Synopsis waste water treatment via bioremediation

7/23/2019 Synopsis waste water treatment via bioremediation

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“Manuciaple Waste Water Treatment ThroughBioremidation ”



INDEX

S.NO ONTENTS !"#E NO.

1. INTRODUCTION

2. REVIEW OF LITERATURE

3. OBJECTIVES

4. METHODOLOGY

5. SIGNIFICANCE

6. REFERENCES



INTRODUCTION

Bioremediation is a pollution control technology that uses biological systems to catalyze the

degradation or transformation of various toxic chemicals to less harmful forms.

Bioremediation is a cost effective and efficient method of decontamination that has become

increasingly popular now-a- days to reduce environmental pollution. In urban and semi-urban

colonies, sewage disposal has become an ecological problem (Moore, !!"#. $he effluents

from residential and industrial discharge constitute a ma%or source of water pollution. $he

industrial effluents were discharged into open drains which finally %oins the rivers (&umari et

al , ')#.

*astewater discharge of industries are ma%or issues of water pollution, contributing to

oxygen demand and nutrient loading of the water bodies promoting toxic destabilized a+uatic

ecosystem (Morrison et al, ' */ and *01, !!2#. 3igh or low p3 values in a river

have been reported to affect a+uatic life which alters the toxicity of other pollutant in one

form or the other (*/, !!)c#. low p3 value in a river impairs recreational uses of

water and affects a+uatic life. decrease in p3 values reduces the solubility of certain

essential element such as selenium and increases the solubility of many other elements such

as l, B, 1u, 1d, 3g, Mn and /e (*/, !!)c#.

*ater +uality characteristic of a+uatic environment arise from a physical, chemical and

biological interactions (euzane, !4! ee, !"!#. +uatic ecosystem balance get upset by

human activities, resulting in pollution which is manifested dramatically as fish 5ill, offensive

taste, odour, colour and unchec5ed a+uatic weeds. $he +uantity of waste in different phases of

a natural a+uatic system is reflected by the level of hardness, al5alinity, free 16' and other

physico-chemical parameters (78, !4)#. 3eavy metals such as lead, cadmium, mercury,

nic5el, zinc, aluminium, arsenic, copper and iron are mentioned as environmental pollutants,

which may cause severe poisoning conditions (ere5, !!! ias et al ., '' Ballantyne et

al ., !!!#.

$he availability of good +uality water is an indispensable feature for preventing diseases

and improving +uality of life (6luduro and dewoye, '4#. 9atural water contains some

types of impurities whose nature and amount vary with source of water for example metals

are introduced into a+uatic system through, weathering of roc5s and leaching of soils,

dissolution of aerosol particles from the atmosphere and from several human activities,

1



including mining, processing and the use of metal based materials (Ipinmoroti and 6shodi,

!!: deyeye, !!; saolu et al., !!4#. Metals after entering the water may be ta5en up

by fauna and flora and eventually, accumulated in marine organisms that are consumed by

human beings (saolu et al., !!"#.

<amuna is one of the important river of India. It is used as a source of drin5ing water and

irrigation but due to rapid industrialization, deforestation and urbanization there is a large

discharge of industrial waste and sewage into the river which is not safe for human beings,

animals, fishes and birds. 9ow a day direct use of river water for drin5ing purpose bears

significant problem (Bari5 and 8atel, ';#. /ertilizer industry is one of the ma%or water

consuming industries responsible for water and soil pollution of considerable magnitude

(=underamoorthy et al, '#. Most of the waste water is being discharged into surrounding

water bodies which disturb the ecological balance and deteriorate water +uality (=ingh et al,

')#. Most of the rivers and fresh water streams are seriously polluted by industrial wastes

which come from different industries such as those of petro-chemicals, fertilizers, oil

refineries, pulp, paper, textiles, sugar mills, steel, tanneries, distilleries, drugs and

pharmaceuticals, fibres, rubber, plastics etc.$he textile industries produce effluents that

contain several types of chemicals such as dispersants, levelling agents, acids, al5alis, carriers

and various dyes ( 1ooper,!!2#.

Many farmers use the effluents of factories for irrigation purpose. $hese effluents contain

many harmful materials. In recent years, the industrial effluents are used after treatment for

irrigation (6m et al , !!;#.

