MATERIALS AND METHODS

2. MATERIALS AND METHODS

2.1. ISOLATION AND SELECTION OF A PHENOL DEGRADING STRAIN BY ENRICHMENT CULTURE

2.1.1 Sample collection

Soil samples were collectc?d from domestic laundry premises of

Kottayam town, Kerala.

2.1.2. Screening of phenol tolerating strains

109 soil collected from the detergent contaminated area was mixed with

90 ml of sterilized distilled water coitaining I mM phenol and was incubated in

a rotary shaker at 150 rpm at room temperature (30 * 2%) for 24 hours. The

soil samples were subjected to serial dilution using sterile distilled water and

were pour plated with nutrient agar medium (appendix V). The plates were

incubated at room temperature for 48 hours and the colony wunt was taken

(Ali et a/., 1998).The same procedure was repeated by the enrichment of the

soil extract progressively with 2wM, 5mM and lOmM phenol concentration

and the respective colony counts were taken.

2.1.3. Screening of the phenol degrading strains

All the 40 * 4 colonies isolated by soil enrichment technique were

individually inoculated into 10n11 of the Mineral Salt Phenol Medium

(MSPM) with ImM phenol conc:entration. The composition of the basal

medium used was l g KHZPO4, 1g(NH4)2S04, 0.5 g Mg S04. 7H20 and

0.001g CaC12 in 1 litre of the distilled water at pH 7. The tubes were

incubated on a rotary shaker at 150 rpm at room temperature (30 * 2%) for 2 days .The isolates, which showed growth in the broth and also on

plating with phenol containing nutrient agar medium were used to inoculate

individually into MSPM with 2mM phenol concentration. The same

procedure was repeated and ihe isolates, which showed growth, were

inoculated into MSPM with 5mM phenol concentration. The same

procedure was repeated with .OmM phenol containing MSPM. The

colonies, which showed growth in MSPM enriched with lOmM phenol

concentration was selected as the phenol degrading strains. The selected

cultures were purified by repeateaj streaking and were stored at 2 0 ' ~ in

50mM KH2POdK2HP04 buffer at pH 7.2 containing 20% (vlv) glycerol.

Working cultures were maintained by sub culturing every two weeks on

mineral salt agar slants plate and broth containing phenol at lOmM

concentration (Ali, et a/. 1998).

2.1.4. Selection of the most efficient strain for the biodegradation of phenol

The selection of the most efficient strain was done by inoculating

the mineral salt phenol medium .at lOmM phenol concentration with the

seven primary screened isolates. Phenol estimation was done at regular

intervals of 12 hours for five consecutive days and the strain, which gave

maximum degradation in minimurn incubation time, was selected as the

most efficient strain. All the isola-ies showed consistent growth upto five

days in MSPM with lOmM phenol concentration and were taken for

identification.

2.1.5. Phenol assay

Phenol concentrations were determined by a modified

spectrophotometric technique, based on a standard method for phenol

estimations (Mordocco et a/, 1999). From the phenol standards of known

concentrations different aliquots were taken in clean test tubes. The

aliquots were made into 5 ml using distilled water. To each of the

standards 50p1 2% Camino phenazone, 50~1, 25N ammonia solution and

25p1 8% potassium ferricyanide were added. The tubes were vortexed and

incubated for 15 minutes at room temperature (30 i 2OC). Optical density

was recorded at 500 nm (Shimadzu spectrophotometer) and a graph was

plotted with Optical Density on Y axis and phenol concentration on X axis.

The graph was used to calculate the unknown phenol concentrations.

2.2. IDENTIFICATION OF M E SELECTED BACTERIAL STRAINS

2.2.1 Morphological characteristics

The cell shape, size, arrangement and the spores of the isolated

bacterium were studied using the nticroscope.

2.2.2. Motility

Hanging drop slides were prepared from 18 h old nutrient broth

cultures and were observed under microscope.

2.2.3 Grams straining

Gram straining of all the isolated bacteria was done by the smear

prepared from 18 h old viable slant cultures. The slides were heat fixed

and treated with crystal violet for :30s. Rinsed gently in a stream of water,

allowed to dry, blotted, flooded with Grams iodine solution for 1 min,

washed with 95% alcohol for 20s and rinsed subsequently with water.

