A ir is the prime supporter of life in the biosphere with dinitrogen as the

major constituent. The inexhaustible 'dinitrogen is useless unless

combined with hydrogen and oxygen. This is made possible through micro-

organisms and physical processes. The manufacture of nitrogenous fertiliser

is another mode of utilisation of dinitrogen. This production of nitrogenous

fertilisers has posed problems such as pollution of freshwaters in developed

countries.

The inert atmospheric nitrogen occupies approximately 78% by

volume of the atmosphere. Lightning enables the combination of dinitrogen

and water vapour to form ammonium nitrite, oxygen and nitric oxide. Both

ammonium nitrite and nitric oxide are introduced into the soil. Ammonium

nitrite is also formed by the oxidation of ammonia, mediated by ozone.

The vast reserves of atmospheric dinitrogen have been continuously

trapped by micro-organisms through biological nitrogen-fixing processes,

asymbiotic as well as symbiotic. The nodulated legumes contribute a

significant proportion to the overall harvest of nitrogen from the atmosphere.

Blue-green algae contribute significantly to the nitrogen economy,

particularly in tropical soils. In addition to their dinitrogen fixing capacity,

the blue green algae in fields also secrete growth promoting substances.

The nitrogen content of soils varies with their geographical location,

climate, vegetation, topography, parental material and age. A major factor

for nitrogen loss from cultivated land is leaching, varying from 20-50% of the

fertiliser nitrogen applied. Soil erosion often removes nitrogen following the

loosening of organic matter by water currents. Volatilisation of ammonia

from soil occurs due to its poor absorption to soil particles, with losses of

5-20% of the fertiliser 'N' applied, to the soil.

Denitrification, i.e., the ability to reduce nitrates and nitrites to gaseous ,. '..' '

products has been attributed to a few bacteria belonging to the genera

Pseudoinonas, Mimococcus, Acl~romobacfer and Bacillus. Autotrophs such as

Thiobacillus denitrijicans and Thiobacillus thioparus are also capable of

converting nitrates to dinitrogen.

The conversion of organic nitrogen to the more mobile inorganic state

is known as nitrogen mineralisation. Although mineralisation and

immobilisation are microbiological processes of opposite character, they may

not be balanced in magnitude. Remineralisation of immobilised nitrogen

may take place slowly resulting in the non-availability of nitrogen to crop

plants.

The intake of protein in different parts of the world differs. Protein of

vegetable origin is the mainstay of developing countries whereas the bulk of

protein in developed countries like Australia and New Zealand is animal in

origin. The intake of protein rich leguminous seeds is highest in India.

Accepted staple foods of developing or underdeveloped countries are

generally lacking in proteins. Pulses or oilseeds contain more protein than

cereah5

Apart from bacterial inoculants, the strategy to increase utilisation o r__--..

biological systems capable of fixing dinitrogen should includefeeking new> .; ', nodulating plants and micro-organisms capable of fixing dinitrogen other

;Ilpdh;,,, -- than those already in use, isolation and propagation of new dinitrogen fixing

micro-organisms from the rhizosphere of plants growing under unusual or

stress conditions.

2.1. Blue green algae

BGA are of world-wide distribution and are often abundant They

constitute an important group of organisms capable of fixing atmospheric

nitrogen. In paddy fields, the relative occurrence of blue green algae varies

within large limits. The BGA constitutes up to 75% of the total algal flora in

Lndian paddy fields. BGA are especially resistant to adverse conditions such

as high temperature, limited water, etc., and thus constitutes the dominant

components of microflora in many cases. BGA comprise unicellular, colonial

and filamentous forms. The free-living diazotrophic cyanobacteria are the

largest contributors to the process of biological nitrogen fixation, the second

most important biological process on the planet. The BGA appears to be the

predominant agent responsible for the maintenance of nitrogen supply in rice

fields and their ecological sigruficance may be immense. Many of the world's

paddy soils receive no artificial fertiliser. Over half the world's population

lives on rice as a staple diet and hence the importance of nitrogen fixing algae

in sustaining rice crops can readily be appreciated.

Most of the nitrogen-fixing BGA belong to the orders Nostocales and

Stigonematales under the genera Anabnena, Anabaenopsis, Aulosirn, Clrloroglwa,

Cylinderosprmum, Nosfoc, Calothrix, Scytonema, Tolypothrix, Fiscl~erelln,

Haplosiphon, Mnstigocladus, Stigonema and Westiellopsis.5

In general, nitrogen fixation is associated with forms possessing

heterocysts, although there are recent reports of fixation by unicellular and

non-heterocystous strains.

2.2. Heterocystous algae

Heterocysts are produced by a group of BGA, of the families

Nostocaceae, Rivulariaceae, Scytonemataceae and Stigonemataceae, which

display an unbranched or branched filamentous vegetative structure.

Heterocysts are large thick-walled, apparently empty cells appearing amidst

normal pigmented cells. In the electron microscope, the heterocysts have

dense cell contents, extensive membrane system and thylakoids have a

reticulate system of membranes. Heterocyst membrane system appears to be

more extensive, constituting a substantial increase of the total membrane

surface area. Heterocysts not only maintain a central nitrogen fixing system,

but also embrace mechanisms which function in order:

(i) to provide a suitable microenvironment for most effective enzyme

function.

(ii) to generate energy and reductants required for nitrogen fixation.

(iii) to bring the nitrogen fixed into organic combination and

(iv) to maintain a dual transport system for the import of carbon and the

export of nitrogen across the heterocyst-vegetative cell boundary.

2.3. Non-heterocystous algae

There are various reports in the literature of nitrogen fixation by non-

heterocystous filamentous blue green algae. Nitrogen fixation by natural

populations of Trichodesmiurn, Oscillatoria sub brevis and Plectoneina and

Lyngbya have been reported.5Jsm

BGA are more common in tropical and subtropical soils than in

temperate areas. The dominant nitrogen fixing species in India belong to the

genera Aulosira, Anabaena, Anabaenopsis, Calotlirix, Campylonettla,

Cylinderospermuin, Fisclwella, Hapalosiphon, Microchaete, Nostoc, Westiella,

Westiellopsis and Tolypothrix. In aquatic ecosystems BGA are abundant in

many neutral and alkaline fresh waters but are rarely found in the sea.

