at 4oC. The supernatant was poured off, and the pellet was resuspended in reagent B. The sample was then subjected to SDS-PAGE.

3.5.6.1. Gel Electrophoresis Analysis of proteins was carried out by SDS-PAGE according to the method of Laemmli (1970). A. Sample buffer 1 M Tris-HCl, pH 6.8 - 60 l -mercaptoethanol - 50 l 10 per cent SDS - 200 l 100 per cent glycerol - 100 l 1 mg ml1 bromophenol blue - 2 l pH was adjusted to 6.8 with 1 N HCl and the final volume was made upto 100 ml with distilled water.

B. Separation gel buffer - 4 x Tris-HCl, pH 8.8 Tris - 18.2 g SDS - 0.4 g Distilled water - 60 ml pH was adjusted to 8.8 with 1 N HCl and the final volume was made upto 100 ml with distilled water. 52

C. Stacking gel buffer – 4 x Tris-HCl, pH 6.8 Tris - 6.0 g SDS - 0.4 g Distilled water - 80 ml pH was adjusted to 6.8 with 1 N HCl and the final volume was made upto 100 ml with distilled water.

D. Acrylamide Stock Acrylamide - 30.0 g N, N-methylene bis acrylamide - 0.8 g Made upto 100 ml with distilled water.

E. Electrode buffer (5 x Tris-HCl) – Stock Tris - 15.1 g Glycine - 72.0 g SDS - 5.0 g Distilled water - 1000 ml The stock was diluted to 1 x before use.

Preparation of separating gel Separating gel was prepared in the following manner. Reagent B - 11.25 ml Reagent D - 18.00 ml Distilled water - 15.75 ml After degassing the acrylamide mixture, 150 l of 10 per cent ammonium persulphate (APS) and 30 l of N, N, N, N-tetramethyl ethylene- diamine (TEMED) were added. Using a gel maker, a linear slab gel was prepared.

53 Preparation of staking gel Reagent C - 3.0 ml Reagent D - 5.0 ml Distilled water - 12.0 ml After degassing the acrylamide mixture, 100 l of 10 per cent APS and 20 l of TEMED were added and mixed well. The solution was layered on the top of the separating gel after introducing a comb. The electrophoresis was run at 100 V for 6 hrs.

Staining and destaining The gel after electrophoresis was stained and destained. The stain was prepared by dissolving 0.25 per cent Coomassie brilliant blue – R 250 (Sigma) in 50 per cent methanol and 7 per cent (v/v) acetic acid. The gel was stained for 4 to 5 hours and destained with 50 per cent methanol and 7 per cent acetic acid mixture.

3.5.7. Amino acid profile by HPLC (Rajendra, 1987) Reagent OPA reagent Prepared by dissolving 50 mg of anhydrous O-Phthaldialdehyde (OPA) in 2 ml of methanol. To this, 8 ml of 0.4 M borate buffer (pH adjusted to 9.5 with 4 N NaOH) and 50 l of 2-mercaptoethanol were added. The reagent was prepared fresh every time.

Procedure To each vial, containing 100 l of amino acid sample, 500 l of OPA-reagent was added. The vials were capped; shaken and kept for 2 min for derivatization. Then, 20 l of each sample was injected separately into HPLC for analysis.

The peaks of the chromatogram were identified and quantified from the retention time (RT) and peak area of the known standard amino acids. 54

Operating conditions of HPLC Column : Isco C18, 4.6 x 250 mm 5 m packing Mobile Phase Solvent A : 0.1 M acetic acid buffer Solvent B : Methanol, Tetrahydrofuran (97:3 v/v) Flow rate : 1.5 ml/min

Detector : Fluorescence, Iso V4, 9 l Flow cell F-L-2 Excitation filter : 305-395 nm Emission filter : 430-470 nm Sensitivity : 0.005 Abs

3.5.8. Lipid Analysis (Sato and Murata, 1988) Extraction of lipid Reagent Chloroform : Methanol – 2:1 (v/v)

Procedure Cultures were centrifuged at 5,000 xg for 10 min. Equal amount of samples on dry weight basis was homogenized in a mortar and pestle with extraction solvent (Chloroform : Methanol 2:1 v/v) and filtered through filter paper. The filtrate was vortexed with sodium sulphate to remove moisture. Then it was taken in a pre-weighed bottle and dried by a steam of nitrogen.

Estimation of total lipids The dried extracts were weighed and the total lipids were estimated by subtracting the initial from the final weight. The amount of total lipid was expressed as mg g1 dry weight.

55 Thin Layer Chromatography (TLC) of lipids Reagents A. Chloroform B. Chloroform : Methanol : Acetic acid : Water – 85 : 15 : 10 : 3.7 (v/v)

The dried lipid samples were dissolved in 0.5 ml of chloroform and safely stored in deep freezer. These were taken for TLC. TLC was done using silica gel G as adsorbent and the reagent B as solvent system for the separation of lipids. Lipids were detected by keeping the plates in iodine chamber and identified using standard lipids.

3.5.9. Fatty acid profile by GLC (Miller and Berger, 1985) Reagents A. Saponification reagent Prepared by dissolving 45 g of NaOH in 300 ml of methanol : water mixture (1:1 v/v). B. Methylation reagent Prepared by mixing 325 ml of 6 N HCl with 275 ml of methanol. C. Extraction solvent Prepared by mixing 200 ml of hexane with 200 ml of anhydrous diethyl ether. D. Base wash Prepared by mixing 10.8 g of NaOH in 900 ml of distilled water.

Sample processing The cultures were centrifuged at 5000 xg for 10 min. From the pellet 100 mg was taken in separate screw cap test tubes. To each tube 1 ml of reagent A was added and tightly sealed with teflon-lined screw cap. Then the tubes were vortexed for 10 seconds and kept in a boiling water bath for 5 minutes. Again the tubes were vortexed 10 seconds. Then the tubes were kept in the water bath for the additional 20 minutes. After a total of 30 minutes of saponification, the tubes were removed from the water bath and cooled to room 56 temperature. To each tube, 2 ml of reagent B was added by uncapping the tubes. After vortexing for 10 seconds, the tubes were placed in the water bath set at 80oC for 10 minutes. Finally 1.25 ml of reagent C was added to each cooled tube.Then the tubes were tightly closed and rotated end-over-end for 10 minutes. From the tubes the lower aqueous phase was removed and discarded. To the upper phase, 3 ml of reagent-D was added and rotated end-over-end for 5 minutes. With the help of clean Pasteur pipette 2/3 of the organic extract from each tube was transferred to GC vials separately and kept in deep freezer by capping with teflon-lined septum. From each vial 2 l of sample was analysed with Hewlett Packard 5890 Gas chromatograph fitted with 10 per cent DEGS column and a flame ionization detector.

1 The carrier gas N2 was supplied at the rate of 30 ml min . The detector 1 1 gas flow rates were 30 ml of H2 min and 300 ml of air min . The chromatograph oven was set at 180oC, with injector and detector temperatures 210oC and 230oC respectively.

From the peak area of fatty acids, the amount of fatty acid was calculated using respective standards.

3.6. Effect of effluent on crop plants In order to find out the effect of treated effluent on crop plants, pot culture experiments were conducted. The seeds of two crop plants such paddy and green chilli were selected.

3.6.1. Pot culture experiment (Fig.63a and b) Experiment was conducted in duplicate using earthern pots. The following treatments were employed. (i) Control 1 – Tap water (ii) Control 2 – Raw effluent (iii) Treated effluent – 1 (with O. salina) (iv) Treated effluent – 2 (with M. aeruginosa) 57 The pots were filled with sterilized soil. The seeds of paddy and blackgram were divided into four sets having 10 numbers in duplicates. Seeds were sown into each pot and were irrigated with different water samples separately. The germination percentage was calculated after 10 days from the date of sowing. Morphometric analysis, such as number of leaves, shoot length, root length, number of seeds and seed weight were carried out. The results were statistically analysed. 4. RESULTS

4.1. Physico-chemical characteristics of effluent The results of physico-chemical analysis of effluent are presented in the table 1. The effluent was slightly alkaline and contained high amounts of nitrate, nitrite and ammonia; total, inorganic and organic phosphate and calcium in all the seasons examined (Table 1). Very low level of dissolved oxygen and high levels of BOD and COD were recorded in the study period. High amount of total suspended solids were recorded in rainy (July – November) and summer seasons (March – June) (Table 1). Total dissolved solids were high in summer followed by rainy and winter seasons (December – February). Nutrients such as nitrate, nitrite, inorganic phosphates, total and organic phosphates were maximum high in summer. BOD was very high during summer, on the other hand high level of COD was recorded during winter. Dissolved oxygen level was very low during summer and high in rainy seasons. Most of the parameters tested were slightly higher in summer than winter and rainy seasons.

