Stress Responses in Cyanobacteria - Spirulina platensis -
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Stress Responses in Cyanobacteria
- Spirulina platensis -
STRESS RESPONSES IN CYANOBACTERIA SPIRULINA PLATENSIS
3.1 Introduction
The balance between world population and world food
production is well known. In an effort to optimize food and forage
production, capital intensive agriculture uses pesticides to reduce
losses to competitive components of the environment: insects,
nematodes. pathogens, weeds, birds and mammals. As the energy
costs escalates, the price of chemical control of pests will also
Increase and even greater use of pesticides may still be expected as
agricultural practice shifts from monoculture to mixed cropping and
l lu l t i~ le rotation
Increased use of pesticides on our land ensures an increased
accumulation of these in our water bodies through agricultural runoff
and aerial dr~fts The interaction of these runoff and drift pesticides
and their metabolites is not only with the aquatic fauna but also with
the aquatic flora. Cyanobacteria being the major component of the
microflora of fields and water bodies adjoining these fields 'take on'
the brunt of the chemical assault.
Cyanobacteria are ubiquitous in the aquatic ecosystem where
they incorporate solar energy into biomass, produce oxygen that is
dissolved in water and used by aquatic organism, function in cycling
and mineral~zation of chemical elements and serve as food of
herbivorous and omnivorous animals. When they die, they sink to the
sed~ment. where their chemical constituents are transformed,
solubiiized and recycled into the water. All these functions are
dependent on the phytoplakton dynamics. The chemical contaminants
including pesticides affects this dynamics causing havoc to the food
chain, food web and thereby the ecosystem.
Like all living cells, Cyancmbacteria are also open system
interacting with their environment by constant exchange of matter,
energy and information. For each eivironmental factor (abiotic, biotic
or xenobiotic), there is a range of lt?vels (the tolerance range) within
which the cells are capable of growh, the rate of growth following an
optimum curve as the levels of the factor changes. However cells are
capable of survival over a larger range of variations, the resistance
range. When the factor goes from the limits of tolerance range in to
the resistance range the factor becomes a stressor.
The response to a stressor is; an adaptation and depending on
the kind and intensly of the factor, the response can persist for hours
or days in Cyanobacteria. It start:; with the destabilization of the
cellular metabolism, by say, to loss of membrane semi-permeability,
denaturation of proteins, changes ill metabolic balance, formation of
radicals etc.. leading to growth inhibition. This is followed by the
acclimation phase initiated by a change in gene expression or non-
genetic mechanisms, which trigger the synthesis of low molecular
stress metabolites, induce certain transport processes, modify the
membrane composition or lead to appropriate metabolic changes etc.
At the same time, changes in gene expression lead to synthesis of
various stress proteins. Some of these having a general protective
function and being concomitants of several stress syndromes can be
considered general stress proteins, t~ut others are stressor specific.
All these processes culminate in a recovery phase in which
growth is gradually resumed until finally, a new steady state is
ach~eved The growth rate of fully acclimated cells is often lower than
the lnit~al growth rate, but it can also be higher (acclimation to sub-
optimal or more optimal environmental conditions). But if the intensity
of the stressor exceeds the resistance limits of the cell, certain
functions breakdown and death may ensue. However successful
accl~mat~on sometimes confers increased resistance to inhospitable
condit~on. It is these acclimation and adaptive processes that serve as
candidates for potential biomarkers in Cyanobacteria.
Cyanobacteria have successfully colonised a wide range of
biotopes in the course of their evolution. Today, they are found in
v~rtually all the aquatic ecosystems (from freshwater through oceanic
to nyper-saline, hot springs and ice), in the soil, on naked rock, in
deserts and even in the air'. They owe their worldwide distribution to
the wide range of morphological and physiological properties they
acqu~red and their remarkable capacity to adapt to changes in a wide
var~ety of environmental factorsz.
In their natural environment, cyanobacteria generally exist
under growth limiting conditions. Several environmental parameters
such as light, salinity, nutrient availability and frequent temperature
changes serve as the growth limiting conditions. Few data is available
on the adaptation of cyanobacteria in natural populations. Most
studies of their adaptations to the various environmental parameters
have beer1 performed with selected model strains in laboratory
cultures under near optimum growth conditions. In the past few years,
many of the adaptation mechanisms used by cyanobacteria have
been explained down to the molecular level, including changes in
response to varying light quality (Chromatic adaptation) and nitrogen
defic~ency (heterocyst differentiati~n)~. "5
3.1.1 Heat Stress
Within a certain temperature ange, the effects of temperature
on all metabolic processes produce an optimum curve bounded by the
temperature tolerances of the various enzymes. Like growth, all
metabolic processes have species-specific minimum, optimum and
maximum temperatures. In cultures, most strains exhibit optimal
growth between 20% and 35OC, but thermophilic strains isolated from
hot springs have a much higher g r~wth optimum and tolerate upto
650C6
A response to heat shock can be elicited in cyanobacteria by
suddenly increasing the growth temperature by 5"-10°C. The response
is very similar in all organisms, from archaebacteria, eubacteria and
plants to animals and includes strongly inhibited growth and inhibition
of most protein synthesis. However, its chief characteristic is the
massive expression of several heat stress specific proteins.
The synthesis of heat stress proteins has been studied in
several cyanobacteria during the past 18 years. They have been
detected in varying numbers in both unicellular and filamentous
cyanobacterial strains. 7.8.9.10,'1,12813 The synthesis of Hsp starts
immediately after heat exposure and can be inhibited by rifampicinI4.
However not all Hsp are exclusively heat induced, many of them being
constitutively expressed and merely being synthesised much more
rapidly after heat shock. Their con:;titutive expression indicates that
they perform essential functions in the normal cellular metabo~ism'~.
Following the initial heat exposu.e, the increased rate of Hsp 18817 synthesis characteristically tails off ater a few hours .
Similarities between cyanobacterial Hsp and those of E.coli
have been noted and these similarities suggest that functional
s~milarities also exist.'= Several Hsp act as molecular chaperones in
cells of widely varying origin. Chaperones are able to protect other
proteins against denaturation, to assist transport and folding, and to
repair damaged proteins by refolding them. In addition, the Hsp's
Include proteases that specifically decompose irreversibly damaged
proteins in heat treated cells.'5
The gene coding for the chaperon groEL, groES and dnaK in
Synechococcus sp. and in Synechocstis sp. has been isolated and
All three genes show similarity to similar genes in
t coll and also in plantsz0
Indirect evidence that Hsp's are involved in the heat tolerance
of cyanobacteria is furnished by the fact that short term heat shock
treatment at sublethal temperatures and long term adaptations to
higher temperatures increases the survival rate at normally lethal
temperaturesq3 "'. In experiments with Synecocystis sp. PCC 6803
culture, growth is accompanied by increased synthesis of groEL
protein even at 3g°C. After heat shock, the increase in Hsp expression
In these cultures was greater than in cells adapted to 22'CZ'. Since
photosynthetic O2 production and PSI1 activity were also less affected
by the heat, the chaperones conceivably associate with thylakoid
structures and protect their structure and function2'. This protective
funct~on has been demonstrated experimentally in E.coli mutants
lack~ng the chaperone genes and therefore capable of growth only in a
very narrow temperature rangeq5. Over expression of an Hsp6O
chaperone also leads to the protection and reactivation of heat labile
enzymes in mitochondria of heat shocked yeasts2'.
Long-term adaptation to different temperatures induces
changes in membrane compostion. Adaptation to relatively high
temperatures increases the propoltion of saturated fatty acids in
cyanobacterial membranes, thus enhancing their stability23a24. The
effect of changes in unsaturated fatly acid content on the heat stability
of photosynthesis apparatus has been studied in Synchocystis sp.
PCC 6803 mutants. It was found tha: the stability of PSI, PSI1 and total
photosynthetic activity after heat shock was independent of the
saturated and unsaturated fatty acid contenf5. However later studies
have shown that the removal of two folds unsaturated lipid molecules
(corresponding to an increase in saturation) tends to reduce heat
tolerance rather than increase ifB.
3.1.2 Salinity Stress
Salinity is one of the most important abiotic factors in aquatic
biotopes. An increase in salinity la reduction has analogous, but
opposite effects) represents a combination of two stress situations for
cyanobacteria. In the first place, the reduction in the water potential of
the surrounding medium causes the cells to lose water. Osmotic and
drought stress also have this effect, and the adaptation mechanisms
of cyanobacteria for coping with ail three stressors therefore share
many common features. Water Ic~sses lead to shrinkage of cells
without cell walls and loss of turgor in those with them. In contrast to
pure osmotic stress (caused by non-permeable organic agents) and
desiccation, an increase in salinitq means a dramatic increase in
concentrations of inorganic ions (especially Na' and CI-) in the
surrounding medium. These ions erter the cells along chemical and in
some cases, electrochemical gradients, thus counteracting the loss of
water since this process reduces the osmotic potential difference6.
However h~gh concentrations of inorganic ions are toxic, interfering
wlth cell metabolism except in halobacteria2'. During adaptation to salt
stress, a oalanced osmotic potential is achieved by extrusion of
excess inorganic ions from the cell and the accumulation of so called
osmoprotective substances8.
3.1.2.1 Salinity and Osrnoprotectants
Osrnoprotective substances are low molecular hydrophilic
compounds that accumulate in large quantities in salt loaded cells.
They do not Interfere with the cellular metabolism because they are
largely Inert and carry no net chargez8. They reduce the osmotic
ootent~al of the cytoplasm to prevent further loss of water and
apparently prevent denaturation of macromolecules by helping them
to retain their natural configuration. Chemically they are grouped into
nonelectrolytes and electrolytes. The nonelectrolytes consist of polyols
and carbohydrates and are common in algae, yeast and cyanobacteria.
Except for 3dimethylsulfoniopropionate and dimethylthetine, the
electrolytes are quaternary compounds of ammonium and are typical of
plants, bacteria and cyanobacteriaZ9.
Although a large number of cyanobacterial strains have been
studied, no link has been found between the kind of osmoprotective
substances accumulated and either taxonomic group or the biotope of
orlgln of the strains. However, it was possible to assign them to three
salt resistance groups, which differ distinctly in the osmoprotective
substances they accumu~ate~~:
1 rhe least halotolerant strains accumulate sucrose and
trehalose and can tolerate up to 0.7M NaCI.
2. Moderately halotolerant cyar obacteria accumulate glucosyl-
glycerol and their tolerance lin~it is 1.8M NaCI.
3. The highest halotolerance is exhibited by strains that
accumulate mainly glycine and glutamate betaine; these
strains can tolerate salt conceltrations up to 2.7M.
In many cases, traces of o.:her osmoprotective substances
have been detected at the same time as the main osmoprotective
substances6.
3.1.2.2 Effects on lntracellular Ionic: Balance
Besides reducing the osn~otic potential, salt stress is
accompanied by a dramatic increase in the concentration of inorganic
ions (especially Na' and Ct) in the s~rrounding medium. Although very
hgh concentrations of inorganic ions are toxic to cyanobacteria, slight
Na' concentrations are essential for many cellular processes, including
the uptake of ammonia, C02, HCOi, and pho~phate~"~'. The
cleavage of water in photosystem I has proved to be another Na-
dependent process besides carbon transport in cyanobacterial
photo~ynthesis~~. The essential role played by low concentration of
Na' ions in maintaining a functioning nitrogen fixation system has
been known for sometime, Na' ions are apparently being needed for
the activation of ni t r~genase~~. More>ver, the transport of Na' is also
involved in regulating internal pH values in ~yanobacteria~~. However
a concentration of 10mM is sufficient to satisfy most of these needs34.
In cyanobacteria the intracelli~lar K' concentration is distinctly
higher than in the surrounding medi~m,, and this is obviously a result
of active accumulation. K' uptake is probably driven by the
transmembrane potential and is at least indirectly dependent on ~ ~ p 3 6 35837 This ion is even probably actively transported in Anabaena
i arrabil~s~" Elevated K' concentrations have oflen been measured in
salt adapted cells of various cyanobacteria and there is no obvious
dramatic change in the Na' IK' ratio in such cases3', 40 41. However,
K' concentration remains fairly constant in Spirulina strains4' or even
decline markedly in cells adapted to higher sa~inities~~. Kt ions
contribute substantially to the osmotic potential of the cells and
therefore can be considered as an osmoprotective s~bstance'~.
lntracellular concentrations of CI- have been studied in
comparat~vely few species. In cyanobacteria, these ions are
accumulated agalnst the membrane potential gradient by means of a
Na' dependent process involving the consumption of AT^"'. Like
the concentrations of Na' and K', the CI- concentration increases
luring salt adaptation but always remains lower than that in the
surround~ng n ~ e d i u m ~ ~ ~ ~ ' ~ ~ ' .
