Characterization of new algae isolated from textile wastewater plant

0 downloads 0 Views 92KB Size Report
Oct 2, 2009 - 4916-7432. 6432. Unit Color Pt-co. 67-225. 147 .... 22Jeffrey, S. W., Mantoura, R.F.C. and Wright, S. W. 1997. Phytoplancton pigments in ...
WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: [email protected]

www.world-food.net

Journal of Food, Agriculture & Environment Vol.7 (3&4) : 700-704. 2009

Characterization of new algae isolated from textile wastewater plant Jihane Cheriaa*, Fadhila Bettaieb, Ikbel Denden and Amina Bakhrouf

*

Laboratory of Analysis, Treatment, Valorization of Environmental Pollution and Products "LR01ES16", Faculty of Pharmacy, Department of Microbiology, Avicenne Street, 5000 Monastir, Tunisia. *e-mail: [email protected], [email protected] Received 11 May 2009, accepted 2 October 2009.

Abstract A new microalga was isolated from a clarifier tank of an industrial textile plant. The main goal of this study was to identify and to determine optimal culture conditions of the isolated alga and to test its involvement in reducing dye pollution. The pollution reduction of four industrial textile dyes (indigo, remazol brilliant orange, direct blue and crystal violet) was evaluated with chemical oxygen demand (COD) exhaustion and with alga color removal measured under shaking conditions. Our results showed that unicellular alga has both autotrophic and heterotrophic growth with a photosynthetic activity producing pigments (chlorophyll a), thus the new isolated unicellular green alga may be Chlorella. For the green alga, optimum culture conditions were pH 8, temperature 25°C and salinity at 15 g L-1. Furthermore, this alga has the capacity to reduce COD and color removal of indigo textile dye at rates of 89% and 46%, respectively, within five days. The results suggest the importance of unicellular green alga as an environmental preservation system. Key words: Autotrophic, dyes, heterotrophic, green microalga, treatment, decolorization, salinity.

Introduction There are more than 100,000 available dyes and different pigments widely used in various stages of dyeing treatments and processing, thus the pollution generated by dye materials is very important 1. Dye presence, as little as 10 to 20 mg L-1, in water affects water transparency and causes a part of aesthetic deterioration 2, 3. In many cases, color is the most easily detected parameter and, also, constitutes the most quickly measurable unit 4. The use of microbial consortia offers considerable advantages over the use of pure cultures in the synthetic dyes degradation 5. Indeed, color removal can be achieved by using different microorganisms belonging to different taxonomic groups of bacteria, fungi such as white-rot fungi, yeasts and algae that have been reported for their ability to decolorize a wide range of dyes 6. Although, bacteria play a key role in the biodegradation of organic pollutants, recent studies have indicated that in addition to providing oxygen for aerobic bacterial biodegraders, microalgae can also biodegrade organic pollutants directly 7, 8. It was reported that more than 30 azo compounds were biodegraded and decolorized by Chlorella pyrenoidosa, Chlorella vulgaris and Oscillatoria tenuis in which azo dyes were decomposed into simpler aromatic amines 9. Microalgae comprise diverse group of prokaryotic and eukaryotic organisms with great ecological importance. Based on numerous biochemical and cellular differences, two major groups of green microalgae are recognized: Chlorophyta and Conjugaphyta 10. Microalgae contain about 50% of global organic carbon fixation11. Many species are source of natural products such as pigments, enzymes, unique fatty acids, vitamins 12 and continue to be used for biotechnological applications 13-15. At first step, the present 700