4.2. Biodiversity of microbes in rubber effluent 4.2.1. Bacteria Bacteria isolated from the effluent were identified based on colony morphology, Gram staining, and various biochemical characteristics. The characteristics of isolated bacteria are given in the table 2. Totally ten different bacteria were isolated from the effluent (Table 3) sample. The species isolated were Escherichia coli, Enterobacter aerogens, Klebsiella pneumoniae, Lactobacillus sp., Pseudomonas putida, P. fluorescens, Proteus vulgaries, Salmonella sp., Bacillus cereus and Xanthomonas fragariae. All the species were recorded in all the seasons.

4.2.2. Fungi Fungi from the effluent sample were isolated based on serial dilution technique. Totally, fifteen different species of fungi belonging to eight genera 59 were isolated from the rubber effluent (Table 4). Among the fungi recorded, Aspergillus was found to be dominant with seven species viz., A. flavus, A. fumigatus, A. luchensis, A. nidulans, A. niger, A. sulphureus and A. ustus. The genus Penicillium was represented by two species viz., P. janthinellum and P. citrinum. The rest of the genera such as Helminthosporium, Trichoderma, Neurospora, Curvularia and Verticillium were recorded with single species each. Among the fungal species, Curvularia and Verticillium were recorded only in summer. Similarly N. crassa was not recorded in rainy season. The rest of the species were recorded in all the three seasons. Of the fungal species isolated, A. niger, A. nidulans was recorded in all the months at all seasons followed by A. flavus (10 months) and T. luchuensis (8 months). The maximum occurrence of these fungal species was noted during summer.

4.2.3. Cyanobacteria Totally 42 species belonging to 9 genera were identified from rubber effluent (Table 5). These genera falling under 3 families. Of these, Oscillatoriaceae was represented by 32 species followed by Chrococcaceae with 8 and Scytonemataceae with 2 species. Among the genera, Oscillatoria with 17 species was found to be the dominant genus followed by Phormidium with 8, Lyngbya with 7, Microcystis with 3, Chroococcus with 3ach 2 species, Aphanothece, Synechococcus, Plectonema and Synechocystis were represented by single species each. Of the total 42 species, 38 were recorded during summer while rainy and winter seasons were represented by 35 and 34 species respectively. Of the cyanobacteria identified, 2 species viz., Oscillatoria okeni and Phormidium uncinatum were recorded only during summer, while P. incrustatum were recorded only winter not in summer and rainy seasons. Similarly O. curviceps, O. princeps, O. formosa and L. confervoides were recorded only summer and winter not in rainy seasons. Altogether 27 species were found in all the seasons examined. Among the species identified, Oscillatoria salina was dominant with 100 per cent occurrence (recorded in all the 12 months), whereas the percentage occurrence of Microcystis aeruginosa, M. flos-aquae, Synechococcus elongates, O. late-virens, O. pseudogeminata and 60 O. willei were more than 75, while the least percentage occurrence (8) was observed with P. incrustatum (Table 5).

4.3. Bioremediation studies Cyanobacteria such as Oscillatoria salina (filamentous form) and Microcystis aeruginosa (unicellular form) were selected for treating the rubber effluent. The reasons for selecting these two species were, based on their dominant occurrence in the effluent during the study period of November 2010 – October 2011 as well as their growth response in the effluent. The cyanobacteria were used in both free and immobilized conditions.

4.3.1. Immobilization For immobilization, polyurethane foam cubes were used. When polyurethane foam pieces were inoculated with cyanobacteria, most of the inoculums got rapidly fixed on the surface and in the internal pores of the matrix, indicating interaction between cell wall and the foam surface, this was followed by a progressive colonization of the foam (Fig.31).

Scanning Electron Microscopic studies on immobilized Oscillatoria and Microcystis were carried out to know the physical interaction between polyurethane foam and cyanobacteria. Cells of the Microcystis species attached over the surface of the polyurethane foam by its extracellular products. The distribution of the cells are not uniform. The cells are attached in groups in many area of the foam (Fig.32b). The density of the cells are depending upon the age of the culture. On the other hand Oscillatoria filaments showed the ramification with surface of the foam (Fig.32a). The filaments were tightly interwoven with the interspace of the cavities because of absence of the proper support and they were unable to cover the cavities of foam due to the interwoving pattern and also the size of the filament. Hence, the association with foam is more than Microcystis species.

61 4.3.2. Growth and influence of cyanobacteria on the physico-chemical parameters of effluent

4.3.2.1. Growth Growth was measured interms of chlorophyll ‘a’ as a biomass component. Cyanobacteria were able to grow well in rubber effluent. However, when compared to control both the tested species recorded slow growth rate (Fig.37).

4.3.2.2. Colour The dairy effluent was white initially. A visual removal of colour from 5th day onwards, was noted in the flasks containing O. salina and M. aeruginosa in both free and immobilized conditions. However, effluent with immobilized cells showed a complete removal of colour on the 10th day itself whereas the free cells took 30 days for the complete removal of colour (Fig.38).

4.3.2.3. pH The initial pH of the rubber effluent was 7.6 (Table 6). In effluent with cyanobacteria, a slight increase in pH was noted from the 10th day onwards. When compared to free cells, effluents with immobilized cyanobacteria recorded higher pH and this was around 8.3. In general the pH of the rubber effluent with cyanobacteria continued to remain higher than the control (Fig.39).

4.3.2.4. Total dissolved solids (TDS) The initial TDS in the effluent was 470 mg l1 (Table 6). On 30th day it was reduced to 82 and 76 mg l1 in effluent with PUF immobilized Oscillatoria and Microcystis respectively. On the other hand effluent with free cells of Oscillatoria and Microcystis showed 76 and 64 mg l1 on the 30th day (Fig.40). The maximum percentage reduction (92) of TDS was observed in effluent with PUF immobilized Oscillatoria followed by Microcystis with 84. The minimum percentage reduction (78) was with free cells of Microcystis (Table 7).

62

4.3.2.5. Free CO2

Fairly higher level of free CO2 was observed initially in the rubber effluent (Table 6). A gradual decline in the level of free CO2 was noted in all the treated effluents from 5th day onwards (Fig.41). A complete removal of Free

CO2 was observed in effluent with free and PUF immobilized Oscillatoria and Microcystis (Table 7; Fig.41).

4.3.2.6. Bicarbonate Initial bicarbonate level in the effluent was 280 mg l1 (Table 6). The level of bicarbonate was gradually reduced from 5th day onwards in effluent with cyanobacteria. A complete removal of bicarbonate was observed in effluent with free and PUF immobilized Oscillatoria and Microcystis (Fig.42).

4.3.2.7. Biochemical Oxygen Demand (BOD) Fairly high level of BOD was recorded initially in the rubber effluent (Table 6). On 30th day, more than 80 per cent reduction of BOD was noted in the effluent treated with PUF immobilized Oscillatoria and Microcystis (Fig.43). The percentage reduction of BOD with free cells (both Oscillatoria and Microcystis) was more than 80 (Table 7). The control did not show much reduction. In general, PUF immobilized cyanobacteria were more efficient in BOD reduction than the other treatments.

4.3.2.8. Chemical Oxygen Demand (COD) The trend of COD reduction was similar to that of BOD reduction. However, the cyanobacteria could reduce COD only around 84 per cent on 30th day. When compared to immobilized Oscillatoria, the COD reducing efficiency of free cells was very low (74%). Among the immobilized cyanobacteria, the PUF immobilized Microcystis recorded 82 per cent reduction of COD as compared to other treatment (Table 7; Fig.44).

63 4.3.2.9. Dissolved oxygen (DO) The level of DO in the untreated effluent was 1.09 mg l1 (Table 6). From the 5th day onwards, a gradual increase in the level of DO was noted both in Oscillatoria and Microcystis treated effluents. On 30th day, this was raised to more than three fold in all the treatments (Fig.45). Whereas the level of DO in the control was not altered much during the experimental period. Among the cyanobacterial treatments, effluent with PUF immobilized Oscillatoria recorded slightly higher level of DO on 30th day over other treatments (Fig.45).

4.3.2.10. Nitrate The initial level of nitrate in the effluent was high (Table 6). There was a gradual reduction noted in all the treatments when compared to control (Fig.46; Table 7). On 30th day the levels of nitrate were 30 and 34 mgl-1 in effluent with PUF immobilized Oscillatoria and Microcystis respectively, whereas in effluent with free cells of Oscillatoria and Microcystis recorded 40 and 45 mgl-1 respectively. The percentage removal of nitrate was maximum (80) in effluent with PUF immobilized Oscillatoria and minimum (70) with free cells of Microcystis (Table 7).

4.3.2.11. Nitrite As in nitrate, nitrite removal was also well pronounced in effluent with PUF immobilized Oscillatoria and Microcystis (Fig.47). It was completely removed on 30th day in effluent with PUF immobilized cyanobacteria whereas, free cells removed nitrite completely only on 30th day (Fig.47). The removal efficiency was more with Oscillatoria in both conditions than with Microcystis in all the days examined. Control recorded 19 per cent reduction on 30th day.