After drying it was flooded with saffranin as counter stain for 30 s, washed

gently with a stream of water, tllotted to dryness and observed under

immersion microscopes.

2.2.4. Biochemical tests

2.2.4.1 Carbohydrate fermentation

The ingredients (appendix I) were dissolved and pH was adjusted

to 7. Medium was sterilized at 15 lbs for 12 min, cooled and were

inoculated with the bacterial strain along with a control. The tubes were

incubated at 30 +- 2% for 48 h and the biochemical changes were noted.

2.2.4.2. Oxidation /Fermentation

Hugh Leifson's medium (appendix II) was melted and poured into

tubes. The tubes were sterilizer1 and cooled. Then the medium was

inoculated by stabbing on hard agar without allowing air bubbles to enter.

The slops were streaked with the same needle, incubated for 48h and

biochemical changes were observt?d.

2.2.4.3. Citrate test

The ingredients of citrate medium (appendix X) were dissolved,

transferred to culture tubes and sterilized at 15 lbs for 15 min. Agar slants

were streaked on the slants with sterile loop, incubated for 48 h at 3oe0C,

and the biochemical changes were noted.

2.2.4.4. lndole test

Tryptone broth was prepared (appendix XIII) poured into test tubes

and sterilized. The medium was inoculated with a loopful of culture and

incubated at 30 + 2OC for 48 h mixed with 3 ml of Kovac's reagent, shaken

well and allowed to stand for 5 mir!. The observations were recorded.

2.2.4.5. Methyl Red test (MR test)

The glucose phosphate broth (appendix Ill and IV) was sterilized

and inoculated with bacterial cult~re and incubated at room temperature

for 48 h. A few drops of methyl red indicator was added and observations

were noted.

2.2.4.6. Voges proskaeur test VP (test)

The tubes of glucose phosphate broth (appendix Ill and IV) were

inoculated with a loopful of bacterial cultures. The tubes were then

incubated at room temp for 48 h and 3 ml of 40% KOH solution was added.

The 0b~e~ations were noted.

2.2.4.7. Gelatin hydrolysis

Gelatin agar medium (appendix XIV and XV) was prepared

sterilized and was poured into pre-sterilized petriplates. The petriplates

were kept inverted for 24h and the bacterial cultures were inoculated by

streaking the surface. It was incubated at 30+iQ°C for 48 h and the medium

was flooded with 10 mi mercuric: chloride reagent. The changes were

noted.

2.2.4.8. Catalase test

A few drops of H2O2 was kept on a clean glass slide. A loopful of

isolated bacterial culture was placed into the drop and the observations

were noted.

2.2.4.9. Starch amylase test

Starch agar medium (appendix XI) was prepared, sterilized and

poured into presterilised petridishes. The petridishes were left for 24 h in

an inverted position for surface drying. The changes were noted. The

medium was inoculated with bacterial cultures by surface streaking

method. The petridishes were incubated at room temperature for two days.

The medium was flooded with geam's iodine solution and the changes

were noted.

2.2.4.10. Hydrogen sulfide production test

Sulfide indole motility aga, medium (appendix IX) was prepared,

poured into tubes and sterilized at 15 lbs for 15 min. The agar tubes were

stabbed with sterile needle containing bacterial culture and kept at 3 w 0 ~

for 48 h. The tubes were examined for presence or absence of black

precipitate along the line of inoculation and the observations were noted.

2.2.4.1 1. Triple sugar iron test

Ingradients of triple sugar iron agar medium (appendix XII) were

dissolved in 11 of dist. H,O and the pH was adjusted to 7.4. They were

distributed in tubes and were sterilized in an autoclave for 15 min at 121°C

under 151bs. The young cultures were stabbed and streaked over the

slop. Inoculated tubes were incubated at room temperature for 48 h and

the biochemical changes were noted.

2.2.4.12. Oxidase test

A piece of Whatman No: 2 filter paper was smeared with oxidase

test reagent (appendix VII). A loooful of organism were smeared over a

small area of the paper and the changes were noted.