9ew technologies are being proposed to access the treatment of waste water. lgae form

one of the components in new technology for waste water treatment. lgal bioremediation

has been well studied over the past ; years by 0yther (!4'#, &uyuca5 (!""#, 0omero-

>onzalez ('# and *ang ('#.1onsiderable research efforts have been devoted to the

development of algal biosorbents to remediate pollutants.

lgae are important bioremediation agents, and are already being used in wastewater

treatment. $he potential for algae in wastewater remediation is however much wider in scope

than its current role (?oles5y, !! *ase and /orster, !!4#. Blue green algae

(1yanobacteria# are considered as a most primitive photosynthetic pro5aryotes which are

supposed to have appeared on this planet during the 8re-1ambrian period (sh and @en5ins,

')#. 8ossibly, these are first photosynthetic microorganisms which persisted over a period

2



of '-: billion years, performing an important role in evolution of higher forms. 1yanobacteria

are a uni+ue assemblage of organisms which occupy a vast array of habitats (bd llah, ')

and 3aande et al , '#.

1yanobacteria are very susceptible to sudden physical and chemical alterations of light,salinity, temperature and nutrient composition (Boomiathan, '2 and =emyalo, '!#.

1yanobacteria show immense potential in waste water and industrial effluents treatment,

bioremediation of a+uatic and terrestrial habitats, chemical industries, biofertilizers, food,

feed, fuel etc (1airns and ic5son, !4#.

Spirulina sp. is a cyanobacterium that grows rapidly (3enri5son, !"!# contains detectable

level of mercury and lead (=lotton et al ., !"!# when grown under the contaminated

condition this implies that it can ta5e up toxic metals from the environment. 1yanobacterial

species such as Oscillatoria salina, Plectonema terebrans, Aphanocapsa sp. and

Synechococcus sp., developed as mats in a+uatic environments, have been successfully used

in bioremediation of oil spills in different parts of the world (0aghu5umar et al., '

0adwan and l-3asan, ' 1ohen, ''#.

Most of the biological treatment technologies involve the use of bacteria, but

microalgae have already been applied for effluent treatment, either as single species, as is the

case of Chlorella, Scenedesmus or Arthrospira (Aee, ' Aima, '; Mulbry, ' and

?oltolina, '2# or as mixed culturesconsortia (Mulbry, ' 6gbonna, ' and $arlan,

''# to treat and remove nitrogen, phosphorus and chemical oxygen demand, from different

types of effluents. $hese organisms are also able to remove and incorporate heavy metals,

such as lead (5su, !!#, 1admium, nic5el or mercury (1hen, !!" and $ravieso!!!#

present in effluents and their use could be potentially more widespread. mong the several

microalgae used to treat effluents Chlorella is found to grow in a mixotrophic environment

(&arlander, !!)#. Investigations conducted by several researchers demonstrated that

Spirogyra sp. is capable of accumulating heavy metals li5e 1opper, 1hromium, Cinc and

/luoride (Bisnhnoi et al, '2#.

/ew species of marine algae such as Ascophyllum and Sargassum are effective in the

biosorption of pollutants (?oles5y and /ourest, !!) <u et al, !!!#. $he ma%or advantage of

this is that concentrations of heavy metals in the polluted environment are reduced to a very

low level. In the past ' years the use of immobilized enzymes or cell components for the

production of a series of metabolites has become a branch of biotechnology of rapidly

3



growing importance.

3uge load of wastes from industries, domestic sewage and agriculture practices find their

way into rivers, pond resulting in large scale deterioration of water +uality leading to the

availability of potable water. $here is an urgent need to screen and develop efficient alga for the bioremediation of waste water. &eeping this fact the research wor5 on D8otential of lgae

in Bioremediation of *aste *aterE will be ta5en.

4



REVIEW OF LITERATURE

Bioremediation is a newer approach directed towards the treatment of decontamination.

Bioremediation primarily deals with the strategies that can employ to clean up thecontaminants biologically. 0emoval and recovery of heavy metals from wastewater is

important for environmental protection and human health. Bioaccumulation process is 5nown

as an active mode of metal accumulation by living cells which depends on the metabolic

activity of the cell (Volesky, 1990; Wase and Foste, 199!".

Microalgae are not uni+ue in their bioremoval capabilities while they offer advantages over

other biological materials in some conceptual bioremoval process schemes. Microalgae

strains purposefully cultivated and processed for specific bioremoval applications and have

the potential to provide significant improvements in dealing with the world-wide problems in

metal pollution (Ed#ad and $ene%ann, 199&"' It is reported that biosorption of heavy

metals by certain types of non-living biomass is a highly cost-effective new alternative for the

decontamination of metal-containing effluents (ato)*+l and Volesky, 199-".Biosorption

of heavy metals from algae can be effective process for the removal and recovery of heavy

metals ions from a+ueous solution (ae#san, .00."'

