- Email: [email protected]
Biodegradation of crude oil by Scenedesmus obliquus and Chlorella vulgaris growing under heterotrophic conditions
International Biodeterioration & Biodegradation 82 (2013) 67e72
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of crude oil by Scenedesmus obliquus and Chlorella vulgaris growing under heterotrophic conditions Mostafa M. El-Sheekh a, *, Ragaa A. Hamouda b, Adnan A. Nizam c a
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Minufyia University, Egypt c Plant Biology Department, Faculty of Science, Damascus University, Syria b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2012 Received in revised form 11 December 2012 Accepted 16 December 2012 Available online 9 April 2013
One of the major environmental problems nowadays is petroleum hydrocarbon contamination, particularly in the zones of petroleum production, and petrochemical industries. This study was carried out to evaluate the potential of two green algae Scenedesmus obliquus and Chlorella vulgaris to degrade crude oil. Experiments were performed by incubating algal cultures with 0.5, 1, 1.5 and 2% crude oil for incubation period of 15 days under heterotrophic conditions. It was found that Scenedesmus obliquus and Chlorella vulgaris performed the highest biodegradation rate of crude oil when 0.5 and 1% oil was applied. The highest growth of S. obliquus was attended with 0.5% crude oil; while it was recorded at 2% for C. vulgaris, under the same heterotrophic conditions. Both algae could grow and degrade oil effectively when incubated with low concentrations of oil. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Biodegradation Heterotrophic Algae Crude oil Scenedesmus obliquus Chlorella vulgaris
1. Introduction Soil and water hydrocarbon pollution, which represents a very serious environmental problem, has been attracting considerable public attention over the last decades. The main sources of hydrocarbon pollution are the spills and leaks of petroleum products (Potter, 1993). Bioremediation is an emerging technology that uses plants and microorganisms to clean up the environment from pollutants and is cheaper than other remediation technologies (Leahy and Colwell, 1990). Numerous microorganisms, including bacteria, fungi and yeasts have the ability to degrade hydrocarbons (Oudot et al., 1993; Chaillan et al., 2004; El-Sheekh et al., 2009). Some species of algae are capable of heterotrophic growth on organic carbon sources (Neilson and Lewin, 1974). The ability of algae to degrade organic pollutants is the reason for their growth in the presence of pollutants. Cerniglia et al. (1980) proved the ability of nine Cyanobacteria, five green algae, one red alga, one brown alga, and two diatoms to oxidize naphthalene. Walker et al. (1975) isolated an alga, Prototheca zopfi which was capable of utilizing crude oil and a mixed hydrocarbon substrate exhibiting extensive * Corresponding author. E-mail address: [email protected] (M.M. El-Sheekh). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.12.015
degradation of n-alkanes and isoalkanes as well as aromatic hydrocarbons. Luther and Shaaban (1990) Luther (1990) confirmed the ability of Scenedesmus obliquus, to utilize naphthalene sulfonic acids as a source of sulfur for biomass production. Tikoo et al. (1997) observed that three species of Chlorella degrade pentachlorophenol. Yan and Pan (2004) reported that more than 30 azo compounds were biodegraded and decolorized by Chlorella pyrenoidosa, Chlorella vulgaris and Oscillateria tenuis, in which azo dyes were decomposed into a simpler aromatic amine. This work aims at studying the potential of microalgae Scenedesmus obliquus and Chlorella vulgaris for crude oil biodegradation, which also includes (i) Biodegradation of crude oil by the microalgae. (ii) The hydrocarbon degradation capacity under the laboratory conditions. (iii) The ability of S. obliquus and C. vulgaris to grow under the heterotrophic condition in the presence of crude oil. 2. Material and methods 2.1. Isolation of algae The green algae Scenedesmus obliquus and Chlorella vulgaris Beijerinck were isolated from water samples collected from River
68
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
OD
0.5
0.1
1.5
Table 1 Effect of different concentrations of crude oil on Chlorophyll-a,b and Carotenoids content of Chlorella vulgaris (mg/ml). Values are mean of 3 replicates standard error of the mean.
2
6 5 4 3 2 1 0
Crude oil 3th day conc.
0
5
10
15
20
Days Fig. 1. Effect of different concentrations of crude oil on growth of Scenedesmus obliquus measured as optical density (560 nm).