2.3. OPTIMIZATION OF THE CONDITIONS OF PHENOL DEGRADATION

2.3.1. lnoculum preparation

One loopful of the culture was inoculated to 50 ml of peptone broth

containing 40 mM phenol. The flasks were incubated over night at room

temperature (30 * 2'~) at 150 rpm. From the culture the cells were

harvested by centrifugation. The pellets were collected and diluted using

physiological saline (0.86% NaCI) till the OD becomes 1. This was used as

the inoculum for process optimizatioai studies.

2.3.2. Effect of phenol concentration on speclfic growth rate

Growth curve of the organism at dierent phenol concentration

were obtained by inoculating 1%( vlv) of 1 OD bacterial suspension in

physiological saline into Mineral salt Phenol Medium at 20mM, 40mM,

60mM, 80mM. 100mM and 120mM phenol concentration and incubating at

room temperature (302+ OC) for five days. Samples were drawn at regular

intervals. Growth was estimated in terms of turbidity resulted in the culture

medium and expressed as optical density (0.D) obtained at 650 nm using

a spectrophotometer. The OD values were plotted against incubation time

in order to draw the growth cuwe. The specific growth rate of the

Alcaligenes sp d2 was determined at each concentration of the phenol and

a graph was plotted with specific growth rate against the respective phenol

concentration.

2.3.3. Effect of Substrate (phenol) concentration on the biodegradation of phenol

5% inoculum was added to the mineral salt phenol medium at different

concentrations of phenol as 2OmM, 40mM, 60mM, 80mM, IOOmM and 120mM.

The flasks were kept at room temperature at 150 rpm along with the control.

The percentage degradation of phenol at each substrate concentration was

monitored for five days and a graph was plotted with % of phenol reduction

against the substrate concentration The highest concentration of phenol at

which 100% phenol degradation txxxlrred was selected as the optimum

substrate concentration for phenol degradation process.

2.3.4. Effect of incubation period on the biodegradation of phenol

The basal medium containing 60mM phenol (optimum substrate

concentration) was inoculated with 3% inoculum and was incubated for 50

hours at room temperature at 150 rpm. Samples were drawn at every 2 h

and the percentage of phenol degradation was calculated in the cell free

supernatant. The minimum incubation period required to effect maximum

phenol degradation was selected as the optimum incubation period.

2.3.5. Effect of pH on the biodegradation of phenol

3% inoculum was added to the basal medium (MSPM) containing

60mM phenol as the substrate. The pH range was from 3.5-10.5. The

flasks were kept at room temperature at 150 rpm for 32 hours. The

percentage of phenol degraded afler 32 hours at each pH was noted. The

pH, which gave maximum pecentage of phenol degradation, was

considered as the optimum pH.

2.3.6. Preparation of immobilized cells

Alcaligenes sp d2, grown in mineral salt phenol medium(MSPM)

for 24 hours were harvested bq centrifugation at 10000 rpm for 30

minutes. These cells were suspenoed in physiological saline (0.86% NaCI)

at a cell concentration of 1 0.D and were mixed with 4% sodium alginate in

the ratio 1:2. The mixture was added drop wise to excess, 0.2M CaCI, to

get alginate entrapped cells (Jayachandran et ab, 1994).

2.3.7. Activation of immobilized viable cells

The immobilized viable beaus were activated for achieving maximal

activity using the mineral salt phenol medium. Prepared immobilized beads

were taken in large 500 ml beaker and immersed with phenol containing

minimal medium for varying time intervals. Optimal activation time that

promoted maximal activity, for immobilized cells was determined in terms

of percentage reduction of phenol.

2.3.8. Growth c u m of free and immobilized cells of Alcaligenes sp dd,

Mineral salt phenol medium (MSPM) was inoculated with 1 O/O

inoculum of 1 OD concentration in the case of free cells and 100

beads/l0Mnl in the case of immobilized cells. The quantification of the

growth of free cells was done by serial dilution of the sample followed by

viable counts at regular intervals of 4 hours. In the case of alginate-

immobilized cells, to recover bacteria for viable counts, known amount of

the beads were immersed in phosphate buffer (lM, pH-7), and dissolved

by vigorous shaking (Muyima and Clorte, 1995).