Chlorella vulgaris and Scenedesmus dimorphus is highly efficient for ammonia and

phosphorous removal during biotreatment of secondary effluents from an agro industrial

wastewater of a dairy industry and pig farming. $hese microalgae were isolated from

wastewater stabilization pond. Both these microalgae removed phosphorous from the

wastewater to the same extent (L/ Estela onale, 199!"' ead dried Chlorella vulgaris

was studied in terms of its performance in binding divalent copper, cadmium, and lead ions

from their a+ueous or 2F vv methanol, ethanol, and acetone solutions. $he percentage

upta5e of cadmium ions exhibited a general decrease with decrease in dielectric constant

values, while that of copper and lead ions showed a general decrease with increase in donor

numbers (Al23/na4t, .009"

lgae have received increasing attention for heavy metal removal and recovery due to their

good performance, low cost and large available +uantities (Wan5 and C*en .00-". S.

incrassatulus was also able to remove all the tested metals to some extent ('2-4"F#, but

bivalent metals were not removed as efficiently as reported in batch cultures, probably due to

the high p3 values there recorded. 1hromium (?I# was more efficiently removed in

continuous cultures than in batch culture, because the upta5e of chromate could be favoured5



by actively growing algae (6e7a2Casto et al .008". Micro-algae can be used for tertiary

treatment of wastewater due to their capacity to assimilate nutrients. $he p3 increase which is

mediated by the growing algae also includes phosphorous precipitation and ammonia

stripping to the air, and may in addition act disinfecting on the wastewater (an

Lasdotte, .00".

lgae have been proven efficient biological vectors for heavy metal upta5e. Biosorption

potential of two strains Spirogyra sp. and Spirulina sp. has been studied under different initial

metal concentrations (:ane and $*osle .01.". $he use of live and dead Spirulina sp. for

sorption of metals li5e 1r:G, 9i'G, 1u'G and 1r)G in form of 1r '6' Spirulina sp. treated with

different metal ions have been employed to understand the sorption mechanism. It is hoped that

live Spirulina sp.will be a strong candidate for management of industrial wastewater

(Dos* et al .00!".

/</an :en5 (.011" reported the biodegradation rates of linear al5yl benzene suffocate

A= by =pirulina platensis increased with Cn (II# and reached the maximum when Cn (II#

was ; mgA. $he %oint toxicity test showed that the combined effect of A= and Cn (II# was

=ynergistic. A= can enhance the biosorption of Cn (II#, and reciprocally, Cn (II# can enhance

A= biodegradation. $ndya et al ( .01." observed the Bioaccumulation of 1admium in

Blue >reen lgae Spirulina (rthrospira# Indica.

Den5 et al (.00!" reported that Cladophora fascicularis green alga is highly efficient for

the biosorption of copper (II# from a+ueous solution. Biosorption is the effective method for

the removal of heavy metal ions from wastewater. 0esults are presented showing the sorption

of 8b (II# from solutions by biomass of commonly available, filamentous green algae

Spirogyra sp (/=ta and Rasto5 .00-"' *ala> (.00-" observed the Biosorption of reactive

dye from textile wastewater by non-viable biomass of Aspergillus niger and

Spirogyra sp.

:onteo et al (.009" observed that strains of the Scenedesmus obliquus microalga tested

have proven effective in removing a toxic heavy metal from a+ueous solutions, hence

supporting their choice for bioremediation strategies of industrial effluents. It was proven, for

the first time, that such a wild microalgae can upta5e and adsorb Cn very efficiently, which

unfolds a particularly good potential for bioremediation. Its performance is far better than

similar (reference# species, especially near neutrality, and even following heat-treatment

(:onteo, .011"'

6



a/s*k et al (.00-" reports on chromium (?I# tolerance of two cyanobacterial strains

Nostoc lincia and Nostoc spongiaeforme isolated from salt affected soils using uni-algal and

bi-algal systems. It was observed that the effectiveness of cyanobacterial concern because

they colour and diminish the +uality of water bodies into which they are released. $he

effectiveness of Oscillatoria was employed for the bioremediation of textile effluents

(A4a*a% and Nanda, .010".