Nile near Tanta city, Egypt. The algae were purified through serial dilution followed by plating. Each algal colony was isolated and inoculated into liquid medium at 25 C 1 Stanier et al. (1971). The purity of cultures was ensured by repeated plating and regular observation under the microscope. 2.2. Algae cultivation with crude oil Crude oil was added to 250 ml Erlenmeyer flasks containing 100 ml BG11 medium with appropriate amount of algal culture to give a total crude oil concentrations (0.5, 1, 1.5 and 2%). The Erlenmeyer flasks were incubated at 25 1 C at a constant shaking rate of 80 rpm under dark condition. 2.3. Assessment of algal growth
Chlorophyll-a 0.5 (mg ml 1) 1.0 1.5 2.0 Chlorophyll-b 0.5 1 (mg ml ) 1.0 1.5 2.0 Carotenoids 0.5 (mg ml 1) 1.0 1.5 2.0
1.24 1.43 1.50 1.61 2.04 2.21 2.07 2.35 1.61 0.70 0.87 1.07
7th day
0.0 3. 30 0.30 2.69 0.34 3.41 0.04 3.22 0.0 1.75 0.03 1.78 0.84 2.62 0.53 2.21 0.0 1.28 0.19 1.13 0.08 1.34 0.51 1.24
11th day 0.06 0.02 0.01 0.34 0.05 0.17 0.26 0.31 0.38 0.06 0.09 0.11
4.55 5.65 5.36 4.82 4.79 5.53 4.94 4.56 0.52 0.20 0.59 0.34
0.23 0.31 0.18 0.52 0.86 0.23 0.45 0.46 0.23 0.01 0.03 0.08
15th day 4.22 5.17 6.13 4.42 4.31 5.14 6.22 4.21 0.11 0 0.24 0.30
0.20 0.26 1.29 0.34 0.27 0.23 1.24 0.43 0.07 00 0.24 0.07
2.5. Biodegradation activity of the algae for crude oil Biodegradation of crude oil was analysed by using GCeMS HP 6890 gas carrier helium (1 ml/min). Capillary Column. 30 m 0.25 rnm ID 0.25 um film and the temperature programming was 70e290 C, 5/15 min. 2.6. Statistical analysis Experiments were conducted in triplicate. Results were expressed as standard error of the mean.
The biomass of algae was determined by measuring the optical density of the algal suspension at 560 nm (Wetherell, 1961) using Unico UV-2000 spectrophotometer.
3. Results and discussion
2.4. Pigments estimation
Results in Figs. 1 and 2 show the heterotrophic algal growth using crude oil as sole carbon source. The growth of S. obliquus increased in the presence of high concentrations of crude oil as compared with C. vulgaris. The highest growth of S. obliquus was attended at 0.5% crude oil, while it was recorded at 2% crude oil for C. vulgaris. This result is in agreement with Gamila and Ibrahim (2004) who indicated that the treatment of algal culture of (S. obliquus, Nitzschia linearis) with crude oil led to prolongation of the growth phase as well as high algal biomass production. Kong
A known volume of culture was centrifuged at speed of (8000 rpm) for 10 min, after that the algal pellets were treated with known volume of ethyl alcohol and kept in water bath for 30 min at 55 C, and then centrifuged again. The colour of pellets must be white to ensure maximum extraction of pigments. If it was not the extraction must be repeated. Absorbance of the pooled extracts was registered on Unico UV-2000 spectrophotometer at 650, 665 and 452 nm. Calculations were made according to the formulae devised by Senger (1970) for chlorophyll a, chlorophyll b and carotenoids.
Table 2 Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of Scenedesmus obliquus (mg/ml). Values are mean of 3 replicates standard error of the mean.
1
Crude oil 3th day conc.
0.8
OD
3.1. Estimation of algal growth
0.6 0.4 0.2 0 0
2
4
6
8
10
12
14
16
18
Days
0.5
0.1
1.5
2
Fig. 2. Effect of different concentrations of crude oil on growth of Chlorella vulgaries measured as optical density (560 nm).
Chlorophyll-a 0.5 (mg ml 1) 1.0 1.5 2.0 Chlorophyll-b 0.5 (mg ml 1) 1.0 1.5 2.0 Carotenoids 0.5 (mg ml 1) 1.0 1.5 2.0
4.97 4.93 4.23 3.23 8.02 7.69 6.04 3.91 0.0 0.0 0.05 1.01
7th day
7.82 1.47 5.95 0.44 3.32 0.08 5.69 0.68 6.93 1.38 6.934 0.10 6.93 2.33 6.93 1.45 0.0 0.67 0.22 0.05 0.0 0.45 0.0 0.10 0.59 0.37 0.77 0.02 0.49 0.70 1.00
11th day
15th day
4.01 0.82 5.60 0.28 5.65 0.08 3.54 0.62 0.65 0.06 0.28 0.077 0.08 0.08 0.51 0.21 3.06 0.45 4.1 0.18 4.05 0.08 2.55 0.40
6.26 0.23 4.56 0.36 6.32 1.58 5.83 0.34 6.70 0.49 4.57 0.25 6.30 2.29 6.19 0.61 0.0 0.2 0.12 0.03 0.03 0.0
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
69
Fig. 3. GC/MS chromatogram of residual crude oil concentrations after 15 days of incubation with BG11 medium (control without algae).