4.3.2.12. Ammonia When compared to nitrate and nitrite, the initial level of ammonia was high (Table 6). With both immobilized Oscillatoria and Microcystis a complete removal of ammonia from the effluent was observed on 30th day (Fig.48). 64 However, the free cells of Oscillatoria and Microcystis completely removed (Table 7). Control exhibited 41 mg l1 (20%) ammonia on 30th day.

4.3.2.13. Total phosphorus A high level (78 mg l1) of total phosphorus was estimated in the effluent initially (Table 6). Throughout the experimental period a gradual reduction in the level of total phosphorus was observed in all the treatments employed (Fig.49). It was completely removed on 30th day in all conditions except control. However, immobilized Oscillatoria and Microcystis removed total phosphorus on 20th day itself. Free cells of cyanobacteria could remove on only 25th day (Fig.49).

4.3.2.14. Inorganic phosphate As in total phosphorus, inorganic phosphate was also completely removed from the effluent on 25th day (Fig.50). The trend of removal of inorganic phosphate was more or less similar to that of total phosphorus. A complete removal in all treatments was observed in 25th day (Fig.50).

4.3.2.15. Organic phosphate The initial level of organic phosphate was 40 mg l1 (Table 6). The trend of removal of organic phosphates was also similar to that of total and inorganic phosphate. The immobilized cyanobacteria could remove organic phosphates on 20th day itself as compared to free cells which took 25th day (Fig.51).

4.3.2.16. Calcium The calcium level in the raw effluent was 130 mg l1. Oscillatoria and Microcystis in both free and immobilized conditions could effectively bring down the level of calcium in the effluent (Fig.52). However, a complete removal was observed on 30th day only with immobilized cyanobacteria, whereas the free cells of Oscillatoria and Microcystis removed only 93 and 90 per cent respectively on 30th day. Among the cyanobacteria, Microcystis was more 65 effective in removing calcium than Oscillatoria throughout the experimental period.

4.3.2.17. Magnesium The untreated effluent recorded 95 mgl1 magnesium (Table 6). The trend of magnesium removal was similar to that of calcium. Free cells of Oscillatoria and Microcystis removed only 90 and 92 per cent on 30th day while immobilized cells removed magnesium completely from the effluent on 30th day (Fig.53).

4.3.2.18. Chloride A very high level (1640 mgl1) of chloride was recorded in the raw effluent (Table 6). Only 50-60 per cent removal of chloride was noticed in effluent with both free and immobilized cyanobacteria on 30th day. Among the cyanobacteria Oscillatoria was more effective in removing chloride than Microcystis. As in other cases, were also immobilized cyanobacteria performed well over free cells (Fig.54).

4.4. Biochemical studies 4.4.1. Pigments Chlorophyll ‘a’, carotenoids and phycobilipigments such as, phycocyanin, allophycocyanin and phycoerythrin were estimated both in controls (C1 and C2) and cyanobacteria grown in effluents (T1, T2). A gradual increase in chlorophyll content was noted in all the treatments including controls from 5th day onwards (Fig.55). However, the level of chlorophyll was well pronounced in C1 and C2 during the experimental period. Among T1 and T2, there was not much difference in the level of chlorophyll ‘a’ through the level th was slightly higher in T2 (Fig.55) on 30 day.

A steady increase in carotenoid content was noted in all the treatments

(Fig.56). However, the levels were higher in C1 and C2 than T1 and T2. Among 66 the cyanobacteria grown in effluent, T1 recorded slightly higher level (Fig.56) of carotenoids than that of T2.

Phycobilins such as phycocyanin, allophycocyanin and phycoerythrin th showed more or less similar trend in their level in C1, C2, T1 and T2 from 5 day onwards (Fig.57-59). In all forms of phycobilins, C1 and C2 exhibited higher levels over T1 and T2. Among the cyanobacteria grown in effluent, Oscillatoria

(T1) recorded maximum levels in all forms of phycobilin pigments over

Microcystis (T2). Of the three pigments, phycocyanin was recorded with higher levels followed by allophycocyanin and phycoerythrin both in Oscillatoria and Microcystis (Fig.57, 58). In general, all forms of pigments showed a reduced level in T1 and T2 when compared to C1 and C2.

4.4.2. Total carbohydrate 1 The total carbohydrate contents of C1 and T1 were 262 and 332 mg g 1 dry wt. respectively. The same in C2 and T2 were 248 and 364 mg g dry wt. respectively (Fig.60a). When compared to C1, T1 recorded more than 20 per cent increase in carbohydrate content, whereas in T2 the percentage increase was more than 25 over control (C2). In general, the cyanobacteria with effluent

(T1 and T2) registered higher levels of carbohydrate over their controls

(C1 and C2).

4.4.3. Total proteins

The total protein contents of Oscillatoria and Microcystis grown in BG11 medium and in effluent were estimated colorimetrically. Of the cyanobacteria tested, Oscillatoria (C1 and T1) recorded higher levels (Fig.60b) of protein over

Microcystis (C2 and T2) Irrespective of the organism tested, the effluent brought down the total protein content (Fig.60b). When compared to C1, T1 reported to have 13 per cent reduction of protein while in T2 the percentage reduction was

20 as compared to C2. This reduction in total protein was also visible in their protein profile with gel electrophoresis (Fig.61). The extraction of proteins in all cases was done on an equal dry weight basis and equal loading on the gel. 67 Protein profile by SDS-PAGE (Fig.61) Protein profile in control and effluent grown cyanobacteria was done by

SDS-PAGE. Totally 18 bands in C1 and 23 in C2 were observed (Fig.61). T1 and

T2 recorded 15 and 16 bonds respectively. Some of the bands below 14.4 kDa observed in C1 were not seen in T1 (Fig.61). Similarly a bands above 94 kDa in

C1 was feable and not clearly seen in T1. When compared to C1 most of the protein bands in T1 were feable. Some of the protein bands which appeared between 14.4 and 20 kDa below 14.4 kDa in C2 were not seen in T2. As in T1 here also most number of bands were feable. Similarly, some of the bands appeared between 14.4 and 20 kDa and between 43 and 67 kDa in T2 were missing in C2. In general, the intensity of many protein bands in both T1 and T2 was very low when compared to C1 and C2. This showed that the quantities of those proteins were less in effluent grown cyanobacteria as compared to controls.

4.4.4. Amino acid profile The amino acid content of Oscillatoria and Microcystis with different treatments was analysed both qualitatively and quantitatively by HPLC. In all 16 different amino acids were traced in different treatments (Table 8). Oscillatoria grown in BG11 medium (C1) recorded 15 amino acids while grown in effluent

(T1) recorded only 11. Amino acids such as serine, glycine, alanine and proline were not detected in T1. With reference to Microcystis, the number of amino acids were 11 in C2 and 12 in T2 where lysine were not detected. Among the amino acids, histidine recorded the maximum level in both cyanobacteria in all conditions followed by arginine and aspartate. Of the amino acids, proline recorded in C1 were not detected from T1. On the other hand valine detected from T1 was not recorded in C1. Amino acids such as phenyl alanine and proline detected in T2 were not recorded in C2. In general, the quantity of aminoacids was maximum in T1 and T2 over their counter part (C1 and C2) (Table 8). The percentage increase was 1.6 and 2.3 in T1 and T2 respectively (Fig.60c).

68 4.4.5. Lipid profile Total lipids in all treatments were estimated by gravimetric method. The th levels of lipid in Oscillatoria and Microcystis, on 30 day, with BG11 medium 1 (C1 and C2) were 132 and 110 mg g dry wt. respectively (Fig.60d). Rubber effluent brought down the total lipid content considerably both in Oscillatoria

(T1) and Microcystis (T2) when compared to controls. The percentage reduction of lipid in T1 was 16 and the same in T2 was 12. Such an overall reduction of lipids compared to control was also evident in the thin layer chromatographic separation of component lipids (Fig.62). The main components such as monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG) and sulphoquinovosyldiacylglycerols (SQDG) although were present in all treatments, their substantial reduction with rubber effluent was clearly evident in the chromatogram, since the TLC was done on equal dry weight and equal loading basis.

4.4.6. Fatty acid profile Fatty acid contents of O. salina and M. aeruginosa with different treatments were anlaysed both quantitatively and qualitatively by gas chromatography. In all, 15 fatty acids from O. salina (C1) and 17 from

M. aeruginosa (C2) were identified. These include long chain, short chain, saturated and unsaturated fatty acids (Table 9). On the other hand, Oscillatoria and Microcystis grown in effluent (T1 and T2) recorded 16 and 15 fatty acids respectively. Fatty acids such as Capric acid (C10:0), Undecanoic acid (C11:0),

Lauric acid (C12:0), Arachidonic acid (C20:4) were detected only from C1 and not in T1. on the other hand fatty acids such as Pentadecanoic acid (C15:0), Behanic acid (C22:0), Lignoceric acid (C24:0), Linoleic acid (C18:2 cis) and G-

Linolenic acid (C18:3 cis) detected in T1 were not detected from C1. Similarly

C2 recorded Lauric acid (C12:0), Eicosadienoic acid (C20:2) and Arachidonic acid (C20:4) which were not detected from T2. On the other hand Palmitic acid

(C16:0) and Lignoceric acid (C24:0) detected in T2 were not recorded in C2.