6ado et al (.010" observed the rate of biosorption of cadmium and copper ions by non-

living biomass of the brown macroalga Sargassum sinicola under saline conditions. $hey see

that presence of salt did not significantly affect the rate of biosorption and there is an

antagonistic effect on biosorption when both these metals are present in the solution.

mong the several microalgae used to treat effluents Chlorella is often found from the

various types of waste water for the treatment of the water (alande and a/ss, 199"'

Ra=oso et al (.010" analyzed the capacity of Chlorella vulgaris and the autochthonous flora

of the effluents to remove some of the compounds present in the effluents. Ce)al et al (.01."

deals with a study of the biosorption of H6' '

ions on two green algaeJ Chlorella vulgaris

and !unaliella salina. By 5inetic investigations it was found that the biosorption process was

greater for Chlorella vulgaris than for !unaliella salina.

annan (.011" seen the detoxification capacity of a variety of microbes especially

cyanobacteria. $hey collected the effluents from tannery industry and added to the

cyanobacterial growth medium in various proportions. $he photosynthetic pigments and

nitrogen status of Anabena flos"aquae were analysed before and after the treatment with

effluents. It showed that Anabena flos"aquae can serve as the potential bioremedial organism

for industrial pollution.

Biodegradation and biosorption capacity of some potential cyanobacterial speciesJ

Oscillatoria sp., Synechococcus sp., Nodularia sp., Nostoc sp. and Cyanothece sp. dominated

the effluents and mixed cultures showed varying sensitivity. 1ontaminants were removed by

all the species either as individuals or in mixtures (D/4ey et al, .011". Lee and C*an5

(.011" observed the biosorption capacity from a+ueous solutions of the green algae species,

Spirogyra and Cladophora, for lead (8b (II## and copper (1u (II##. In comparing the analysis

of the Aangmuir and /reundlich isotherm models, the adsorption of 8b (II# and 1u (II# by

these two types of biosorbents showed a better fit with the Aangmuir isotherm model.

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:anda et al (.01." observed two species of cyanobacteria, Oscillatoria laete"virens

(1rouan K 1rouan# >omont and Oscillatoria trichoides =zafer which were isolated from a

polluted environment and it is studied for their 1r )

removal efficiency from a+ueous

solutions.

Bioaccumulation is the effective method for removal of heavy metal ions from

wastewaters. Bioremediation, the use of algal to extract, se+uester and or detoxify heavy

metals and other pollutants. $hey use filamentous alga of Pithophora sp. for the removal of

cadmium, chromium and lead from industrial wastewater ($a*%4*att et al .01."' Mercury

is higher in !unaliella alga as compared to those of cadmium and plumb. $his is vivid that

!unaliella is highly tolerant to the ascending concentration of heavy metals and their

absorption in a+uatic media. $his approves the usage of !unaliella as useful e+uipment for

the elimination of heavy metals environment (I%an et al .011".

ao and ?an (.01." observed the 0esponse of Chara globularis and #ydrodictyon

reticulatum to lead pollutionJ their survival, bioaccumulation, and defense they observed that

#. reticulatum exhibited higher tolerance to 8b pollution than C. globularis. =ome wor5ers

determine the feasibility of using algae growing in wastewater lagoons to absorb residual

heavy metals for subse+uent complete removal by algae-intermittent sand filtration system

and they found that this techni+ue is very helpful in removal of certain heavy metals from

wastewater (Danel et al, 19!9"'

$he ma%or problem in utilization of microorganisms in any industrial or waste water

treatment is harvesting of the biomass. $his is solved by the strategy of immobilization.

Immobilization techni+ue is essential not only in waste water treatment but also in various

industries (6akas*a% and Ra%aks*na 199-"'

6ne of the main interests for microalgae in biotechnology is focussed on their use for

heavy metals removal from effluents and waste water (:all)k, .00."'=ome research has

been done dealing with the immobilization of microalgae for different purposesJ morphology

studies, the production of fine chemicals, energy production, wastewater treatment etc.

Immobilization strains of microalgae is been used for sewage treatment. 7fficiency of

depuration was highest when a fluidized bed and Chlorella vulgaris were used (Ta+eso,

199."'

Hse of immobilized algae in wastewater treatment and heavy metal removal processes

efficient and offer significant advantages in bioreactors (a%eed and E4a*%, .00!"' Ta%

et al, 1998 used chlorella vulgaris cells immobilized in alginate beads for removing 9 and 88

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from wastewater and they achieved significant reductions in wastewater ammonia and

phosphate. Spirulina platensis, a cyanobacterium of economic important was studied for the

tolerance to cadmium.