2.3.9. Biodegradation of phenol by immobilized cells of Alcaligenes sP d2

The mineral salt phenol medium was taken as 300 ml aliquots in

llitre flasks and was inoculated with 300 activated immobilized beads

(Jayachandran et a/, 1994). T7ese flasks were incubated at room

temperature (30* 2 OC) on a shak,sr at 150 rpm under optimized conditions

of phenol degradation. Samples were withdrawn at regular intervals of 4

hours for phenol estimations frorn the mineral salt medium and a graph

was plotted with% of phenol reduction against incubation period.

2.4. ANALYSIS OF THE PRODUCTS OF PHENOL DEGRADATION

2.4.1 Ortho lMeta cleavage test

100 ml of the MSPM was inoculated with 3% of the inoculum

(mentioned in 2.3.1 section) of Alcaligenes sp d2 and was incubated for 32

hours at optimized conditions 'of phenol biodegradation. lOml samples

were taken at an interval of 4 hours and were centrifuged at lOOOOg for 15

minutes at 4OC. From the concentrate 0.5 ml was resuspended in two ml of

0.2M tris buffer (pH8) and was added to 0.5 ml of toluene to solubilize the

membrane. This was shaker1 with 0.2ml of 1M catechol solution.

Appearance of yellow color wittiin few minutes was the indication of meta

cleavage activity. To 2.5ml of the cell suspension l g ammonium sulphate

was added and incubated for one hour at 30'~. The pH was adjusted to 10

with 0.5 ml ammonia (5N) and a drop of 1% sodium nitroprusside was

added to the mixture. Appearance of deep blue colour was observed for

the orthocleavage activity.

2.4.2. Analysis of the products of biodegredation by GClMS and FTnR

lOOOml of the MSPM was taken as equal aliquots in three IOOOml

flasks and were inoculated with the Alcaligenes sp d2 at 3% inoculum.

(Mentioned under 2.3.1 section).The flasks were incubated for 32 hours

under the optimized conditions of phenol biodegradation. The medium was

centrifuged at IOOOOg and the pellet was separated. The supernatant was

repeatedly extracted with the solvent ether and the extract was

concentrated by evaporation. This extract was used as the extract of

biodegraded MSPM (biodegraded phenol). A control was also kept by

incubating lOOOml of MSPM (without inoculation) under similar conditions

for 32 hours. It was also extracted similarly and was concentrated by

evaporation. This extract was used as the extract of uninoculated MSPM

(Phenol control). The two extract5 prepared under similar conditions were

subjected to GCIMS and FTIIR analysis at STIC, Cochin University,

Cochin, Kerala. The conditions used in the above analysis are specified in

appendix XVI and XVII.

2.5. ISOLATION AND PURlflCATlON OF POLYPHENOL OXIDASE FROM Ahdi~e- SP d,

2.5.1. Enzyme production in the MSPM by Alcaligenes sp d2

The Alcaligenes strain was inoculated to 500 ml of mineral salt

phenol medium (MSPM) and wa:; allowed to grow for 32 h under the

optimum conditions of phenol bicxlegradation. Atter 32 h the culture was

centrluged at 10000 rpm for 10 minutes. The cell free supernatant was

collected and used for estimating the enzyme activity.

2.5.2. Assay of polyphenol oxidase

The enzyme activity of the supernatant was determined by measuring

the oxidation of 40mM ABTS (2,2' azinobis benzthiazoline 6 sulfonate) in

50mM phosphate citrate buffer, pH 5 (Hublik and Schinner, 2000).

2.5.3. Preparation of partially purified enzyme concentrate

500 ml of the MSPM was inoculated with Alcaligenes sp d2 and

incubated for 32hours.The cell free supematant was collected after 32 h of

inoculation by centriiuging at a speed of 10000 rpm for 10 minutes at 4OC.

2.5.3.1. Ammonium sulphate precipitation

The cell free supernatani was subjected to ammonium sulfate

fractionation at different saturation concentrations starting from 30°h

saturation. The finely powdered ammonium sulphate was added very

slowly into the crude enzyme with continuous stirring at 4% in an ice bath.

The precipitated protein was removed by centrifugation at 10000 rpm for

20 minutes at 4Oc.~he same procedure was repeated for successive

saturation concentrations up to 90% ammonium sulphate saturation. The

enzyme assay (Hublik and Schinner, 2000)) and protein assay (Bradford,

1976) of all pellets at different anlmonium sulphate concentrations and the

corresponding supernatants were performed. The pellet, which gave

maximum activity for polyphenol oxidase, was taken. It was dissolved in

5ml of the 0.04M phosphate citrate buffer (pH 5) and was subjected to

dialysis.