3.1.2.3 Effects on Photosynthesis
In photo-autotrophic cells, the photosynthesis usually
correlates quite well with the growth rate. Since high salinities oflen
~nhibit cyanobacterial growth, the growth rate of cyanobacteria
adapted to high salinities, unlike their respiration is generally
d e p r e ~ s e d ~ ~ . ~ " ~ ~ . The photosynthetic activity of Synechocystis sp.
PCC 6803 and Spirulina platensis decreases quickly and drastically
during the first few hours following salt shock, but has almost reached
a steady state after 12 In contrast in Synechocystis sp.
FCC 631 1 stimulated photosynthetic activity, measured 24 hours after
exposure to salt shock even thougt growth had been inhibited during
the intervening period5'.
Apart from affecting overall ohotosynthetic activity, changes in
salinity also leads to adjustments in the photosynthetic characteristics
of cells of Synechocystis sp. PCCfi803. The PSlllPSl ratio is lower
after salt adaptations2, mainly owin3 not only to an increase in PSI
activity, but also to an increase in PSI content53. As in the case of
respiration, the increase in PSI activ~ty is greater than in the amount of
PSI. The higher PSI activity is us?d mainly to increase the cyclic
electron transport, which might be a source of additional energy for ion
extrusion by means of ATPases. Changes have been found in
pigmentation50854 and the transmission of energy between
photosystems and also in the phycobilisomes on P S I I ~ ~ ' ~ ~ .
3.1.2.4 Salt Induced Changes in Gene Expression
The cyanobacterial metaboli!;m is regulated principally at the
transcription level, i.e., by the activation or repression of the
expression of certain genes to suit the needs of cell growth. In view of
the manifold physiological and biochemical changes that take place in
salt-stressed cyanobacteria, it is scarcely surprising that signs of a
change in gene expression have also been reported.
In cyanobacteria exposed to salt, osmotic or drought stress,
three basic groups of proteins can be identified by their response to
environmental factors: 1) proteins whose expression remain relatively
unaffected by variations in environmerital factors ("household proteins);
2) proteins whose synthesis is turned off or inhibited specifically when
environmental conditions change; anJ 3) proteins whose synthesis is
Increased or induced specifically under unfavorable environmental
conditions. The members of the group 3 are called stress proteins
because one may assume they perform certain functions during
adaptation. which permit the cells to achieve optimum growth in the
changed environment.
Many cyanobacterial strains from various biotopes and with
varlous salt tolerances synthesize the same stress proteins in
response to salt stress '',56.577.58859 . Close study of the k~netics of stress
protelns have revealed that overall protein synthesis is strongly
depressed immediately after addition of salt to the medium5' and that
stress protetns predominate among newly synthesized proteins during
the flrst few hours of s t r e ~ s ~ ~ . ~ ~ . Normal protein synthesis is then
gradually resumed as osmoprotective substances accumulate during
further adaptation process. Certain stress proteins are fund only
during early stages of adaptation, i.e. their expression or induction is
only trans~enfly stimulated, whereas the synthesis rates of others are 56858 also. or only, hlgh in salt adapted cells .
Comparison of the stress proteins synthesized by various
cyanobacterlal strains show that their numbers and molecular weights
vary from one strain to another, proteins with a low molecular weight
predom~natlng. Most stress proteins are soluble, but several of them
are also found on the cytoplasmic membrane41.56859.
The synthesis of several stress proteins produced by
Anabaena strains is also stimulated by osmotic stress56860. However,
slnce other proteins are synthesized specifically in response to salt or
osmotic stress, the pathways for sensing and transmitting the stimulus
obviously differ, although the responses are partly identicale0.
Like interactions between salt, osmotic and drought stress,
heat shock is also able to induce ;a few salt stress proteins 14,56861
Stress proteins found after salt loading can therefore be grouped into
general stress proteins (response to both salt and heat shock),
general water stress proteins (response to salt, osmotic and drought
stress, respectively) and those stress proteins that are specific to a
given stressor.
Apart from their molecular wetghts, the sequence in which they
are synthesized during adaptation and the effects of interactions
between stressors on their expression, little is known about the
function of most stress proteins. Those induced even by heat shock
probably have a non-specific protective (Chaperone) function for
macromolecules in the presence of high ion concentrations. Those
stress proteins that are permanently and strongly expressed, probably 56 a61 play some direct role in the adaptation process .
3.1.3 Effects of Pesticides on Cyanobacteria
Biologists are increasingly becoming aware of the importance
of determining deleterious effects C I materials used in and around
living systems. The pesticides and agrochemicals are major among
these toxic substances ofien used ill the ecosystems. Of course, the
wide spread use of biocides has greatly aided man, but at the same
time the indiscriminate and extensive use in modern agriculture in
particular, has created lot of problenis in ecosystems. Their effect on
soil and water microorganism's, among which cyanobacteria occupy
an important position are alarming. The importance of cyanobacteria
as a major group under microflora is due to :
1. Their ability to grow under ex:remes of environmental stresses,
2 Thelr position at the first trophic level of the food chain and
3 Thew ability to fix atmospheric nitrogen autotrophically
The work in the context during the last few decades includes
pesticide degradation, bioaccumulation and algal bioassay but
major~ty of the work has dealt with pesticide toxicity. The interesting
aspect of pesticide - cyanobacteria interaction is the influence of these
toxicants on the population which indicates either they stimulate or
decrease or have no effect on the growth of these microbes.
There are a number of pesticides like 2, QD, DDT, parathion,
malathion endosulfan, dimethoate, paraquat, etc which after their
appl~catlon in the fields, undergo transformation to different intermediate
products both biologically and abiotically in nature. Microorganism's
Including cyanobacteria perform the biotic transformation of these
pest~ctdes. Subsequently these organisms are exposed to the toxicants in
the~r orig~nal, degraded or transformed forms. In such cases of interactions
with toxlc chemical, cyanobacteria can exhibit a variety of different
reactions like negligible to sublethal effects, reduction in survivability and
growth, low metabolic activity or ultimately death.
3.1.3.1. Effect on Survival 8 Growth
Interaction of cyanobacteria with agrochemicals continues to
be the maln theme of modern day cyanobacterial research and also
much attention has been devoted to pesticides especially insecticides.
The response of cyanobacteria have been studied to a combination of
pest~c~de compoundse263pe4. Insecticides constitute around 83 percent
of the total pestic~des, which are in use today. With reference to their
Impact on cyanobacteria and as a group, organochloride insecticides
are hazardous by virtue of their tox~city and persistence in the soil of
these organisms. It is known th.at microorganism's exposure to
organochloride compounds inhibit :heir photosynthetic and enzyme
activity, altering membrane structure, permeability, integrity and
interfere with the synthesis of DNA, I?NA and proteid5.
Kapoor and SharmaS6 studred the effect of 0.001 to 0.006
percent endosulfan on Nostoc muscorum and Anabaena dolidurn and
found that these concentration were able to sustain growth. While
Kapoor and ~ r o r a ' ~ ~ ' found endosulfan to be inhibitory and even
lower concentration. Tandon et al." observed the inhibitory effect of
endosulfan on Anabaena sp. Aulosira fertilissima and observed that
not early but in late photosynthesi:; and nitrogenase activity where
inhibited by endosutfan. Goyalss noted significant reduction in the
growth of Anabaena iyengarii, Haplosiphon intricatus and Calothrix
bhardwaja. These results become significant because Endosulfan is
widely in use in developing countries including lndia.
Recent trends in insecticide research lay emphasis on the
interaction of paddy field cyanol~acteria with organophosphate
pesticides. The work has been done n many parts of lndia because of
the fact that lndia is a tropical country and major rice producing
country. These chemicals are less persistent in the soil, so, farmers
prefer these compounds. Gangwane7' and Tandon eta/.? studied the
effect of Malathion on Anaebaena rip. and Aulosira fertilissima and
found that the former was able to tolerate Malathion upto 500 ppm
while the later was inhibited at 10 ppm of this pesticide.
3.1.3.2 Effect on Metabolic Processes
3.1.3.2.1 Photosynthesis and Respiration
The effect of agrochernicals on photosynthesis and respiration
of cyanobacteria has been studied by a few workers. Kurnar7'
reported the effect of pesticide on the pigment formation of unicellular
cyanobacteria Anacystis nidulans and concluded that amitriazole had
adverse effects on the photosynthetic process of cyanobacteria by
~nhibiting the photosynthetic pigment formation. Similarly Lazaroff and
~ o o r e " found that thiocarbarnate pesticides interfered with
photoinduced development of Nostoc rnuscorurn. Vosiliva and
~ inev i sh '~ reported that diuron and hydroxylamine denatured the PS I
and PS II of Anacystis nidulans. They noted that photosynthetic
oxygen evolution was inhibited and functioning of photosynthetic
apparatus was disturbed by different concentrations of both the
agrochernlcals tested. Inhibition of photosynthetic process of algae by
methyl parathion has also been Mehta and Hawxby7'
reported that the lethal action of simazine at higher concentrations on
A.n~dulans is due to imbalance in functions of thylakoids by making
these polyhedral bodies incapable of performing their function.
Agrochemlcals are known to interfere with chlorophyll and carotenoid
synthes~s by inhibiting the formation of porphyrin and the biosynthetic
steps in the carotenoid synthetic pathway, which leads to chlorosis and
accumulation of carotenoid precursors. This ultimately inhibited
photosynthesis and photosynthetic oxygen evolution76. Papst and !30yer~~
reported the inhiMion of chlorophyll-a synthesis of fresh water algae at
hgher concentrations of both the insecticides. Mohapatra et
reported that the organophosphate insecticide dimethoate had adverse
effect on the chlorophyll synthesis of Anabaena doliolurn. However,
C-N medium they found the effect to be more significant even at low
concentrations. They also found that the insecticide endosulfan also
had similar effect on A. doliolum
3.1.3.2.2 Nitrogen Fixing Ability
The cyanobacteria that produc:e heterocyst have the ability to
fix nitrogen. Besides them non-hetrocystous cyanobacteria, both
unicellular and filamentous are also capable of nitrogen fixation. Most
of these diazotrophs could fix N2 only microaerobically or even
anaerobically. The nitrogen fixing ability of cyanobacteria is regulated
when the organism is exposed to a(lrochemicals. 2,443, one of the
most commonly used herbicide, at its normal field doses reduces the
nitrogen fixing ability of Nostoc punctitorme and cylindospennum sp."
and Tolypothrix tenuiss'. Pesticides like diquat, paraquat, limuron,
MCPA. Malathion and manuron hamper N2 fixing potential of
cyanobacteria at higher con~entratio?'~. Das and ~ i n g h ' ~ " ~ work~ng
with four fresh water cyanobacteria reported increase in nitrogen
fixation by 2,4-D (10mglL) and inhibition of the process by HCH at
lower concentration(l0mglL). Suppression of heterocyst frequency
with application of this pesticide at 50k100mg/L in 10 days incubation
was due to its utilization by cyanobarferia as the nitrogen source. On
the other, 10 and 25 mg/L of carbofuran increases the heterocyst
frequency and total nitrogen fixed by Nostoc muscorum. However
higher concentrations (50-1000 mg/-) have adverse effects on the
nitrogen fixing ability of the cyanobacteriums5. From this it is obvious
the field application rate (0.5kglha) which is usually 2-4 mg/L in the
fields probably has no effect on the I& fixing ability of cyanobacteria.
The pesticides at higher concentration have significant effect on the
respiratory oxygen uptake and production of energy rich compounds
like ATP. This deficiency in energy regulates the activity of Glutamine
synthetase, a key enzyme for nitrogen assimilation86a87 decreasing the
total nitrogen content of the culture. Thus pesticides have a significant
role in N2 fixing of cyanobacteria. The type of inhibition observed in the
above studies with respect to energy sources suggests that ATP may
b~nd at a site, which interacts with the binding site of adenine
nucleotide on the enzyme. Nitrogenase activity, as well as synthesis,
may be regulated by the availability of ATP and reductants inside the
cell. Artific~al manipulation of the ratio of ADP to ATP (the D/T ratio) in
cultures, as a consequence of compounds that interfere with ATP
synthesis, showed that nitrogenase activity declined as the DIT ratio
increased above 0.6, which is the average value in illuminated
cu~tures'~
Stimulation of nitrogen fixation has also been reported when
organophospate pesticides like nigercin increased the P content in
culturesag S~ngh and Bisoyigo, found that organophosphate pesticide
at low concentrations encouraged the growth and nitrogen yield of
Aulos~ra sp Nirmal Kumar et a?' studied the response of nucleic acids
ot Anabaena sp, to pesticide Bevistin and found that the pesticide
enhances the RNA as well as DNA content at 100-300 microgram per
rnl dose favouring the growth of the cyanobacteria.