$he Biosorption studies showed that the algae had a great potential for adsorbing the heavymetal on to the cell. $he immobilized cell of Spirulina platensis was able to be more effective

in absorbing the metal to the cell (://5esan et al , .00-"'

$he process of biosorption of trivalent chromium (1r :

# by live culture of Spirulina

platensis and the sorption potential by the dried biomass, in both free and immobilized states

have been investigated for simulated chrome li+uor in the concentration range of L;2

ppm. Both live and dried biomass were very good biosorbents as they could remove high

amounts of chromium from tannery wastewater (@*as*ek*a et al , .00-"



O$ECTIVE@

1' 1ollection of waste-water samples from waste water treatment plant in rcew.

.. $o study the wastewater samples collected from different sites with respect to L

2. a. 8hysico-chemical aspects

i. 1olour and p3

ii. cidity and l5alinity

iii. 3ardness

iv. $== ($otal =uspended =olids#, $= ($otal issolved =olids# and

$= ($otal =olids#

v. 6 (issolved 6xygen#, B6 (Biological 6xygen emand# and 16

(1hemical 6xygen emand#

vi. 6rganic 1arbon and mmonical 9'

'. b. Identification of lgae

3. 1ollection, Identification, Isolation and 1ulture of specific algae $Spirulina sps.,

#ydrodictyon sps., Spirogyra sps., Chlorella sps#

4. ssessment of the specific algal isolates ( Spirulina sps., #ydrodictyon sps.,

Spirogyra sps., Chlorella sps., its mixed culture etc# in the bioremediation of the waste

water samples and screening the most efficient algal isolate.

1!



:ETODOLO?

1. Colle)ton o> #aste2#ate sa%=les >o% d>>eent stes o> A5a'

*ater samples will be collected from different sites of gra in a ' litre bottle which

will previously washed with F 396: for ;" hr, labelled these bottles and few

drops of 396: will add to prevent loss of metals.

.' To st/dy t*e #aste2#ate sa%=les )olle)ted >o% d>>eent stes #t* es=e)t to B

.' a' 6*ys)o2)*e%)al as=e)ts

$he physico-chemical parameters of collected waste water samples will be

determined before and after treatment by following the =tandard Method

7xamination of *ater and *aste *ater given in D7nvironment and 8ollutionE of

mbast (!!# and 83 (!!"#. $he data will also be statistically analysed by

ta5ing the value of standard error (1handel, !!!#.

i) Colo/ and =

Colo/- the colour intensity of water will be observed from na5ed eyes.

=2 the p3 will be measured by the digital p3 meter. 1alibration of the

p3 meter will be accomplished by p3 electrode submerged in a p3 4 buffer

solution. p3 measurement will be made by placing p3 electrode tip )-"cm into

the sample and then recording the meter reading after the stabilization.

ii) A)dty and Alkalnty

A)dty2

Rea5ents2

o :et*yl Oan5e

o NaO

11



o 6*elo=*t*alen

6o)ed/e2

ml of water sample will be ta5en in a flas5 in which ' drops of methyl orange

is added it turned pin5 and then titrate it with .29 9a63 solution. t the end

point the pin5 colour change to light yellow. 9ow added :-; drops of

phenolphthalein and continued to titrate it until the end point from yellow to pin5

is changed. /ollowing formula is used to calculate the acidity-

cidity (mgl# as 1a16: 9a63 total titration vol. in mlN .29N N 2

ml of sample ta5en

Alkalnty2

Rea5ents2

o 6*enol=*t*alen nd)ato L .2gm phenolphthalein indicator will be

dissolved in 2ml of !2F ethyl alcohol and 2ml of distilled water .29

9a63 was added drop by drop until colour becomes %ust li5e pin5.

o Ind)ato %et*yl oan5e- .2gm methyl orange is dissolved in ml

distilled water.

o 0'1N Cl- it was prepared by ta5ing ".:;ml 31l and diluted it in litre

distilled water.

6o)ed/e2

$a5e ml water sample and add ' drops of phenolphthalein indicator. =olution

turns pin5 and titrated with the dil. 31l. $he end point come with sharp disappear of

pin5 colour volume of dil. 31l will be noticed. 9ow in same flas5 '-: drops of

methyl orange will be added and the colour of solution turns yellow. /urther titration

continued and a new end point reached when a solution in the flas5 is %ust turns to

pin5. $otal al5alinity will be calculated by following formula-

12



l5alinity $otal 31lN .9 31lNN2

ml of the sample

iii) adness- the hardness of water sample will be measured by 7$$itrimetric Method (mbast, !!#.