In India, rice is the most important staple food and its nitrogen

nutrition is associated with nitrogen fixing BGA. The soil conditions in rice

fields provide a congenial environment for the growth of nitrogen-fixing

BGA. Besides fixing atmospheric nitrogen, BGA synthesise and excrete

several vitamins and growth substances (Vitamin B12, auxins and ascorbic

acid) which contribute towards better growth of rice plants. The observed

increased yields of rice due to algal inoculation, even under heavy doses of

nitrogenous fertilisers, could be attributed to the combined effect of

biologically fixed nitrogen and the growth substances secreted by BGA.

The free-living BGA, Lyngbya sp. occur as a black encrusting film on

the rock. Below 35"C, masses of Nostoc and very tough mats of Lyngbya occur

in some streams. Lyngbya has been shown to have good growth in nitrogen

deficient medium.21 Lyngbya are filamentous forms in which the trichomes

contain only vegetative ~eIls.2~

Lyngbya aesturii occurs as a cryptoendolith layer in buckled crusts of

desert sediments at the extreme upper edge of the supralittoral, whereas

lower down, where wetting increases, the species forms thick mats.21 It was

suggested that L. aestuarii in the surface layer was protected from excessive

radiation by the brown extracellular sheath pigment 'Scytonernin'. The

brown colouration apparently takes place only in direct sunlight and is

effective in reducing incident light by as much as 95% within the first

0.5-1 mm of algal mats. Extracts of sheath material from marine Lyngbya mats

show strong UV abso rp t i~n .~~

2.4. Free-living bacteria

It is now clearly understood that a diverse range of microbial species

are capable of biological nitrogen fixation. This capacity allows them to

pioneer the colonisation of nitrogen-poor niches in ecosystem because of the

selective advantage they enjoy.

The free living bacteria having the ability to fix molecular nitrogen can

be distinguished into obligate aerobic, facultative aerobic and anaerobic

organisms. Obligate aerobic bacteria belong to the genera Azotobacter,

Beijerinckia, Derxia, Achromobacter, Mywbacterium, Arthrobacter and Bacillrrs.

Among the facultative anaerobic bacteria are the genera-Aerobacter, Klebsiella

and Pseudornonas. Anaerobic nitrogen fixing bacteria are represented by the

genera Closhidium, Chlorobium, Chromatiurn, Rhodonricrobiliin,

Rhodopseudomonas, Rlwdospirillurn, Desulfovibrio and Metlmtlobacferiunr.

Bacteria of the family Azotobacteriacae constitute the majority of

heterotrophic free living nitrogen-fixing bacteria. They are grouped into

three genera-Azotobac ter, Beijerinckia and Derxia.

Azotobacters are aerobic, mainly soil dwelling organisms with a unique

array of metabolic capabilities in addition to nitrogen fixation. They have

various abilities to synthesise alginates, poly-P-hydroxybutyrate, pigments,

and plant hormones. A unique feature of Azotobacters is their extreme

tolerance to oxygen while fixing nitrogen.5

Heavy metals are one of the major contaminants in various habitats.

'Contamination' is the release of substances into the environment at

measurable concentrations, while pollution implies that these substances have

measurable effect on living 0rganisms.2~ Among ecotoxicologists, the term

'heavy metals' is generally used to refer to metals that have been shown to

cause environmental problems.~ Appreciable amounts of heavy metals are

contributed to domestic effluents from waste, corrosion of water pipes (Cu,

Pb, Zn and Cd) and consumer produck like detergent formulations

containing Fe, Zn, Mn, Cr, Ni, Co, B and As.23

The evolution of metal-based industries has led to the contamination of

environment with heavy metals. Among these myriad of heavy metals,

cadmium, merits a special reference as a potentially toxic element The use of

Cd in fertilisers and pesticides as well as Cd from sludge contribute to

environmental pollution. Most of it gets deposited in soil or water.9

Ernakulam District in Kerala accounts for more than 70% of the

chemical industries situated on the banks of the rivers Periyar and

Chitrapuzha. The effluents from these industries reach the estuary by tides

and fresh water discharges. Pesticides, insecticides, fungicides, oil spills from

ships and boats, land drainage, trade effluents, sewage disposal, etc., make

the estuary more polluted.1~

To understand the effect of toxicity of heavy metals in the massive

environment, it is important to distinguish between particulate and dissolved

species. The Periyar brings heavy metals in the form of fine grade suspended

particulate matter from the effluent discharge point. Contamination of rivers

and estuaries with Hg, Cd and Cu have been documented in numerous

surveys."

A number of heavy metals are required as micronutrients in biological

systems to act as cofactors and/or as part of prosthetic group of enzymes in

a wide variety of metabolic pathways. Although contaminated soils

adversely affect the growth of plants and soil-living microbes, these

environments are not totally devoid of all flora and fauna as a number of

species have been adopted to tolerate the increased metal concen t r a t i~n .~~ , "~~~

Arndt-Schulk law s t a h that toxic substances in low concentrations

have a universal tendency to stimulate rather than to depress

Vagchi-Lange also reported that low levels of metals may not be phytotoxic

in most natural and agricultural environments, but the low level exposure can

result in significant concentration of certain metals in plant tissues.Z6

Some algal cells or phytoplankton are also known to accumulate

metals, some of which are essential micronutrients. The essential nutrients are

transported from cell surfaces into their cells to make use of them. Similarly

some toxic metals, at their high concentrations, once get associated with the

cells, are bioconcentrated though the toxicity is elicited only after uptake.

Various reports are available on the apparent presence of metallothionein-like

protein in plank. The metal inducibility of these proteins was also

demonstrated in plants.",","

Under normal conditions, minimum energy is used to maintain the

detoxification systems. However, when presented with a metal challenge, the

prokaryotic cells can respond quickly by synthesising proteins that

specifically detoxify the inductive metal. Several workers have reported the

synthesis of heavy metal-binding peptides by p l a ~ ~ t s , ' ~ . ~ ~ algae and fungi.17

Synthesis of heavy-metal binding is one mechanism used

to alleviate stress imposed by exposure to excess heavy-metals. The function

of the polypeptides is to sequester and detoxify excess metal i0ns.'4,~4 These

heavy metal-binding polypeptides are known as phytochelatins because of

their biological origin and their capacity to bind heavy metals. Synthesis of

phytochelatins can be induced by a variety of metals including Ag, Au, Bi,

Cd, Cu, Hg, Pb and Zn.16

Like metallothioneins, phytochelatins have a high cysteine content,

contain Cys-X-Cys sequences, and have spectra characteristic of metal

thiolates. Biochemical analyses of these peptides, revealed that they consist

of a group of GSH-related peptides with a general structure of (y-glutamyl

cysteine),,-glycine in which n = 2 to 10.1*,16,24,27

Among the common metals, Cadmium is the strongest inducer, while

Zn appears to be weak, as it requires very high levels for induction.'+,24

Phytochelatin (PC) synthesis can be observed within 5-15 min of exposure to

excess metals. PC synthesis is catalysed by PC synthase, which is activated

by the heavy metal cations and is completely inactive in their absence.2'

