Use of aquatic fern for textile dye decolorization

Efficient decolorization and detoxification of textile industry effluent by Salvina molesta in lagoon treatment

By

SULAIMAN ISHAQ MUKTAR

20162418

12.16.2016

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Table of content

• Introduction

• Materials

• Methods

• Results

• Conclusion

• References

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Introduction

• Dye processors use about 3500 different dyes, out of which, 84% iscontributed by sulphonated azo dyes

• 10–15% of the wastewater is discharged to the environment

• Dye effluents contain organic compounds, metals, salts directly affectwater color,COD,BOD,TDS,TSS and pH.

• Treatment methods like filtration, flocculation, coagulation,adsorption, chemical oxidation, photodegradation designed for thetreatment of textile effluents containing dyes are costly and producesecondary wastes.

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• Modern biological treatment using microorganisms are needed forthe treatment of wastewater which also has its own drawback.

• The use of phytoremediation of T.Dyes on a large scale remains scares

• The decolorization and degradation of dye Rubine GFL, a simulateddye mixture and a real textile effluent by Salvinia molesta wasstudied.

• S. molesta which is an aquatic fern,has dense root system spreadingover water was explored in a constructed lagoon for large scaletreatment

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• It has the ability to grow naturally It is native of Brazil

• Requires 20–30 °C temperature and within a pH 4 and 9.

• Can grow in high salt concentration.

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2. Materials

• 2.1. Chemicals

• 2, 2-Azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) and riboflavin.

• 2, 6-dichlorophenol indophenol (DCIP),

• nicotinamide adenine dinucleotide (di-sodium salt),

• n-propanol,

• Catechol,

• veratryl alcohol

• Tartaric acid

• The textile dyes Rubine GFL, Remazol Black B, Red RBL and effluent,

• The seeds of T. aestivum (monocot) and P. mungo (dicot) and S. molesta plant

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Methods1.Decolorization studies with S. molesta

• S. molesta plants with root of 60±2 g was used for phytoremediationstudies

• S. molesta plants roots were washed with tap water and submergedin 500 mL dye solutions in 1000 mL beaker.

• Dyes namely Rubine GFL, Remazol Black B, Red RBL, Bottle GreenNo.9, Navy Blue Rx and Scarlet RR were used at concentration of100 mg/L separately.

• Similarly, plant were exposed to 500 mL of the simulated dye mixturecontaining Rubine GFL, Remazol Black B and Red RBL at aconcentration of 100 mg/L.

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• Aliquots of 2 mL taken from dye solution at intervals of 12 h, over theperiod of 72 h.

• Centrifuged at 4561×g for 10 min

• Clear solution of Rubine GFL was taken at 530nm

• % decolorization was calculated as

• %Decolorization=(Initial absorbance−Final absorbance/Initialabsorbance)×100.

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3.2 Characterization of dye, dye mixture and textile effluent

• Rubine GFL

• Rubine GRL +Remazol Black B + Red RBL

• Textile effluent

• Characterize ADMI, BOD5 ,COD,TSS,TDS (APHA 1995),Heavy meatals(AAS).

• Samples where collected and stored at 4°C until use

• Dilluted sample was put into a 300mL BOD bottle

• Then 1mL phosphate buffer +1 mL MgSO4+1 mL CaCl2+1 mL FeCl3• Neutralized to pH 7 by using 1NaOH or H2SO4

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• One set of BOD bottle incubated at 20°C for 5 days

• Other sets were used for DO at the same time.

• 1mL KI and MgSO4 +2mL Conc H2SO4 stirred to dissolve ppt

• Aliqout of 50mL sample taken into conical flask and titrated againstsodium tiosulphate,with starch as indicator.

• After 5days incubation DO was measured,using the same procedure.

• Distilled water was used as blank.

• BOD5(mg/L)=(D0–D5)−(B0–B5)×dilution factor.

• where, D0 – 0 d DO, D5 – after 5 d DO, B0 – 0 d blank and B5 – after 5 dblank.

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3.3. Anatomical studies of stem during dye degradation

• The transverse sections of stem were taken at 12, 24, 36 and 48 htime interval after the exposure of Rubine GFL.