2.5.3.2. Dialysis

The dialysis was performed in the cellulose acetate semi permeable

dialysis bag in excess of 0.04M phosphate citrate buffer (pH 5) taken in a

500 ml beaker. The dialysis was continued up to 24 hours at 4% with

frequent mixing and buffer changes. The protein and enzyme estimations

were done as mentioned earlier

2.5.3.3. Ion exchange chromatography

The dialyzed ammonium sulfate fraction was subjected to ion

exchange chromatography with tbe anion exchange resin, DEAE cellulose

(Sigma, USA).

109 of DEAE cellulose was weighed and equilibrated in 0.04M-

phosphate citrate buffer (pH 5) for 24 h. The pre swollen DEAE cellulose

was packed without trapping air bubbles into a column of 3x20 cm size.

The column was stabilized and equilibrated by pre running with the same

buffer for 1 h (0.5 mllminute flow rate) at 4%. The dialyzed fraction was

taken and the pH was made to 5. This was introduced from the top of the

column and was allowed to bind with the column. The column was washed

with the same buffer and the eluant was tested for enzyme activity and

protein activity as mentioned earlic!r. Elution of the enzyme was done with

a phosphate citrate buffer of a 'concentration ranging from 45 mM to

360mM.The collected fractions were analyzed for enzyme activity and

protein activity as mentioned earlie[,.

2.5.3.4. Native Polyacrylamide gel 13lectrophoresis (Native PAGE)

The purified enzyme was subjected to electrophoretic studies to

confirm purity. Electrophoresis was performed as suggested by Hames

(1990) using polyactylamide.

Stock solutions

1. Actylamide - bisactylamide (30:0.8) was prepared by dissolving 30 g

of acrylamide and 0.8 g of bis.acrylamide in a total volume of 100 ml

of distilled water. The solutiori was filtered through Whatman No: 1

filter paper and stored at 4OC i I a dark bottle.

2. TEMED (N,N,N',N'- tetrametlryl ethylene diamine) was used as such

which was stored in a dark bottle at 4' C.

Stacking gel composition (2.5%)

Acrylamide- bisacrylamide -2.5 ml

(30:0.8 g/ 100 ml distilled water)

Stacking gel buffer stock - 5.0 ml

10 % Ammonium per sulphate

Distilled water

TEMED

Resolving gel composition (1 0%)

Acrylamide - bisacrylamide

Resolving gel buffer

10 % Ammonium per sulphate

Distilled water

TEMED

- 10.0 ml

- 3.75 ml

- 0.15 ml

- 14.45 ml

- 0.02 rnl

Stacking gel buffer stock -Tris-tic1 (pH 6.8)

6.0 g Tris was dissolved in 40 ml of distilled water and titrated to a

pH of 6.8 with 1.0 M HCI and rnade upto a final volume of 100 ml. It was

filtered through Whatman No: I filter paper and stored in a refrigerator.

Resolving gel buffer stock - Tris; - HCI (pH 8.8)

36.3 g Tris was dissolve~-l in 48 ml of 1 .O M HCI and made up to a

final volume of 100 ml. It was filtered through Whatman No: I filter paper

and stored in a refrigerator.

Reservoir buffer

3.03 g Tris and 14.4 g g1yc:ine were dissolved in distilled water and

made up to 1000 ml with distilled water (pH 8.3).

Sample Preparation

Sample Buffer

Distilled water : 3.0 ml

Stacking gel buffer : 1.0ml

Bromophenol blue (0.5 %) : 0.4 ml

Sucrose (1 0%) : 1.6 ml

Enzyme solution and sample buffer are mixed in the ratio 1:l.

Pre-running of the gel

The glass plates with the gel were clamped to the electrophoresis unit

and connection was made to catt~ode and anode. The tanks were filled

with rese~oir buffer, which contain tris base and glycine. pH was

maintained as 8.3 and the pre-running was continued for '/2 h.

