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.
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.
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