• The sections were mounted in glycerine and observed on amicroscope.

• The plants again transfer to fresh water after 48 h experiment andwere again studied for anatomical changes at 60 h.

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3.4. Analysis of photosynthetic pigments

• 5g of each control and treated plants leaves were taken in separatemortar and pestle.

• 50 mL of acetone (80%) was added at the time of crushing along witha pinch of MgCO3 powder

• After crushing, extract was filtered and then centrifuged at 2000×gfor 10 min

• For the estimation of chlorophyll content, Abs of supernatant wasmeasured at 663 and 645 nm; while carotenoids were estimated at470 nm.

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3.5. Preparation of crude extracts of root and stem tissue and enzyme assay

• 2 g of each root and stem of S. molesta were excised and cut into finepieces

• They were then separately suspended in 50 mM potassiumphosphate buffer of pH 7.4.

• Root and stem pieces were crushed

• Centrifuged for 20 min at 8481×g at 4 °C.

• Supernatant was used as an enzyme source.

• Dye degrading enzymes were assayed spectrochemically.

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Lignin peroxidase (LiP)

Veratryl alcohol oxidase

Tyrosinase

Azo reductase

DCIP reductase

Catalase

Superoxide dismutase (SOD)

Riboflavin reductase

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• 3.0 mL reaction mixture contains (Tyrosinase)

2.7mL potassium phosphate buffer (50mM,pH 6.8).

0.1mL catechol (1.5nM)

0.1mL L-ascorbic acid (2.5mM)

0.1mL crude extract & abs at 265nm

• All assays were done at room temp. Except crude extract.

• Enzyme sets were done in triplicate, average rate of test calculated

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3.6. Analysis of metabolites

• Decolorization of Rubine GFL was examined by UV–visiblespectrophotometric analysis using crude extract, whereas metaboliteswere examined using HPLC,FTIR and GC-MS.

• For the extraction of the metabolites after the dye decolorization,plants were removed from dye solution and then centrifuged

• The solution was extracted with equal volume of ethyl acetate.

• The extract was evaporated and dried. Obtained residue wasredissolved in small quantity of HPLC grade methanol and used foranalytical study.

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• The metabolites were examined using

HPLC

FTIR

GC-MS

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3.7. Analysis of phytotoxicity and total bacterial count before and after treatment

• Toxicity of effluents containing textile dyes has direct effects on thephotosynthetic reaction and also arrests the growth of the plant.

• 50 seeds of each T. aestivum (monocot) and P. mungo (dicot) weretaken separately in petri plate containing blotting paper

• Phytotoxicity assay was done at 30±2 °C.

• Daily application of 5 mL untreated and treated Rubine GFL, dyemixture and textile effluent on separately above seeds to assess theirphytotoxicity

• Distilled water was kept as control

• Shoot & root lengths were measured after 6 days.

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• 1mL sample textile effluent was collected before and after treatment.

• This sample was diluted ( 7 times), using 0.9% saline

• Sample was spread on nutrient agar medium & incubated at 37°C.

• The bacterial count was measured in terms of CFU

• N.agar composition g/L

Yeast extract 1.5,Peptone 5,NaCl 5,beef extract 1.5% ,1.5% agar.

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3.8. Phytotreatment in lagoons

• After the Lab studies ,S.molesta was used for Phytoremediationtreatment process of textile effluent at lagoon scale.

• The plants were stored in a lagoon for 15d containing tap water (5-8cm) and later 10-12 cm at the time of treatment.

• 7m*5m*2m of surface area 35m2

• The lagoon was mulched using mulching paper (for reducing loss ofeffluent).

• Inlet and outlet were provided opp each other.

• 52,500L of effluent & S.molesta were spread on the lagoon.

• Sample collected from inlet as 0h20

• Sample collected after treatment at 192h.

• Lagoon stired with steel rod before collection of samples.

• Effluent Parameters were analysed up to 8 days (192 h) with 24 hrsinterval.