Reports have shown that GSH serves as a precursor for PC

biosynthesis; PC formation being catalysed by PC synthase which is activated

by metal ions and uses GSH as a substrate.'4,*6,24,25,28 Tomsett et aI. described

phytochelatins as large or rapidly synthesised pool of compounds rich in

sulfhydryl groups and have the ability to sequester metal ions when they

enter in plant cells.Z5 suggested PC act as sulphur carrier during

sulphate reduction.'4

Animals require certain essential. amino acids including methionine

and are unable to reduce oxidised form of sulphur (e.g., %2-, a2-). Plants

on the other hand, derive the Samino acids almost entirely hom sulphur in

soils, and perhaps have a greater capacity for handling-SH groups through

the proposed harrier." -.

Steffens et $?reported that failure to synthesise the PC results in ----,--

growth inhibition or cell death. It is reported that depending on amount of

labile sulphur, Cd can be displaced from PC at pH values of 5 to 3.5 where

vacuolar pH ranges from 3.5 to 6.0.14 Thus from photosynthetic algae, and the

plant Kingdom-Gymnospermae, Dicotyledonae and Monocotyledonae up to

the most advanced order of Orchidales-plants inactivate the heavy metal

ions by a cytoplasmic detoxification mechanism, the PCs. Several other

reports are also available on the distribution of PCs among monocots and

dicots to the red, green and brown algae. Studies indicated that

measurements of PC in natural phytoplankton populations at

environmentally relevant concentration supported the idea that PC might be

good quantitative indicator of metal toxicity."

Free living bacterial cells have evolved resistance mechanisms to cope

with heavy metal pollution. These resistance mechanisms are highly specific.

The genes determining resistance mechanisms occur on plasmids. These

resistance plasmids also have genes controlling resistance to most known

antibiotics.31

The mercury cycle is the best known case of microbial metabolism

affecting the chemical form of a h e a y metal. Microbial activity is associated

with mercury methylation, demethylation and oxidation-reduction of

inorganic mercury.

Mercury-resistant micro-organisms are found in much higher

frequencies in polluted waters than in cleaner waters. Bacteria with plasmids

showed a smaller number of patterns of resistance to organomercurials. HgZC

resistance in bacteria resulk from enzymatic detoxification of mercury

compounds. Mercury is volatalised as metallic Hg and the enzyme

responsible is called mercuric reductuse. Several organomercurials are also

enzymatically detoxified to volatile compounds. Benzene is produced from

phenyl mercury, methane from methyl mercury and ethane from ethyl

mercury. The enzymes responsible for cleaving Hg-C bond are

organomercurial lyases.31

Plasmid determined Cd resistance has been found only in

Staphylococcus aurerrs. There is no evidence for chemical transformation

associated with resistance. In Stnphylococci, Cd2+ is accumulated by a

membrane transport system utilising the cross membrane potential.31

Two separate plasmid genes are responsible for the Cd2+ resistance of

S.aureus strain: Cad A and Cad B genes confer respectively a large and a small

increase in Cd2+ resistance. Cd2+ resistance is due to lessened accumulation of

Cd2' by resistant cells. The Cad A gene product causes this lessened

Cd2+ accumulation. A plasmid encoded efflux system rapidly excretes Cd2+

from resistant cells. It seems plausible that Cad A gene product might also

cause ZnZ+ eff1ux.j'

The mechanism of Cad B gene function is not known. The clues at the

moment include the significant resistance to ZnZ+ conferred by Cad B and an

inducible Cd2+ binding activity governed by this gene. It might be due to a

membrane component analogous to metallothionein, the small CdZ+ binding

protein synthesised by both mammals and microbes.

Several other mechanisms for heavy metal resistances are there, but

todate not much is known about the mechanisms of resistance to Bi, 8, Co, Pb,

Ni or Te ions. Cupric ion resistance has been found both with plasmids in

E.coli and S.aureus, but no studies have been reported on the mechanism of

this resi~tance.~~

Ghosh et al. reported that five nitrogen fixing bacterial strains were

resistant to mercury.32 The optimum concentration for the induction of

mercuric reductase was 20 pM HgCl2.. Even in the presence of a higher

concentration of the inducer HgCl2 the percentage of Hg volatilisation did not

increase. However, the cells could survive even at high concentration of Hg. , , , ,

Earlier reports by indicated that at high concentration of Hg, .

the free reduced glutathione levels and glutathione reductase activity were

increased within the cell.=

Nickel is required for growth of some prokaryotic organisms and may

be beneficial to some plants and animals. The biological importance of nickel

is multi-faceted. It is a component of urease in higher plants and rumen

bacteria. Urease activity in soybean leaves has been shown to depend on

nicke1.3

Several reports indicate that Ni has a stimulatory effect on the process

of nitrogen fixation by micro-organisms, plants and blue green algae.% Ni

was later found to be a micronutrient element for hydrogen dependent

growth of Rlziwbiunr japnicurn, Azotobacter chroowccurr~, Azospirillrrrtr l)rsiler[se,

Azospirillrrttt lipo@urrf and Derxia guntmosa. It was also found that Ni has a

positive effect on active hydrogenase formation in Anabnetza.

Bacteria require Ni for the synthesis of nickel hydrogenase(s), nickel

containing carbon monoxide dehydrogenase(s), methyl Co-M reductase,

urease, and/or for unknown reasons. All hydrogenases contain iron; many in

addition contain nickel. The latter enzymes are here referred to as nickel

hydrogenases. Growth of Oscillatotin and Clilorella has been reported to be

stimulated by Ni."

. Rai et al. also reported the existence of Ni-dependent hydrogen

production and uptake hydrogenase activity in the two cyanobacteria

Anabaena C A and Anabaena variabilis.'O

Daday et al. reported that higher concentrations of Ni (0.68 ph4)

resulted in yellowing of cyanobacteria) cultures indicating toxicity. Although

algae and cyanobacteria are amongst the most sensitive organisms to nickel, it

is not toxic as substantiated by other reports.36

Dalton et al. reported that the efficiency of N2 fixation was improved

by lower concentrations of nickel.% Low concentration of Ni also stimulated

microbial urease and hydrogenase activities in soybeans. These reports

substantiate the biological significance of nickel at lower concentrations,

though it is toxic at elevated concentrations. Rai et al. reported that

cyanobacteria showed better growth in the presence of Ni which may be due

to the tolerance of the alga to Ni. Since Ni is known to stimulate hydrogen

uptake, it is likely that excess hydrogen uptake by the test algae may offer

reducing power to nitrogenase thereby stimulating the activity of the enzyme.