69 Short chain fatty acids such as capric (C10:0), undecanoic (C11:0) and lauric (C12:0) acids were detected from C1, whereas C2 recorded lauric (C12:0) acid. Arachidonic acid (C20:4). On the other hand T1 and T2 recorded Tridecanoic acid (C13:0) and Palmitic acid (C16:0).

Though, large number of fatty acids found in control were not detected from cyanobacteria grown in effluent, some of the fatty acids showed an increase in their content over control (Table 9). The fatty acids with notable increase in their content over control in O. salina were Tridecanoic acid (C13:0), Palmitic acid (C16:0), Heneicosanoic acid (C21:0), Myristoleic acid (C14:1),

Palmitioleci acid (C16:1) and Eicosenoic acid. Similarly T2 recorded Elaidic acid (C18:1), Oleic acid (C18:1), Eicosenoic acid (C20:1), Diocosahexaenoic acid (C22:6) and Nervonic acid with increase in their content over C2. Among the cyanobacteria, O. salina with effluent (T1) recorded maximum reduction in both number and quantity of fatty acids when compared to M. aeruginosa (T2). Most of the long chain unsaturated fatty acids which were detected from the controls, have not been detected from cyanobacteria with effluent (both in Oscillatoria and Microcystis).

In general, there was a significant reduction of unsaturated and short chain fatty acids with slight increase in some of the long chain fatty acids in T1.

On the other hand in T2 there was an increase in the level of most of the long chain saturated and unsaturated fatty acids over control (Table 9; Fig.60e).

4.5. Effect of effluent on crop plants Effect of effluent on seed germination is given in the table 10. Among the two plants (paddy and green chilli), the percentage of seed germination was more in paddy (80-90) than black gram (65-75). Of the treatments, seeds irrigated with untreated effluent (raw effluent) showed better response in seed germination in both crops than other treatments (Table 10).

70 Untreated effluent registered a better response with reference to various morphometric parameters tested both in paddy as well as green chilli when compared with all other treatments (Table 11). Among TE1 and TE2, the latter produced more number of seeds per plants and they also weighed more in both crop plants. 5. DISCUSSION

Now-a-days urban people are facing many problems and water pollution is one of them. Industrial wastes and sewage of the area either accumulate in the form of ponds in the cities or are being let into near by rivers. There is general interest in studying, the diversity of indigenous microorganisms capable of degrading pollutants. Identification of the key organisms that play a role in pollutants degradation processes is relevant to the development of optimal in sites biodegradation strategies (Abed et al., 2002). Large numbers of different organisms including microbes have been used for the evaluation of polluted habitats. In comparison to freshwater systems, microbes in waste water are exposed to different environmental stress and a study on the biological parameters of such water bodies certainly paves the way for future waste water programmes, using the indicator species. Since effluents always abound with natural populations of microbes, an attempt has been made to explore the nature of microbial flora such as bacteria, fungi and cyanobacteria in rubber effluent in order to understand their utility in waste water treatment.

Several reports are available on the occurrence of algae in polluted waters (Somashekar and Ramasamy, 1983; Pandey and Tripathi, 1988; Vijayakumar et al., 2005, 2007 Ganapathyselvan, 2011 and Senthil et al., 2012a). However, the diversity in physical, chemical and biological characteristics of industrial effluents is so great that each waste water habitat requires a separate study. A thorough knowledge of the physical, chemical and biological characteristics of an industrial waste is a preliminary and essential requirement for any attempt in the chemical and or biological treatment of the waste. Hence in the present investigation, the effluent from rubber industry has been analysed in various angles.

72 5.1. Environmental studies 5.1.1. Physico-chemical characteristics of effluents The physicochemical analysis of the effluent revealed its slightly alkaline nature and also presence of high quantity of both organic as well as inorganic nutrients in all the seasons examined (Table 1). Values of DO were very low indicating highly obnoxious conditions. Though BOD and COD levels in the present study were high as per IS standards, their levels were not so much high as compared to other types of effluents such as paper (Manoharan and Subramanian, 1992b) tannery (Manoharan and Subramanian, 1993a, b) distillery (Jain et al., 2001; Ganapathy et al., 2011), Dairy (Boominathan et al., 2007), chemical (Senthil et al., 2010), dye (Vijayakumar et al., 2005, 2007 and 2012) and rubber effluent (Senthil et al., 2012a). Most of the parameters tested were slightly higher in summer than in winter and rainy seasons, Vijayakumar et al. (2007) reported similar results with dye effluent at different seasons. They recorded objectionable amounts of BOD, COD, algal nutrients such as ammonial nitrogen, nitrate nitrogen, phosphates and calcium. Such a trend was observed in the rubber effluent also. Senthil et al. (2010) analysed pollution load of four different effluents such as chemical, Sago, oil and distiellery. Among these, highly objectionable amounts of various pollutants including BOD, COD were recorded chemical followed by distiellery, oil and sago. While studying the seasonal variations in physicochemical parameters of sugar and paper mill effluent, Vijayakumar et al. (2007) found a direct relationship between temperature and organic matter. High organic matter was observed during summer and low during winter. This is in keeping with the observations that the course of decomposition of organic matter depends on temperature (Ganapathiselvam et al., 2011 and Senthil et al., 2012a). In the present study also, a higher amount of organic matter was observed during summer followed by winter and rainy seasons (Table 1). In general the results observed in the present study coincided with earlier findings.

73 5.1.2. Biodiversity of microbes Bacterial diversity has not been studied in detail waste water. However, a few reports are available on the bacterial flora of certain waste water. Abed et al. (2002) isolated bacteria different groups mainly the Cytophaga – Flavobacerium – Baterioides group,  and  subclass of the class proteobacteria, and the green and non sulphur bacteria, from a heavily polluted site in a coastal steam. Similarly, Boominathan et al. (2007) and Ganapathyselvam et al. (2011) isolated eight and six different bacterial genera respectively from dairy and distiellary wastes, in order to carry out biodegradation of dairy and distiellery effluent. In the present study also, nine different genera were isolated from the rubber effluent in all seasons (Table 3). Most of the isolated genera are potential pathogens. Some of these bacteria have previously been reported to be present in waste water (Cho and Kim, 2000) and oil polluted sites (Macnaughton et al., 1999; Whitely and Bailey, 2000). However, Sulaiman et al. (2002) isolated only two bacterial genera such as Derxia and Beijerinckia from dye effluent drenched soils. The lessen number of bacterial community was apparently due to the environmental stress caused by the high level of pollutants, which allowed only a restricted number of species that tolerated such conditions.

Fungi occurred over different seasons of the year but some of them showed restricted distribution (Table 4). They found in maximum numbers and diversity during summer with 15 species followed by winter with 11 and rainy seasons with 9 species. Among the fungi species, isolated Curvularia and Neurospora species were not reported from winter and rainy seasons. Of the fungal genera, Aspergillus was dominant with seven species. Boominathan et al. (2007) reported 11 species of fungi from dairy effluent dranched soil and found Aspergillus, Penicillium, Curvularia, Verticillium and Trichoderma were the dominant genera. Ganapathyselvam et al. (2011) also observed six pathogenic fungi from the effluents of distiellery with Aspergillus as the dominant genus with three species, since it occurred in most of the months studied thereby lending support to the present investigation. Higher concentration of chlorides, total dissolved solids and BOD were the seasons for their frequent occurrence 74 (Kousar et al., 2000). Most of the aquatic fungi preferred low temperature between 15 to 30°C has been reported by Hasija and Khan (1987). In the present study also, the temperature of the rubber effluent range between 25 and 28°C there by favouring the growth of fungi.

BOD of effluent was higher during summer than rainy and winter seasons (Table 1). This has direct correlation with distribution of fungi (Hasija and Khan, 1987). In the present investigation, during summer maximum number of species recorded, which might be due to higher level of organic matter. The highest nitrogen content of water during summer coincided with the highest number of fungi (Table 4). Goldstein (1960) reported that nitrogen content of the water along with other seasonal changes in the environment effects the occurrence and distribution of fungi.

Freshwater fungi generally grow at pH 7.0 to 8.5 (Hasija and Khan, 1987). Cantrell and Dowler (1971) and Fowles (1976) found that pH affected the growth of Pythium irregulare and P. vexans. Berner and Chapman (1977) the occurrence of Saprolegria parasitica in a pH range of 7.7 to 8.3. Saxena et al. (1990) observed species of Aspergillus, Fusarium and Curvularia between pH 7.9 and the present study range from 7.3 to 7.8 and fungi such as Aspergillus flavus and species of Curvularia were recorded. This agrees with the findings of Saxena et al. (1990).