Sample loading

The crude sample, dialysed ammonium sulfate fraction and the

anion exchange fraction were loaoed. The 260 mM column fraction that

showed the maximum activity was used as the purified enzyme fraction for

loading. 20pI of the sample was mixed with the sample buffer, which

consists of glycerol and the tracking dye, bromophenol blue. The pre

running was stopped and the sariple was loaded into the wells. The

marker (Genei, Banglore) was loaded separately .The marker contained

four protein markers of molecular weights 66, 43,29 andl4 kilodaltons.

Gel running

After sample loading the electrophoresis was performed for 5 h at

4OC at a voltage of 80V. After running the gel was carefully taken out as

and was subjected to silver staining.

Silver staining

The gel was transferred to a clean plastic container and washed in

the washing solution with slow shaking for 10 min. The washing solution

was discarded and the gel was rir~sed with plenty of water for 2 minutes.

The gel was immersed in sodium :hiosulfate solution for 1-2 min. The gel

was washed with water twice, each time 1-2 min. and the washed water

was drained off. The gel was soaked in silver nitrate solution for 10' with

gentle shaking .The gel was washed with water twice, each time 1-2

minutes. The washed water was drained off. The developer solution was

poured to the container and the gel was slowly shaken. The proteins

reduced the silver nitrate and yellow-brown colored bands appeared.

When sufficient intensity was dt?veloped for the bands of the bands

developed, citric acid solution was added to stop the reaction. The

protein-banding pattern was recorded by photography.

2.6. TREATMENT OF THE PHENOLIC PAPER FACTORY EFFLUENT

2.6.1 Sample

Raw effluent from Hindustan Newsprint Factory, Velloor,

Kottayam, and Kerala, India was used in the present study as phenolic

effluent

2.6.2 Characterization of the effluent

2.6.2.1. Analytical methods

The phenolic effluent was a~ialysed for Total solids, Chemical Oxygen

Demand (COD) and Biological Oxygen Demand (BOD) (APHA, 1989).

2.6.2.2. Total solids (TS)

Total solids (both dissolved and suspended solids) present in the

effluent were determined based on the method suggested by

APHA (1 989).

Total Suspended Solids (TSS)

1. A known volume of sample (100 ml) was filtered through a tarred

crucible ignited to constan weight (w,) and the crucible was dried

at 130°C for 1 h.

2. Later, the crucible with the contents was cooled in a desiccator and

the weight of the crucible was recorded (w2).

3. Suspended solids were calculated using the formula.

w , - w , x106 Total suspended solids (mg/l)=

volume of the sample

Total dissolved solids (TDS)

1. A known volume of the filtrate (100 ml) obtained from the above

experiment was taken in a tarred dish ignited to constant weight (w,).

2. The dish with the contents was dried at 130°C for 1 h, cooled in a

desiccator and weighed (W2).

3. Dissolved solids were calc~~lated using the formula

w, -w,x106 Total dissolved solids (mg/l)=

volume of the sample

2.6.2.3. pH

Measurement of pH was carried out using a pH meter (Systronics

digital pH meter).

2.6.2.4.Biochemical Oxygen Demcind

The biochemical oxygen demand was estimated as per the official

method in APHA (1989).

1. The collected samples wore diluted before incubation to bring the

oxygen demand and supoly into an appropriate balance. One litre

of distilled water was miwed with nutrients. 1 ml each of buffer,

calcium chloride magnesium sulfate and ferric chloride.

2. Samples were neutralized to pH 6.5-7.5 with 1 M H2S04 or 1M

NaOH.

3. The DO of the sample w3s determined initially and after 5 days of

incubation in a BOD incubator at 20°C.

4. A blank was also carried out simultaneously.

5. The BOD5 was then calculated by the following formula.

BOD5 at 20°C in mg/l= (I)o-D5)-(Co-C5)xdilution factor

Dilution factor= lOOC -

vol.of sa rnple

Where

Do - - DO content of the sample on the 1" day

D5 - - DO content of the sample on the tith day

co - - DO content of the blank on the IS' day

c5 - - DO content of the blank on the 5'h day

2.6.2.5. Chemical Oxygen Demana (COD)

Chemical Oxygen Demancl was determined following the official

method mentioned in APHA (1989).

1. 10 ml of the sample was diluted to 500 ml using distilled water.

2. 50 ml of the diluted sample was taken in a round bottom flask

(R. 6. Flask) for COD determination.