study aims to identify a new unicellular alga in textile wastewater; secondly, to determine the effect of some parameters, pH, salinity and temperature, on alga growth and the color removal capacity of four industrial textile dyes (ITD), indigo (IND), direct blue (DB), remazol brilliant orange (Rbo) and crystal violet (CV) used in Tunisian textile dyeing industry. Materials and Methods Water sampling:Samples of water were purchased from the secondary clarifier (SC) textile wastewater Tunisian plant. The main characteristics of water samples were indicated in Table 1. For the first time we isolated the alga on Chlorella agar medium, pH 4.5 ± 0.2 (Sigma, Product of India, C9720), in Petri plates incubated at 25°C over one week under daylight. Cells were plated on nutritive agar (Institut Pasteur, France) growth medium in Petri plates and allowed to develop colonies at same conditions. Green colonies were selected for further analysis of morphology cells and pigments production, to determine cells growth conditions and their ability to reduce dyes pollution. Morphological observations: Fresh samples culture of isolated alga and a Hücker modified Gram stain, were preliminarily investigated light microscopy (LM), using a microscope (Orthoplan, Leitz Wezler, Germany). Scanning electron microscopy (SEM): Cells algal cultures were harvested by centrifugation (5000 rpm × 10 min), washed with PBS and fixed with 0.5% glutaraldehyde and 1% formaldehyde,

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009

dehydrated by successive passages through 50, 80 and 100% ethanol (three times), washed with water and spread over supporting glass squares and dried at room temperature 16, 17. Qualitative analysis of pigments: The profile pigment analysis was studied to verify if the isolated alga is photosynthetic or not. Alga cells were cultivated on nutritive agar medium, during seven days at 25°C. The extraction of pigments was carried out from colonies obtained after culture as described by Jin et al. 12. The absorbance of the supernatant was measured at (400-800 nm) by using a UV-Visible spectrophotometer (Beckman Coulter, Inc. Fullerton, USA). Optimization of cell growth conditions: To found the optimal T°C, pH and salinity growth conditions of the isolated microalgae species, cells were cultivated on Chlorella broth flasks at variable temperatures (20, 25, 30, 35 and 40°C) and at pH tested (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10) in an orbital incubator at 110 rpm for seven days. The pH of Chlorella broth was adjusted with HCl and NaOH solutions (1 N). To test the effect of salinity on alga cells growth, sodium chloride salt was used at different concentrations (0, 5, 10, 15, 20, 25, 30, 35 and 40 g L-1). All experiments were carried into 250 ml erlenmeyer flasks containing 100 ml Chlorella broth medium. The pellet cells were suspended in Chlorella broth (Sigma, Product of India, C9845) medium at a density ranged between 105-106 cells/ml. Cells were incubated at 25°C with agitation at 110 rpm in an orbital incubator (SI-600 shaker, Lab Companion-Jeio Tech, Korea) under daylight. After three days of each culture period, the growth was determined spectrophotometrically at 665 nm as pigments extracted by methanol (90°C) from the cells mass according to De Marsac and Houmard 18 procedure, Test of unicellular alga activity on industrial textile dyes: The new isolated microalgae from textile wastewater were used as biological material to reduce pollution of ITD. We tested four different dyes: indigo (indigoid dye), remazol brilliant orange Rbo (azo-dye), direct blue (reactive dye) and crystal violet (basic dye), purchased from industrial textile corporations (SITEX, Ksar-Hellal, Tunisia), which showed maximum absorbance (λmax), respectively, at 602, 617, 594 and 592 nm. The industrial textile dyes were prepared and used at 10 mg L-1 in erlenmeyer flasks containing 100 ml of microalgae seeded in sterile Chlorella broth medium adjusted between 105-106 cells/ml. Growth of unicellular green alga was evaluated daily during eight-days by using a spectrophotometer at 665 nm wavelength. This growth was compared to cells cultivated in medium without dyes. The percentage of chemical oxygen demand (COD) removal was calculated as following % COD = [(COD at t0) – (COD at t1)]/ (COD at t0) × 100. The COD was measured after removing algal cells by centrifugation at 3000 rpm during 10 minutes 19. The decolorization experiments were performed at the same time. Aliquot was centrifuged at 5000 rpm during 10 minutes in order to precipitate the cell mass 20 and the supernatants were evaluated at respective maximum absorbance (λmax) of the tested dyes. The percentage of decolorization was calculated as following % decolorization = [(Absorbance at t0) - (Absorbance at t1)]/ (Absorbance at t0) × 100. At t1 the COD and color absorbance were measured after five days.