Among the species Aspergillus nidulans and A. niger was the dominant, which occurred in all the months in all seasons. In stagnant water bodies, the temperature may raise during summer, as a results, the species diversity could be reduced as pointed out by Hasija and Khan (1987). Contrary to this, in the present investigation, the temperature of the effluent was not varied much (Table 1) as it was collected from the running stream and hence the observed variation.

Several important publications deals with the ecological distribution of cyanophyceae (Fritsch, 1907, Prescott, 1938, Vijayakumar et al., 2007, 75 Boominathan, et al., 2007, Ganapathyselvam et al., 2011, Senthil et al., 2010, Gomathy et al., 2011 and Vijayakumar et al., 2011). Many of them emphasized the importance of light, temperature, pH carbondioxide, organic matter, alkalinity, nitrates and phosphates as factors important in determining the distribution of blue green algae. The above observations have been made in natural freshwater lentic and lotic systems. In the man made systems such as the present one, the above factors remain more or less uniform throughout the year. In the present investigations, it was observed that cyanophycean members dominated the effluent streams during all the seasons. During the summer 37 species were observed, while their number during rainy and winter, seasons was 35 and 34 species respectively (Table 5). Of the genera Chroococcus minor, Microcystis robusta, Oscillatoria chlorina and Lyngbya majuscula observed both in summer and rainy seasons were not recorded in winter. Similarly, O. curviceps, O. formosa and Lyngbya confervoides were not founding rainy seasons. On the other hand O. claricentrosa, Phormidium corium, P. incrustatum and P. jadinianum was not recorded in summer. The periodicity of blue green was found related to changes in the physico-chemical nature of the effluent. Although the values of physico-chemical factors did not vary much (Table 1) its dilution by rain water during rainy seasons also played a role. As a whole, condition in the effluent appeared to be favourable for the cyanophycean members.

Rich blooms of some cyanobacteria such as Microcystis aeruginosa, Synechococcus elongatus, Oscillatoria earlei, O. salina and O. willei were observed in all seasons in throughout the year. This abundance is attributed to favourable conditions of oxidizable organic matter, less dissolved oxygen and high calcium content (Table 1) an observation which supports Venkateswaralu (1969b) Manoharan and Subramanian (1993a, b). Observation of Muthukumar et al. (2009) and Gomathy et al. (2011) suggest that cyanophyceae grow luxuriously with great variety and abundance in ponds rich in calcium. High level of orhophosphates favoured the development of cyanobacterial bloom (Khan and Seenaya, 1982). 76 The poisitive correlation between phosphate and cyanobacteria was observed by Sarojini (1996). Muthukumar et al. (2009) and Vijayakumar et al. (2011) reported that low concentration of oxygen and high concentration of nitrogen and phosphates, were favoured the luxuriant growth of cyanobacteria. Similar observations were also made in the present study with reference to various nutrients (Table 1). Boominathan et al. (2007) and Kannan (2008) observed non hetrocystous forms in the polluted waters rich in nitrogen. Present investigation also showed dominance of non heterocystous forms and not single species of heterocystous cyanobacteria (Table 5).

Genus Oscillatoria has bee found to be tolerant to pollution which frequently inhabits the polluted waters (Senthil et al., 2010 and Vijayakumar et al., 2011). Present study confirmed their observations as Oscillatoria was found dominating the rubber effluent represented by 17 species. Kashthuri (2008) emphasized the use of algae as reliable indicators of pollution. In the present study, percentage value of algae above 65 (Table 5) should be considered as indicator species of rubber effluent. Their high representation indicates their capacity to thrive in this type of man made habitat, such species can be used as ‘Marker species’ or indicators of particular habitats (Vijayakumar et al., 2005). Besides Oscillatoria many other cyanobacteria aso were found to be tolerant to rubber effluent as evidenced by this occurrence (Table 5).

5.1.3. Bioremediation of rubber effluent Water pollution is essentially a biological phenomenon. The chemical methods measure the concentration of pollutants and the biological indicators show the degree of ecological imbalance. Large number of different organisms have been used for the evaluation of polluted habitats. However algal test systems have gained special significance since most of them are used as biomonitoring agents to control pollution the indicator species, otherwise represent the actual survivors of the habitat and their abundance indicate their ‘adaptation’ to a known habitat. Many of the studies conducted in various laboratories have also shown that under defined conditions indicator species 77 actively take part in the degradation of organic matter (Sengar and Sharma, 1987; Dash and Mishra, 1999a; Vijayakumar et al., 2005; Sanjay et al., 2011). It is, therefore, aimed that the dominant taxa ecountered in the present study such as Oscillatoria salina (Filamentous) and Microcystis aeurginosa (Unicellular) used to treat rubber effluent.

Inoculated cyanobacteria are known to grow fairly efficient in different types of effluents and generally this growth is measured with the chlorophyll a as the biomass component (Vijayakumar et al., 2005; Kannan, 2008; Kasthuri, 2008 and Ganapathyselvam et al., 2011). In the present study also both Oscillatoria and Microcystis were found to grow in rubber effluent (Fig.37). However, growth was well pronounced in control when compared with effluent. Among cyanobacteria, Microcystis recorded marginally better growth than Oscillatoria. The slow growth rate of Oscillatoria and Microcystis in rubber effluent might be due to higher COD, Magnesium and Chloride. Moreover the colour of the effluent was white initially which could prevent easy light penetration into the effluent and it probably contained some toxic substrances also from fungi which are abundant in this effluent (Table 4). Boominathan et al. (2007) noticed similar observations in dairy effluent. Such slow growth rate in cyanobacteria with different effluents such as oil refinery (Jeganathan, 2006) Sago (Kannan, 2008) pharmaceutical and textile (Sanjay et al., 2011) and dye (Vijayakumar et al., 2012) has already been reported.

Initially the effluent was turbid and white in colour (Fig.38). This turbidity and white colour was due to total suspended solids (TSS). Sharma et al. (2003) reported, carbohydrates and protein may be responsible for total ISS and turbid nature of the effluent. Turbidity of effluent showed continuous decrease and by 10th day the effluent became almost clear when treated with cyanobacteria in immobilized conditions, whereas with free cells it took 15 days to clear the turbidity. Sharma et al. (2003) and Boominathan (2005) points out, organic substances are being broken down into simple inorganic forms for absorption by growing cyanobacteria. Decrease in BOD, COD and dissolved 78 organic matter (Fig.43, 44) further confirms that carbohydrates and proteins are being broken down before absorption.

The initial pH of the effluent was 7.6 and was raised to 8.3 on 30th in effluents with free and immobilized cyanobacteria (Fig.39). Manoharan and Subramanian (1992a, b and 1993a) and Vijayakumar et al. (2005) found a rise in pH value upto 10th day in various effluents inoculated with free BGA and after that it decreased. Similarly Sanjay et al. (2011) reported a rapid rise in pH while tertiary waste water was treated with cyanobacteria. They attributed the fact that the rise in pH might be due to the photosynthetic activity of cyanobacteria (from 7.6 to 9.3). Vijayakumar (2005) also noticed a steep rise in pH when dye effluent with PUF immobilized Oscillatoria and Westiellopsis. On the other hand, Dash and Mishra (1999b) observed almost no change in pH when paper mill effluent was treated with Westicllopsis prolifica. However, the typical capacity of cyanobacteria to bring about changes in the pH to suit their requirement was evident in this study also.

There was fairly good correlation between the initial levels of biocarbonate and free CO2 in the effluent. There was no carbonate in the effluent during the experimental period, but fairly high levels of bicarbonate and free th CO2 were present. A gradual reduction in bicarbonate from 5 day onwards was noticed in the effluent treated with Oscillatoria and Microcystis in both free and immobilized conditions. However the reduction was maximum in effluent with immobilized conditions than with free cells (Fig.42). On the other hand CO2 was completely removed from the effluent on the 25th day itself (Fig.41). The - relative proportion of CO2 and HCO3 depends on the pH of the medium such - that HCO3 is the predominant from between 7.0 to 10.5. Thus cyanobacteria may have a competitive advantage over chlorophytes since the former are - capable of assimilating HCO3 as source of inorganic carbon for photosynthesis and they have high CO2 affinity and low CO2 compensation point (Colman, - 1989; Boominathan, 2005). In the present investigation, though HCO3 was not completely from the effluent, their initial level was very high when compared to 79

CO2 level (Table 6). This could be the reason for not complete removal of - HCO3 from the effluent. However, the removal of these carbon sources effectively by cyanobacteria both in free and immobilized conditions as could be expected was observed in the present investigation.