He suggested that since Ni stimulated carbon fixation and nitrogenase

activities, Ni is to be included in the category of essential mi~roelements.'~

The efficiency of nitrogen fixation in some legumes is decreased by the

evolution of hydrogen from nodules. The efficiency of nitrogen fixation can

be improved by recycling of the hydrogen within the nodule by an uptake

hydrogenase system in which ATP is generated by the oxidation of hydrogen.

It has been reported that hydrogenase activity in soybean was

diminished when the supply of Ni was inadequate. Ni was found to

stimulate urease at 5 pM/g dry weight, But stimulatory effects of nickel on

hydrogenase were not evident at the final harvest perhaps due to

accumulation of toxic levels of nickel at the highest application rates. Toxicity

symptoms were visible at the l W M level. These plants became moderately

chlorotic and produced significantly lower dry weight yields of roots and

shoots at final harvest.34

A pH value of 6.0 or above seems to be critical in limiting availability

of Mn, Zn, Cu and Ni. Microbial sensitivity to high Ni levels is also

dependent on pH. Ni toxicity to diverse micro-organisms is greatly reduced

when the pH is above 6.5, presumably because of reduced availability

associated with changes in chemical form.%

Some species such as Alyssum bertolinii are Ni-tolerant, i.e., they are

able to accumulate Ni to a greater extent on a Ni-enriched soil. Plants that

contain more than 1 mg nickel per gram dried leaves are designated as

hypuccumulators. In Ni-accumulating plants, the metal is present as a water-

soluble polar complex with citric, malic or malonic acids. It is suggested that

hyperaccumulation is a kind of xeromorphic adaptation and that these plants

can tolerate unfavourable nickel-rich soils by producing organic acids as

chelating agents to prevent Ni toxicity. In hyperaccumulators, Ni may inhibit

a key metabolic enzyme causing an overproduction of an organic acid which

renders Ni non-toxic by chelation and compartmentalisation of vacuoles.35

Many bacteria appear to transport Ni by a Mg transport process.

Translation of Ni was dependent upon physiological temperatures and

oxidizable substrates (e.g., glucose, pyruvate, succinate, etc.). Ni was

transported by the same system responsible for Mg transport and that toxicity

arose from direct interaction with respiratory chain. In methanogens, Ni

transport was optimal at pH 4.9 and 49°C (e.g., Methunobacterir~rrr brygantii).

Mid log phase cells exhibited maximal capacity for Ni transport.=

The symptoms of Ni toxicity appears to be the combination of induced

Fe deficiency, chlorosis and foliar necrosis. Ni excess induces a predisposing

condition in areas of the leaf before the emergence of the coleoptile. These

areas give rise to chlorotic bands which develop on the leaf 24 h after

emergence.3'

The toxicity of Ni to micro-organisms is well known. Ni is used as a

fungtcide especially for the control of cereal rusts. Algae are known to be

rather more sensitive to the presence of Ni in water bodies. Ni has been reported to enhance the growth of blue green algae.37

In 1965, Bartha and Ordal demonstrated the Ni requirement for

chemolithotropic growth of Alcaligenes eutrophicus HI and H16.38

confirmed that Ni is intact as a constituent of the soluble

hydrogenase of A.eutr0phicus.~9 The optimal growth was obtained at a level

of 0.1 pM NiS04. This level is equal to that reported for Alcaligenes eutropl~trs.

The increase in hydrogenase activity on Ni supplementation was completely

inhibited by chloramphenicol, an inhibitor of protein synthesis, indicating

that Ni is required for the hydrogenase synthesis. Ni has been shown to be a

component of hydrogenases in many micro-organisms in addition to

Alcaligenes eutroplr~s.4~

Shuttleworth and Unz reported that ITlriothrix strain absorbs Ni and Zn

very quickly. Most of the metal ions which were taken from the solution

were bound within 10 min and there was no increase in the level of bound

metal after 1 h. Metal uptake was also a function of cell-age and was not

linear with cell protein concentration. They also indicated a concenbation of

0.7 to 0.8% Ni or Zn per cell dry weight of Thiothrix A1 strain, when the

bacterium was exposed to 100 pM metal c~ncentration.~~

Introduction of higher than natural levels of Ni into aquatic systems

may have either two consequences for the associated algal community. At

high Ni concentration, species diversity and biomass may be seriously

reduced. An alternative effect involves shifts in community, composition and

may be mediated by sublethal doses of nickel.

Studies on Ni toxicity indicates that variability exist in inhibition of

growth at relatively low concentration. Ni inhibited growth of Chlorella

vulgmis less than Hg, Cu and Cd.42 Ni tolerance in algae apparently depends

on previous exposure to nickel as well as innate tolerance.43

Stokes et al. compared the growth of a lab strain of Scendesmus

(S.accuminatus) and a strain from a Ni contaminated lake (S.acutijimis urn

alternans). The results showed that the lake strain tolerated higher levels of

Ni. S.acuminatus grew well at 0, 0.05, 0.15 and 0.25 mg NiS04/L. Growth

was impaired (less than 5% of control levels) at 0.5 and 1.5 mg/L. The lake

strain grew at all these concentrations, achieving growth equivalent to 60 and

22% of the controls at 0.5 and 1.5 mg NiSO4/L, respectively.43

Limited data suggested that organic chelators ameliorate nickel's toxic

influence on algal growth43 Maier et al. studied Ni accumulation and storage

in Bradyrhizobiunr japonicum.44

Hydrogenase derepressed (chemolithotropic condition) and

heterotrophically grown cultures of ~radyrhizobiurn japonicum accumulated Ni

to an equal extent over a 3 h period. Both types of cultures accumulated Ni

primarily in a form that was not exchangeable with NiCIL and they

accumulated much more Ni than would be needed for the Ni-containing

hydrogenase. The Ni accumulated by heterotropically grown cultures was

associated with soluble proteins rather than particulate material, and this Ni

was not lost upon dialysing an extract containing the soluble proteins against

either Ni-containing or EDTA containing buffers, but was lost upon pronase

or low pH treatments.