Results and Discussion Isolation and morphology of microalga: Alga cells cultivated on nutrient agar medium gave green colonies. The size of the microalgal colonies was between 0.1-0.2 cm with circular shapes (Fig. la), similar to colonies obtained by eubacteria. The examination of exponential culture by light microscopy showed spherical to oval shapes with a thin cell wall, a bright green single parietal chloroplast, the multiplication mode is by autosporulation and the autospores are released through a rupture of the mother cell wall (Fig. 1b-c). The observation by transmission electronic microscopy confirmed the oval shape with a 3 µm diameter cells size (Fig. ld). The isolated microalga was able to grow in nutrient broth, media adapted to eubacteria. This result showed that isolated microalgae can grow heterotrophically. The ability of alga to grow both autotrophically and heterotrophically was also showed by Oh-hama and Miyachi 21.

Figure 1. Algae isolated from textile wastewater: (a) color appearance of alga colonies on nutritive agar plates grown at 25°C, under daylight, (b) alga cells under culture conditions (c) appearance of cells algae after Gram stain, modified by Hücker method (b and c) were observed by light microscopy at 1000× magnification, (d) SEM showing the size of algae cells.

Pigments production: The analysis of profile pigments (Fig. 2) produced by the isolated microalga cells after culture on nutrient agar medium, showed two maximum peaks obtained at 420- 460 nm corresponding to the carotenoid. Peaks of absorbance were between 400-500 nm and 600-700 nm correspond to chlorophyll (Chla) and chlorophyll (Chlb), respectively. As indicated by Jeffrey et al. 22 and from the profile (Fig. 2), we could deduce that the isolated Chlorella-like alga can produce also carotenoid. It should be noted that, carotenoid, Chla and Chlb are produced under heterotrophic condition. Moreover, photosynthetic activity producing oxygen by alga can relieve biological oxygen demand in wastewater 19. According to Jenkins et al. 23, few works indicate the presence of photosynthetic algae in water charged with color because of the weak penetration of solar rays. Indeed, we often isolated microalgae in textile industrial wastewater after treatment from the SC. Optimal conditions of algal cells growth: The optimal culture conditions of the green unicellular alga growth were temperature 25-28°C, pH 8.0, salinity at 15 g L-1 equivalent to 0.25 M of NaCl (Fig. 3A-C). Growth of alga cells depends on temperature range,

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009

701

0.7 0.6

Absorbance at 665 nm

Relative absorption

0.8

0.5 0.4 0.3 0.2 0.1 0 0 4 8 2 6 0 4 4 8 0 40 42 44 47 496 520 54 56 592 616 64 664 688 712 73 76 78 Wavelenght at nm

Effects of green alga on textile dyes: Using these optimized parameters, we have studied the effect of industrial dyes on the growth of isolated green microalga in Chlorella broth. The growth curves of alga cells (Fig. 4) shows that alga growth was remarkably reduced compared to the control group (alga without

Table 1. Characteristics of the secondary clarifier of industrial Tunisian textile plant. Parameter

Range

Mean value

T (°C)

20-32

27

pH

7.7-8.1

7.9

SS (mg L-1) COD (mg L-1) BOD5 (mg L-1)

9-35

18

120-240

161

12-40

21

Salinity (mg L-1)

4916-7432

6432

Unit Color Pt-co

67-225

147

SS suspended solids, BOD5 Biochemical oxygen demand during 5 days, COD Chemical oxygen demand, T Temperature.

702

Absorbance at 665 nm

pH and salinity concentration. Optimal pH for the alga growth is 8; this result explains in partly its presence in SITEX textile wastewater, because the average value of the pH is 7.9 (Table 1). Sodium chloride is also a key growth factor in medium for microalgae. The isolated green alga may be qualified as halotolerant species; cells may grow at 2-2.5% of NaCl. These culture conditions confirm the previous reports. Indeed, Häubner et al. 24 demonstrated that selected aeroterrestrial microalgal strains grew between 1 and 30°C with optimum rates at 20-23°C. Optimal pH has been reported at 7±0.2. Chlorella vulgaris grows significantly at a high pH (8.0), populations of Chlorella vulgaris co-immobilized with Azospirillum brasilense grow more remarkably compared to their growth at a lower pH. Furthermore, salinity improved microalgae growth. Salt tolerance of algae Dunaliella tertiolecta has been reported to be in the range of 0.17-1.5 M of NaCl 25. However, other investigators working on Dunaliella tertiolecta have gone far beyond these limits by quoting a NaCl tolerance range of 0.053 M 26. According to the results obtained, we could draw that this unicellular green alga belongs to Chlorella genus. The optimal values (pH, salinity and temperature) obtained for alga growth resemble to the physicochemical parameters of the textile wastewater plant from which alga cells were isolated. In this case, alga can be considered as acclimated.