BOD and COD are the parameters, which will determine the strength of waste water. COD is generally as a major indicator of organic pollution in water (Vijayakumar et al., 2005). The rubber industry waste water analysis in India have been reported to show higher BOD and COD that Bureau of Indian standard (BIs) permissible limits (Jayachandran et al., 1994). Vimalamma et al. (2007) in his study, on the effluents arising from three rubber plant units in Kerala state, manufacturing latex, Dry rubber in addition to other produces has reported BOD and COD ranging from 500 to 1200 mgl-1 and 750 to 1450 mgl-1 respectively. In the present study, also the initial levels BOD and COD were 360 and 820 mgl-1 respectively. Inoculation of cyanobacteria in both in free and immobilized conditions brought down the BOD and COD levels considerably when compared to control (Figs. 43, 44). The reduction percentage of BOD ranges from 82-86 with free cells and 90-92 with immobilization cyanobacteria. Similarly, COD reduction ranges from 72-74 per cent with free cells and 80-82 per cent with immobilized cells (Table 7). The BOD reduction of more than 35 per cent of rubber effluent using actinobacter sp. has been reported (Jayachandran et al., 1994).

Use of acclimatized algal cultures in considerably reducing BOD and COD with different effluents including dye industry has been reported. (Govindan, 1983, 1984 and 1985; Manoharan and Subramanian, 1992a, b and 1993; Sharma et al., 2003; Boominathan, 2000, 2005; Ganapathyselvam et al., 2011 and Vijayakumar and Manoharan, 2012). Efficiency of immobilized cyanobacteria in removing BOD and COD over free cells has already been reported (Patnaik et al., 2001; Vijayakumar and Manoharan, 2012). Considering the above observations, the results obtained in the present investigation is quite conceivable. 80

Determination of the amounts of O2 dissolved in water at studied sites is undoubtedly of great importance since it is considered as one of the best toxic substances (Lester, 1975, Manoharan and Subramanian, 1992a, Boominathan, 2005). As revealed from the results herein obtained (Fig.45) the initial DO content of rubber effluent was very low (1.09 mgl-1). A gradual increases in DO level was noticed from 5th day onwards in free and immobilized cyanobacteria treated effluents. At the end of 30th day more than three fold increase in DO level was noticed in all treatments. Vijayakumar et al. (2005) observed similar increases in DO level with dye effluents when treated with Oscillatoria brevis. While treating distiellery effluent with Nostoc muscorum, Ganapathyselvam et al. (2011) noticed considerable increases in DO level from 5th day onwards. The correlation between the initial increases in DO and removal of BOD and COD observed in the study agree with observations by Vijayakumar et al. (2005), Jeganathan (2006), Gopalakrishnan (2007) and Sanjay et al. (2011).

Common inorganic nutrients present in waste water include nitrite, nitrate, ammonia and phosphate. All these compounds are essential requirement for growth of cyanobacteria. They have nutrient uptake capabilities as they can accumulate inorganic phosphate and nitrogen and store them as polyphosphate and cyanophycin, respectively (Fay, 1983). Suspended cultivation of microalgae is one of the biological processes which have been employed to eliminate residual inorganic nutrients as a tertiary treatment step from secondary treatment effluents (Prakashan and Ramakrishnan, 1998, Vijayakumar, 2012).

In the present study, also both Oscillatoria and Microcystis effectively removed all forms of inorganic nitrogen from rubber effluent in both free and immobilized conditions (Fig.46-48). Among inorganic nitrogen, ammonia was completely removed from the effluent first, followed by nitrite. Though nitrate level was also observed, the cyanobacteria could not remove nitrate completely from the effluent. In most of the effluents ammonium, nitrate and nitrite remain together. Under such conditions, cyanobacteria utilize first ammonium, the nitrite and nitrate. This is mainly because, ammonium is required to be least 81 processed to incorporate into cell constituents, whereas nitrate requires special enzyme for transport of nitrate into cell and then converted to nitrite and then ammonium by the sequential action of nitrate and nitrite reductase. Hence nitrate assimilation requires more energy (Lee et al., 1995; Vijayakumar et al., 2005). This could be reasons for the least absorption of nitrate by cyanobacteria in the present investigation. The specific use of cyanobacteria, both free and immobilized forms, in the efficient removal of different forms of combined nitrogen has also been reported (Boominathan, 2005). Vijayakumar et al. (2005) reported that Oscillatoria brevis could remove more than 90 per cent of all forms of inorganic nitrogen from the dye effluent. Similarly, Ganapathyselvam et al. (2011) found that there was nearly 78 per cent removal of nitrite and ammonia and 80 per cent removal of nitrate from distillery effluents using Nostoc muscorum.

Entrapment matrices play a major role in the change of rate of removal of inorganic nitrogen by cyanobacteria. It was observed that, chitosan immobilized Anabaena dioliolum is more efficient in removing nitrate and nitrite over ammonia, whereas carrageenan immobilized cyanobacterial cells efficiently remove ammonia compared to other inorganic nitrogen compounds (Vijayakumar and Manoharan, 2012). In the present study, PUF immobilized Oscillatoria and Microcystis removed ammonia efficiently and completely, within short duration (25 days), from the effluent, when compared to other inorganic nitrogen compounds, which showed that the removal efficiency was more with immobilized cyanobacteria than with free cells (Table 7). Contrary to this, Grabisu et al. (1991) reported that polyvinyl and polyurethane immobilized Phormidium laminosum cells showed more than 90 and 60 per cent reduction of nitrate uptake to that of free cells. However, in the present study, it was observed that the immobilized cyanobacteria were more efficient in removing all forms of inorganic nitrogen when compared to free cells.

Oscillatoria and Microcystis both in free and immobilized conditions could bring down the level of all forms of phosphate in the effluent (Fig.49-51). 82 However, the percentage removal of inorganic phosphate was higher than that of organic phosphate (Table 7). The capacity of cyanobacteria to remove large amount of phasphorus from industrial waste water has been demonstrated by several workers (Tam and Wong, 1989; Manoharan and Subramanian, 1993a; Vijayakumar et al., 2005; Gopalakrishnan, 2007; Ganapathyselvam et al., 2011, Sanjay et al., 2011 and Vijayakumar and Manoharan, 2012). Further, the cyanobacteria are known to absorb and store large amount of phosphorous as polyphosphate granules (Reynolds, 1984; Boominathan, 2005). Vijayakumar et al., 2005 and Ganapathyselvam et al., 2011 found a total removal of all types phosphate by Oscillatoria and Nostoc from dye and distiellery effluent respectively. Tang et al. (1997) also reported a higher phosphate uptake, despite low biomass of cyanobacteria. Vijayakumar and Manoharan (2012) also observed 100 per cent removal of phosphate from the dye effluent while treating Oscillatoria and Westiellopsis. In he present investigation, immobilized cyanobacteria, effectively removed all form of phosphate from the effluent when compared to free cells (Fig. 49-51). Contrary to the present findings, Robinson et al. (1988) and Mallick and Rai (1994) earlier reported that inorganic phosphate removal by microalgae is found to be more effective in free cells rather than in immobilized conditions. However, de la Noe and Proulx (1988) found chitosan-immobilized Phormidium cells were able to remove phosphate upto 90 per cent with the retention time of 24 h from urban waste water. Similarly, Markov et al. (2001) observed that PUF immobilized Anabaena variabilis was more efficient in and uptake than free living cells. Their observations support the present findings in phosphate removal efficiency of immobilized cyanobacteria. Phosphate uptake is more pronounced in growing cells to that of stationary phase cells. Robinson (1995) has found that exponentially growing cells eliminate phosphate from the medium five times more rapidly than the cells of older age indicating the culture age is an important factor in phosphate removal from waste water.

Calcium and Magnesium constitute the total hardness of the waste water. In the present study, a complete removal of both calcium and magnesium on 30th 83 day by both cyanobacteria in immobilized condition was observed. Similarly, free cells were also effective, though not complete, in removing these divalent cations from the effluent (Table 7; Fig.52, 53). Manoharan and Subramanian (1992a, b and 1993a) studied the reduction of both calcium and magnesium in domestic, sewage, ossein and paper mill effluents by O. pseudogeminata. They observed more than 70 pr cent reduction of calcium and magnesium with retention time of 15 days. Similarly, Vijayakumar et al. (2005) and Ganapathyselvam et al. (2011) studied the effective use of cyanobacteria in dye and distiellery effluent respectively, which has high level of calcium and magnesium. They found more than 65 per cent reduction of calcium with in 8 days when the effluent was treated with Oscillatoria and Nostoc. Dash and Mishra (1999b) observed 50 per cent reduction of calcium in paper mill effluent by Westiellopsis (retention time of 15 days). Kannan (2008) and Ksthuri (2008) reported more than 80 per cent removal of calcium and magnesium from sago and coirpith industry effluent when treated with Oscillatoria and Synechococcus respectively.