3. l g HgS04 was added to the above sample to overcome the

difficulties caused by chloricle ions.

4. 5 ml of con. HzSO4 was added to dissolve the HgSO.,.

5. 1 g AgS04 was then added to the above mixture as a catalyst.

6. To the above solution 25 n ~ l of 0.25 N potassium dichromate was

added.

7. The RB flask was attached to the condenser and the water was

allowed to flow.

8. 70 ml of con. H2S04 was added through the open end of the

condenser and swirling was continued while the acid was being

added.

9. The contents in the flask were refluxed for 2h, cooled, washed into

a 500 ml beaker and was suitably diluted and made upto 140 rnl.

10. 3-4 drops of ferroin indicator was added and the contents were

titrated against ferrous ammonium sulfate (0.25 N).

11. The end point of the titration was the first sharp colour change from

the blue-green to reddish t,rown.

12. A blank was also run sin~ultaneously in the same manner using

distilled water.

13. The COD then calculated using the formula.

( A - B ) X N O ~ Fe(NH,),SO,x8~1000 COD mg/l=

voll~me of sample

Where A= volume of F:e (NH& SO4 consume for blank (ml)

B= volume of Fe (NH4), SO4 consumed for sample (ml)

2.6.3. Preparation of immobilized cells

lmmobilised viable cells were prepared as mentioned in the section

2.3.6. and 2.3.7.

2.6.4. Growth curve of free and immobilized cells in effluent

The effluent was inoculated with 1 % inoculum of 1 OD concentration

in the case of free cells and 100 beadsJ100ml in the case of immobilized

cells. The quantification of the growth was done by serial dilution of the

sample followed by viable counts at regular intervals of 4 hours. In the case

of alginate-immobilized cells, to recover bacteria for viable counts, known

amount of the beads were immersed in phosphate buffer (lM, pH-7). and

dissolved by vigorous shaking (Muyima and Clorte, 1995).

2.6.5 Treatment of the effluent by free and immobilized cells of Alcaligenes sp d, under batch process

The phenolic effluent samples were taken as 300 ml aliquots in llitre

flasks and were inoculated with the 18 hour old culture at 5% inoculum level

in free cell treatment and 300 activated immobilized beads in immobilized

cell treatment (Jayachandran et ai, 1994). These flasks were incubated at

room temperature (30 * 2 OC) on a shaker at 150 rpm. Samples were

withdrawn at regular intervals of L. hours for phenol and chemical oxygen

demand estimations. The graph w,as plotted with %reduction in phenol and

COD along Y axis and incubation period along X axis.

2.6.6. Treatment of the effluent by immobilized cells of Alcaligenes sp d2 under continuous process

Continuous treatment of the phenolic effluent was performed with a

packed bed reactor in a glass column (30 cm length and 6cm diameter).

lmmobilised cells in beads (number of beads- 1050 approximately) were

packed to a height of 25 cm in the glass column carefully without trapping

any air bubble. After activation of the immobilised beads with phenolic

effluent, the packed bed reactor was exposed to the effluent at varying

flow rates of 2.5 ml /hour, 5 mWttour, 7.5 rnl/ hour and 10 ml /hour. The

percentage reduction in phenol and chemical oxygen demand of the

respective samples were also estimated.

2.6.7. Treatment of the phenolic pager factory effluent by the bioaeactsr

2.6.7.1. Monitoring of the colour of the effluent

The monitoring of the colour of the phenolic paper factory effluent

was performed by finding out the absorption maxima. The coloured effluent

was scanned in a wavelength range from 200 nm to 1100nm and the

wavelength which showed maximum absorbance was taken as the

absorption maxima. The intensity of the colaur of the effluent was

expressed as the optical density at the absorption maxima (Edwards et a/.,

1999).

2.6.7.2. Designing of the bioreactor

The bioreactor was made with poly acrylic sheet of 4mm thickness

as three separate reactors of same dimension of 9.5cm x9.5 cm. x 13cm.

Each reactor was equipped with one removable plastic filter of Imm pore

size at both bottom and top of the reactor. These three reactors could be

used as individual units and also in association with each other. One of

these reactors was equipped with an inlet (First reactor-plate 1) and.

another was equipped with an outlet (Third reactor- plate 3). The remaining

one was considered as the second reactor (Second -plate 2).The second

reactor was used only in the three stage process as the final stage reactor

with chitosan coating.