(A)

0.12 0.10 0.08 0.06 0.04 0.02 0 20

25 30 35 Temperature (°C)

40

0.6 0.5

(B)

0.4 0.3 0.2 0.1 0

5

5.5

6

6.5

7 7.5 8 pH

8.5

9 9.5 10

0.35 Absorbance at 665 nm

Figure 2. UV-visible profile of total pigment extracted by methanol solvent from algae isolated from textile wastewater plant. The peaks at 420-460 nm are corresponding to carotenoid and the peaks located in 400-500 nm and 600-700 nm to Chl a with Chl b.

0.16 0.14

(C)

0.30 0.25 0.20 0.15 0.10 0.05 0

0

10 15 20 25 30 35 5 Concentration of NaCl gL -1

40

Figure 3. Growth curves of green alga at different conditions of (A) temperatures, (B) pH and (C) salt concentrations.

dyes). So, little concentration of dyes has negative effect on Chlorella cells growth. Nevertheless, the industrial dyes effect was different from one type to another. Indeed, growth of isolated alga decreased significantly with crystal violet, remazol brilliant orange and direct blue compared to indigo dye as shown in Fig. 4. While limited growth of green microalga was accompanied by COD reduction (Fig. 5), the high percentage of COD removal for direct blue was 60.1% and 46.7% for indigo. The percentage of decolorization for tested dyes (Fig. 6), showed that alga color removal efficiency values varied from dye to another, indigo (89.3%); direct blue (79%); remazol brilliant orange (75.3%) and crystal violet (72.5%). Results showed that isolated alga contribute to remove color with a significant reduction of COD in different samples of industrial textile dyes. The active role of isolated alga belonging to Chlorella genus, to reduce the pollution of indigo dye may be due to the acclimatization of alga since it was isolated from an effluent charged in indigo dye. Indeed, several groups showed that microalgae may serve as a solution to emerging environmental problems such as greenhouse effect and waste treatments 27-29. Biological remediation by unicellular green microalga was different from one dye to another; this may be

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009

Absorbance at 665 nm

0.30 0.25 0.20 0.15 0.10 0.05 0

1

2

4 5 6 3 Incubation period (days)

7

8

Figure 4. Effect of dyes concentration (10 mg L-1) on green alga growth incubated at 25°C, under agitation 110 rpm conditions in Chlorella broth medium.  cells culture without dyes,  indigo,  remazol brilliant orange (Rbo),  direct blue and  crystal violet.

% Average of COD removal

70 60 50 40 30 20 10 0 IND

Rbo

D.B.

CV

Dyes

Figure 5. Percentage of chemical oxygen demand removal by green alga.

% Average of decolorization

attributed to the adsorption or/and to the biodegradation by microalgae. Consequently, the dye chemical structure, the pollutant charge induced by each dye and the acclimated period were very important to enhance bioremediation as demonstrated by Acuner and Dilek 30. However, biodegradation depends strongly on the nature and properties of the molecule considered; properties in turn depend on molecule/environment interaction 31. The color removal of azo-dyes, such as remazol brilliant orange, was related to the presence of an enzyme; in fact several works reported that Chlorella and Oscillatoria algae both have been demonstrated to reduce azo dyes through the activity of unspecific, soluble cytoplasmic reductases known as azo reductases. These enzymes convert azo dyes into aromatic amines leading to a decrease of environmental rates 32, 33. Microalga could contribute to a valuable procedure that might be associated with pre-existing treatment methods23. Thus, Peralta-Zamora et al. 34 showed that aerobic and/or anaerobic procedures offer an extensive range of valid choices for improving industrial wastewater.