et al. (2011) denoted the rapid growth rate of the culture of C. vulgaris under mixotrophic and heterotrophic and reached stationary phase a head of the control compared with autotrophic growth. Perez-Garcia et al. (2010) reported C. vulgaris grown heterotrophically had slightly larger populations and higher growth rates than under autotrophic regime. Under heterotrophic growth conditions, respiration rates of algal cells are equal or exceed the theoretical minimum cost of their biomass. Under some heterotrophic growth conditions, the microalgal biomass yields were consistent and reproducible; reaching cells’ densities of 50e100 g of dry biomass per liter (Radmer and Parker, 1994; Gladue and Maxey, 1994a,b), much higher than the maximum (30 g l 1 of dry cell biomass) in autotrophic cultures (Javanmardian and Palsson, 1991). 3.2. Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of Chlorella vulgaris (mg ml 1) In addition the main photosynthetic pigment chlorophyll, microalgae contain auxiliary photosynthetic pigments which improve the use of light energy (phycobiliproteins) and protect against solar radiation (carotenoids) (Cohen, 1986; Pulz and Gross, 2004; Del Campo et al., 2007). Naturally, all pigments are produced under autotrophic growth conditions, but surprisingly some are produced, and in large quantities, under heterotrophic dark conditions. Results in Table 1 show the subsequent increase of chlorophyll content of Chlorella vulgaris from the first day of growth to day eleven of incubation with 0.5, 1, 1.5 and 2% crude oil concentrations. The highest chlorophyll content of Chlorella vulgaris (6.13 mg ml 1) was revealed after 15 days of incubation with 1.5% crude oil. On other hand, the carotenoids of Chlorella vulgaris decreased after seven days of incubation with different concentrations of crude oil. 3.3. Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of S. obliquus (mg ml 1) Results in Table 2 show the effect of crude oil on chlorophyll a,b and carotenoids contents of heterotrophic cultures of Scenedesmus obliquus after 15 days of incubation. These results indicated that, the chlorophyll a,b and carotenoids contents decreased with
increasing concentrations of crude oil. The maximum chlorophyll a content (7.82 1.47 mg ml 1) of S. obliquus was obtained at 7 days with 0.5% crude oil. Chlorophyll-b content of S. obliquus was reduced after 7 days of incubation with different concentrations of crude oil. Highest carotenoids content of S. obliquus was obtained after 11 days of incubation and subsequently decreased after this period. 3.4. Biodegradation activity of the algae The results obtained by GCeMS analyses (Fig. 3 control without algae and Fig. 4) show that, the highest biodegradation rate of crude oil was observed at oil concentration of 0.5%. Indole-3-acetic acid completely disappeared in all the concentrations of crude oil incubated with algae for 15 days, also Decane, Indole-3-acetic acid, p-Phenyltoluene, Naphthalene, 3 ethyl, Tridecane, phenanthracene, 1-methyl, Benzene, decyl, phenanthracene, 2-methyl, cyclohexane undecyl, b-pregnane and Octacosane were totally absent when C. vulgaris incubated with 2% crude oil. Naphthalene, 3 ethyl, phenanthracene, 1-methyl, Pentadecane and b-pregnane were degraded after 15 days of incubation of Scenedesmus obliquue with 2% crude oil. These results indicated that algae are more efficient to degrade polycyclic aromatic hydrocarbons (PAHs) under heterotrophic conditions. Both Cyanobacteria and eukaryotic algae are capable of biotransforming aromatic compounds including phenols (Semple and Cain, 1996; El-Sheekh et al., 2012). Although the biomass production of C. vulgaris was less than S. obliquue, C. vulgaris showed higher degradation activity of crude oil. Sometimes the percentage of some remaining hydrocarbon (HC) was noticed to be above the control level. This may be due to the ability of studied algae to utilize crude oil and convert it to intermediate compounds (Okoh, 2006). Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all (Atlas and Bragg, 2009). The removal percentage of n-alkanes by S. obliquue cells ranged from 46% (after 1st week of incubation) to 88% (after 6th week), while it ranged from 41% to 87% for PAHs, through the same incubation period (Gamila and Ibrahim, 2004). Chlorella pyrenoidosa grown in sterilized sewage could use some of the organic matter, as indicated by a decrease in soluble BOD and dissolved volatile solids in cultures of short retention times (Pipes and Gotaas, 1960).
70
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
Fig. 4. GC/MS chromatogram of residual crude oil concentrations after 15 days of incubation with liquid cultures of C. vulgaris (A 0.5, B 1.0, C 1.5 and D 2%) and S. obliquus. (E 0.5, F 1, G 1.5 and H 2%).
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
Fig. 4. (continued).