Although, calcium is undoubtedly required for cyanobacterial growth (Jaganathan, 2006), substantial reduction in calcium and magnesium are known to be essential for flocculation and would coflocculate Uma and Subramanian (1990). Fogg et al. (1973) and Boominathan (2000) observed the excretion of organic acids by microbes and especially cyanobacteria and their capacity to solubilize magnesium in the waste water, which could explain observed reduction.

Chloride are generally considered to be one of the major pollutants in effluents which are difficult to be removed by conventional biological treatments. However, in the present study 50-60 per cent removal of chloride from the effluent by both cyanobacteria under different conditions (free and immobilized) was observed (Table 7; Fig.54). Maximum level of chloride removal was noticed in effluent with PUF. immobilized cyanobacteria (both Oscillatoria and Microcystis) and minimum (50%) with free living cells of 84 Oscillatoria. When compared to Oscillatoria, Microcystis responded well in the removal of chloride Uma and Subramanian (1990). Studied the effective use of cyanobacteria in ossein effluent which has a very high level of chloride content, which could not be treated successfully by the conventional treatment systems consisting of settlement tanks and aerobic digester. They aged a halophilic bacterium, Halobacterium sp. US101, a marine filamentous cyanobacterium, Oscillatoria sp. BDU 10142 and a calcicolous unicellular cyanobacterium Aphanocapsa sp. BDU16, both in laboratory and field conditions. They observed 50 and 25 per cent in laboratory and field conditions respectively. Ganapathyselvam et al. (2011) also observed more than 60 per cent removal of chloride from distiellery effluent using Nostoc muscorum. A similar observation attributing 65 per cent chloride reduction under laboratory conditions by O. brevis was also reported in dye effluent (Vijayakumar and Manoharan, 2012).

From the above discussion, it is clear that cyanobacteria can successfully be used for the treatment rubber effluent in both free and immobilized conditions. It is also concluded that PUF immobilized cyanobacteria are of potential value for biological removal of various chemicals including nitrogen and phosphates.

5.2. Biochemical studies on Oscillatoria and Microcystis Scientists interested in the use of algal or cyanobacterial systems have concentrated more on the influence of these systems on the removal of nutrients from the effluents but only few have investigated (Manoharan and Subramanian, 1995, 1996 and Vijayakumar et al., 2007) the effect of effluents on the biochemistry of the cyanobacterial systems. To develop suitable and efficient treatment systems, it is obligatory to understand the mutual influence and interactions between the effluents and the organisms, so that manipulations to improve the treatment system become feasible and hence the present investigation on the biochemistry of effluent grown cyanobacteria was carried out.

85 Along with chlorophyll-a, phycobiliproteins such as phycocyanin, phycoerythrin and allophycocyanin from the main light-harvesting pigments of photosynthesis in cyanobacteria (Ranjitha and Kaushik, 2005). In the present study all forms of pigments including carotenoids were extracted and quantified. A significant reduction in the level of all forms of pigments was observed in effluent grown cyanobacteria when compared to control (Fig.55-59). Among cyanobacteria, Microcystis showed maximum reduction of pigments when compared to Oscillatoria. Variations in pigments levels in many cyanobacteria at different environmental conditions such as temperature (Chaheva et al., 2007), salinity (Senthilkumar, 2004), nitrogen supply (Gordillo et al., 1999) and herbicide (Vijayakumar, 2012).

Anand and Hopper (1987) and Senthilkumar (2004) observed that increased salinity (70 to 100%) reduced the phycobilin pigments and proteins considerably. Nitrogen limitation and higher CO2 concentration reduced the pigment levels in Spirulina plantensis. The influence of CO2 was very similar under N sufficiency and N limitation. Chlorophyll-a was clearly diminished by

20-25 per cent at high CO2 concentration. Similarly a significant decreases of total carotenoids concentration to less than 25 per cent was observed at high

CO2 concentration (Gordillo et al., 1999 and Manojkumar et al., 2011). In the present study, though, all forms of inorganic nitrogen were high (Fig.46-48), higher initial CO2 was also observed. This could be one of the reasons for reduced pigments levels as reported by earlier workers (Sakamoto and Bryant, 1998). Olguin et al. (2001) reported that the pigment synthesis was adversely affected in nitrogen deficient and high nitrogen cultures. Boominathan (2005) also observed a reduction in chlorophyll and phycobiliproteins when Oscillatoria acuminata and Aphanocapsa pulchara were grown in dairy effluent. Considering the above facts, the reduced pigment levels in the pigment investigation might be due to various factors including CO2 concentration and nitrogen enhancement.

86 The total carbohydrate content showed considerable enhancement in both cyanobacteria with rubber effluent over controls. Among the cyanobacteria, Microcystis with effluent recorded higher level of carbohydrate (Fig.60a) over other treatments. Nitrogen limitation caused photoassimilated carbon to be directed towards the synthesis of carbohydrates in stead of proteins and chlorophyll. This response has been widely observed in many algal species (Karanth and Madaiah, 2011). But in the present investigation, considerable levels of all forms of inorganic nitrogen (Figs.46-48) were observed. This could not explain the observed variation in carbohydrate content. However, protein and chlorophyll decrease and carbohydrate increase by CO2 enrichment have been previously observed in a number of species (Vijayakumar, 2005). In the present investigation also, there was fairly a high level of CO2 in the rubber effluent (Fig.41). This confirms the earlier findings of Loehle (1995). Increase in carbohydrate level in cyanobacteria, over control has already been reported with different effluent (Reddy et al., 1983; Manoharan and Subramanian, 1992a, b, 1993a and 1996; Boominathan, 2005 and Vijayakumar, 2005).

The total protein level in both cyanobacteria, grown in rubber effluent, showed significant reduction compared to controls (Fig.60b). Proteins and chlorophyll decrease and carbohydrate increase by CO2 enrichment has been previously observed in a number of species (Loehle, 1995). This was supported by a substantially higher level of carbohydrate accumulation (Fig.60a). There was an inverse correaltion between carbohydrate and protein level among the cyanobacteria grown in rubber effluent. The level of protein reduction was minimum (13%) in Oscillatoria grown in effluent and conversely the carbohydrate enhancement was also less (20%) as compared to control. On the other hand Microcystis with effluent recorded higher percentage (20%) of protein reduction while carbohydrate enhancement was also higher (Fig.60a, b). Similar observations with decreases in protein level and increases in carbohydrate content in Oscillatoria psedogeminata with different effluents including paper mill and ossein (Manoharan and Subramanian, 1992a, b and 1993a), Oscillatoria brevis with dye effluent (Vijayakumar, 2005) have already 87 been established. Renuga (2005) reported an increased concentration of tannery effluent (less diluted) decreased the biochemical contents such as proteins and carbohydrate in blue green algae as compared to control. However, increased dilutions significantly increased the biochemical contents. In the present study, 100 per cent effluent was used to grow cyanobacteria. This could be reason for the reduced biochemical components of test organisms. The significant reduction of the above biochemical metabolites suggests the effluent affects algal metabolism at multiple sits (Reddy et al., 1983 and Boominathan, 2005). The substantial decrease in protein levels in the test organisms with rubber effluent were clearly reflected in the density of staining of the protein profile by electrophoresis.

Reduction in protein content was also visualized in the density of staining of the protein profile by electorphoresis (Fig.61). Some of the protein bands which appeared in C1 and C2 were missing T1 and T2. Moreover, most of the bands appeared in C1 and C2 were feable in T1 and T2. Though, it is difficult to pin point either the cause or the nature of the missing protein, considerable alteration in protein profile resulting in the inhibition of the synthesis of several proteins and synthesis of certain specific proteins for enhancement of certain other under different types of stresses including effluent stress in cyanobacteria has been well documented (Manoharan and Subramanian, 1992a; 1996; Boominathan, 2005; Vijayakumar, 2005).

The aminoacid content of Oscillatoria and Microcystis grown in rubber effluent showed a substantial increase both qualitatively and quantitatively as compared to controls (Table 8; Fig.60c). Various amino acids reported in the present study have already been reported by earlier workers (Vigna, 1967 and Vijayakumar et al., 2007). Contrary to this observation Boominathan (2005) reported a decrease in the level of amino acids in both qualitative and quantitative when Oscillatoria and Aphanocapsa treated with dairy effluent. Rzharova (1968) found arginine as the dominant aminoacid in Phormidium uncinatum followed by hisditine, lysine, leusine and -alanine in fairly good 88 quantity. However, in the present investigation it was found that histidine was dominant in both Oscillatoria and Microcystis which is followed by arginine, aspirate, asparagines, glutamine and so on (Table 8). Similar observation with higher content of histidine has already been reported (Boominathan, 2005 and Vijayakumar et al., 2007). A noteworthy observation, most of the aminoacids recorded T1 and T2 showed higher in their content cover C1 and C2. Similarly, some of the amino acids detected effluent grown Microcystis were not detected in control (grown in BG11 medium). Hence comparison with results obtained with other cyanobacteria is neither feasible nor possible. This may be concluded that the observations in amino acids in the present case might be due to effluent stress.