Rea5ents2

o $/>>e sol/ton (=10"

).!gm ammonium chloride will be dissolved in ;:ml of concentrate

ammonium hydroxide.

.4!gm di sodium 7$ (7thylene ditetra cetic cid# and .4"gm

Mg=6;.43'6 will be dissolved in 2ml distilled water. Both prepared solution

is mixed and volume it up to '2ml by adding distilled water.

o E)*o%e $la)k T nd)ato (E$T" L .2gm dye will be dissolved in ml

nitrotriethanol.

o EDTA Ttant (0'01:" - :.4': gm di =odium salt of 7$ will be dissolved in

distilled water and raised a volume of it upto litre with distilled water.

6o)ed/e2

2ml of sample will be ta5en in a conical flas5. -' ml of buffer solution and -'

drops of 7B$ indicator will be added into the flas5. $he solution turns wine red.

$he sample will be titrated against standard 7$ $itrant. $he sample will be

titrated up to the end point till the colour turn from wine red to blue and notice the

titrant reading. $he following formula will be used-

3ardness (mgl# 7$ used (ml# N

ml of sample.

13



iv) T@@ (Total @/s=ended @olds" TD@ (Total Dssol+ed @olds" and T@ (Total

@olds"

Total s/s=ended solds (T@@"

/or the measurement of $== a 5nown volume of sample will be titrated

through oven dried pre-weighted filter paper and the residue containing filter

paper was oven dried at O1 and again weighted.$== of the sample will be

calculated by following formula-

$== (mgl# initial weight of filter paper- final weight of filter paper

Total dssol+ed solds (TD@" L

*ater sample will be ta5en and then filtered it to remove suspended particles.

'2ml of clear filtrate will be evaporated in an oven at P1 in porcelain

disc. Measurement will be observed by-

$= (mgl# *'-*N

?

*here,

* weight of empty disc

*' weight of oven dried disc

? volume of sample ta5en (ml#

• Total @olds (T@"

$otal solids include both suspended and dissolved solids. It is calculated by

using the formula-

$= (mgl# $==$=

14



v) Dssol+ed Oy5en (DO", $olo5)al Oy5en De%and($OD" and C*e%)al Oy5en

De%and(COD"2

• Dssol+ed Oy5en (DO"

Rea5ents2

o Con)' .@O8

o :an5ano/s s/l=*ate sol/ton2 it will be prepared by dissolving :);gm

Mn=6; in distilled water and dilute to litre.

o Alkal odde ade sol/ton2 it will be prepared by dissolving 4gm

&63 and 2 gm &I in litre of distilled water. $hen gm sodium azide

(9a3:# will be dissolved in ;ml distilled water and added to above

solution.

o @ta)* sol/ton2 prepared by forming an emulsion of .2gm starch in a

bea5er with a small +uantity of distilled water. $his emulsion will be

poured in ml boiled distilled water and solution will be boiled 2-)

minute and settled overnight. =upernatant was ta5en as starch indicator.

o @od/% t*os/l=*ate sol/ton (0'1N" " ';."'gm 9a='6: will be dissolved

in boiled distilled water and on cooling diluted it in litre distilled water.

.'29 9a='6: will be prepared by diluting '2ml 9a='6: stoc5 to

ml distilled water.

6o)ed/e2

*ater sample will be collected without bubbling in the '2ml glass bottle. 'ml

each of mangnous sulphate and al5ali iodide azide solution will be added right at

the bottom of the bottle with separate pipettes. $he bottle will sha5e at least six

times. $he brown precipitate formed allowed to settle, 'ml concentrate sulphuric

acid will be added and sha5en the bottle to dissolve the brown precipitate. 2ml15



of solution is ta5en in a flas5 and then titrate it with sodium thiosulphate solution

(ta5en in burettes# till the colour change to pale straw. ' drops of starch solution

is added to the above flas5. $his changed the colour of the content from pale to

blue solution that is titrated again with thiosulphate solution till the blue color

disappears. $he total amount of sodium thiosulphate will be observed and

dissolved oxygen content in water (mgl# is calculated by following formula-

6 (mgl# ("Q N N 9# Nv

?