71
72
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
4. Conclusion This work highlights the ability of the two green microalgae S. obliquus and C. vulgaris to grow under heterotrophic conditions using crude oil as sole carbon source. This study also proved the ability of the studied algae to degrade n-alkane and PAHs. Using microalgae to degrade crude oil pollutant is considered a promising technique and an economically feasible method which could be used at large scale. Acknowledgements The authors gratefully thank Prof. Adel Selem, Chemistry Department, Faculty of Science, Tanta University, Egypt for his help in discussing GC/MS analyses. The authors also thank Prof. John Kennedy for his critical reading of the Manuscript. References Atlas, R., Bragg, J., 2009. Bioremediation of marine oil spills: when and when not e the Exxon Valdez experience. Microbial Biotechnology 2, 213e221. Cerniglia, C.E., Gibson, D.T., VanBaalen, C., 1980. Oxidation of naphthalene by cyanobacteria and microalgae. General Microbiology 116, 495e500. Chaillan, F., Lefleche, A., Bury, E., Phantavong, Y., Grimont, P., Saliot, A., Oudot, J., 2004. Identification and biodegradation potential of tropical aerobic hydrocarbondegrading microorganisms. Research in Microbiology 155, 587e595. Cohen, Z., 1986. Products from microalgae. In: Richmond, A. (Ed.), Handbook for Microalgae Mass Culture. CRC Press, Boca Raton FL, USA, pp. 421e454. Del-Campo, J.A., Garcia-Gonzalez, M., Guerrero, M.G., 2007. Cultivation of microalgae for carotenoids production: current state and perspectives. Applied Microbiology and Biotechnology 74, 1163e1174. El-Sheekh, M.M., Gharieb, M.M., Abou-El-Souod, G.W., 2009. Biodegradation of dyes by some green algae and cyanobacteria. International Biodeterioration & Biodegradation 63, 609e704. EI-Sheekh, M.M., Ghareib, M.M., Abou-El-Souod, G.W., 2012. Biodegradation of phenolic and polycyclic aromatic compounds by some algae and cyanobacteria. Journal of Bioremediation and Biodegradation 3, 133. http://dx.doi.org/10.4172/ 2155-6199.1000133. Gamila, H.A., Ibrahim, M.B.M., 2004. Algal bioassay for evaluating the role of algae in bioremediation of crude oil:1-isolated strains. Bulletin of Environmental Contamination and Toxicology 723, 883e889. Gladue, R.M., Maxey, J.E., 1994a. Microalgal feeds for aquaculture. Applied Phycology 6, 131e141.
Gladue, R.M., Maxey, J.E., 1994b. hydrocarbon pollutants. Biotechnology and Molecular Biology Review 1, 38e50. Javanmardian, M., Palsson, B.O., 1991. High-density photoautotrophix algal cultures: design, construction, and operation of noval photobioreactor system. Biotechnology and Bioengineering 328, 1182e1189. Kong, W., Song, H.Y., Cao, Y., Yang, H., Hua, S., Xia, C., 2011. The characteristics of biomass production, lipid accumulation and chlorophyll biosynthesis of Chlorella vulgaris under mixotrophic cultivation. African Journal of Biotechnology 10, 11620e11630. Leahy, J.G., Colwell, R.R., 1990. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54, 305e315. Luther, M., 1990. Degradation of different substituted aromatic compounds as nutrient sources by green alga Scenedesmus obliquus. Dechema Biotechnology Conference 4, 613e615. Luther, M., Shaaban, M.M., 1990. Abbau Von. 1,5-Naphthlindisul fonsaure (1,5-NDS) drush eine Algen-Bakterien Mischkulture. Forum Mikrobiology 1, 2e42. Neilson, A., Lewin, R., 1974. Uptake and utilization of organic carbon by algae. Phycology 13, 229e257. Okoh, A.I., 2006. Biodegradation alternative in the cleanup of petroleum. Biotechnology and Molecular Biology Reviews 1 (2), 38e50. Oudot, J., Dupont, J., Haloui, S., Roquebert, M.F., 1993. Biodegradation potential of hydrocarbon-assimilating tropical fungi. Soil Biology and Biochemistry 25,1167e1173. Perez-Garcia, O., Bashan, L.E., Hernandez, J.P., Bashan, Y., 2010. Efficiency of growth and nutrient uptake from wastewater by heterotrophic, autotrophic and mixotrophic cultivation of 1 C. vulgaris immobilized with Azospirillum brasilense. Phycologia 46, 800e812. Pipes, W.O., Gotaas, H.B., 1960. Utilization of organic matter by Chlorella grown in seawage. Applied Microbiology 8, 163e169. Potter, J.L., 1993. Analysis of petroleum contaminated soil and water: an overview. In: Calabrese, E.J., Kostecki, P.T. (Eds.), Principles and Practices for Petroleum Contaminated Soils. Lewis Publishers, Chelsea, pp. 11e14. Pulz, O., Gross, W., 2004. Valuable products from biotechnology of microalgae. Applied Microbiology and Biotechnology 65, 635e648. Radmer, R.J., Parker, B.C., 1994. Commercial application of algae opportunities and constraints. Journal of Applied Phycology 6, 93e98. Semple, K.T., Cain, R.B., 1996. Biodegradation of phenols by the alga Ochromonas danica. Applied and Environmental Microbiology 62, 1265e1273. Sengar, H., 1970. Charakterlsiarung einer synckronkultur yon Scenedesmus obliquus. Planta 90, 243e266. Stanier, R.Y., Kunisawa, R., Mandel, M., Cohen-Bazire, G., 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriology Review 35, 171e205. Tikoo, V., Scragg, A.H., Shales, S.W., 1997. Degradation of pentachlorophenol by microalgae. Journal of Chemical Technology and Biotechnology 68, 425e431. Walker, J.D., Colwell, R.R., Vaituzis, Z., Meyer, S.A., 1975. Petroleum degrading achlorophyllous alga Prototheca zopfii. Nature 54, 423e424. Wetherell, D.F., 1961. Culture of fresh water algae in enriched natural seawater. Plant Physiology (Copenh), 14e16. Yan, H., Pan, G., 2004. Increase in biodegradation of dimethyl phthalate by Closterium lunula using inorganic carbon. Chemosphere 55, 1281e1285.