The overall increases in the total carbohydrate level in both organisms is reflected not only in the reduction of total protein but also in the reduction of total lipids and fatty acids (Table 9; Fig.60d). The level of reduction of total lipids in Oscillatoria with effluent was higher (16%) as compared to control, while in Microcystis with effluent the same was minimum (12%). Reduction in lipid content level in Oscillatoria with different effluent has been well documented (Manoharan and Subramanian, 1992a, b; 1995; 1996 and Boominathan, 2005). Alteration in the lipid content of an organism is of major importance in response to environmental stree (Olie and Potts, 1986; Rittere and Yopp, 1993). Variation in lipid contents and composition under different environmental conditions including light and dark has been observed in a number of cyanobacteria (Al-Hasan et al., 1989). The reduction in lipid content with dairy effluent compared to control was also clearly visualized by thin layer chromatographic analysis of the different lipids (Fig.62). The different classes of lipids also present in both in control and effluent treated cyanobacteria, different slightly in their intensity. All the classes of lipids observed in the present study, have been reported in cyanobacteria by different workers (Al-Hasan et al., 1989; Boominathan, 2005; Vijayakumar et al., 2007 and Karanth and Madaiah, 2011).

89 The lipids of cyanobacteria are generally esters of glycerol and fatty acids. They may be either saturated or unsaturated. Some of the filamentous cyanobacteria tend to have large quantities of unsaturated fatty acids (Kenyon et al., 1972; Vargas et al., 1998; Safonova and Reisser, 2005 and Karanth and Madaiah, 2011). In the present investigation 20 different fatty acid were detected from Oscillatoria (C1 and T1) and 19 from Microcystis (C2 and T2). These include short chain, long chain saturated and unsaturated fatty acids

(Table 9). Among the treatment C1 and T1 recorded 15 and 16 fatty acids respectively, while C2 T2 observed with 17 and 15 respectively. However, the number unsaturated fatty acids, in both test organisms, was more when compared to other classes. This confirms the earlier findings of Vijayakumar et al. (2007). In the present study, long chain fatty acids such as behenic, linoceric and unsaturated fatty acids g-linilenic and linoleic acids were detected from T1 and not in C1. Similarly, in C2 lignoceric was not recorded which could otherwise detected in T2. Moreover in both T1 and T2 most of the unsaturated fatty acids, particularly g-linolenic acid were recorded significantly with higher levels. Environmental and nutritional conditions leading to enhanced production of unsaturated fatty acids particularly linolenic acid has been reported in micro algae (Cohen and Heimer, 1991; Manoharan and Subramanian, 1993b; Boominathan, 2005). These findings may thus explain the observed changes in the levels of different fatty acids in the present study.

From the above discussion, the converse effects of rubber effluent on cyanobacteria reveals that (a) the rubber effluent had an inhibitory effect on the biochemical contents of cyanobacteria; (b) in a few cases the effluent had actually enhanced the level of some of the individual biochemical contents. The conditions leading to such enhanced level need to be studies further in order to exploit them commercially.

5.3. Effect on effluent on crop plants When paddy and green chilli seeds were germinated using tap water and effluents, untreated effluent enhanced percentage of germination in both crops 90 (Table 10). The ability of the rubber effluent to enhance the germination of seeds could be due to the heavy load of organic matter present in the effluent as was evident from the work of Chaiprapat and Sdoodee (2007). They found that rice and vegetable such as Chinese green mustard and cucumber responded well in seed germination and other morphometric and biochemical characteristics when treated with undiluted effluents, over other treatments. Similar observations were also made by Orhu et al. (2005a) in Dialium guineensis seed when irrigated with different dilution of rubber effluent. However, there have been a number of reports by different workers who have shown that high concentration of various effluents adversely affect the seed germination and growth of different plant species (Rajula and Padmadevi, 2000; Khatrt et al., 2003; Orhue et al., 2005b; Arora et al., 2006; Sharma et al., 2011).

In the present investigation, crops irrigated with cyanobacteria treated effluents (TE1 and TE2) showed poor response in seed germination and other morphometric analysis when compared with untreated (raw) effluent. This might be due to low availability of various nutrients including nitrogen and phosphorous which were either partially or completely removed from the effluent due to the inoculation Oscillatoria and Microcystis (Table 10).

As raw effluent from the rubber industry has a profound influence on seed germination and morphometric characteristics of important crop plants like paddy and green chilli, it can be potentially employed for irrigation purpose, so that the effluent can be effectively disposed off. As this is only a preliminary study, attempts should be made to have an indepth study on various other aspects of using the rubber effluent for irrigation purposes such as nutritional qualities of seed, impact of effluent on soil (both physico-chemical as well as biological) and soon before arriving at any definite conclusion.

91 Conclusion Based on the overall discussions, the following conclusions can be made.  The effluent itself continuous various indigenous microbes which can be tried for the treatment of various effluents.  Cyanobacteria in a potential group of organisms for bioremediation of rubber effluent.  Both O. salina and M. aeruginosa (test organisms) were employed and found to be successful.  The test organisms were more effective in their performance under immobilized conditions rather than free cells.  The influence of rubber effluent on cyanobacteria suggests that it has an inhibitory effect on various biochemical components.  The effluent from the rubber industry in its untreated condition is a potential source of irrigation and it had a positive response on seed germination and morphometric characteristics of crop plants tested. However, cyanobacteria treated effluents registered a better seed production and weight. 6. SUMMARY

In view of increasing awareness of waste water treatment, an attempt has been made to treat rubber effluent using indigenous microorganisms. Since effluents always abound with natural populations of microbes, biodiversity of microbes, such as bacteria, fungi and cyanobacteria, in rubber effluent has been investigated in order to understand their utility in waste water treatment. The effluent for the present study was collected from Nigavalli latex situated at Cochin, Kerala, India. Physicochemical analysis and biodiversity of microbes were carried out for a period of one year. Among the cyanobacteria isolated from the natural habitat, dominant species, such as Oscillatoria salina (filamentous) and Microcystis aeruginosa (unicellular) were used for the treatment of effluent.

Cyanobacteria were used in both free and immobilized conditions. For the immobilization, polyurethane foam cubes were used. The experiment was conducted for a total period of 30 days under controlled conditions. During the experimental period, effluent samples (control and treated) were periodically (every 5th day) analysed for physicochemical characteristics.

In order to understand the mutual influence and interactions between the effluent and the organisms, the biochemical studies, such as pigments, total carbohydrates, total proteins, aminoacids, lipids and fatty acids were carried out in both control and cyanobacteria grown in effluent. Moreover, to find out the effect of treated effluent on the growth and morphometric characteristics of crop plants, commercial crop such as Green chilli and careal such as paddy have been selected and irrigated separately with raw effluent (untreated effluent), cyanobacteria treated effluent and tap water. Morphometric analysis such as number of leaves per plant, shoot length, number of seeds per plant and weight of seeds (100), in addition to percentage of seed germination were studied and statistically analysed. 93 The study revealed the following:  Totally ten different bacteria were isolated from the effluent sample. All the species were recorded in all the months.  Altogether fifteen species of fungi were isolated from the effluent with Aspergillus as the dominant genus with seven species.  In cyanobacteria, totally 42 species falling under nine genera were isolated. Among the genera, Oscillatoria alone represented with seventeen species followed by Phormidium with eight, Lyngbya with seven, Microcystis with three, Plectonema with two and unicellular cyanobacteria chroococcus with two Aphanothece, Synechococcus and Synechocystis with sing species each.  Of the cyanobacteria, O. salia and M. aeruginosa were selected as indicator species since they were represented in all the seasons.  Both cyanobacteria responded well in removing various nutrients and reducing BOD and COD levels from the effluents. They also increased the DO content of the effluent.  Immobilized cyanobacteria were more efficient to treating the effluent than that of free cells.  Of the cyanobacteria, O. salina has a little edge one M. aeruginosa in treating the effluent.  The influence of rubber effluent on the biochemistry of test organisms showed that some of the biochemical contents of cyanobacteria such as carbohydrate, aminoacids and fatty acids showed their enhancement over controls, while others registered a decline in their level.  The percentage germination of seed was maximum with untreated effluent and minimum with treated effluent.  Significant increase in growth and morphometric characteristics were also observed when the crops were irrigated with untreated effluent over their treatments.

94 The crop grown using untreated effluent registered a better response with reference to various morphometric parameters tested. However, when cyanobacteria treated effluent was used for irrigation, the crops produced more number of seeds per plant and also weighed more.

This study revealed that indigenous cyanobacteria can be successfully employed under immobilized conditions for treating the rubber effluent. The converse effect of effluent on the test organisms revealed that it had an inhibitory effect on most of the biochemical contents of those organisms. Raw effluent can be used as a potential source of irrigation which when exploited properly can solve the problem of disposal of rubber effluent.