3.1.4 Use of Cyanobacteria in Pollution Abatement
The bioaccumulation and biomagnification of residual
insecticides in cyanobacteria, which constitute the major chunk of the
primary producers in the food chain is biologically and toxicologically
significanty2 It is well established that algal biomass have larger
surface area attracting the biophilic pesticide molecules thus helping
in predicting impact of pollution in aquatic systems. Depending on the
type, biological property and the concentration of pesticides and the
algal strains, their effect could be inhibitory, selective or even
stimulatoryg3. It has been observed that cyanobacterial forms used in
biofertilizers are capable of tolerat~ng pesticide levels recommended
for field applicationsw. Flavoprotein systems isolated from algae seem
to play a important role in pesticide degradation in aquatic
environments. Such flavoprotein sy:;tems are active in the degradation
of xenobiotics under aerobic and anaerobic conditions by promoting
photochemical and reductive degradation activitiesg5. Rath and
Adhikarysa studied the effect of Furudan on two species of Anabaena
with relation to irradiation and pH. They found that the organisms grew
comparatively better and synthesizt?d higher amount of chlorophyll at
higher irradiance levels. The toxicity EC50 dose of the pesticides
gradually decreased with the increased irradiance. The toxic effect of
Furudan was larger when the initial cyanobacterial population
concentration was low and vice-versa.
3.2 Materials and Methods
3.2.1 Maintenance of Spirulina platensis
Sptrulina platensis (ARM 730) procured from NCCUBGA, lARl
New Delh~ was used for all the experiments. Stock culture of this was
maintained in our laboratory. It was grown in CFTRl medium having
the followrng composition.
CFTRl Medium
The media was prepared in tap water and this was autoclaved
at 1 2 1 ' ~ for 20 minutes at 151bs. CaCI*, MgSO4 and FeSO, were
autoclaved separately and after cooling added to the medium
aseptically The medium was adjusted to pH8.5 with O.1NaOH. 50ml
Sprrulina from a mid log phase growth culture was inoculated into 1 .OL
Components I-- - NaHC03
KZHPO4
NaCl (sea salt) , NaN03
MgSO4 CaCI,
FeS04
KzSOI
of the media. Then 200ml from this was dispensed aseptically into
cotton plugged 500 ml sterile Conical flasks. These were maintained
at 25' .c 2% under 24 h light in an illuminated chamber at 2.5 Klux.
The cultures were thoroughly shaken 2-3 times daily to prevent mat
Quantity (glL) 4.5
0.5
1 .O
1.5
1.2
0.04
0.01
1 0
3.2.2 Stress Induced Growth lnhibition Studies
A 96 hour toxicity assay was performed to study the inhibition
of growth and also to determine sub lethal concentration of stressors.
The stressors were natural stressots like temperature. Salinity and
agrochemical stressors like Endos~lfan, Paraquat, Malathion and
Dimethoate. A control was also run simultaneously.
Inhibition of growth was studied by taking varied concentration
of commercial grade agrochemicak; and analytical grade NaCl for
testing salinity stress and by placing ihe cyanobacterial in a water bath
at fixed temperatures for studying temperature stress effects on
growth
Six replicates of 200 ml of l4/15-day log phase cultures were
exposed for 96 h to the various stressors as detailed below. The initial
doses were chosen by referring tc phytotoxicity data available on
these agrochemicalsS7 and from other cited references.
Temperature
Salinity
Endosulfan 1 S P P ~ I I O P P ~ I 15ppm I ~ O P P ~ 1 Paraquat
The growth inhibition was noted by estimating the chlorophyll
Dimethoate
content at every 24 hours for 96 hous
I I I I
0.1 ppm
~ O P P ~
0.25ppm
~ O P P ~
0.5ppm I .Oppm
30ppm 4Oppm
59
3.2.2.1 Chlorophyll Estimation
Chlorophyll estimation was performed according to the method
of ~rnon''and modifications of Witham ef a1 ".
Reagent
Ice cold 80% aaueous acetone
Procedure
1 25 0ml of media was withdrawn from the culture and
centr~fuged at 5000 rpm for 10 minutes.
2 Then 5.0 ml ice cold 80% acetone was added to it.
3 Thls was sonicated at 90 watts for 5 minutes with 30-40 sec
bursts with intermittent cooling on ice. Then centrifuged again
at 5000 rpm for 10 minutes. The supernatant was transferred
to 25 0 rnl std. flask.
4 Repeated the above procedures (step 2 &3) three more times
with fresh 5.0 ml of ice cold buffer.
5 Made up the volume in the flask up to the mark with 80%
acetone
6 Reaa the O.D. of the above solution at 645nm and 663nm
agalnst a solvent blank of 80% acetone.
7 Calculated the amount of chlorophyll as per the following
formula
12.7(0.D66,)-2.69(0.D64,) mg Chlorophyll iml sample =
vol .ofacetone
3.2.3 Sample Preparation
For the various biochemical assays and Hsp6O detection by
SDS-PAGE, a single high sublethal concentration determined by data
obtained from the above toxicity assay was utilised. The stressors
were 33% temperature, 1.5gm% salinity, 2.5ppm paraquat. 15ppm
endosulfan. 20ppm malathion and 30 ppm dimethoate. The time
periods of exposure for the various biochemical assays were 1 hour, 3
hour. 6 hour, 9 hour and 12 hour.
For ELlSA studies in a.jdition to the high sublethal
concentrations mentioned above, ower sublethal concentration of
30°C temperature, I .Ogm% salit~ity, 1.0ppm paraquat, 10ppm
endosulfan lOppm malathion and 20 ppm dimethoate were also used.
The time periods for this study were 3, 6 and 12 hours.
3.2.3.1 Determination of Stress Proteins
Cultures of Spirulina plater,sis were exposed for 6 hours to
sublethal concentrations of various stressors. SDS- PAGE followed by
western blotting was performed to cetect the expression of Hsp6O. For
this, procedures explained in chapter 2, section 2.2 and section 2.3
were followed.
Weighed samples in fixed amount of sample buffer with final
concentration of ImM Phenyl Metl~yl Sulfonyl Fluoride (PMSF), were
ultra-sonicated at 90 watts for 5 minutes (30-40 second bursts with
intermittent cooling on ice. Centri'uged at 5000 rpm for 10 minutes
and the supernatants taken for the analysis. An aliquote was taken for
protein estimation by Bradford 4ssay (chapter 2, section 2.1.1).
Samples of 1 mglrnl protein concer~tration were loaded on to the wells.
3.2.3.2 Quantification of Hsp 60 Expression
To quantify Hsp6O expression on a time and concentration
scale ELlSA as described in chapter 2, section 2.4 was followed.
Weighed samples in fixed amount of coating buffer were ultra-
sonlcated at 90 watts for 5 minutes (3040 second bursts with
~ntermittent cooling on ice. Centrifuged at 10,000 rprn for 10 minutes
and the supernatants were taken for the assay. Aliquotes of the
supernatants were taken for protein estimation by Bradford Assay
(chapter 2 section 2.1.1). Samples of 3 mglml protein concentration
were loaded on to the wells. The results are expressed as pg of
human Hsp6O equivalent Img protein.
3.2.4 lsoenzyme Analysis
For this. procedures explained in chapter 2. section 2.7.1 and
2 7 2 were followed. Weighed samples in fixed amount of sample
buffer w~th ImM Phenyl Methyl Sulfonyl Fluoride (PMSF), were ultra - sonlcated at 90 watts for 5 minutes (3040 second bursts with
Intermittent cooling on ice). Centrifuged at 10,000 rpm for 10 minutes
and the supernatant were taken for the analysis. Aliquotes of the
supernatants were taken for protein estimation by Bradford Assay
(chapter 2 section 2.1.1). Samples of Imglml protein concentration
were loaded on to the wells.
3.2.5 Effects of Free Radicals
3.2.5.1 Protein Carbonyl Estimation
Samples were extracted in 0.067M Phosphate buffer pH7.0 by
ultra-sonlcat~on and centrifuged at 5,000 rpm for 10-15 minutes. The
supernatants were used for the assay as described in chapter 2,
section 2.5.2. Aliquotes were also taken for protein estimation by
Lowry et a1 method (chapter 2, zection 2.1.2). The results are
expressed as nMlmg protein.
3.2.5.2 TBARS Estimation
The weighed samples were extracted by ultra-sonication in
known volume of 0.025M Tris-HCI buffer (pH 7.5) and centrifuged at
5,000 rpm for 10-15 minutes. Protein concentration of the aliquotes of
supernatants were estimated by Low/ et a1 method. The supernatants
were used for the assay described n chapter 2. section 2.5.1. The
TBARS content is expressed as mM of TBARS I mg proteins.
3.2.6 Antioxidant Systems
3.2.6.1 SOD Estimation
Known quantity of samples was extracted by ultra- sonication in
known quantity of 0.25M s m s e and centrifuged 10,000 rpm for 10-15
minutes. The supernatants were used for the assay as per procedure in
chapter 2, section 2.6.1. Aliquotes were taken for protein estimation by
Lowry et a1 method. The SOD activity is expressed as Ulgm protein.
3.2.6.2 Catalase Estimation
Basic procedure as detailed in chapter 2, section 2.6.2 was
followed. The samples were extracted in 0.067 M phosphate buffer
(pH 7.0). The extracts were centr.fuged at 5,000 rpm for 10-15
minutes. Aliquotes of the supernatants were estimated for protein
63
content by Lowry et a1 method (chapter 2, section 2.1.2). The catalase
activity IS expressed pM of H202 consumed lminlmg protein.
3.2.6.3 Glutathione (GSH) Content
GSH content was estimated by the procedure detailed in
chapter 2. section 2.6.4. The sample extract was prepared by ultra
sonlcating weighed samples in 0.2M phosphate buffer (pH8.0). The
extracts were centrifuged at 5,000 rprn for 10-15 minutes. The GSH
content 1s expressed as mg1100g sample.
3.2.6.4 Peroxidase Activity
The method of Putter, J was followed for estimating peroxidase
adi~ity.' '~
Reagents
Phosphate Buffer 0.1M (pH. 7.0)
20mM Guaiacol in D.W
1 1 3mM hydrogen peroxide
Procedure
I The samples were extracted in phosphate buffer by ultra-
sonicatlon at 90W for 5 minutes (30-40 second bursts with
~ntermlttent cooling on ice).
2 Centrifuged the resultant extract at 10,000 rprn for 10 minutes.
3 Allquotes of the extract were taken for protein estimation by Lowry
et a1 method (chapter 2, section 2.1.2).
€4
4. To 3 ml of phosphate buffer in a spectrophotometric cuvette added
50p1 guaiacol, O.lml of enzyme extract and 3 0 ~ 1 of hydrogen
peroxide solution and mixed well.
5. Placed in a spectrophotometer set at 436nm and waited till an
increase of 0.05 in Absorbance was noted. Then noted the time
needed for an increase in Absorbance of 0.1.
6. The enzyme activity is expressed as Ulmg proteins.
3.2.6.5 GST Estimation
Basic procedure as detailed in chapter 2, section 2.6.5 was
followed. The samples were extracted i r ~ 0.05 M phosphate buffer (pH 6.5).
The extracts were centrifuged at 5,000 rpm for 10-15 minutes.
Aliquotes of the supernatants were estimated for protein content by
Lowry et a1 method (chapter 2, secti'm 2.1.2). Values are expressed
in nM of CDNB complexed I minl mg protein.
3.3 Results
3.3.1 Stress Induced Growth Inhibition Studies
Chlorophyll content per ml of culture was used to assay the
growth of Spirulina platensis. Table C1 shows effect of various
temperature regimes on the growth of Spimlina platensis.
