Use of aquatic fern for textile dye decolorization

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  1. 1. Efficient decolorization and detoxification of textile industry effluent by Salvina molesta in lagoon treatment By SULAIMAN ISHAQ MUKTAR 20162418 12.16.2016 1
  2. 2. Table of content Introduction Materials Methods Results Conclusion References 2
  3. 3. Introduction Dye processors use about 3500 different dyes, out of which, 84% is contributed by sulphonated azo dyes 1015% of the wastewater is discharged to the environment Dye effluents contain organic compounds, metals, salts directly affect water color,COD,BOD,TDS,TSS and pH. Treatment methods like filtration, flocculation, coagulation, adsorption, chemical oxidation, photodegradation designed for the treatment of textile effluents containing dyes are costly and produce secondary wastes. 3
  4. 4. Modern biological treatment using microorganisms are needed for the 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 simulated dye mixture and a real textile effluent by Salvinia molesta was studied. S. molesta which is an aquatic fern,has dense root system spreading over water was explored in a constructed lagoon for large scale treatment 4
  5. 5. It has the ability to grow naturally It is native of Brazil Requires 2030 C temperature and within a pH 4 and 9. Can grow in high salt concentration. 5
  6. 6. 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 6
  7. 7. Methods 1.Decolorization studies with S. molesta S. molesta plants with root of 602 g was used for phytoremediation studies S. molesta plants roots were washed with tap water and submerged in 500 mL dye solutions in 1000 mL beaker. Dyes namely Rubine GFL, Remazol Black B, Red RBL, Bottle Green No.9, Navy Blue Rx and Scarlet RR were used at concentration of 100 mg/L separately. Similarly, plant were exposed to 500 mL of the simulated dye mixture containing Rubine GFL, Remazol Black B and Red RBL at a concentration of 100 mg/L. 7
  8. 8. Aliquots of 2 mL taken from dye solution at intervals of 12 h, over the period of 72 h. Centrifuged at 4561g for 10 min Clear solution of Rubine GFL was taken at 530nm % decolorization was calculated as %Decolorization=(Initial absorbanceFinal absorbance/Initial absorbance)100. 8
  9. 9. 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 4C 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 9
  10. 10. One set of BOD bottle incubated at 20C 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 against sodium tiosulphate,with starch as indicator. After 5days incubation DO was measured,using the same procedure. Distilled water was used as blank. BOD5(mg/L)=(D0D5)(B0B5)dilution factor. where, D0 0 d DO, D5 after 5 d DO, B0 0 d blank and B5 after 5 d blank. 10
  11. 11. 3.3. Anatomical studies of stem during dye degradation The transverse sections of stem were taken at 12, 24, 36 and 48 h time interval after the exposure of Rubine GFL. The sections were mounted in glycerine and observed on a microscope. The plants again transfer to fresh water after 48 h experiment and were again studied for anatomical changes at 60 h. 11
  12. 12. 3.4. Analysis of photosynthetic pigments 5g of each control and treated plants leaves were taken in separate mortar and pestle. 50 mL of acetone (80%) was added at the time of crushing along with a pinch of MgCO3 powder After crushing, extract was filtered and then centrifuged at 2000g for 10 min For the estimation of chlorophyll content, Abs of supernatant was measured at 663 and 645 nm; while carotenoids were estimated at 470 nm. 12
  13. 13. 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 fine pieces They were then separately suspended in 50 mM potassium phosphate buffer of pH 7.4. Root and stem pieces were crushed Centrifuged for 20 min at 8481g at 4 C. Supernatant was used as an enzyme source. Dye degrading enzymes were assayed spectrochemically. 13
  14. 14. Lignin peroxidase (LiP) Veratryl alcohol oxidase Tyrosinase Azo reductase DCIP reductase Catalase Superoxide dismutase (SOD) Riboflavin reductase 14
  15. 15. 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 15
  16. 16. 3.6. Analysis of metabolites Decolorization of Rubine GFL was examined by UVvisible spectrophotometric analysis using crude extract, whereas metabolites were 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 was redissolved in small quantity of HPLC grade methanol and used for analytical study. 16
  17. 17. The metabolites were examined using HPLC FTIR GC-MS 17
  18. 18. 3.7. Analysis of phytotoxicity and total bacterial count before and after treatment Toxicity of effluents containing textile dyes has direct effects on the photosynthetic reaction and also arrests the growth of the plant. 50 seeds of each T. aestivum (monocot) and P. mungo (dicot) were taken separately in petri plate containing blotting paper Phytotoxicity assay was done at 302 C. Daily application of 5 mL untreated and treated Rubine GFL, dye mixture and textile effluent on separately above seeds to assess their phytotoxicity Distilled water was kept as control Shoot & root lengths were measured after 6 days. 18
  19. 19. 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 37C. 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. 19
  20. 20. 3.8. Phytotreatment in lagoons After the Lab studies ,S.molesta was used for Phytoremediation treatment 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 of effluent). Inlet and outlet were provided opp each other. 52,500L of effluent & S.molesta were spread on the lagoon. Sample collected from inlet as 0h 20
  21. 21. 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 hrs interval. 21
  22. 22. 22
  23. 23. 4. Results 4.1 Decolorization of dye Rubine GFL by S. molesta Wild plants of S. molesta was observed to decolorize various screen dyes such as Remazol Black B, Red RBL, Bottle Green No.9, Navy Blue Rx, Scarlet RR and Rubine GFL up to 48%, 61%, 58%, 64%, 69% and 76%, respectively within 60 h Looking at maximum decolorization with Rubine GFL it was taken for further studies Absorbance of withdrawn supernatant was measured at 530 nm which is the wavelength of its maximum absorbance. Within 72 hrs Rubine GFL has % decolorization of 97%. 23
  24. 24. 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 mixture Textile 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 24
  25. 25. 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. 25
  26. 26. 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 24.480.36 8.010.25 32.760.38 12.520.34 Test 27.730.26 10.010.23 37.520.31 17.790.27 26
  27. 27. Analysis of enzyme activity of S.molesta during dye degradation 27
  28. 28. Enzymes Salvinia molesta root cell Salvinia molesta stem cell Control Test % Induction Control Test % Induction Lignin peroxidase 4.00.01107 3.260.28108*** 716 2.720.01107 1.390.05108* 411 Veratryl alcohol oxidase 2.910.26108 8.561.16108* 193 2.890.48108 1.070.71107** 269 Laccase 1.990.75109 2.