100 90 80 70 60 50 40 30 20 10 0 IND

Rbo

D.B.

CV

Dyes

Figure 6. Percentage of decolorization of textile dyes by green alga.

Conclusions The present study showed that green microalga may grow in textile wastewater plant in spite of the high polluted charge of dyes and can be cultivated in media like bacteria. The results of optimal conditions indicate that microalga was naturally acclimated and contribute to reduce dyes pollution with efficiency values. Indeed, biological treatment by activated sludge is better to reduce chemical pollution from textile industry because it is inspired from natural system of purification. Photosynthetic microalgae may be introduced as a secondary stage for industrial textile effluent treatment. Finally, for our study it is very interesting to realize molecular and cytological analysis in order to determine exactly the kind and the species of the isolated alga. Acknowledgements This study was supported by operating grants from “SITEX” society. Many thanks to Professor Mahmoud Rouabhia, Faculté de Médecine Dentaire, Bureau 1728, Université Laval Québec, Qc, Canada, G1K 7P4. References 1

Pala, A. and Tokat, E. 2002. Technical note: Color removal from cotton textile industry wastewater in an activated sludge system with various additives. Water Res. 36:2920-2925. 2 Manu, B. and Sanjeev, C. 2002. Anaerobic decolorization of simulated textile waste water containing azo dyes. Biores. Technol. 82:225-231. 3 Alinsafi, A., Da Motta, M., Le Bonté, S., Pons, M. N. and Benhammou, A. 2006. Effect of variability on the treatment of textile dyeing wastewater by activated sludge. Dyes and Pigments 69:31-39. 4 Sangyong, K., Chulhwan, P., Tak-Hyun, K., Jinwon, L. and Seungwook, K. 2003. COD reduction and decolorization of textile effluent using a combined process. Journal of Biosci. and Bioeng. 95(1):102105. 5 Junnarkar, N., Murty, D. S., Bhatt, N. S. and Madamwar, D. 2006. Decolorization of diazo dye remazol brilliant orange 81 by a novel bacterial consortium. World J. Microbiol. Biotechnol. 22:163-168. 6 Anjaneyulu, Y., Sreedhara, C. N. and Samuel, S.R.D. 2005. Decolorization of industrial effluents-available methods and emerging technologies-a review. Reviews in Environ. Sci. Biotechnol. 4:245-273. 7 Mallick, N. 2002. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: A review. Biometals 15:377-390. 8 Tam, N. F., Chong, A. M. and Wong, Y. S. 2002. Removal of tributyltin (TBT) by live and dead microalgal cells. Mar. Pollut. Bull. 45:362371. 9 Zhang, Z. B., Liu, C. Y., Wu, Z. Z., Xing, L. and Li, P. F. 2006. Detection of nitric oxide in culture media and studies on nitric oxide formation by marine microalgae. Med. Sci. Monitor 12:75-85.