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4. Results

4.1 Decolorization of dye Rubine GFL by S. molesta

• Wild plants of S. molesta was observed to decolorize various screendyes such as Remazol Black B, Red RBL, Bottle Green No.9, Navy BlueRx, Scarlet RR and Rubine GFL up to 48%, 61%, 58%, 64%, 69% and76%, respectively within 60 h

• Looking at maximum decolorization with Rubine GFL it was taken forfurther studies

• Absorbance of withdrawn supernatant was measured at 530 nmwhich is the wavelength of its maximum absorbance.

• Within 72 hrs Rubine GFL has % decolorization of 97%.

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4.2 Involvement of S.molesta in treatment of dye mixture and textile effluent Characterization of dye mixture and textile effluent before and after their treatment at lab scale

Dye mixtureTextile effluents

Parameter Untreated treated % reduction Untreated treated % reduction

ADMI 534 98 81 694 107 84

BOD5 (mg/L) 1490 492 66 1845 573 68

COD (mg/L) 1367 418 69 1652 576 65

pH 8.5 7.2 15 9.9 7.6 23

TDS (mg/L) 18 4 77 4380 794 81

TSS (mg/L) 25 12 52 640 235 63

Turbidity (NTU)

34 18 47 278 54 80

Hardness 280 110 60 540 190 64

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4.3 Anatomical analysis of S.molesta

• Anatomy of stem of S. molesta a) control plant, exposed to Rubine GFL b) 12 h, c) 24 h, d) 36 h, e) 48 h, f) after 48 h plant exposed to normal water and observed anatomical changes at 60 h.

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4.4 Alteration of photosynthetic pigment during decolorization

Chlorophyll and carotenoid content of S. molesta leaves before and after exposure to 100 mg/L with Rubin GFL over a period of 72 h.

Chlorophyll a Chlorophyll b Total chlorophyll Carotenoid

Sample (mg/mL) (mg/mL) (mg/mL) (mg/mL)

Control 24.48±0.36 8.01±0.25 32.76±0.38 12.52±0.34

Test 27.73±0.26 10.01±0.23 37.52±0.31 17.79±0.27

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Analysis of enzyme activity of S.molestaduring dye degradation

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Enzymes Salvinia molesta root cell Salvinia molesta stem cell

Control Test% Induction

Control Test% Induction

Lignin peroxidase

4.0±0.01×10−7 3.26±0.28×10−8*** 716 2.72±0.01×10−7 1.39±0.05×10−8* 411

Veratryl alcohol oxidase

2.91±0.26×10−8 8.56±1.16×10−8* 193 2.89±0.48×10−8 1.07±0.71×10−7** 269

Laccase 1.99±0.75×10−9 2.67±0.90×10−9* 34 1.98±0.21×10−8 9.77±0.90×10−8** 392

Tyrosinase 3.03±1.50×10−9 6.28±6.87×10−9* 106 3.14±1.17×10−9 5.99±1.32×10−9* 90

Catalase 5.92±0.04×10−7 1.74±0.14×10−8* 194 7.52±0.11×10−7 1.88±0.14×10−8* 151

Riboflavin reductase

6.72±0.48×10−8 3.71±0.27×10−8** −44 4.11±0.14×10−8 9.12±0.14×10−7** −77

NADH-DCIP reductase

3.73±12.22×10−10 6.32±34.91×10−10*

* 69 4.54±13.53×10−10 8.38±22.09×10−10*

** 84

Superoxide dismutase

3.44±0.44×10−8 6.09±0.06×10−8** 77 2.76±0.12×10−8 4.86±0.20×10−8* 75

Azo reductase 2.27±0.28×10−8 4.97±0.31×10−8*

119 9.32±0.19×10−8 4.83±0.53×10−8** -48

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Phytotoxicity testing of Rubine GFL, mixture of dyes and textile effluent before and after 72 h treatment in initial experiments using S. molesta in glass beakers

Parameters Water Rubin GFLDye metabolite

Untreated dye mixture

Treated dye mixture

Untreated textile effluent

Treated textile effluent

Triticum aestivum

Germination %

100 50 90 40 80 30 70

Plumule (cm)