*here,

? volume of the sample ta5en

(ml# v volume of the titrant used

9 normality of the titrant

"Q it is the constant since .ml of .'2 sodium thiosulphate solution is

e+uivalent to .'mg of oxygen.

• $olo5)al Oy5en De%and($OD"2

Rea5ents2

o 6*os=*ate 4/>>e(= !'."2 ".2 gm &3'86;, '.42gm & '386;, ::.;gm

9a'386;.43' and .4gm 93;1l dissolved in litre of distilled water.

o :5@O8 sol/ton2 '.'2gm Mg=6;.43'6 will be dissolved in ml

distilled water.

o CaCl. sol/ton2 '.42gm 1a1l' will be dissolved in ml distilled water.

o FeCl& sol/ton- .'gm /e1l:.)3'6 will be dissolved in litre distilled

water.

o @od/% @/l=*te @ol/ton (0'0.N" 2 .242gm 9a=6: will be dissolved in

litre of distilled water.

16



6o)ed/e2

$he dilution water will be prepared by adding ml of each phosphate buffer

solution, magnesium sulphate solution, calcium chloride solution, ferric chloride

solution to litre distilled water. 'ml water sample will be added and aerated. $he

6 of undiluted sample will be determined which designated as 6!. $he desired

percentage mixture was prepared by adding sample in dilution water. 6ne bottle

will be filled with the mixture and designated as 6 and the other one with

dilution water (blan5# designated as 6'. Both bottles will be incubated at 'ᵒ1 for

2 days and after incubation, the 6 will be determined. $he B6 will be obtained

by the following formula-

B6 (mgl# RS(6'-6# NT (6'-6!#TU

C*e%)al Oy5en De%and(COD"

Rea5ents"

o 0'1: 6otass/% d)*o%ate sol/ton- :.)4)gm & '1r '64 will be

dissolved in litre distilled water.

o @od/% t*os/l=*ate (0'1:" 2 2."gm 9a='6: will be dissolved in

'litre of distilled water.

o @/l=*/) a)d (.:" 2 ."ml of concentrate 3'=6; will be dissolved in

ml distilled water.

o 10G o> 6otass/% odde sol/ton- gm &I will be dissolved in ml

distilled water

o 1G @ta)* sol/ton- gm starch will be dissolved in ml distilled water.

6o)ed/e2

2ml of water sample will be ta5en in triplicates of ml flas5 and triplicates of blan5

will also prepare. 2ml of & '1r64 solution will be added to each ) flas5s. $hen 5ept

these flas5 at O1 in water bath for hr. the samples will be allowed to cool for

minutes and then 2ml of &I will be added then add ml of 3'=6; in each flas5.

1ontent of each flas5 will be titrated in .M 9a'='6: till the appearance of pale

yellow color. ml of starch solution is added due to which the solution turns

17



pale yellow to blue and titrated it again until the blue colour disappear completely.

16 will be calculated by applying the formula-

16 of the sample (mgl# "N 1N (B-#

=

*here,

1 concentration of the titrant (mMl#

?olume of the titrant used for blan5 (ml#

B ?olume of the titrant used for sample (ml#

= ?olume of the water sample ta5en

+" O5an) Ca4on and A%%on)al N.

• O5an) Ca4on

Rea5ents2

o @/l=*/) a)d

o 0'1N Iodne sol/ton

o 6otass/% s/l=*ate

o C/@O8

o 0'1N @od/% t*os/l=*ate

6o)ed/e2

ml water sample will be ta5en in a round bottom flas5 (&%eldahlVs /las5#. :ml

of concentrate 3'=6; will be added. $hrough rubber stopper a thistle funnel will be

inserted into &%eldahl flas5, which dipped into sulphuric acid and water mixture.

$he side tube of the 5%eldahl flas5 will be connected to two flas5s arranged in

series containing 42ml of .9 iodide solution and the flas5s will be connected to

suction pump. Before inserting the thistle funnel, ;gm of potassium sulphate and

2gm of 1u=6; will be added to the mixture of sulphuric acid and water sample.

$he 5%eldahl flas5 will be heated with the help of burner till the clear blue colour

obtained. $he iodine present in flas5 will be titrated against .9 sodium

thiocyanate and the organic carbon will be estimated by using the formula-

18



ml of .9 iodine used.: gm of organic carbon

A%%on)al N.

In water the nitrogen content will be estimated by &%eldahl method (83, !!"#.