Table C l : 96 hours assay of Chlorophyll content (pglml) of Spirulina platensis under varied temperature stress
. ~ ~
STRESS I--- ] CONTROL
~ ~
T1 -30°C + - . - -
I ~ 2 . 3 3 ' ~
~ 3 - 3 5 ' ~ I 1~~ ..
From the results it can be seen that there was significant
decrease in chlorophyll content and hence decrease in growth of
Sp~rul~na platensis during the assay period at 35'C and 38%. At 30°C
the growth of Spirulina platensis was following a similar pattern like
that at control temperatures. There was slight reduction only at 33'C
out not to the levels at 35OC and 38 '~ .
The effect of various concentrations of NaCl on the chlorophyll
content and hence growth of Spirulina platensis is shown in Table C2.
Table C2: 96 hours assay of Chlorophyll content (pglml) of Spirulina platensis under varied salinity stress
Although there was a slight reduction at 1.5g%, the growth of
Spidina platensis was not significantly affected up to 1.59% and upto
this concentration growth of Spirulina j~latensis followed more or less a
similar pattern to that of the control group. Growth was significantly
inhibited at 2.09% concentration of NaCI.
The effed of a bipyridyl herbicide paraquat on the growth of
Spirulna platensis was assayed and the results are shown in Table C3.
96h
8.631 + 0.151
8.505 + 0.460
8.239 + 0.233
7.61 3 + 0.279
3.052 + 0.059
48h
6.702 + C.182
6.720 + (1.199
6 558 + 0.100
5 482 2 11.166
3.286 2 3.264
Uh
5.449 + 0.194
5.486 2 0,175
5.180 + 0.292
4.451 2 0.218
4.033 2 0.223
STRESS
S1-0.5g%
S2-1.@%
S3-1.5g0/.
S4-2.0g%
72h
7.557 + 0.220
7.486 + 0.238
7.406 2 0.176
6.725 2 0.136
3.172 5 0.212
Oh
4.082 + 0.087 - 4.062
+ 0.087 -
4.062 + 0.087 -
4.062 + 0.087 - 4.062
+ 0.087 -
Table C 3: 96 hours assay of Chlorophyll content (pglml) of Spirulina platensis under varied paraquat concentration
~ ~~-
A concentration of 0.5ppm was sufficient to induce significant
~nh~bition in the growth of Spirulina platensis. There was sl~ght
.rih~blt~on of growth at 0.25 pprn. Below this concentration the growth
rate was not significantly altered from the control. At a concentration of
Opprn, there was complete inhibition of growth.
The effect of various concentrations of organochloride
pesticide endosulfan on the growth of Spirulina platensis is detailed in
Table C4 Based on the result, it is clear that significant reduction in
growth of Spirulina platensis was induced at 30 pprn. Above this
concentrat~on ie., 5, 10 and 15ppm the growth pattern was similar to
that of control, albeit at somewhat lower levels
Table C4: 96 hours assay of Chlorophyll content (Clglml) of Spimlina platensis under varied endosulfan concentration
The effect organophosphate pesticide malathion on the growth
of Spirulina platensis is shown in Table C5.
Table C5: 96 hours assay of Chlorophyll content (pglml) of Spirulina platensis under varied malathion concentration
96 h 8.565
+ 0.218
8.775 + 0.214 7.969
+ 0.238
1.282 + 0.207
0
72h 7.474
+ 0.154
7.868 + 0.220
7.326 2 0.253
2.549 + 0.232
0
STRESS
'ONTRoL
MI-lOppm
M2-20ppm
M3-30pprn
M4-40pprn
Oh 4.193
+ 0.185
4.193 + 0.185
4.193 + 0.185
4.193 + 0.185
4.193 + 0.185
24h 5.398
+ 0.165
5.102 + 0.321
5.001 + 0.318
3.934 + 0.273
3.546 + 0.327
48 h 6.730
+ 0.145
6.830 + 0.31 1
6.163 + 0.272
3.302 20.336
1.204 + 0.276
On referring the results obtained it was clearly seen that upto
2Oppm of malathion, growth of Spirulina platensis followed a similar
pattern to that of the control. At 30 ppm and above there was a
significant inhibition of growth.
The effect of varied concentrations of another organophosphate
~estlclde dlniethoate on the growth of Spirulina platensis is
represented in Table C6.
Table C6: 96 hours assay of Chlorophyll content (pglml) of Spirulina platensis under varied dimethoate concentration
48h 72h 96 h
From the results, it is clear that at lower concentrations of
dlmethoate, there was enhanced growth at lOppm of dimethoate At
20 ppm and 30 ppm the growth pattern followed a slm~lar pattern to
that of control but for a sllghtly reduced growth at 30ppm There was
slgn~flcant ~ n h ~ b ~ t ~ o n of growth at 40ppm
From the above results it was considered to take 33OC, 1.5g%,
O 25ppm paraquat, 15ppm endosulfan, 20ppm malathion and 30ppm
dlmethoate as the temperature and concentration of the various
stressors for the further studies undertaken. These were considered
as paints at which growth was affected ie., reduced but not inhibited.
3.3.2 Stress Protein Induction in Spirullna plafensis Under Various Stressors
3.3.2.1 SDS PAGE and Western Blot Analysk
The molecular weight analysis of SDS PAGE electrophenograph
of the extracts of Spirulina platensis subject to various stresses and the
subsequent western blot analysis of the gel (Plate GI & C2
respectively) revealed a human HspGO equivalent was being
expressed at 53.8 KDa.
3.3.2.2 Dose and Time Dependent Expression of Hsp6O
T~me and Dose dependent expression of human Hsp6O
equivalent in Spirulina platensis was studied using ELISA. The
concentration and time dependent expression of Hsp6O under two
temperatures (30°C and 33'C), two salinity concentrations (1.09% and
1 59%). two paraquat concentrations (0.10 and 0.25pprn), two
endosulfan concentrations (10 and 15 pprn), two malathion
concentrations (10 and 20 pprn) and two dimethoate concentrations
(20 and 30ppm) respectively are detailed in table C7.
Tabk C7: Time and dose dependent expression of Hsp6O (in pg lisp60 equivalent/ mg protein) in Spirulina platensis under various stress conditions.
Average of three values in each case 2 SD. Pc0.0025 T- Temperature. S- Salinity, P-Paraquat E- Endosulfan, M-Malathion, D- Dimethoate
An enhanced expression of a stress protein equivalent of
human Hsp6O was clearly seen undr.r sub lethal stress in Spirulina
platensis. The expression was seen to have a clear dose dependent
and time dependent relationship. Under a lower temperature stress at
30°C, the expression was seen to be enhanced as early as 3 hours
with significant increase by 6 hours and at the higher temperature
stress of 33'C, significant increase was seen as early as 3 hours.
Under salinity stress of igm% and 1.5 gm% and sublethal Paraquat
concentration of O.lOppm and 0.25 ppm the expression scheme was
seen to be similar to that of temperature stress. However under stress
induced by lower concentration of E~idosulfan (10ppm) a significant
increase was noted only at 12 hoi~rs even though a perceivable
increase was noted as early as 6 hours. But under higher
concentration of 15 ppm of endosulfran an increase in expression of
Hsp6O was seen as early as 3 hours and a significant increase was
seen at 6 hours. Under stress from organophosphate pesticides
Malathion and Dimethoate, at lower concentrations of 10 and 20 ppm
respectively, an increase in expression was seen from 3 hours
onwards with the expression levels leaching significant levels at 12
and 6 hours respectively. At higher concentrations of 20 ppm
Malathion and 40 ppm Dimethoate s~gnificant increase was noted at
6 hours itself.
3.3.3 Stressor Induced Potential for Oxidative Stress
The potential of the various stressors to induce oxidative
damage and therefore to cause oxidative stress in Spirulina platensis
was studied. The oxidative stress iriduced damage was studied by
measuring the in vivo protein oxidation through increased protein
carbonyl formation and the lipid peroxidation by products that were
Th~obarb~tur~c reactive substances (TBARS). There are a number of
antioxidant systems that are useful in the controlling the oxidant
induced damage. These are antioxidants like reduced glutathione
(GSH). and enzymes like superoxide dismutase (SOD), Catalase,
Peroxidase and Glutathione- S- Transferase (GST). The results of the
various stressor induced effects on the above mentioned systems are
detalled below.
3.3.3.1 Temperature Stress
The result of the sublethal temperature stress (33' C) induced
Oxidative damage on Spirulina platensis is detailed in Table C8a.
Table C8a: Effect of Temperature induced oxidative stress
:arbonvls * I TRARS 1
i 6hours 111 ,"::;; I 0.186'
~ - - + 0.006
9 hour 1 0.575'
+ 0.010 1 2 0.013
J -
12 hour / 0.588' 0.240' + n ni7
~ .. -.
...-~ - Protein ( < I . --. .-
"CU.UU25 A - nM/rng protein
Controi I 0.521 0.133
- I
From the above table it can be seen that there was a steady
increase from control, in protein carbonyl formation and accumulation
of TBARS with respect to various timtt periods. There was a significant
increase in protein oxidation and TBARS accumulation from 3 hours
onwards.
The response of the various antioxidant systems in Spirulina
platensis when subjected to temperatlire stress is detailed in table C8b.
Table C8b: Antioxidant systems under Temperature induced stress
C - Ulgm protein. D - pM of H202 wnsumi dlminlmg prote~n, E - mgl100gm wet Weight, F - Ulmg protein. G - nM of CDNR complexed lmin Img protein
GSH PeroxidaseF
2.566
+ 0.595 - 3.208
+ 0.481 - 4.338'
+ 0.733 - 4.721'
+ 0.557 - 5.739*
+ 0.734 - 6.572'
+ 0.495 -
From the table C8b, it call be seen that there was a steady
increase in SOD with significant increase from control from 3 hours
onwards, with a slight decline at 12 hours. Catalase and Peroxidase
also had a similar increase with significant increase from 3 hours
onwards, but there was no declins as seen in the SOD. GST showed
GST
8.499
- + 0.466 9.168
+ 0.531 - 9.836
- + 0.685
11.025'
- + 0.960
13.219'
- + 1.167 12.963*
- + 1.290 Average of six values in each case + SD.
P<0.0025
an increase during the study period with a significant increase from 6
hours onwards and then a small decline at 12 hours when compared
tc; the value at 9 hours. The antioxidant GSH showed a steady
decline, with significant decrease from 3 hours onwards.
3.3.3.2 Salinity Stress
Salinity stress in Spirulina platensis was induced by 1.5gm% of
NaCl The result of this on the protein oxidation and lipid peroxidation
is shown in table C9a.
Table C9a: Effect of Salinity induced oxidative stress
Like under temperature stress, salinity stress also caused an
increased protein carbonyl formation with a significant increase as
early as 3 hours. Lipid peroxidation also showed an increase as
~~~ ~ ..
Control , ~ .... ~ .
i hour ~
3 hours I .
i Ghours ~~
i 9 hour i I
12 hour I L~ .~ .~ ~-~ . ~~ ~
Average of six values in each case 2 SD. ' ?<0.0025 A - nM1rng proteln
Protein carbonyls A
0.521
+ 0.011 - 0.543
+ 0.010 - 0.562*
+ 0.012 - 0.580*
+ 0.010 - 0.587-
+ 0.012 0.592'
+ 0.01 1 -
TEARS
0.133
- + 0.005
0.136
+ 0.006 0.142
- + 0.003
0.152'
- + 0.005
0.157'
- + 0.005
0.166'
- + 0.008
76
evidenced from the increased product on of TBARS, but unlike protein
oxidation there was a significant increase only from 6 hours onwards.
The Antioxidant systems have also shown quite interesting
results, which are shown in table C9b
Table C9b: Antioxidant systems under Salinity induced stress
* Pc0.0025 C - Ulgm protein, D - (IM of Hz02 consurned/min/mg protein. E - mg1100gm wet. Weight. F - Ulmg protein, G - nM of CDNB complexed lrnin Img protein
The antioxidant enzymes like SOD, catalase and peroxidase
all showed an increased induction There seems to be an significant
increase in all three enzymes as ~,arly as 3 hours. The GST enzyme
also showed an increase, but there seem to be significant increase
only at 6 hours onwards. GSH showed a steady decline but the
decline increased signif~antly only at 9 hours onwards.