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009

703

10

Pulz, O. and Gross, W. 2004. Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65:635-648. 11 Field, C. B., Behrenfeld, M. J., Randerson, J. T. and Falkowski, P. 1998. Primary production of the biosphere: Integrating terrestrial and oceanic components. Sci. 281:237–240. 12 Jin, J., Guan, M., Sima, J., Gao G., Zhang, M., Liu, Z., Fant, J. and Ma, J.X. 2003. Decreased pigment epithelium-derived factor and increased vascular endothelial growth factor levels in pterygia. Cornea 22:473477. 13 Apt, K. E. and Behrens, P. W. 1999. Commercial developments in microalgal biotechnology. J. Phycol. 35:215-226. 14 Richmond, A. 2003. Handbook of Microalgal Culture. Biotechnol. Appl. Phycol., Blackwell Publishing. 15 Leôn-Banares, R., Gonzalez-Ballester, D., Galvan, A. and Fernandez, E. 2004. Transgenic microalgae as green cell-factories. Trends Biotechnol. 22:45-52. 16 Huss, V., Ciniglia, C., Cennamo, P., Cozzolino, S., Pint, G. and Pollio, A. 2002. Phylogenetic relationships and taxonomic position of Chlorella-like isolates from low pH environments (pH < 3.0). BMC Evolut. Biol. http://www.biomedcentral.com/1471-2148/2/13. 17 Makni, H., Bettaieb, F., Dhaouadi, H., M’Henni, F. and Bakhrouf, A. 2006. The anaerobic treatment of sewage and granule formation in upflow anaerobic sludge blanket reactor. Environ. Technol. 27:10311036. 18 De Marsac, T.N. and Houmard, J. 1988. Complementary chromatic adaptation: Physiological conditions and action spectra. Methods in Enzymology, Vol. 167, Academic Press, San Diego, CA, USA, pp. 318-328. 19 Lee, K. and Lee, C.-G. 2001. Effect of light/dark cycles on wastewater treatment by microalgae. Biotechnol. Bioproc. Eng. 6(3):194-199. 20 Demet, Ç. and Göonöul, D. 2006. Decolorization of reactive dyes by mixed cultures isolated from textile effluent under anaerobic conditions. Enzyme Microb. Technol. 38:926–930. 21 Oh-Hama, T. and Miyachi, S. 1986. Chlorella. In Borowitzka, M. A.and Borowitzka, L. J. (eds). Microalgal Biotechnology. Cambridge University Press, Cambridge, UK, pp. 1-26. 22 Jeffrey, S. W., Mantoura, R.F.C. and Wright, S. W. 1997. Phytoplancton pigments in oceanography: Guidelines to modern methods. Part II. Experimental of scor working Group 78 workshops. pp.179-361. Part IV. Data for the identification of 47 key phytoplancton pigments. pp. 447-554. Monographs on Oceanographic Methodology 10. SCOR and UNESCO, France. 23 Jenkins, D., Richard, M.G. and Daigger, G.T. 1993. Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Lewis Publishers, Inc., Chelsea, Michigan, pp. 66-68. 24 Häubner, N., Schumann, R. and Karsten, U. 2006. Aeroterrestrial microalgae growing in biofilms on facades-response to temperature and water stress. Microb. Ecol. 51(3):285-293. 25 Oliver, B., Caumettte, P., Garcia, J.-L. and Mah, R. A. 1994. Anaerobic bacteria from hypersaline environments. Rev. Microbiol. 58:27-38. 26 Jahnke, L. S. and White, A. L. 2003. Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta. J. Plant Physiol. 160:1193–1202. 27 Craggs, R. J., Mc Auley, P. J. and Smith, V. J. 1997. Waste-water nutrient removal by marine microalgae grown on a corrugated raceway. Water Res. 31:1701-1707. 28 Korner, S. and Vermaat, J.E. 1998. The relative importance of Lemna gibba L., bacteria and algae for the nitrogen and phosohorus removal in duckweed-covered domestic wastewater. Water Res. 32:3651-3661. 29 Parikh, A. and Madamwar, D. 2005. Partial characterization of extracellular polysaccharides from cyanobacteria. Biores. Technol. 97: 1822-1827. 30 Acuner, E. and Dilek, F.B. 2004. Treatment of tectilon yellow 2G by Chlorella vulgaris. Process Biochem. 39:623–631. 31 Ferrier, M. D., Butler, B.R.Sr., Terlizzi, D. E. and Lacouture, R. V. 2005. The effects of barley straw (Hordeum vulgare) on the growth 704

of freshwater algae. Biores. Technol. 16:1788-1795. Jinqi, L. and Houtian, L. 1992. Degradation of azo dyes by algae. Environ. Pollut. 75:273-278. 33 Robinson, T., Mc Mullan, G., Marchant, R. and Nigam, P. 2001. Remediation of dyes in textile effluent: A critical review on current treatment technologies with a proposed alternative. Biores. Technol. 77:247-255. 34 Peralta-Zamora, P., Esposito E., Groto R., Reyes J. and Duran, N. 1998. Effluent treatment of pulp and paper, and textile industries using immobilised horseradish peroxidase. Environ. Technol. 19:5563. 32

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009