Muiioz et al. studied the effects of the addition of two heavy metals

and a support material (purified sepiolite) on the methanogenesis have been

evaluated in two types of domestic sewage sludges.45 A higher toxic effect of

the metals was observed on the production of methane from loading sludge

than from the anaerobic digester sludge, Ni being more toxic than Pb in all

cases studied.

Daday et al. studied the role of Ni in cyanobacterial Nz fixation and

growth via cyanophycin metab0lism.3~ The growth was not observed in the

presence of NiSO4 (0.68 wM). It was associated with delayed heterocyst

differentiation, delayed nitrogenase synthesis and inhibition of pigment

synthesis. It is proposed that Ni depletion results in a diversion of N& from

protein synthesis into cyanophycin, thereby delaying denouo synthesis of

proteins and enzymes required for the synthesis of active nitrogenase.

Copper smelting or refining industries, manufacturers of copper

products and sewage disposal processes all contribute to the man-made input

of copper to the biosphere. Use of copper based agrochemicals such as using

copper sulphate as an algicide can lead to excessive accumulation of soil

copper in localised areas. Use of copper as a supplement to the animal feeds

may introduce copper to farmland."

Copper is required by biological systems as structural and catalytic P A 1 - f l

dL" components of proteins and enzymes and as cofactors essential to normal j o "

growth and development. in e x c i , copper i i toxic to c e l l s . 4 ~ _ ~ l ~ h i k b r ; ; ; f X reported that the deficiency of micronutrient element C; producedT -

.---

unfavourable effects on growth and nitrogen fixation in ~ z o l 1 a . c 69, , r r b \ r n '

.. Several reports are available on the toxicity of copper. According to Z

Lepp)copper toxicity in plants is generally manifested as chlorosis and " - --,._,-

stunting of growth.46 Crop stunting due to excess Cu may be due to its

antagonistic effects with other nutrients, or reduced root growth and

penetration into the soil.

Hunter and @ 3 s of opi"on that copper toxicity can be reversed

by EDTA, soon after treatment with copper; 6-12 h after onset of copper

treatment, EDTA is ineffective.48 More specific effects of Cu have been

postulated in relation to the sensitivity of various enzyme systems to excess

Cu. Effects of Cu on rice seedlings, include reduced nucleic acid content,

reduced activity of a-amylase, RNase and reduced protease activity in the

endosperm." Excess Cu may also reduce metabolic activities in the soil. Cu

has specific effects on photosynthetic reaction.

Reckendorfer suggested that leaf scorch, arising as a result of excessive

application of metal based pesticides (including copper) could be due to

metal replacement of Mg in the chlorophyll m0lecule.4~ Gross et al. observed

that photosynthesis in green alga C h l d l a was inhibited by Cu and

coincident with this was a change in the absorption spectrum of the algal

chlorophyll.^

Rajarathinam et al. studied the effect of copper algicide on growth and

nitrogen fixation in Azolla p f"natar R.BR.=l Most common method for

chemical control of free-living algae is the application of 1% CuSO4 solution.

Copper in C S 0 4 solution completely controlled snails which affect the

growth of Azolla. They reported that increased Cu concentration in the

medium resulted in the reduction of biomass, chlorophyll content, nitrogen

content and GH2 reduction activity.

the effec't of some heavy metals on the

cobalt nutrition of Rhizobiunr meliloti. They found that no significant growth

inhibition was observed by 0.01 or 0.1 pM Cu, but growth reduction was

observed at 1 pM Cu. The reduction was not large, but would be quite

significant with solutions very low in cobalt (Co). At high level of Co (10 pM),

the growth reduction produced by 1 pM Cu disappeared.52

Extracellular ligands are used to increase bioavailability of an essential

metal. Studies indicated that certain phytoplankton species may have the

capacity to reduce Cu uptake by limiting the free cupric ion concentration

[CU~'] in near-surface sea water through the production and secretion of

extraceuular Cu-binding ligands.16

Most phytoplankton species are extremely sensitive to [CuZ+], which is,

on the one hand, an essential micro-trient and on the other, a strong u phytotoxin. Copper toxicity in phytoplankton appears to be primarily due to

competition with MnZ+ which plays an essential role in the oxidation of

water to oxygen in photosynthesis. Cu has been demonstrated to

competitively inhibit the accumulation of Mn by algal cells, with concomitant

reduction in cell growth.x6

It has been seen that the *lgae are especially sasceptible to Cu

toxicity, primarily because of the inhibition of atragen fixation. Cu has also

been reported to be highly toxic to the (presumably non-nitrogen fixing) blue

green alga Spirulina platensis in Lake Nakurn, Kenya, as a result of laboratory

assays using natural lake water and it.; associated algal population.15

It is reported that alkaline pH and high organic carbon levels in the

water may detoxify Cu, so that it is not only an ineffective algicide but it may

actually stimulate growth. Such conditions are common in lakes that have

excessive algal growth, but sensitivity of nitrogen fixation to Cu suggests that

Cu treatment is suitable for lakes subject to large blooms of nitrogen fixing

blue-green algae.l5 Rijstenbil et a1 studied the effects of the algal cellular N

status on the defense against Cu toxicity in batch cultures of coastal diatom

ThaJassiosira pseudunana grown in coastal sea water.%

Binding and uptake of Cu in algae is a dynamic process that depends

on competition for ions between ligands and cells that are continuously

internalising metals. It was expected that Cu uptake would be independent

of the nitrogen status, however, N-depleted cells synthesise less

phytochelatin and were more sensitive to C U . ~

Intra and interspecies variability in copper content of marine plants is

extremely large. There is some evidence that algae reared or found in high

copper environments can tolerate additional levels of this element more

readily than algae from low copper en~ironments.5~

Xue and Sigg studied the interactions of Cu(Q with algal surfaces and

exudates in metal-Nitrilotriacetic acid buffers by a combination of several

analytical techniques. Results indicated that the binding of Cu(Q to algal

exudates has a more significant effect on copper speciation than the binding

to the algal surfaces. These extracellular ligands may play an important role

in decreasing the concentration of free copper ion and thus mitigating the

potential toxic effects in organisms.S6

Several reports are available on the binding of copper to extracellular

agents of algae.57-s9 McKnight and Morel characterised copper complexing

properties of organic compounds released by 21 algal species. Of seven blue

green algal species studied, four produced strong copper-complex agents of

5 x 10-7 M concentration, two produced complexing agents similar to those

produced by eukaryotes. In time-course experiments with three chlorophytes

and two cyanophytes the release of measurable concentrations of

Cu-complexing agents occurred principally during stationary phase.S7

From their experiments, McKnight and Morel found that iron

limitation greatly increases extracellular concentration of strong Cu-

complexing agents in cultures of Anabaena Posaquae and Anabaenn cylindrica

and that iron-algal exudate complex was much more stable than, Cu complex

and they concluded that strong Cu-complexing agents released by

filamentous BGA were siderophores. Further experiments demonstrated that

BGA overcome toxic effects of micromolar copper additions. From estimates

of concentration of Fe, Cu and siderophores in Fe-limited BGA blooms, it was '