GSTG
8.499
+ 0.466 - 9.342
+ 0.853 - 10.771
- + 1.213
11.995'
- + 1.228
13.254'
+ 1.080 - 13.666'
+ 1.545 -
GSH 18.193
+ 2.132 - 17.013
+ 2.133 - 15.844
+ 1.590
14.383
+ 1.258
11.715'
+ 1.253 - 10.819'
+ 1.471 - Average of six values in each case 2 SD.
peroxidaseF 2.566
- + 0.595
2.630
- + 0.542
4.41 4' - + 0.633
5.661'
- + 0.347
6.603-
- + 0.525
5.729*
- + 0.521
3.3.3.3 Paraquat Induced Stress
The results of 0.25ppm Paraquat induced oxidative damage in
Sp~ruhna platensis is shown in table C l Oa.
Table ClOa: Effect of Paraquat induced oxidative stress
. ~
Control .
1 hour
3 hours
6hours . ~ ~-
9 nuur -
A PCO M)25 -
nMImg protein
Protein carbonyk * 0.521
+ 0.01 1 - 0.549
+ 0.01 1 -
12 hour ~ -~
H - mM1 mg protein
TEARS ' 0.133
- + 0.005
0.160'
- + 0.012
0.576'
+ 0.009 - 0.593'
+ 0.012 - 0.600'
+ 0.010 -
Paraquat, a potent inducer of oxidative stress in various
organisms has shown enhanced protein oxidation and lipid
peroxidat~on A high level of protein carbonyl formation is seen from 1
hour onwards with significant protein carbonyl formation from 3 hours
onwards Significantly increased accumulation of TBARS is seen from
1 hour rtself and continues through out the study period under
paraquat induced stress.
0.180'
- + 0.014
0.249'
- + 0.008
0.255-
- + 0.010
Average of sixvalues in each case + SD.
0.613'
+ 0.012 - 0.279-
- + 0.013
The details of the antioxidant systems under paraquat stress is
shown in table ClOb.
The above data indicates that SOD, catalase and peroxidase
enzyme systems, all showed signi.'icant increase in activity from the
first hour itself. While continued in,:reased expression were noted in
the SOD and catalase enzyme systems through out the 12 hour
period, peroxidase was slightly reduced at 12 hours, even though the
level was significantly higher than that of control. GST showed
significant increased levels from 3 hours and the increase continued
through out the remaining time periods. The antioxidant, GSH showed
significant decline in levels from that of control from 3 hours and this
decline in levels continued upto 12 hours, where a slightly increased
level from that at 9 hours was detected.
Table ClOb: Antioxidant systems under Paraquat induced stress
Average of six values in each case + SD. * PCO.0025 C - Ulgm protein, D - pM of H202 con~umeclmidmg protein. E - mg1100gm wet. Weight. F - Ulmg protein, G - nM of CDNB complexed Imin Img protein
Peroxidase 2.566
- + 0.595
4.336*
- + 0.605
4.695*
- + 0.794
6.397'
- + 0.394
7.71 3*
- + 0.445
7.21 1'
- + 0.657
Control
I hour
3 hours
6hours
9 hour
12 hour
GST
8.499
- + 0.466
10.751
- + 1.228
12.656'
- + 1.292
13.754'
+ 1.108 - 14.141'
- + 1.283
14.901'
+ 1.038 -
Catalase 268.546
59.302
288.108'
+ 12.767
319.392'
+ 12.307
341.679'
+ 1 1.030
360.443'
+ 9.894
386,278'
+ 1 1 .873
SOD
1.808
+ 0.451 - 2.804'
+ 0.437 - 3.663'
+ 0.605 - 4.365*
+ 0.605 - 5.1 15*
+ 0.423 - 5.319'
+ 0.591 -
GSH
18.1 93
+ 2.132
14.438
+ 1.309
12.595'
+ 1.341
10.837' + 1.286
8.380'
+ 1.954
8.588'
+ 2.107
3.3.3.4 Endosulfan Induced Stress
The effect of 15ppm of organochloride endosulfan induced
oxidative stress is detailed in table C l la.
Table C l I a: Effect of Endosulfan induced oxidative stress
Protein carbonyls TEARS ' 0.0521 0.133
Control C
+ 0.01 1 + 0.005
q hour + 0.012
0.580' 12 hour --I-- + 0.010
I hour !
~
3 hours ~
L--- - I - Average of SIX values in each case 2 SD ' PcO 0025 A nM/my protein €3 - mMi my proteln
+ 0.007 - 0.549
+ 0.01 1 -
By far the lowest levels of effects of oxidative stress were
induced by endosulfan. The protein carbonyl content was increased
sltghtly at 1 hour and 3 hours but thereafter, significant increased
levels were detected at 6 hours to 12 hours. Similar trend was noticed
In the accurn~~lation of TBARS.
- + 0.006
0.140
- + 0.005
The activity of the antioxidant systems was also studied and
the results are shown in table C l 1 b.
Table Cl lb: Antioxidant systems under Endosulfan induced stress
;SH Peroxidase ' GST
Average of six values in each case + SD. P<0.0025
C - Ulgrn protein. D - pM of Hz02 wnsumedlrainlmg protein. E - rngl100gm Wet. Weight. F - Ulmg protein. G - nM of CDNB cmplexed Irnin Img protein
The SOD enzyme system sh~wed an increase throughout the
study period, with significant increase noted as early as 3 hours. The
catalase system also showed an increase but it reached significant
levels only at 6 hours and the increase continued throughout the study
period. Although there was increase in both peroxidase and GST
levels, they reached significant levels only at 12 hours. The
antioxidant GSH showed a stead!! decline through out the study
period but unlike other stressors, GSH levels during endosulfan stress
did not decrease significantly during the study period.
3.3.3.5 Malathion Induced Stress
The effects of 20ppm organophosphate malathion induced
ox~dative stress were studied and the results are shown in table C12a.
Table C12a: Effect of Malathion induced oxidative stress
I Protein carbonyls A I TEARS '
3 hours + 0.01 1
+ 0.005
' ~<0.<025 A - nM/mg protein B - mM/ mg protein
The protein oxidation and lipid peroxidation levels showed an
- + 0.006
O.20Oe - + 0.008
Y hour + 0.013 -
Increase during the study period, with the increased accumulation of
Average of six values in each case 2 SD.
12 hour ,~ ...
both the protein carbonyls and TBARS respectively. The increase of
0.588' + 0.015 -
both these by products reached significant levels from that of control
at 6 hours and continued thereafter up to the end of the study period.
The role of the antioxidant sy!items was also studied under the
same stress conditions and the res~~lts of the same are as given in
table C12b.
Table C12b: Antioxidant systems under Malathion induced stress
I SOD I Catalase I GSH 1 PeroxidaseF 1 GST I -
Control
. -.---- C - Ulgm Protein. D - pM of Hz02 wnsumedl~ninlrng protein, E - mg1100gm wet. Weight. F - Ulmg prdein. G - nM of CDNB complexed Imin /mg protein
I hour
3 hours
6hours
9 hour
12 hour
The SOD and peroxidase t!nzyme system showed a rapid
increase with significant levels whei compared to the control being
reached as early as 1 hour. This increase in both these systems
continued up to the 9 hour of the study period, but there after at
12 hours there was a slight declin,?. Through out the study period
catalase enzyme system showed a decrease in activity when
compared to control with a significalt decrease being noted as early
as 3 hours, which continued therea'ter through out the study period.
GST levels increased with the increase reaching significant levels
when compared to control at 6 hours and a slow increase was also
1.808
+ 0.451 -
Average of six values in each case + SD. Pcn nn76
2.668'
- + 0.574
3.306' + 0.475 - 3.853'
+ 0.500 - 5.006'
+ 0.542 - 4.718'
+ 0.574 -
268546
+ 9.302 - 252.554
+ 11.651
230.489'
+ 11.048
212.723'
+ 10.866
195.148*
2 10.880
192.997'
+ 8.302
18 193
:: 2.132
17 469
:: 1.445
14.513
:: 1.396
13.251
:: 1.463
' 0.817'
:: 1.821
' 0.212'
:: 1.657
2 566
+ 0 595
8 499
- + 0.466
3.491'
- + 0.574
4.323'
- + 0.471
5.189'
- + 0.541
6.135'
- + 0.591
5.996'
- + 0.687
9.218
- + 0.988
10.694
- + 1.457
13.272'
- + 1.009
13.802'
+ 1.154
13.442'
- + 1.292
noted thereafter through out the study period. GSH levels showed
cont~nued decline, with it reaching significant levels at 9 hours and a
cont~nued decllne was noted till the end of the study period
3.3.3.6 Dimethoate Induced Stress
The effects of 40 ppm of organophosphate pesticide Dimethoate
Induced ox~dative damage in Spirulina platensis was studied and the
results are as given in table C13a.
Table C13a: Effect of Dimethoate induced oxidative stress
TEARS ' 0.133
l hour i
Like in all other stress conditions, the protein carbonyl content
and TBARS accumulation also increased during the study period. The
3 hours ~ I ~
6hour:j b~ -
9 hour . ~
12 hour L.~
increase in both these byproducts reached significant levels at 6 hours
when compared to their respective controls and the increase was
noted upto the end of the study period.
0.537
- + o.O1 I 0.134
+ 0.010 -
Average of SIX values in each case 2 SD. ' P<0.0025 A - nMlmg protein 8 - rnM1 mg protein
0.544
+ 0.01 - 0.570'
+ 0.010 - 0.579'
+ 0.01 1 -
0.587'
+ 0.012 -
0.145
- + 0.005
0.162-
- + 0.005
0.189'
- + 0.009
0.199'
- + 0.008
The levels of the various antioxidant systems was also studied
under similar stress conditions and the results of these are detailed in
table C13b.
l2 hour - + 0.645 2 11.234 ! 1.907 + 0.681 + 0.751
Averaae of six values in each case 2 SD.
Table C13b. Antioxidant systems under Dimethoate induced stress
-~
~<0.0025 C - Ulgm protein, D - pM of Hz02 consumel/min/mg protein. E - mgI100gm wet. Weight, F - Ulmg protein, G - nM of CDNB wmplexed Imin lmg protein
Like the response under malathion, dimethoate induced stress
also induced a rapid increase in both the SOD and peroxidase
enzyme systems, with both reaching significant levels as early as
3 hours and this increase continued upto the end of the study period.
GST I SOD I Catalase ( GSH
However there was a small reduction in peroxidase levels at 12 hours
when compared to the 9 hour leve s. The catalase enzyme levels also
decreased during the study period with significant decrease from
control being noted from 6 hour:; onwards. The GST levels where
enhanced when compared to the control, which significantly increased
pemxidaseF
at 6 hobrs and then increased to level off at 9-12 hours. The GSH
levels slowly decreased when compared to control, with significant
reduction seen only at 12 hours.
3.3.4 Antioxidant lsoenzymes Analysis
The antlox~dant enzymes superoxlde dlsrnutase and catalase
*ere analyzed for therr lsoenzyme expression after 6 hours under
s$eal )m~t~ons and the electrophenograph of the same are seen in
Pt.te CB and Plate C4 respectively
From the electrophenographs it is clear that there are two
for superoxide dismutase in Spimlina platensis. The higher
r w d g M isozyme (marked 1) was expressed even under
‘X.ntUi mmdkns. . M e . . . as the lower molecular weight (marked 2)
ardy- a e s s conditions or in the controls it was
at uery~firvels: that it could not detected.
The cat~tpgs-enograph , . shows that only one isozyme
\n% mp3Smd BF -platensis and that it showed variation in
a with respW to o w i v e alterations caused by stress. ~. . ,
Plate C3: Superoxide dismutase isozyme expression after 6 hours of sublethal stress. C-Control, T-Temperature, S-Salinity, E-Endosulfan, P-Paraquat, D- Dimethoate, M- Malathion
Plate L,. 3atalase enzyme expression after 6 hours of sublethal stress. C-Control, T-Temperature, S-Salinity, E-Endosulfan, P-Paraquat, D- Dimethoate, M- Malathion
3.4 Discussion
The presence of chemical contaminants has become a
pervasive threat to many natural aquatic ecosystems. These
contaminants can have toxic effects on many different types of
organisms and affect biological processes at the cellular, population,
community and ecosystem levels of organizations. These chemical
contaminants are often called environmental contaminants, which
arise, from a variety of sources that usually classified as point source
or non-point source of pollutants. The effects of toxic chemicals have
been measured on a wide variety of algal species by using different
cultural methods and number of different biological responses. Algae
commonly tested ranges from common, to rare and toxic fresh water
and marine species of various divisions. Cultural methods vary in
composition of solutes in the nutrient medium, in nutrient
concentration; in temperature and in intensity, periodicity and quality
of light Various measurements of algal response to chemical
contaminants ~nclude but is not limited to, I) Photosynthetic uptake of
radiolabelled carbon dioxide, an indication of the functioning of
Photosystem I associated with chlorophyll-a: 2) evolution of oxygen, a
measure of the rate of the Hill reaction in Photosystem II associated
with Chlorophyll-b; 3) measures of relative population growth in time
based on changes in cell numbers, amounts of chlorophyll-a extracted in
vitro, turbidity of a cell suspension and changes in the dry weight of the
culture; and 4) measures of critical physiological and biochemical rates,
such as synthesis of lipids, proteins and nucleic acids, as well as the
uptake of organic and in organic nutrients from nutrients medial0'.