9.11±0.46 2.98±0.35* 6.88±0.11$ 2.66±0.16* 4.88±0.18$ 2.28±0.09* 4.66±0.24$

Radicle (cm)

5.95±0.27 2.33±0.21* 4.56±0.33$ 1.96±0.37* 4.31±0.23$ 1.46±0.20* 3.68±0.14$

Phaseolusmungo

Germination %

100 40 90 30 90 40 80

Plumule (cm)

8.23±0.40 3.11±0.34* 6.28±0.16$ 2.30±0.11* 4.70±0.32$ 2.11±0.28* 4.63±0.19$

Radicle (cm)

6.43±0.30 1.93±0.32* 5.10±0.23$ 2.08±0.10* 3.93±0.08$ 1.53±0.22* 3.60±0.15$29

Characterization of untreated and treated textile effluent in lagoon grown with S. molesta.

Parameter Untreated effluent Treated effluent

pH 9.0 7.2

COD (mg/L) 1185 283

BOD5 (mg/L) 1440 249

ADMI 950 180

TDS (mg/L) 7560 2480

TSS (mg/L) 4730 1720

Cadmium (ppm) 0.03 0.01

Mercury (ppm) 0.0 0.0

Chromium (ppm) 3.45 0.80

Lead (ppm) 0.30 0.15

Nickel (ppm) 0.0 0.0

Arsenic (ppm) 1.70 0.49

Bacterial count (CFUs) 28×10−7 93×10−7 30

Conclusion

• S. molesta showed the potential to decolorize and degrade textileeffluent at laboratory scale as well as on field application in aconstructed lagoon.

• Further research on treating textile effluents on actual dye disposalsite is in progress by the authors.

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Thank you

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References

• APHA, 1995. Standard Methods for the Examination ofWater andWastewater, 19th edition. American Public Health Association,Washington D. C.

• Arnon,D.I.,1949 .Copper enzymes in isolated chloroplasts .Polyphenol oxidase in Beta vulgaris. Plant Physiol.24,1–15.http://dx.doi.org/10.1104/pp.24.1.1.

• Davies et al., 2005 L.C. Davies, C.C. Carias, J.M. Novais, S. Martins-Dias Phytoremediation of textile effluents containing azo dye byusing Phragmites australis in a vertical flow intermittent feeding constructed wetland Ecol. Eng., 25 (2005), pp. 594–605http://dx.doi.org/10.1016/j.ecoleng.2005.07.003

• Ferreira et al., 2014 R.A. Ferreira, J.G. Duarte, P. Vergine, C.D. Antunes, F. Freire, S. Martins-Dias Phragmites sp. physiologicalchanges in a constructed wetland treating an effluent contaminated with a diazo dye (DR81) Environ. Sci. Pollut. Res., 21 (2014),pp. 9626–9643

• Upadhyay and Panda, 200 R.K. Upadhyay, S.K. Pand Salt tolerance of two aquatic macrophytes, Pistia stratiotes and Salviniamolesta Biol. Plant, 49 (2005), pp. 157–159

• Rane et al., 2015

• N.R. Rane, V.V. Chandanshive, A.D. Watharkar, R.V. Khandare, T.S. Patil, P.K. Pawar, S.P. Govindwar Phytoremediation of sulfonatedRemazol Red dye and textile effluents by Alternanthera philoxeroides: an anatomical, enzymatic and pilot scale study Water Res.,83 (2015), pp. 271–281

• Patil et al., 2016 S.M. Patil, V.V. Chandanshive, N.R. Rane, R.V. Khandare, A.D. Watharkar, S.P. Govindwar Bioreactor with Ipomoeahederifolia adventitious roots and its endophyte Cladosporium cladosporioides for textile dye degradation Environ. Res., 146(2016), pp. 340–349

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Enzyme activities during dye degradation

Enzyme Root Stem

LiP 8.1 5.1

VAO 2.9 3.6

SOD 1.7 1.7

Laccase 1.3 4.9

Tyrosinase 2.0 1.9

Catalayse 2.9 2.5

DCIP 1.6 1.8

Azo reductase Increase in the root Decrease in the stem

Riboflavin reductase less less

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