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of crude oil by Scenedesmus obliquus and Chlorella vulgaris growing under heterotrophic conditions Mostafa M. El-Sheekh a, *, Ragaa A. Hamouda b, Adnan A. Nizam c a
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Minufyia University, Egypt c Plant Biology Department, Faculty of Science, Damascus University, Syria b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2012 Received in revised form 11 December 2012 Accepted 16 December 2012 Available online 9 April 2013
One of the major environmental problems nowadays is petroleum hydrocarbon contamination, particularly in the zones of petroleum production, and petrochemical industries. This study was carried out to evaluate the potential of two green algae Scenedesmus obliquus and Chlorella vulgaris to degrade crude oil. Experiments were performed by incubating algal cultures with 0.5, 1, 1.5 and 2% crude oil for incubation period of 15 days under heterotrophic conditions. It was found that Scenedesmus obliquus and Chlorella vulgaris performed the highest biodegradation rate of crude oil when 0.5 and 1% oil was applied. The highest growth of S. obliquus was attended with 0.5% crude oil; while it was recorded at 2% for C. vulgaris, under the same heterotrophic conditions. Both algae could grow and degrade oil effectively when incubated with low concentrations of oil. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Biodegradation Heterotrophic Algae Crude oil Scenedesmus obliquus Chlorella vulgaris
1. Introduction Soil and water hydrocarbon pollution, which represents a very serious environmental problem, has been attracting considerable public attention over the last decades. The main sources of hydrocarbon pollution are the spills and leaks of petroleum products (Potter, 1993). Bioremediation is an emerging technology that uses plants and microorganisms to clean up the environment from pollutants and is cheaper than other remediation technologies (Leahy and Colwell, 1990). Numerous microorganisms, including bacteria, fungi and yeasts have the ability to degrade hydrocarbons (Oudot et al., 1993; Chaillan et al., 2004; El-Sheekh et al., 2009). Some species of algae are capable of heterotrophic growth on organic carbon sources (Neilson and Lewin, 1974). The ability of algae to degrade organic pollutants is the reason for their growth in the presence of pollutants. Cerniglia et al. (1980) proved the ability of nine Cyanobacteria, five green algae, one red alga, one brown alga, and two diatoms to oxidize naphthalene. Walker et al. (1975) isolated an alga, Prototheca zopfi which was capable of utilizing crude oil and a mixed hydrocarbon substrate exhibiting extensive * Corresponding author. E-mail address: [email protected] (M.M. El-Sheekh). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.12.015
degradation of n-alkanes and isoalkanes as well as aromatic hydrocarbons. Luther and Shaaban (1990) Luther (1990) confirmed the ability of Scenedesmus obliquus, to utilize naphthalene sulfonic acids as a source of sulfur for biomass production. Tikoo et al. (1997) observed that three species of Chlorella degrade pentachlorophenol. Yan and Pan (2004) reported that more than 30 azo compounds were biodegraded and decolorized by Chlorella pyrenoidosa, Chlorella vulgaris and Oscillateria tenuis, in which azo dyes were decomposed into a simpler aromatic amine. This work aims at studying the potential of microalgae Scenedesmus obliquus and Chlorella vulgaris for crude oil biodegradation, which also includes (i) Biodegradation of crude oil by the microalgae. (ii) The hydrocarbon degradation capacity under the laboratory conditions. (iii) The ability of S. obliquus and C. vulgaris to grow under the heterotrophic condition in the presence of crude oil. 2. Material and methods 2.1. Isolation of algae The green algae Scenedesmus obliquus and Chlorella vulgaris Beijerinck were isolated from water samples collected from River
68
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
OD
0.5
0.1
1.5
Table 1 Effect of different concentrations of crude oil on Chlorophyll-a,b and Carotenoids content of Chlorella vulgaris (mg/ml). Values are mean of 3 replicates standard error of the mean.
2
6 5 4 3 2 1 0
Crude oil 3th day conc.
0
5
10
15
20
Days Fig. 1. Effect of different concentrations of crude oil on growth of Scenedesmus obliquus measured as optical density (560 nm).