In the reactors the inlet was slightly projecting into the bottom

space of the reactor below the filter thereby giving a; enclosed bottom slot.

Powdered Activated Charcoal (PAC) was packed in one such bottom slot

(plate 4). The immobilized cells were packed over the filter of the reactor

(plate 5).The pretreated chitosan (plate 6, plate7) was layered as a $oat

over the filter of reactor.

Plate I

First Reactor

Plate 2

Second Reactor

Plate 3

Third Reactor

plate 4

The Bottoms Space for PAC Packing in the Reactor

Plate 5

The Filter Bed of the Reactor for packing Immobilized Cells

Plate 6

The Space for Coating Chitosan in the Reactor

Plate -7- .. . . .

Chitosan Coating in the,Reactor

2.6.7.2. Treatment of the effluent with PAC in the first stage of the reactor

Treatment of the effluent with powdered activated charcoal (PAC)

was done individually with the first reactor. The PAC was packed in the

bottom space of the first reactor up to a height of and the effluent was

passed slowly from the top of the reactor so that it falls along the side of

the reactor (plate 8). The effluent on reaching the bottom space of the

reactor slowly rises along the packed PAC and gets eluted along the inlet

pipe projecting into the bottom space (plate 4). The experiment was

conducted at different flow rates of 25mllhour, 50ml/hour, 75 mllhour,

100ml/hour and 125 milhour with a peristaltic pump (Pharrnacia). The

%reduction in colour, phenol and COD were evaluated and a graph was

plotted with %reduction in colour, phenol and COD alo,ng Y axis and flow

rates along X axis.

Plate 8

Treatment of the Effluent with PAC packed In the Reactor

2.6.7.3. Treatment of the effluent with Immobilized cells

Treatment of the effluent with imobilized cells was done individually

with the second stage of the reactor. The immobilized cells

(1500approximately) were packed on the filter of the reactor and the

effluent was passed from .the bottom of the reactor so, that it slowly rises

through the immobilized bed (plate 9). The experiment was conducted at

different flow rates of 25ml/hour, 50ml/hour, 75 mllhour, 100mllhour and

125 mllhour with a peristaltic pump(Pharmacia). The %reduction in colour,

phenol and COD were evaluated and a graph was plotted with %reduction

in colour, phenol and COD along Y-axis and flow rates along X axis.

Plate 9

Treatment of the Effluent with Immobilized Cells

2.6.7.4. Treatment of the effluent with chitosan coated bioreactor

Treatment of the effluent with chitosan coating was done

individually with one of the reactors. The chitosan coating was made on

the filter bed of the reactor and the effluent was passed from the bottom of

the reactor so that it slowly rises through the chitosan coating (plate 10).

The experiment was conducted at different flow rates of 25ml/hour,

50ml/hour, 75 mllhour, 100ml/hour and 125 mllhour with a peristaltic

pump(Pharmacia). %Reduction in colour, phenol and COD were evaluated

and a graph was plotted with %reduction in colour, phenol and COD along

Y-axis and flow rates along X axis.

Plate 10

Treatment of the Effluent with Chitosan Coating

2.6.7.5. Treatment of the effluent with the three stage bioreactor.

Treatment of the effluent was also attempted with the three stages of

tha reactor connected in series (three stage reador) as PAC packed reactor,

immobilized cells packed reactor and chitosan coated reactor. The effluent

was passed slowly from the top of the first reactor witfithe help of a peristaltic

pump so that it falls along the side of the reactor into the bottom space of the

first reactor packed with PAC. The effluent coming out from the first stage of

the reactor was slowly rising from the bottom of the second reactor. It slowly

rises through the immobilized cells in the second stage of the reactor and then

through the chitosan coating in the third stage of the reactor (plate I I). The

experiment was conducted at flow rates of 25mllhour using a peristaltic

pump(Pharmacia). The %reduction in color, phenol and COD were evaluated

and a graph was plotted with %reduction in color, phenol and COD along

Y-axis and flow rates along X-axis.

Plate 11

Treatment of the Effluent with the Three Stage Reactor