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

BOD, dissolved CO2, NO3, SO4

2, Ca2+, 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

not be found during summer. High SO42 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 Noe and Basseres, 1989),

Scenedesmus (Martin et al., 1985; Tam and Wong, 1989; de la Noe and

Basseres, 1989), Chlamydomonas (Taylor et al., 1988) and the blue green algae

– Spirulina (Kosaric et al., 1974), Phormidium (de la Noe 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,

with a maximum value of 4.9 mg P-PO43/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

indigenous rubber company rich in PO43- 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 Noe, 1985; de la Noe 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 Noe, 1985), Chitosan (de la Noe 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

were more efficient at removing NO3, NO2

, PO43 and Cr2O7

2 from waste

waters than free cells or cells immobilized on agar, alginate and carrageenan

(Mallick and Rai, 1994). Carrageenan-immobilized cells, however, were better

in removing NH4 and NO2. The PO43 uptake capacity was significantly

increased in cells starved in PO43 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

immobilized Anabaena CH3 were pH 7-9 and temperature 30-40oC.

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 ml1 was obtained for Chlorella in the effluent (pH 7.4). While 2.7 mg

ml1 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 gl1 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.

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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.