Nile near Tanta city, Egypt. The algae were purified through serial dilution followed by plating. Each algal colony was isolated and inoculated into liquid medium at 25 C 1 Stanier et al. (1971). The purity of cultures was ensured by repeated plating and regular observation under the microscope. 2.2. Algae cultivation with crude oil Crude oil was added to 250 ml Erlenmeyer flasks containing 100 ml BG11 medium with appropriate amount of algal culture to give a total crude oil concentrations (0.5, 1, 1.5 and 2%). The Erlenmeyer flasks were incubated at 25 1 C at a constant shaking rate of 80 rpm under dark condition. 2.3. Assessment of algal growth
Chlorophyll-a 0.5 (mg ml 1) 1.0 1.5 2.0 Chlorophyll-b 0.5 1 (mg ml ) 1.0 1.5 2.0 Carotenoids 0.5 (mg ml 1) 1.0 1.5 2.0
1.24 1.43 1.50 1.61 2.04 2.21 2.07 2.35 1.61 0.70 0.87 1.07
7th day
0.0 3. 30 0.30 2.69 0.34 3.41 0.04 3.22 0.0 1.75 0.03 1.78 0.84 2.62 0.53 2.21 0.0 1.28 0.19 1.13 0.08 1.34 0.51 1.24
11th day 0.06 0.02 0.01 0.34 0.05 0.17 0.26 0.31 0.38 0.06 0.09 0.11
4.55 5.65 5.36 4.82 4.79 5.53 4.94 4.56 0.52 0.20 0.59 0.34
0.23 0.31 0.18 0.52 0.86 0.23 0.45 0.46 0.23 0.01 0.03 0.08
15th day 4.22 5.17 6.13 4.42 4.31 5.14 6.22 4.21 0.11 0 0.24 0.30
0.20 0.26 1.29 0.34 0.27 0.23 1.24 0.43 0.07 00 0.24 0.07
2.5. Biodegradation activity of the algae for crude oil Biodegradation of crude oil was analysed by using GCeMS HP 6890 gas carrier helium (1 ml/min). Capillary Column. 30 m 0.25 rnm ID 0.25 um film and the temperature programming was 70e290 C, 5/15 min. 2.6. Statistical analysis Experiments were conducted in triplicate. Results were expressed as standard error of the mean.
The biomass of algae was determined by measuring the optical density of the algal suspension at 560 nm (Wetherell, 1961) using Unico UV-2000 spectrophotometer.
3. Results and discussion
2.4. Pigments estimation
Results in Figs. 1 and 2 show the heterotrophic algal growth using crude oil as sole carbon source. The growth of S. obliquus increased in the presence of high concentrations of crude oil as compared with C. vulgaris. The highest growth of S. obliquus was attended at 0.5% crude oil, while it was recorded at 2% crude oil for C. vulgaris. This result is in agreement with Gamila and Ibrahim (2004) who indicated that the treatment of algal culture of (S. obliquus, Nitzschia linearis) with crude oil led to prolongation of the growth phase as well as high algal biomass production. Kong
A known volume of culture was centrifuged at speed of (8000 rpm) for 10 min, after that the algal pellets were treated with known volume of ethyl alcohol and kept in water bath for 30 min at 55 C, and then centrifuged again. The colour of pellets must be white to ensure maximum extraction of pigments. If it was not the extraction must be repeated. Absorbance of the pooled extracts was registered on Unico UV-2000 spectrophotometer at 650, 665 and 452 nm. Calculations were made according to the formulae devised by Senger (1970) for chlorophyll a, chlorophyll b and carotenoids.
Table 2 Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of Scenedesmus obliquus (mg/ml). Values are mean of 3 replicates standard error of the mean.
1
Crude oil 3th day conc.
0.8
OD
3.1. Estimation of algal growth
0.6 0.4 0.2 0 0
2
4
6
8
10
12
14
16
18
Days
0.5
0.1
1.5
2
Fig. 2. Effect of different concentrations of crude oil on growth of Chlorella vulgaries measured as optical density (560 nm).
Chlorophyll-a 0.5 (mg ml 1) 1.0 1.5 2.0 Chlorophyll-b 0.5 (mg ml 1) 1.0 1.5 2.0 Carotenoids 0.5 (mg ml 1) 1.0 1.5 2.0
4.97 4.93 4.23 3.23 8.02 7.69 6.04 3.91 0.0 0.0 0.05 1.01
7th day
7.82 1.47 5.95 0.44 3.32 0.08 5.69 0.68 6.93 1.38 6.934 0.10 6.93 2.33 6.93 1.45 0.0 0.67 0.22 0.05 0.0 0.45 0.0 0.10 0.59 0.37 0.77 0.02 0.49 0.70 1.00
11th day
15th day
4.01 0.82 5.60 0.28 5.65 0.08 3.54 0.62 0.65 0.06 0.28 0.077 0.08 0.08 0.51 0.21 3.06 0.45 4.1 0.18 4.05 0.08 2.55 0.40
6.26 0.23 4.56 0.36 6.32 1.58 5.83 0.34 6.70 0.49 4.57 0.25 6.30 2.29 6.19 0.61 0.0 0.2 0.12 0.03 0.03 0.0
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
69
Fig. 3. GC/MS chromatogram of residual crude oil concentrations after 15 days of incubation with BG11 medium (control without algae).