predicted that Cu-siderophore complex was the major copper species. The

Cu-siderophore complex was probably not toxic and its presence in fresh

water may be advantageous to cyanophyte population.58

c@3eported the production of strong extracellular Cu chelators by

marine cyanobacteria in response to Cu ~tress.5~ Cultures of cyanobacteria

Synechococcus-sp., a ubiquitous and important group of phytoplankton highly

sensitive to Cu toxicity, were previously shown to produce chelators Moffet

showed that cultures of Synechococcus exposed to toxic concentration P: o Cu

~roduced an extracellular ligand." Co-ordination of Cu by this compound

decrease the concentration of free cupric ion (the toxic form) in culture media

to levels that do not inhibit growth. He suggested that cyanobacteria modify

copper chemistry in sea water, creating conditions more favourable for

growth. Hawkins and Griffiths reported that there was an immediate decline

in algal density after copper addition, followed by an increase in numbers of

more copper-tolerant chlorophyte sp.60 Zoo plankton and other aquatic fauna

were also affected by the copper treatment But any possible long term

effects of Cu were marked by flow induced rapid destratification which

interrupted and reset algal succession. He concluded that Cu treatment is not

an effective control of cyanobacterial growth.60

According to Metaxas and Lewis, Skeletonenza exhibited increased

growth rate and lag phase with increasing Cu concentration. On the other

hand, Nittschia demonstrated decreased growth rate and there was no effect

on lag phase.61

Collins et al. studied the effect of copper on Methylomonas albus BG 8

increased cell yield and methane monoxygenase activity. Intracytoplasmic

membrane was formed only in cells grown 'with copper supplementation.

Additionally, abundance. of two major membrane proteins were affected by

Cu in growth medium. These findings indicated the effects of Cu on the

physiology of methanotropic bacteria BG 8.62

~ a r w o o d k q a n d Gordon reported that growth of marine bacterium

Vibrio alginolyticus was temporarily inhibited by micromolar levels of copper.

During Cu-induced stationary phase, compounds which complex and

detoxify Cu are produced. In this study they identified two Cu-inducible

supernatant proteins having molecular masses of 21 and 19 KDa.63

Cadmium is an environmental pollutant not only because of its

phytotoxicity, but also because of its uptake and assimilation in plants may

introduce it into the food chain. Cd2+, a non-essential element, enters the

environment through various industrial processes and to lesser extent from

natural weathering. Cd2+ is emitted to the atmosphere from coal fired plants,

steel mills, metal smelting and roasting operations, incineration of wastes and

electroplating process. In soils, Cd2+ accumulation occurs either by

deposition from the atmosphere, or from phosphate fertilisers and sewage

sludge.64~65

Several reports are available on the biochemical studies related to the

effects of Cadmium. Pollution with Cd does not seem to be widespread.

Serious consequences have been encountered thus far largely in

circumscribed geographic areas, consequent to industrial use.27 dr l..pgcr c t . k-8 $> #-*

According to heavy metals a requ~red by biological

systems as structural and catalytic components of proteins and enzymes and

as cofactors essential to normal growth and development In excess, they

become extremely toxic to cells. Toxic or lethal levels are experienced by

plants growing near mining or smelting operations, industrial and municipal

waste disposal sites, on some natural soil types and on some agricultural

soils.14

But Vallee and Ulmer reported that'the growth of Clilorella was

stimulated by low concentration of Cd, inhibited by higher concentration and

finally ceased when both protein and chlorophyll concentration decreased.

According to them, labelled Cd was taken by Staphylococclcs allreus and 40%

remained fixed after repeated washings of the cells.Z7

Chugh et al. studied the effect of Cd on enzymes of nitrogen

metabolism in pea seedlings. They reported that there was a substantial

increase in number and mass of nodules of the plants during the course of the

experiment. There was significant inhibition of the rate of Nz fixation per

plant and 35% decrease occurred with 2.5 mM cadmium. There was 30%

decrease in fresh weight of nodules when compared to the controls. Thus Cd

treatment caused marked perturbations in nitrogen metabolism of plants by

influencing activity of key enzymes. It also showed Cd exerted an adverse

effect on GS/GOGAT and GDH.& .... ~..,

i:. , , b

: ~ s s c h h e t al. reported a reduction in biomass and inhibitional quality \..

in Phaseolus vulgaris L. when crops grown in soils were contaminated with

heavy metals, viz., Zn and Cd. These elements were found to inhibit

photo~ynthesis.~7

Another important 0bSe~ation was increase in activity of several

enzymes, e.g., peroxidase, glucose-6-phosphate dehydrogenase, glutamate

dehydrogenase. There was an increase in protein concentration in roots and

leaves after toxic Cd and Zn treatment. Cd inhibits root water uptake in i

corn.67 r&- *&t, - , v , '~ " 4 c-

heavy metal toxicity induces premature

plant sene~cence.~~ A general increase of hydrolytic (protease, RNase-,

DNase) activity after application of Pb and Cd is in favour of this hypothesis.

Keshan and Mukherji studied the phytotoxic effects of cadmium sulphate on

nitrogen content, nitrogen fixation, nitrate reductase in root nodules of

mungbean. According to them Cd reduced N content, inhibited the N-ase

and nitrate reductase activities. Cdz+ has been reported to cause iron

deficiency.%

Smith et al. studied Cd sensitivity of soybean related to efficiency in

iron utilisation They postulated that the most iron efficient cultivar would

be least injured by Cd. Growth reductions in presence of excess Cd follow

nutrient imbalance and result primarily from a loss in the production of

carbohydrates in the treated plants.68

There are many reports concerned with biochemical and physiological

changes caused by Cd in cyanobacteria. Pinas et al. studied the influence of

Cd on the ion balance of cell and also the interactive effect of Cd and calcium.