Rea5ents2

o @od/% *ydode

o 0'08N .@O8

o 6*enol=*t*alen nd)ato

6o)ed/e2

2 ml of water sample will be ta5en in 5%eldahl flas5 and neutralized it to p3 4. ml

of concentrate 3'=6;, ).;gm & '6; and .'ml 3g=6; solution will be added

to flas5. /ew glass beads also added into the flas5 to prevent bumping. ll the

material will be mixed and heated under a hood until white fumes will be

observed. $he material will be digested until the turbid samples will be turned

into straw colour. fter digestion, :ml of distilled water and 2ml of 9a63

solution will be added into the flas5. $he flas5 then connected to the distillation

unit. 6ne end of the distillation unit connected to &%eldahl flas5 and another end

to distillate containing 2ml of .;9 3'=6; solution. gain 5%eldahl flas5 is

heated for half an hour. blan5 reagent will also carried using all the steps of

procedures. $he nitrogen will be estimated by titration method, using

phenolphthalein as an indicator. $he nitrogen content present in the sample will

be calculated by using the formula-

9itrogen (mll# -B N '"

ml of sample used

*here,

volume of the titrant used for sample

B volume of titrant used for blan5

.'4' Ident>)aton o> Al5ae

Identified the algae present in these water sample collected from different sites of

gra.

1



3. Colle)ton, Ident>)aton, Isolaton and C/lt/e o> s=e)>) al5ae (Spirulina s=s',

Hydrodictyon s=s', Spirogyra s=s', Chlorella s=s"

ifferent types of algae will be sub%ected for the bioremediation of water.6ut of these,

few are collected locally ( #ydrodictyon sps.# from this water samples and identifies on

the basis of their morphological characteristics. =pecies of Spirulina will be previously

identified in Spirulina Aaboratory, epartment of Botany, .7.I, ayalbagh, gra while

remaining will be procured from I0I. $hen we will culture these algal samples in

different media. =ome will be cultured directly in water. *hile some algae will be

cultured in prescribed medium such as 1/$0I Medium and B>- Medium.

" CFTRI :ed/%

C*e%)als 5Hl

9a316: ;.2

& '386; .2

9a96: .2

& '=6; .

9a1l .

Mg=6;.43'6 .'

1a1l' .;

/e=6; .

*ater litre

2!



" $211 :ed/%

C*e%)als 5%Hl

9a96: .2 g

& '386; .; g

Mg=6;W43'6 .42 g

1a1l'W'3'6 .:) g

1itric acid .) g

/erric ammonium .) g

citrate

7$ (disodium salt# . g

9a16: .' g

$race metal mix 2 . ml

istilled water . A

p3 should be maintain 4. after sterilization

Ta)e %etal % A

C*e%)als 5%sHl

3:B6: '.") g

Mn1l'W;3'6 ." g

Cn=6;W43'6 .''' g 9aMo6;W'3'6 .:! g

1u=6;W23'6 .4! g

1o(96:#'W)3'6 ;!.; mg

21



4. Assess%ent o> t*e s=e)>) al5al solates ( Spirulina s=s' Hydrodictyon s=s'

Spirogyra s=s', Chlorella s=s', ts %ed )/lt/e et)" n t*e 4oe%edaton o> t*e

#aste #ate sa%=les and s)eenn5 t*e %ost e>>)ent al5al solate'

:ml of different water samples will be inoculated with :ml algal isolates of

particular density in a flas5 and 5ept it under illumination at :O1 then observed it

after the interval of days upto :days under aerobic condition. /or first ;" hr of

incubation, the flas5 will be 5ept in a sha5er at rpm for the purpose of uniform

mixing of algae and effluents. $hen periodically monitoring of the samples will be

done for investing the physiochemical characteristics and biodegradability of the

effluents. 6n the basis of 8hysico-chemical analysis the most efficient algal isolate

will be screened on the basis of their reduction efficiency in B6, 6 and 16.

22



@INIFICANCE

lgal bioremediation is considered as an efficient and environmentally safe technology for

inexpensive decontamination of polluted systems. It is widely used for heavy metal

removal from waste water. $he ob%ective of the proposed study is to isolate the most

efficient naturally occurring algal species with high bioremediation capabilities of water

bodies in gra.

23



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?olume ', Issue ! , 8ages 2'!4L2:;

<u X, Matheic5al @$, 8inghe <in, &aewsar 8 (!!!#. 3eavy metals upta5e capacities of

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