et al. (2011) denoted the rapid growth rate of the culture of C. vulgaris under mixotrophic and heterotrophic and reached stationary phase a head of the control compared with autotrophic growth. Perez-Garcia et al. (2010) reported C. vulgaris grown heterotrophically had slightly larger populations and higher growth rates than under autotrophic regime. Under heterotrophic growth conditions, respiration rates of algal cells are equal or exceed the theoretical minimum cost of their biomass. Under some heterotrophic growth conditions, the microalgal biomass yields were consistent and reproducible; reaching cells’ densities of 50e100 g of dry biomass per liter (Radmer and Parker, 1994; Gladue and Maxey, 1994a,b), much higher than the maximum (30 g l 1 of dry cell biomass) in autotrophic cultures (Javanmardian and Palsson, 1991). 3.2. Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of Chlorella vulgaris (mg ml 1) In addition the main photosynthetic pigment chlorophyll, microalgae contain auxiliary photosynthetic pigments which improve the use of light energy (phycobiliproteins) and protect against solar radiation (carotenoids) (Cohen, 1986; Pulz and Gross, 2004; Del Campo et al., 2007). Naturally, all pigments are produced under autotrophic growth conditions, but surprisingly some are produced, and in large quantities, under heterotrophic dark conditions. Results in Table 1 show the subsequent increase of chlorophyll content of Chlorella vulgaris from the first day of growth to day eleven of incubation with 0.5, 1, 1.5 and 2% crude oil concentrations. The highest chlorophyll content of Chlorella vulgaris (6.13 mg ml 1) was revealed after 15 days of incubation with 1.5% crude oil. On other hand, the carotenoids of Chlorella vulgaris decreased after seven days of incubation with different concentrations of crude oil. 3.3. Effect of different concentrations of crude oil on Chlorophyll-a,b and carotenoids content of S. obliquus (mg ml 1) Results in Table 2 show the effect of crude oil on chlorophyll a,b and carotenoids contents of heterotrophic cultures of Scenedesmus obliquus after 15 days of incubation. These results indicated that, the chlorophyll a,b and carotenoids contents decreased with
increasing concentrations of crude oil. The maximum chlorophyll a content (7.82 1.47 mg ml 1) of S. obliquus was obtained at 7 days with 0.5% crude oil. Chlorophyll-b content of S. obliquus was reduced after 7 days of incubation with different concentrations of crude oil. Highest carotenoids content of S. obliquus was obtained after 11 days of incubation and subsequently decreased after this period. 3.4. Biodegradation activity of the algae The results obtained by GCeMS analyses (Fig. 3 control without algae and Fig. 4) show that, the highest biodegradation rate of crude oil was observed at oil concentration of 0.5%. Indole-3-acetic acid completely disappeared in all the concentrations of crude oil incubated with algae for 15 days, also Decane, Indole-3-acetic acid, p-Phenyltoluene, Naphthalene, 3 ethyl, Tridecane, phenanthracene, 1-methyl, Benzene, decyl, phenanthracene, 2-methyl, cyclohexane undecyl, b-pregnane and Octacosane were totally absent when C. vulgaris incubated with 2% crude oil. Naphthalene, 3 ethyl, phenanthracene, 1-methyl, Pentadecane and b-pregnane were degraded after 15 days of incubation of Scenedesmus obliquue with 2% crude oil. These results indicated that algae are more efficient to degrade polycyclic aromatic hydrocarbons (PAHs) under heterotrophic conditions. Both Cyanobacteria and eukaryotic algae are capable of biotransforming aromatic compounds including phenols (Semple and Cain, 1996; El-Sheekh et al., 2012). Although the biomass production of C. vulgaris was less than S. obliquue, C. vulgaris showed higher degradation activity of crude oil. Sometimes the percentage of some remaining hydrocarbon (HC) was noticed to be above the control level. This may be due to the ability of studied algae to utilize crude oil and convert it to intermediate compounds (Okoh, 2006). Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all (Atlas and Bragg, 2009). The removal percentage of n-alkanes by S. obliquue cells ranged from 46% (after 1st week of incubation) to 88% (after 6th week), while it ranged from 41% to 87% for PAHs, through the same incubation period (Gamila and Ibrahim, 2004). Chlorella pyrenoidosa grown in sterilized sewage could use some of the organic matter, as indicated by a decrease in soluble BOD and dissolved volatile solids in cultures of short retention times (Pipes and Gotaas, 1960).
70
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
Fig. 4. GC/MS chromatogram of residual crude oil concentrations after 15 days of incubation with liquid cultures of C. vulgaris (A 0.5, B 1.0, C 1.5 and D 2%) and S. obliquus. (E 0.5, F 1, G 1.5 and H 2%).
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
Fig. 4. (continued).
