review of journal

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. Table of content
• Introduction
• Materials
• Methods
• Results
• Conclusion
• References
2

3. Introduction
• Dye processors use about 3500 different dyes, out of which, 84% is
contributed by sulphonated azo dyes
• 10–15% 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. • 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. • 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.
5

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. Methods
1.Decolorization studies with S. molesta
• S. molesta plants with root of 60±2 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. • Aliquots of 2 mL taken from dye solution at intervals of 12 h, over the
period 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/Initial
absorbance)×100.
8

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 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
9

10. • 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 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)=(D0–D5)−(B0–B5)×dilution factor.
• where, D0 – 0 d DO, D5 – after 5 d DO, B0 – 0 d blank and B5 – after 5 d
blank. 10

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. 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 2000×g
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. 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 8481×g at 4 °C.
• Supernatant was used as an enzyme source.
• Dye degrading enzymes were assayed spectrochemically.
13

14. Lignin peroxidase (LiP)
Veratryl alcohol oxidase
Tyrosinase
Azo reductase
DCIP reductase
Catalase
Superoxide dismutase (SOD)
Riboflavin reductase
14

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. 3.6. Analysis of metabolites
• Decolorization of Rubine GFL was examined by UV–visible
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. • The metabolites were examined using
HPLC
FTIR
GC-MS
17

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 30±2 °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. • 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.
19

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. • 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

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. 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. 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. 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
26

27. Analysis of enzyme activity of S.molesta
during dye degradation
27

28. 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
28

29. 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 GFL
Dye
metabolite
Untreated
dye
mixture
Treated dye
mixture
Untreated
textile
effluent
Treated
textile
effluent
Triticum
aestivum
Germinatio
n %
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$
Phaseolus
mungo
Germinatio
n %
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

30. 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

31. Conclusion
• S. molesta showed the potential to decolorize and degrade textile
effluent at laboratory scale as well as on field application in a
constructed lagoon.
• Further research on treating textile effluents on actual dye disposal
site is in progress by the authors.
31

32. Thank you
32

33. 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 by
using Phragmites australis in a vertical flow intermittent feeding constructed wetland Ecol. Eng., 25 (2005), pp. 594–605
http://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. physiological
changes 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 Salvinia
molesta 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 sulfonated
Remazol 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 Ipomoea
hederifolia adventitious roots and its endophyte Cladosporium cladosporioides for textile dye degradation Environ. Res., 146
(2016), pp. 340–349
33

34. 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
34