3.4.1 Growth Inhibition Studies
We followed growth response of Spirulina platensis using the
chlorophyll content as an index of growth. To decide on the toxicity
levels of various stressors 96 hour growth inhibition studies was
followed. Under temperature stress, it was found that Spirulina
platensis could grow uninhibited at even 30' C. Growth was slightly
reduced at 33%. Fatma et a/,102 studied the growth response of
Spirulina platensis in various culture media under varied conditions
and found that at 30' C growth was maximum in CFTRl medium when
compared to other culture media. This was in consonance with our
findings. At higher temperatures we found growth was inhibited slightly
at 33' C, significantly reduced at 35% and completely inhibited at
higher temperatures. In large scale Spirulina cultivation maintenance
of optimum temperature of the cultlire is essential for maximum output
of bi~mass"'~. High temperatures enhance the evaporation rates of
water from open culture systems or reactors leading to the substantial
increase in the salinity or osmoticiim of the culture mediumiD4. This in
turn poses a great threat to the via~ility of Spirulina cultures. .
Salinity intrusions into freshwater water system from the sea in
coastal areas and estuarine areas are quite natural. In these areas,
salinity is one of the important environmental factors that can affect
growth of cyanobacteria. In general cyanobacteria exhibit considerable
tolerance to salt and osmotic stressto5. This could be seen with respect to
the high dose of 2.0gm% of NaCI necessary causing significant
reduction1 inhibition of growth. Fron 0.5gm% - 1.5gm% there was only
slight decrease in growth of the c~lture. Although the molecular basis
of the mechanism involved in cyzmobacterial salt tolerance is not fully
understood, some concepts have emerged in recent years. Prominent
among these are (1) curtailment of Na' influx and prevention of
lntracellular Na' accumulation1068107, (2) accumulation of internal
osmoticurn in the form of inorganic ions, such as K + 1088109 , or organic
solutes such as glycopyronosylglycero1"0, sucrose"', trehalosellz or
glycine betalne113 and (3) metabolic adjustments to tune the cellular
activities to function at higher internal osmoticuml". Obviously, atleast
some or all of the above mechanism may have played a role in the
high salinity resistance of Spirulina platensis as seen from our results.
Paraquat is a non- selective contact broad spectrum herbicide,
recogntsed for use in aquatic and terrestrial weed control. The effects
of paraquat on growth of Spirulina platensis have not been studied.
From our data it was concluded 0.5 ppm was sufficient to induce
significant growth inhibition and that even at lower concentration of
0.25pprn growth was reduced. Similar results have been seen in
studles with different algae. The chlorophyll a and carotenoids
contents in Scenedesmus quadricauda and S. dimorpha was found
to progressively decrease with increasing Paraquat concentration and
thereby effect on their growth and metabolic activityTf5. It was seen
that there was a wide range in the sensitivity of algae to paraquat.
0.lpprn was reported to be sufficient to cause growth inhibition in
Navicula osteraria while 100ppm was needed to stop growth in
Phaeodactylum tricornutum 'I6. It was earlier found that 53% decrease
In carbon fixation occurred in estuarine phyotoplankton after 4 hours
of exposure to lppm of paraquat"7. Similar inhibitory results were
observed In the growth parameters such as growth rates and
generat~on times of algae Scenedesmus quadricauda, S. acutus,
Selenastrum caprcornutum and Chlorella vulgaris with increasing
concentrat~ons of paraquat118. Marked inhibition was also observed in
the nitrogen fixing ability of certain cyanobacteria. All these inhibition
may be due to damage caused by paraquat on key enzyme systems
or due to the reduction of paraquat lo toxic radicals. which interfere
with photosynthesi~"~.
The organochlorine pesticides are used in greatest tonnage in
rice fields in India. Due to their efficiency as insecticides, these
compounds were considered a boon to agriculture. Several
researchers have studied the toxicity of organochlorine pesticides on
cyanobacteria. Unfortunately, none oi these studies involved Spirulina
platensis. We used endosulfan as a representative of this group of
compounds to study its effects on Spirulina platensis. It was found that
30 ppm of endosulfan caused significant reduction in the growth of
Spirulina platensis. Tandon et found in their studies on
Anabaena and Aulosira that lpgfml of endosulfan caused reduced
growth, with 50% inhibition at 20 fijfml. Complete bleaching in the
cultures occurred at 50flglml. This inhibition was attributed to its
effects on photosynthetic apparatus. Prompt inhibition of
photosynthesis by endosulfan was earlier reported in Chlorella
protothecoides by Subbaraj and ~ose'", while Singh &
VaishampayamlP, attributed this to the prevention of choloroplast
electron flow through Photosystem-ll. Such a mechanism might have
come into play in the inhibition of growth in Spirulna platensis by
endosulfan noted by us.
The organophosphate group of insecticides have also been
used extensively for testing their influence on cyanobacteria. Again
none of these studies involved Spirjlina platensis. From our studies
on Spirulina platensis using malathion, we found that 40 ppm of
malathion caused complete inhibition of growth of Spirulina platensis.
At a lower concentration of lOppm there was enhanced growth.
Malath~on was found to have differential toxicity to Anabaena sp. and
Aulosira fertislissima; the former survived upto 300ppm, where as the
later succumbed at 10ppmB3. No conclusive explanation was given on
th~s disparity of toxicity. Subramanian et a/'23, found that Aulosira
fertilrssima and Nostoc muscurom could utilise low concentration of
malathton for growth. They concluded that the pesticide could be used
as source of phosphorus, resulting in enhanced growth at low
concentration. This could be the reason for the enhanced growth
observed in our study in Spirulina platensis at a low concentration of
Malathion
D~methoate is another organophosphate pesticide used in the
study. We noted that upto 30 ppm there was no significant inhibition in
growth but at 40 ppm there was pronounced growth reduction. As in
the case of malathion there was enhanced growth at lower
concentration of 10ppm. Wong et a/.lZ4, found that high concentration
of dimethoate and malathion was required to effect an inhibition of
growth in cyanobacteria. The photosynthetic activity was also inhibited
only at high concentrations and that the inhibition was due to the
~nfluence, synthesis of Chlorophyll a had on photosynthesis. The
difference in toxicity of malathion and dimethoate or among the dierent
organophosphate pesticides may be due to the differential ability of
cyanobacteria to phosphorylate different pesticides and whether they are
diethyl phosphorothioate or dimethyl phosphorothioate, with the diethyl
ones being more toxic than the dimethyls.
3.4.2 Stmss Protein Induction Studies
In the present age of industria ization and agriculture, like other
organisms, cyanobacterial cells are also constantly facing different
kind of stresses in their external ard internal environment and are
struggling to survive through morpho,ogical and metabolic alterations.
Stress point is that metabolic stale where regulation of cellular
pathways towards positive direction for organisms fitness is at its
limits. Such a state is reached when the extent of pollutant inputs
exceeds organism's degradation anti transformation capacities. Like
other organisms cyanobacterial genome also responds in a
programmed manner to cope up with various stresses. This may
include alterations in protein synthesis by selective increase or
decrease in the expression of specific genes resulting in increase or
decrease of already existing proteins or synthesis of new sets of
proteins and enzymes'z5.
Stress in Spirulina platensis was induced by using sublethal
temperature, salinity or concentration of paraquat, endosulfan,
malathion and dimethoate as the various stressors. The preliminary
aim of the study was to investigate whether heat shock protein (Hsp)
60 could be used as biomarker for toxicity studies in various
organisms including Spirulina platensis. For this SDS PAGE and
western blot was done to detect the presence of Hsp6O and ELlSA
used to quantify Hsp6O on a time and concentration scale. From the
results obtained it was seen that ;I human Hsp6O equivalent was
expressed at 53.8 KDa. More over. :he control group also expressed
this protein, thereby indicating that this protein was constitutive
expressed in Spirulina platensis ancl that all the stressors enhanced
the expression of this protein. Thus clearly it can be seen that this
Hsp6O equivalent could be used as a general stress indicator.
Constitutive expression of a 64KDa (Hsp6O) protein has been shown
in cyanobacteriurn Synechocystis PCC 6803 and has been suggested
to have a chaperonin functionlB. Chlamydomonas reinhardtii has also
been shown to possess a Hsp6O protein and has been suggested to
have a stabilizing effect on the photosynthesis system under thermal
stressqz7
Moreover, the antibody used in the experiments was earlier
found to be immunogenic against Hsp6O from E.coli, rats, mouse and
humans""his clearly indicates that this 53.8 KDa protein from
Sp~rulina platensis was evolutionarily conserved and structurally
related to the Hsp6O in the above organisms. However, the degree of
conservation can be known only after a detailed sequence analysis,
as there are no previous studies involving HspGO expression in
Sp~rulina platerisis to give such information. Another facet is that the
response time for the induction of Hsp6O in response to a diverse
group of stressors happen to be vety short (ie., within hours of facing
stressful conditions), thus helping us in identifying it as a early marker
ot cellular stress in Spirulina platensis.
The hallmark of any biomarker is that, it should be intracellular
and the blornarker should have a dosetconcentration and time
response From the results of the ELSA studies it can be clearly seen
that, this ~ndeed was the case of Hsp6O in Spirulina platensis. Hence
we conclude that Hsp6O can be used as a biomarker of stress in
Sprrulrna platensrs
3.4.3 Influence of Oxidative Stress, in Stress Protein Induction in Spirulina platensis
The second aspect of our study in Spirulina platensis involved
studying the possible reason for the induction of Hsp6O under these
diverse stressors. Studies on Hsp incluction using in vivo and in vitro
approaches have indicated that accumulation of abnormal or
denatured proteins was involved in the enhanced expression of Hsp's
in various organism^'^^^'^^'^' . Oxidative stress happens to be one
such mechanism that can induce intracellular denaturation of
proteins13'.
Even under optimal conditio~ls, many metabolic processes,
including the chloroplastic, mitochotidrial, and plasma membrane-
linked electron transport systems of cyanobacteria, produce reactive
oxygen species (ROS). Furthermort?, the imposition of biotic and
abiotic stress conditions can give rise to excess concentrations of
ROS, resulting in oxidative damage at the cellular level. Therefore,
antioxidants and antioxidant enzymes function to interrupt the
cascades of uncontrolled oxidation in each organelle'33.
The effects of intracellular oxidative stress on proteins and
lipids can be gauged by the increased protein oxidation and lipid
peroxidation byproducts. Protein carbonyl formation and TBARS is an
index of protein oxidation and lipid pe~.oxidation respe~tively'~~.
Under high sublethal tempemture stress induced at 33OC, it
was seen that both protein carbonyl: and TBARS were increased as
time progressed. Significant differences were noted from controls at
time periods as early as 3 hours. This was despite, increased
expression in antioxidant enzymes ike, SOD, catalase, peroxidase
and GS7 Furthermore, there was a corresponding depletion of
ant~ox~dant, GSH. All this is indicative of Spirulina platensis cells being
ilnder ox~dative stress.
Stm~larly under high salinity stress induced by 1.5gm% of
NaCI, it was seen that both protein carbonyls and TBARS content was
Increased with significant differences in protein carbonyl content being
noted at 3 hours onwards and TBARS accumulation being noted from
6 hours onwards. The antioxidant enzymes studied also showed an
Increase, wrth a corresponding depletion of cellular GSH content. Thus it
could be conduded that salinity stress had also induced oxidative stress in
SphIina platensis. Results obtained by Singh et in Anabaena sp.
with graded concentrations of NaCl (20-200 mM) showed a decrease in
the chlorophyll 'a' contents of Anabaena with increasing concentration
of NaCl except at extremely low concentration of NaCl (5-20 mM). The
rates of H~li activity and oxygen evolution were stimulated by lower
concentrations of NaCI, but not at higher concentrations of NaCI. Their
results had demonstrated that the O2 evolution process was relatively
more sensitive to NaCl stress than the Hill activity. Further, their
results showed that NaCl induced an increase in the rate of bleaching
and loss of total thiol (-SH) contents. Taken together, these results
suggest a NaCI-induced general oxidative stress. Results on the effect of
oxygen radical quenchers reveal a predominant role of singlet oxygen in
the NaCI-induced general oxidative stress. However, unlike our results
on antioxidant enzymes and lipid peroxidation, the rate of lipid
peroxidation and SOD activity in Anabaena as studied by them showed
a declining pattern in response to increasing concentrations of NaCI.