71
72
M.M. El-Sheekh et al. / International Biodeterioration & Biodegradation 82 (2013) 67e72
4. Conclusion This work highlights the ability of the two green microalgae S. obliquus and C. vulgaris to grow under heterotrophic conditions using crude oil as sole carbon source. This study also proved the ability of the studied algae to degrade n-alkane and PAHs. Using microalgae to degrade crude oil pollutant is considered a promising technique and an economically feasible method which could be used at large scale. Acknowledgements The authors gratefully thank Prof. Adel Selem, Chemistry Department, Faculty of Science, Tanta University, Egypt for his help in discussing GC/MS analyses. The authors also thank Prof. John Kennedy for his critical reading of the Manuscript. References Atlas, R., Bragg, J., 2009. Bioremediation of marine oil spills: when and when not e the Exxon Valdez experience. Microbial Biotechnology 2, 213e221. Cerniglia, C.E., Gibson, D.T., VanBaalen, C., 1980. Oxidation of naphthalene by cyanobacteria and microalgae. General Microbiology 116, 495e500. Chaillan, F., Lefleche, A., Bury, E., Phantavong, Y., Grimont, P., Saliot, A., Oudot, J., 2004. Identification and biodegradation potential of tropical aerobic hydrocarbondegrading microorganisms. Research in Microbiology 155, 587e595. Cohen, Z., 1986. Products from microalgae. In: Richmond, A. (Ed.), Handbook for Microalgae Mass Culture. CRC Press, Boca Raton FL, USA, pp. 421e454. Del-Campo, J.A., Garcia-Gonzalez, M., Guerrero, M.G., 2007. Cultivation of microalgae for carotenoids production: current state and perspectives. Applied Microbiology and Biotechnology 74, 1163e1174. El-Sheekh, M.M., Gharieb, M.M., Abou-El-Souod, G.W., 2009. Biodegradation of dyes by some green algae and cyanobacteria. International Biodeterioration & Biodegradation 63, 609e704. EI-Sheekh, M.M., Ghareib, M.M., Abou-El-Souod, G.W., 2012. Biodegradation of phenolic and polycyclic aromatic compounds by some algae and cyanobacteria. Journal of Bioremediation and Biodegradation 3, 133. http://dx.doi.org/10.4172/ 2155-6199.1000133. Gamila, H.A., Ibrahim, M.B.M., 2004. Algal bioassay for evaluating the role of algae in bioremediation of crude oil:1-isolated strains. Bulletin of Environmental Contamination and Toxicology 723, 883e889. Gladue, R.M., Maxey, J.E., 1994a. Microalgal feeds for aquaculture. Applied Phycology 6, 131e141.
Gladue, R.M., Maxey, J.E., 1994b. hydrocarbon pollutants. Biotechnology and Molecular Biology Review 1, 38e50. Javanmardian, M., Palsson, B.O., 1991. High-density photoautotrophix algal cultures: design, construction, and operation of noval photobioreactor system. Biotechnology and Bioengineering 328, 1182e1189. Kong, W., Song, H.Y., Cao, Y., Yang, H., Hua, S., Xia, C., 2011. The characteristics of biomass production, lipid accumulation and chlorophyll biosynthesis of Chlorella vulgaris under mixotrophic cultivation. African Journal of Biotechnology 10, 11620e11630. Leahy, J.G., Colwell, R.R., 1990. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54, 305e315. Luther, M., 1990. Degradation of different substituted aromatic compounds as nutrient sources by green alga Scenedesmus obliquus. Dechema Biotechnology Conference 4, 613e615. Luther, M., Shaaban, M.M., 1990. Abbau Von. 1,5-Naphthlindisul fonsaure (1,5-NDS) drush eine Algen-Bakterien Mischkulture. Forum Mikrobiology 1, 2e42. Neilson, A., Lewin, R., 1974. Uptake and utilization of organic carbon by algae. Phycology 13, 229e257. Okoh, A.I., 2006. Biodegradation alternative in the cleanup of petroleum. Biotechnology and Molecular Biology Reviews 1 (2), 38e50. Oudot, J., Dupont, J., Haloui, S., Roquebert, M.F., 1993. Biodegradation potential of hydrocarbon-assimilating tropical fungi. Soil Biology and Biochemistry 25,1167e1173. Perez-Garcia, O., Bashan, L.E., Hernandez, J.P., Bashan, Y., 2010. Efficiency of growth and nutrient uptake from wastewater by heterotrophic, autotrophic and mixotrophic cultivation of 1 C. vulgaris immobilized with Azospirillum brasilense. Phycologia 46, 800e812. Pipes, W.O., Gotaas, H.B., 1960. Utilization of organic matter by Chlorella grown in seawage. Applied Microbiology 8, 163e169. Potter, J.L., 1993. Analysis of petroleum contaminated soil and water: an overview. In: Calabrese, E.J., Kostecki, P.T. (Eds.), Principles and Practices for Petroleum Contaminated Soils. Lewis Publishers, Chelsea, pp. 11e14. Pulz, O., Gross, W., 2004. Valuable products from biotechnology of microalgae. Applied Microbiology and Biotechnology 65, 635e648. Radmer, R.J., Parker, B.C., 1994. Commercial application of algae opportunities and constraints. Journal of Applied Phycology 6, 93e98. Semple, K.T., Cain, R.B., 1996. Biodegradation of phenols by the alga Ochromonas danica. Applied and Environmental Microbiology 62, 1265e1273. Sengar, H., 1970. Charakterlsiarung einer synckronkultur yon Scenedesmus obliquus. Planta 90, 243e266. Stanier, R.Y., Kunisawa, R., Mandel, M., Cohen-Bazire, G., 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriology Review 35, 171e205. Tikoo, V., Scragg, A.H., Shales, S.W., 1997. Degradation of pentachlorophenol by microalgae. Journal of Chemical Technology and Biotechnology 68, 425e431. Walker, J.D., Colwell, R.R., Vaituzis, Z., Meyer, S.A., 1975. Petroleum degrading achlorophyllous alga Prototheca zopfii. Nature 54, 423e424. Wetherell, D.F., 1961. Culture of fresh water algae in enriched natural seawater. Plant Physiology (Copenh), 14e16. Yan, H., Pan, G., 2004. Increase in biodegradation of dimethyl phthalate by Closterium lunula using inorganic carbon. Chemosphere 55, 1281e1285.