They found that Ca was able to counteract the toxic effect of Cd towards

growth, photosynthesis, nitrogenase activity and pigment content of the

cyanobacteria Nostoc sp. UAM 208. They also reported that there was a

significant increase in the cellular content of nearly all of the elements studied

after 4 h exposure to cadmium.69 F - ,L4" Two important food tuff& rice and wheat are able to take up L,

considerable quantities of Cd from the soil. Also wheat grains accumulated c 7'

more ,than rice. Concentration above 10 pg/g in soil brought less yield of

both ;ice and wheat It has been shown that increased soil acidity resulted in

higher Cd concentration in plants. Addition of Cd to soil always increased

the Cd content in grain of cereals or in edible portion of vegetables.

Considerable Cd uptake occurs in wheat when sewage sludge is used as a

plant nutrient source. The lower the pH, the greater was the Cd

concentration in wheat and sludge containing Cd more than 25 pg/g (dry wt)

should not be used at all for food cr0ps.~0

Foliar applied Cd has been found to be associated with cellular

fraction and very small amounts of the element were found in nuclear,

mitochondria1 and microsome fractions of soybean plants grown on Cd-

containing sludge. Cd has been found to stimulate the activity of GDH."

Weigel an Jager eported that Cd in bean plants was accumulated in cytosol 41.3 of the cell and was bound to peptides or low molecular weight pr0teins.n

9 Metals are an intrinsic component of earth's crust With the rapid

L, development, the evolution of metal-based industries has led to the

contamination of environment with heavy metals. Among these myriad of

environmental pollutants, Cd, a heavy metal, merits a special reference as a

potentially toxic element Most of Cd, introduced by the use of fertilisers,

pesticides and sludge treatment, gets deposited in the soil or water.

Ever since man started to produce metals, he also started to pollute the

environment. Cd and its compounds have been increasingly used in

industries causing a sharp increase in environmental contamination. Sources

of environmental pollution-electroplating, Cu-Cd alloys, cadmium sulphide

and cadmium sulfoselenide-colour pigments in plastics and various types of

paint. Cd serve as an electrode component Under natural conditions, the

amount of Cd absorbed by plants are usually low.

Cd present in the sediments is bioconcentrated by planktonic algae.15

The ability of algae and macrophytes to accumulate Cd has been well

d o ~ u m e n t e d . ~ ~ , ~ , ~ ~ , ~ Wang et al, reported Cd removal by a new strain of

Pseudornonas aeruginosa in aerobic cu1ture.n

Mercury pollution is a global problem due to its adverse effect on all

living systems. Though the use of mercury containing pesticides is banned

world wide, many developing countries including India still routinely use

mercury-containing pesticides. In India, a number of mercurial compounds

are routinely used in agriculture to control seed-borne and soil-borne fungal

diseases. Mercury compounds like phenyl mercury, merthiolate,

mercurochrome are extensively used as household and hospital

d i s i n f e ~ t a n t s . ~ ~ , ~ ~ , ~ ~

A substantial proportion of the annual production eventually reaches

the environment. About half the mercury is lost from the chloralkali 1 ----.--- ,

industry. Chlorine and caustic soda are produced electrolytically using

mercury electrodes and it has been common with this process for 150-200 g of

mercury to be lost to the atmosphere and in waste waters for each tonne of

product. Most of the remaining half of the lost mercury comes from uses

which inevitably disperse mercurial compounds into the environment. In

areas where mercury has been discharged from anthropogenic sources the

local concentration have been considerably higher.p The prevalent use of

organomercury compounds as fungicides and slimicides has also contributed

to the increased concentration." Mercury is present in slirnicides used in the

timber and paper pulp industries to prevent fungal growth, antifouling

paints for ship hulls, pesticides and seed dressings in agriculture and in

pharmaceuticals.76 Mercury loss associated with the amalgamation process of

gold recovery from stamp mill mining has produced occasional levels of

mercury ~ontamination.~

These inputs are estimated to be about 7000 tonnes per year world

wide, but a further 3000 tomes per year is derived from burning fossil fuels.

Crude oil and coal contain only traces of mercury. They are however burned

in such large quantities that their input to the atmosphere is ~onsiderable.'~

Major sources of mercury in coastal waters are rivers, marine outfalls

and wastes dumped directly into the sea. Dissolved mercury in the sea is in

the form of Hg (0H)z or HgCl2 with chloride predominating, but to a

considerable extent, Hg is adsorbed on to particulate matter and is not in

solution. It also forms stable complexes with organic compounds occurring

in the sea, especially sulphur containing proteins and humic substances.76

Microbial systems in the sea can convert all these inorganic forms of

mercury into methyl mercury. Organic forms of mercury are more toxic than

inorganic salts. Mercury toxicity to humans has been known for centuries.

Mad hatters in Victorian England got their name from convulsions and loss of

neuro-muscular co-ordinations, symptomatic of chronic poisoning by the

mercury used in the treatment of felt in the manufacture of hats. In this

century, mercury has been the only contaminant introduced by man into the

sea that has certainly been responsible for human deaths (Minimata

disease).76

Mercury is not an essential element, instead it is considered to be toxic

to biological systems. Mercury has a strong affinity for ligands such as

phosphates, cysteinyl and 'stidy side chains of proteins, purines, pteridines 0 and porphyrins. Adsorption of heavy metals to bacterial surfaces has been

reported and it affects the free movement and active transport of

electrolytes."

Ellis et al. supported that .the accumulation of heavy metals by

agricultural crops is a public health c0ncern.n Peles is of opinion that

agricultural species grown in sludge-amended soil take up heavy metals.78

Gadd also reports that heavy metals such as Hg can be accumulated by

micro-organisms by non-specific physicochemical interactions as well as

specific mechanisms of sequestration or transport A variety of resistance

mechanisms have been documented in all microbial groups.T9

Vallee et al. has reported that HgZ+ reacts weakly but instantaneously

with NADH, forming a 1:l complex, broken down by EDTA with

concomitant loss and restoration of the 340 nrn absorption of NADH. HgZ+

also reacts with S S bonds. Hg2+ and organic mercurials interact with -SH

and S-S groups of proteins in a multitude of systems. The existence of S-Hg-S

bond in proteins has been demonstrated through simultaneous elimination of

Hg by EDTA and carboxymethylation of -SH groups thus released. Hg also

interacts with phosphate groups of all membranes and with amino and

carboxyl groups of enzymes."