They suggest the possibility of a NaCI-induced decrease in the rate of
lipid peroxidation when the SOD activity is low. But the NaCI-induced
decline in the SOD ad i i t y sugges:s, that symptoms of general
oxidative stress at elevated levels of NaCl were apparently owing to
collapse of intracellular defense of the cells against the toxic oxygen
radicals, induced by decoupling of the ~hotosynthetic system.
0.5ppm of Paraquat, a known oxidative stress generator in
organisms showed the fastest increase in protein carbonyls and
TEARS in our study with significant increase as early as 1 hour.
Similar increase in all the antioxidard enzymes and corresponding
significant decrease in GSH content suggest that paraquat had
induced oxidative stress in Spirulir a platensis. Paraquat (1 0-30
microM) exerted a dose-dependent ;and light-dependent toxicity on
Chlorella sorokiniana. Paraquat wa:i also seen to increase the
superoxide dismutase content of these cells136. Paraquat was found to
increase the photoproduction of Or in C. sorokiniana and the increase
in the cell content of superoxide dismutase was found to be an
adaptive response which provided protection against this herbicide.
Paraquat was also found to cause a tiine-, dose-, and light-dependent
bleaching of the halophilic green alga Dunaliella ~alina'~'. Sublethal
levels of paraquat elicited increases ir cell content of both superoxide
dismutase and catalase. We also observed similar results.
15 ppm of Endosulfan, an organochlorine pesticide induced
significant increase in protein oxidation and lipid peroxidation by 6
hours. A study of the antioxidant systt?m also reveals a slow increase
in all antioxidant enzyme systems sludied and a slow decrease in
GSH. This is an indication that oxidative stress plays a role in the
toxicity of endosulfan in Spirulina platc!nsis. Organochlorine pesticides
have been known cause bleaching of chlorophyll and to prevent
chloroplast electron flow through Photosystem-ll thereby inhibiting
photosynthesis"8. The most direct route for photoproduction of a
variety of active species, results in the production of singlet oxygen
('0,) through the interaction of O2 with excited triplet state chlorophyll
In the light ha~esting complex of photosystem I and II (PS I And PS 11).
Protection against the formation of singlet oxygen usually involves the
quenching of the excited state of chlorophyll by carotenoids before it
becomes triplet chlorophyll 138 8139 . The second O2 activation pathway
results in the production of the superoxide anion radical (0;) by the
~nteractron of 02, via a Mehler type reaction, with electron acceptors of
PSI'^^ Alterations in chlorophyll structure (resulting in bleaching,
which 1s a delayed end result) or alteration in the photosynthetic
system can cause these perfectly fine tuned systems to collapse,
thereby leading to increased generation of oxy radicals. This is in addition
to oxy radicals formed via, the respiratory and biotransformation systems.
A11 these leading to oxidative stress in the organism.
Ihe gganophosphate pesticides malathion and dimethoate
were also seen to cause increased lipid peroxidation and protein
ox~dat~on, with an increase in the antioxidant enzymes SOD,
peroxidase. GST and corresponding with a depletion of intracellular
GSH. Unlike in other stressors, under the influence of these two
pestlcldes a decrease in catalase activity was noted. This decrease
was corroborated by the isozyme analysis study as seen in plate C4.
Thus it can be concluded that either the pesticide directly or the highly
~ncreased peroxide production by SOD affected the structure of the
catalase enzyme causing destruction of the active site thereby
~nh~biting catalase.
The electrophenogram of SOD revealed the presence of two
achrornatlc bands signifying, the presence of two electromorphs, one
that was constitutively present and the second that was induced or
had increased expression under stress conditions. Superoxide
dismutases are metalloproteins 2nd are ubiquitous in aerobic
organisms and catalyses dismutation of 0 2 - to H202 and 02. There are
3 types of SOD enzymes with molecular ranges of 17 -84 KDa, which
can be distinguished by the prosthetic metal present at the active site
and are thus accordingly named a!: Fe SOD, Mn SOD and CuIZn
SOD'418142. Fe SOD occurs in higher plants, cyanobacteria and other
prokaryotes. The Mn SOD though widely distributed in prokaryotes
and eukaryotes, is observed in only some cyanobacteria. In bacteria
FeSOD andlor Mn SOD occur in the cytoplasm and have very similar
protein ~tructure'~~. Some cyanobacteria have cytoplasmic Fe SOD
and thalakoid membrane associated Mn SOD'".
Studies conducted under paraquat stress in ChloreMa
sorokiniana by Rabinowich et a1.'32s110w the presence of one catalase
electromorph (consistent with our finding) and the appearance of a Mn
SOD as a new electrophoretically distinct isozyme. Cells grown in the
absence of paraquat contained one manganese-superoxide
dismutase and two iron-superoxide dismutases, while the paraquat-
grown cells contained an additional manganese-superoxide
dismutase. Similar may be the case in our study but a more detailed
study on the prosthetic metal group is needed to discern the type of
SOD newly expressed. Moreover this may be used as a indicator of
oxidative stress in Spirulina platensis.
3.5 Summary
lnd~scrlminate use of pesticides on land for increased crop
production and food storage has resulted in an increased
accumulat~on of pesticides in our water bodies. Cyanobacteria, are the
pnotosynthesizing blue green algae, which has great potential as
o~ofertilizer 'These organisms are under stress1 threat due to the
~ntolerable levels of pesticides and herbicides in our environment. The
stressors like temperature, salinity and pesticides caused oxidative
stress in Spirc~lina platensis and thereby enhanced protein oxidation
and lipid peroxidation. There was increased production of antioxidant
enzymes like SOD, catalase, peroxidase and GST with simultaneous
~lepletion of ir~tracellular antioxidants. The SOD isoenzyme analysis
revealed the presence of one isoform that was expressed only under
stress conditions. This may be a special mechanism in cyanobacteria
to overcome stress. Photosynthetic activity was also inhibited.
Increased lipid peroxidation caused membrane destabilization.
Protein denaturation is the end result of increased protein oxidation.
The structurally stable and active proteins could become destabilized
or denatured. The chaperonins are assigned with the function of
renaturatlon of the denatured proteins. Hsp6O is thought to have a
chaperonln function. It is possible that due to increased oxidative
stress and thereby protein denaturation, increased Hsp6O expression
mas necess~tated to stabilize them and also to stabilize destabilized
membrane structures. New protein synthesis is also known to take
place in stressed cells and Hsp6O is known to aid in these proteins
attalnlng thelr preferred tertiary or quaternary structures as part of its
chaperonln function. Thus the time and dose related synthesis of
53.8 KDa equivalent of human HspGO seen in Spirulina platensis could
be used as a biomarker for oxidative stress.
3.6 References
Fogg GE. Stewart WDP, Fay P. Walsby AE (eds.) 1973: The blue green algae. Academic press. London and New York. pp 10-16.
Stanier RY and Cohen Bazire G. 1977: Ann. Rev. Microbiol. 31: 225-274.
Tandeau DeMarsac N.1991: Chronatic adaptation by cyanobacteria. In Bogard L & Vasil IK (4s.) Cell :ulture and somatic cell genetics in plants. Vol7B. Academic press. NeN York. pp 417-446.
Buikema WJ and Haselkom R. 1993: Ann. Rev. Physiol. 44: 33-52.
Tandeau DeMarsac N and Houmiird J. 1993: FEMS Microbiol. Rev. 104: 119-190.
Hagemann M 8 Erdmann N 1997 Environmental stresses. In Ashwini K R (ed.) Cyanobacterial nitrogen metabolism and biotechnology. Narosa pub. New Delhi. pp155-221
Borbely G. Surayani G, Korcz P , and Palfi Z. 1985: J. Bacteriol. 161:1125-1130.
Suranyi G. Korcz A. Palfi Z and l3orbely G. 1987: J. Bacteriol. 169: 632-639.
Bhagwat AA and Apte SK. 1989: J. Bacteriol. 171:5187-5191.
Webb R, Reddy KJ and Sherman LA 1990; J. Bacteriol. 172: 5079- 5082.
Hagemann M, Techel D and Resin.3 L 1991: Arch. Microbiol. 155: 587- 592.
Nicholson P. Varley JPA and Hov~e CJ 1991: FEMS Microbiol. Lett. 78:109-114.
Blondin PA, Kirby RJ and Burmum jR. 1993: Curr. Microbiol. 26:79-84
Borbely G, Suranyi G and Kos P 1990: FEMS Microbiol. Ecol. 74: 141-152.
Parsell DA and Lindquist S. 1993: Ann. Rev. Genet. 27: 437- 496.
Lehel C, Wada H, Kovacs E. Torol: Z, Gombos 2, Hovrath I, Murata N and Vigh L. 1992: Plant. Mol. Biol. 18 : 327-336.
Yura T, Nagai H and Mori H 1993: Ann. Rev. Microbiol. 47: 321-350.
Cookson MJ, Baird PN, Hall LM(: and Coates ARM. 1989: Nucleic Acid Res. 17: 6392.
. , ,' ).,, .,..' we% R, RWKI~KJ a d e r r n a n LA. 1990; J. Bacteriol. 174: 61456192.
Ch~tn~s PK and Nelson N. 1991: J. Biol. Chem. 266: 58-65.
Lehel C. Torok 2, Gombos Z and Vigh L.1993: Plant Physiol. Biochem. 31. 81-88.
Mart~n J Howich AL and Hartl FU. 1992: Science 258: 995-998.
Sato N and Murata N. 1980: Plant Mol. Biol. 24: 819-823
Murata N. 1989: J Bioenerg. Biomem. 21: 61-75,
Gombos 2, Wada H and Murata N 1991: Plant Cell Physiol. 32: 205-21 1
Gombos Wada H, Heidig E and Murata N 1994: Plant Phsiol. 104: 563-567
Kuschner DJ. 1985: The Halobacteriacae. In : Woese CR, Wolfe RS (eds) Bacteria: a treatise on structure and function. Archaebacteria, VoIP Academic Press. Orlando. pp171-184.
Browr~ AD. 1976: Bacteriol. Rev. 40:803-846
Csonka LN and Hanson AD. 1991: Ann. Rev. Microbiol. 45: 596-606.
Reed Rti , Borowitzhka LJ, Mackay MA Chudek JA, Foster R. Warr SRC. Moore DJ and Stewart WDP. 1986: FEMS Microbiol. Rev. 3951-56.
Esp~e GS, Miller AG and Cavin DT. 1988: Plant Physiol. 88: 757-763.
Fernandez VE and Avendano MC. 1993: Pbnt Cell Phpiol. 34: 201-207.
Zhao J and Brand JJ. 1988: Arch. Biochem. Biophys. 264: 657-664
Thomas J and Apte SK ,1984: J Biosci 6: 771-794
Padan E and Vitterbo A. 1988: Cation transport in Cyanobacteria. In Packer L and Glazer AN (eds) Methods in Enzymolgy. Vol 167. Academic press San Deigo, pp 561-571.
Paschinger H 1977: Arch Microbiol. 113: 285-291
Ritch~e RJ. 1992: J. Plant Physiol. 139: 320-330.
Reed RH. Rowell P and Stewart WDP. 1981: J Biochem. 116: 323-330.
Blumwald E. Mehlhorn RJ and Packer L. 1983: Plant Physiol. 73: 377-380.
Reed RH. Warr SRC, Richardson DL, Moore DJ and Stewart WDP. r985 FEMS M~crobiol. Lett. 28: 225-229.
Hagemann M, Fuda S. And Schubert H. 1994: Curr. Microbiol. 28: 201 -20'7.
Gabbay-Azaria R. Schonfeld M. Tel-Or S, Messinger R and Tel-Or E. 1992 Arch. Microbiol 157: 183-190.