Hg appears in industrial discharges in five principle forms:

(1) divalent mercury Hg2+, (2) metallic mercury HgO, (3) phenyl mercury GI%

Hg', (4) alkoxy alkyl mercury CH3-OCHrCH2- Hg+ and (5) methyl mercury

C&Hgf. All mercury compounds are cytotoxic to cells. Hg2+ readily change

cell membrane permeability and organomercurials influence the permeability

of chloroplast membrane^.^^

Mercury reduces or inhibits growth, ' photosynthesis and nitrogen

fixation. Several reports are available on the effects of mercury on nitrogen

fixation and photosynthesis. 74,75 Trehan ef al. reports that higher concentration

of Hg2+ inhibited the activity of enzymes of nitrogen assimilation nitrogenase,

besides heterocyst production in Nostoc muxorum. Accordingly, the

inhibition of GOGAT was relatively slow in contrast to glutamine

syntheta~e.2~ The undifferentiated cells in presence of Hg2+ is responsible for

imbalance in carbon and nitrogen supply for carrying on functional nitrogen

fixation.24 Boyle points that industrial contaminants especially heavy metals

such as Hg reduce photosynthesis by causing structural damage to

chloroplasts.'5

According to Ghosh and co-workers the nitrogen fixing bacteria, being

abundantly present in the soil may be exposed to mercury compounds which

enter into soil systems through the use of mercury based pesticides and

fungicides during agriculture and seed dressing and thereby they become

mercury re~istant.~2,~

Ghosh et al. reported that all the bacterial strains resistant to Hg were

not equally resistant (sensitive) to all organomercurials ~ s e d . ~ 4 , ~ Growth

studies indicate that N2 fixation activities of all Hg 11-resistant bacteria were

not inhibited completely in the presence of higher amounts of HgC12.

According to them, nitrogenase of cells grown in the presence of HgC12

showed inhibition of nitrogen fixation in the presence of HgClz (10 and

50 pM/L). Inhibition of nitrogen fixation might be due to either direct

inhibition of the nitrogenase protein complex by Hg or disruption of the flow

of energy. Hg has high affinity towards thiol compounds and if Hg

accumulates intracellularly, it could interact with numerous thiol containing

enzyme systems of the cells.

It is possible that in the case of cells which were grown in the presence

of HgCla nitrogenase may have some protection meted out by Hg(II)

reductase. In the presence of H@z, the synthesis of nitrogenase may be

partially affected. Ghosh et al. suggested that mercuric reductase may play an

important role against Hg(II) toxicity as GSH and NADPH are cofactors of

this enzyme (when GSH and NADPH are added in the assay mixture).74

The earliest reports of the presence of bacteria resistant to mercury and

organomercurial compounds came from Japan, when resistant bacteria were

isolated from soil polluted with organomercurial compounds. It was also

found that many of the antibiotic resistance plasmids in both gram negative

and gram positive bacteria have the genes to determine resistance to Hg2+ and

occasionally to organomercurials. The resistance to Hg is inducible and

resistance to any one of the organomercurial compounds confers resistance to

all.BU The basic mechanism of resistance appears to be conversion of Hg2' to

HgO.

C&Hg+ + CH4 + Hg2+ -+ RS Hg+ or RSHgSR -+ HgO

Pseudomaas species was reported to be used in removing mercury by

volatilisation fkom effluents before they are discharged into water bodiesg

Marine algal species have also been suggested to be the indicators of

pollution.81 Some workers have reported the effect of Hg on in si tu nitrogen

fixation in an algal community and in rhizobium system.

Rapid accumulation of Hg, especially organomercury compounds, by

various species of algae under controlled conditions is documented. Marine

algae showed great inter-species variations in sensitivity to marine insults.

Phytotoxic effects include reduced growth, developmental abnormalities,

photosynthesis, inhibition and death.55

Chatuwedi et al. studied the DNA adduct formation of ceresan-a

mercury fungicide on Azotobactev vinelandii-free living nitrogen fixing

bacterium. Whether the mercury moiety of ceresan binds at the A-T sites or

not could be ascertained through the use of ceresan and W separately and in

combination. They found that at lower concentration of ceresan, repair

mechanism was quite efficient and is able to counteract the stresses to a

considerable extent At high doses, the repair mechanism was ineffective and

consequently the survival fails.82

Studies by Chatumedi et al. also indicated that continuous treatment of

bacterial cells with ceresan decreases the nitrogenase activity. It has been

shown that Hg, the active moiety present in ceresan binds with DNA bases.

Viscosity and melting temperature studies of Awtobacter vinelandii DNA also

indicated that Hg moiety present in ceresan intercalates with A-T base pairs

of DNA. It is possible that long duration treatment with ceresan cause

impairmat of structural and functional integrity of DNA in uiuo.82

@jpulse treatment of bacterial cells with ceresan induces nitrogenase

activity. This may be due to the action of ceresan on the membranes which in

turn might induce CAMP cascade?

Ghosh et al. studied the Hg volatilisation patterns of five nitrogen-

fixing bacterial strains, the effect of different inducers on mercuric reductase,

and the pattern of substrate utilisation by organomercurial lyase. It is

significant that the total viable count of Hg-resistant, nitrogen-fixing bacteria

increased with the increase in the Hg content of the It was also found

that some amount of Hg remained bound to the cellular constituent even after

several washes with deionised water.

At low concentration of Hg compounds, Hg-resistant bacteria could

detoxify the toxic HgZ+ by completely eliminating all the Hg from the

bacterial system. But at low concentration, intracellular sequestration by

metal-binding components may also take place. They also indicated that Hg-

resistant bacteria have limited capacity for Hg-volatalisation.32

Tsai and Oslon stressed that Hg and its toxicity has become an

excellent chemical model for the study of transformation of metal salts.

According to them, the complete detoxification of Hg involves the reduction

of inorganic (Hg2+) or organomercurial ( a - H g + [R-Hg+]) form to the less

toxic elemental mercury (Hgo).@ They reported that mercury resistant

bacteria isolated from Hg- polluted sediments have been found to transform

organic and inorganic mercury to Hgo.@

Iverson and Brinckman also pointed out that, of the several forms of

mercury found in the environment, including elemental Hg, mercuric or

mercurous ions and organomercury compounds, an organic form of Hg,

methyl mercury, is the most toxic." 'According to them, several strains of

bacteria such as E.coli, and Pseudo~nonas species convert inorganic Hg(I1) to

elemental Hg."

Fisher and Reinfelder reported that Hg can be methylated by bacteria

and that methylated mercury were supposed to display greater

bioaccumulation than inorganic forms, partly because the methylated metals

are lipophilic and diffuse through biological membrane.17

Rapid accumulation of mercury, especially organomercury

compounds, by various species of algae under controlled conditions is

documented. Marine algae showed great inter-species variations in

sensitivity to marine insults. Phytotoxic effects include reduced growth,

developmental abnormalities, photosynthesis inhibition and death."