Voshank A, GuyR and Guy M. 1!&88: Arch. Microbiol. 150: 417420
Dewar MA and Barber J. 1974: f'lanta 11 7: 163-1 72
Ritchie RJ. 1992: Plant Cell Envi.on. 15: 163-177.
Reed RH, Richardson DL and S:ewart WDP. 1985 Biochem. Biophys. Acta. 814: 347-355.
lncharoensakdi A and Takabe T.1988: Plant Cell Physiol. 29: 1073-1075.
Tel-Or E.1980: Appl. Environ. Mirsobiol. 40:689-593
Erdmann N, Fuda S and Hagemann M. 1992: J. Gen. Microbiol. 138: 363-368.
Rai AK and Abraham G. 1993: Bul. Environ. Contam. Toxicol. 51: 724-731.
Blumwald E and Tel-Or E 1984. F'lant Physiol. 74: 183-185.
Schubett Hand Hagemann M 19!30. FEMS Microbiol. Lett 71:169-172.
Jeanjean R, Matthijis HCP. Onaia B. Havaux M and Joset F. 1993: Plant Cell Physiol. 34: 1073-1079
Rai AK. 1990: FEMS Microbiol. Lett. 69: 177-180
Schubert H, Fulda S and Hageriann M. 1993:L. Plant Physiol. 142: 291-295.
Apte SK and Bhagwat AA. 1989: .I. bacteriol. 171: 909-91 5.
Hagemann M. Wolfel L and Kmger B. 1990: J. Gen. Microbiol. 136:1393-1399.
Hagemann M, Techel D and Re~sing L. 1991: Arch. Microbiol. 155: 587-592.
Hershkovitz N, Oren A. Post A and Cohen Y 1991: FEMS Microbiol. Lett 83: 169-172.
Fernandes TA, lyer V and Apte SK.1993: Appl. Environ. Microbiol. 59399-904.
Molitor V. Kunter 0, Sleyter UB and Peschek GA. 1986: FEBS Lett. 204: 251-256.
Stratton GW. 1983: Bull. Environ. Oontam. Toxicol. 31: 129-143.
Tandon RS, Lal R and Rao WSN. 1988 Environ. Pollut. 52:l-9.
Kapoor K and Arora L . 2000: Ind. J.Env. Sci. 4(1): 89-96
Lal R and Saxena DM. 1980: Residue Rev. 73: 49-86,
Kapoor K and Sharma VK. 1980: 5 . Allg.Mikrobiol. 20: 465469.
Kapoor K and Arora L. 2000: Ind. J Env. Ecoplanning. 3(2):219-226
Kapoor K and Arora L. 1996: Poll. Res. 15(4): 343-351.
Goyal S K. 1986: Interaction between pesticides and cyanobacteria. Proc Natl. Symp., Harayana agricultural University. Hissar. pp93-96.
Gangwane L V. 1979: Pesticides. 13: 33-34.
Kurnar HD. 1963: Ind. J.PI.Physiol. 6: 150-155.
Lazaroff N and Moore RB. 1966: J. Phycol. 2:7-10.
Vosiliva VE and Pinevich W. 1970: Soviet. Plant Physiol. 17(6): 990-994.
Edwln S. 1977: Action of Methyl Parathion on Algal metabolism. MK Un~versity. M Phil Dissertation
Mehta RS and Howxby KW. 1979: Bull. Environ. Contam. Toxicol. 23: 319-326.
Morelarid DE. 1980:Rev. Plant Physiol. 31:397-638.
Papst MH and Boyer MG. 1980: Hydrobiol. 69(3): 245-250.
Mohapatra PK, Sethi PK and Mohanty RC. 1990: Proc. Natl. Symp. CNF 487-492.
Mohapatra PK, Sethi PK and Mohanty RC. 1990: Trends in Ecotoxicol. 90(2) 245-251
Lundkvist 1. 1970: Svensk. Bot. Tidskr. 64: 460-461.
Hamdi YA, El-Nawaway and Tewfic AS. 1970: Acta Microbiol. Biomia. 2 53-56.
Da Silva EJ, Henrikson LE and Henrikson E. 1975: Arch. Environ. Contam. Toxicol. 3: 193-204.
Das B and Singh PK. 1978: Zeit. F. Allg. Mikrobiol.18: 161-167.
Das Band Singh PK. 1977: Arch. Environ. Contam. Toxicol. 5:437-445.
Kar S and Singh PK 1978. Bull. Environ. Toxicol. 20: 707-714.
McMoster BJ, Danton MS. Storch TA and Dunham VL. 1980:Biochem. B~ophys. Res. Commun. 96: 197-203.
Reddy GN. 1985: Studies on ammonia assimilation and its regulation In cyanobacteria Gloeocapsa sp. Ph D. Thesis. Univ. Madras.
Gallon JR. 1989: Phykos. 28(1-2): 18-46.
Hawkesfoed MJ, Reed RH. Rowell P and steward WDP. 1982: Eur. J. Blochem. 127: 63-66
Stngh PK and Bisoyi KM. 1989: Phykos. 28(1-2) 181-195.
Nirmal Kumar JL, Nirmal R and Ran.3 BC.1996: Pol. Res.15(2):147-150.
Dash AK and Mishra PC.1999: Role of cyanobacteria in water pollution abatement. In Fatma T (ed) Cyanobacterial and algal Metabolism and Environmental Biotechnology. Narosa pub. New Delhi. pp196-207.
Roger PA and Kulasoonia SA. 1980: Blue green algae and rice. Intl. Rice Res. Inst. Los Banos. Phillipires. pp 2340.
Venkataraman LV and krishna Kuinari MK. 1992: Exploitation of algae for biomonitoring and abatemen: of pollution in aquatic systems. Limnological Reveiws. Vo12. Narendra pub. New Delhi. pp 12-15.
Booth GM and Ferell D(eds)1998: In: Pesticides In the aquatic environment. Pelmun press. New ''ork. pp221-223.
Rath H and Adhikary Sp. 1996: Biiogica Plantarum. 38(4): 563-570.
Worthing. CR and Walker BS(eds:.1987. The Pesticide manual: World compendium. The British crop protection council. UK.
Arnon Dl. 1949: Plant Physiol. 24: 1-7
Witham FH, Blaydes DF and Deklin RM (eds). 1971. Experiments in plant physiology. Van Norstad pub New York. pp 245-248.
Putter. J. 1974. In Bergmeyr (etl.) Methods of enzymatic analysis. Vo1.2. Academic press. New York. pp 685687.
101. Mohapatra P K and Mohanty. R C 1993. Interaction of Agrochemicals with the cyanobacteria In Kudesia, V P (ed) Pesticide Pollution. Pragati ~. ~rakasha-n pub. Meemt, India. pp 137-98.
102. Fatma T., Sarada R. and Venkdaraman, LV. 1999. Evaluation of selected strains of Spirulina for their constitutents. In Fatma T (ed.) Cyanobacterial and algal metabolism and environmental biotechnology. Narosa pub. New [)elhi. pp 113-119.
103. Singh S. Patel R and Datta P. 2C01. Growing Spirulina Outdoors. An Overview In Fatma T (ed.) Cynot~acterial biotechnology. Narosa pub. New Delhi. pp 80-90.
104. Torzillo G.. Sacchi A. and Materassi R. 1991. Bioresource. Technol. 38: 95-100.
105. Apte SK, and Bhagwat AA. 1989. 1 Bacteriol. 171 (2): 909-91 5
106. Apte SK., Reddy BR and Thom~s J. 1987. Appl. Environ. Microbiol. 53:1934-1939.
107. Apte SK and Thomas J. 1986. EUI. J. Biochem. 154: 395401.
108. Miller, DM., Jones, JH., Yopp, JH., Tindall, DR and Schmid, WD. 1976. Arch. Microbiol. 11 1: 145-149.
' L O
121
122
123
Reed, RH and Stewart, WDP. 1985. Biochim. Biophys. Acta. 812: 155162.
Borow~tzka, LJ., Dimmerie, S., Mackay, MA and Norton, RS. 1980. Sc~ence. 210: 650-651.
Blumwald, E.. Melhorn, RJ and Packer L. 1983: Proc. Natl. Acad. Sci USA 80: 2599-2602.
Reed, RH., Richardson, DL., Warr, SRC. and Stewart, WDP. 1984. J. Gen Microbial. 130: 1-4.
Reed. RH., Chudek, JA, Foster R and Stewart, WDP. 1984. Arch. Microbtol. 138: 333-337.
Blumwald, E.. and Tel-Or, E. 1983. Plant Physiol. 74: 183-185.
Ibrahlm. EA. 1990. Water Airand Soil Pollu. 51: 89-93.
Butler, GL. 1977. Residue Rev. 66: 19-62.
Ware RW and Roan. CC. 1970. Residue Rev. 33: 15-45.
Saenz. ME., Alberdi, JL., Di Marzio, WD., Accoriniti, J. and Tortorelli, MC 1997. Bull. Environ. Contam. Toxicol. 58:922-928.
DaSilva, EJ., Henrilsson, LE.. anf Henrickon E. Arch. Environ. Contam. 1975 3(2): 193-204.
Tandon, RS., Lal. Rand Rao. WSN. 1988. Environ. Pollut. 52:l-9.
Subbara~, GS and Bose. S. 1983. Pesfcide Biochem. Physiol. 20: 188-193.
Singh. HN and Vaishampayam, A . 1978. Environ. Expt Bot 18: 87-94.
Subrarnanian. G., Sekar. S and Sampoornam. S. 1994. Int. Biodet. Biodeg. 33: 129.143.
Wong. F'Kand Chang. L ,1988. Environ Pollut. 55: 179-189.
Fatma I'., and Sultan S 1999. Cyanobacteria and heavy metal stress. In Fatrna T (ed.) Cyanobacterial and algal metabolism and env~ronrnental biotechnology. Narosa pub. New Delhi. pp 150-157.
Lehei C. Wada H, Kovacs E, Torok 2, Gombos Z, Howath I, Murata N and V~gh. L. 1992. Plant Mol Biol. 18(2): 327-336.
lanaka Y, Nishiyama Y, and Murata N. 2000. Plant Physiol. 124(1): 44 ' ~449
Stressgen Vancovor, Canada, technical note 608.
Ananthan J, Goldberg AL and Voellmy R 1986. Science. 232: 522-524.
Beckrnan RD. Mizzen LA and Welch WJ 1990. Science 248: 850-851
Rothrnan JE. 1989. Cell. 59: 591-602.
132. Dean RT, Shanlin FU, Stocker F and Davies MJ. 1997: Biochem. J. 324:l-18.
133. Shigeoka S, lshikawa T, Tamoi h4, Miyagawa Y, Takeda T, Yabuta Y and Yoshimura K. 2002. J Exp. Bot. 53(372): 1305-1319.
134. Faure, P and Lafond, JL. 1995. Measurement of plasma sulhydrl and carbonyl groups as possible indir:ators of protein oxidation. In Favier RG (ed.) Analysis of free radicals in biological systems. Birkhauser Verlag. Basel. pp 237-248.
135. Singh DP, Kshatriya K. 2002. Curl'. Microbiol. 44(6):411417.
136. Rabinowitch HD, Clare DA, Cra3o JD, and Fridovich 1. 1983. Arch Biochem Biophys 225(2): 640-648.
137. Rabinowitch HD, Privalle CT and Fridovich 1. 1987. Free Radic. Biol. Med. 3(2):125-131.
138. Demming-Adams, B., and Adams WW.1994. Light stress and photoprotection related to the xanthophyll cycle. In Foyer, CH. and Mullineaux (eds). Causes of phottmxidative stress and amelioration of defence systems in plants. CRC press. London. pp 105126.
139. Campbell. WS and Laudenbach, [)E. 1995. J. Bact. 177(4): 964-972.
140. Krause, GH. 1994. The role of oxygen in photoinhibition of photosynthesis. In Foyer, CH. and Mullineaux (eds). Causes of photooxidative stress and amelioQtion of defence systems in plants. CRC press. London. pp43-76.
141. Campbell, WS and Laudenbach. DE.1995. J. Bact. 177(4): 964-972.
142. Chadd, HE., Newman J., Man NH and Carr, NG. FEMS Microbiol. Lett. 138: 161-165.
143. Okada.S., Kanematsu, S. and Asaca, K1979. FEBS Lett. 103: 106-110.
144. Canini, A., Civitaraete, P. Marini, S., Caiola, MG. and Rotillo G. 1992. Planta Berl. 187: 438-444.
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