Bioremediation of Al-Sayyadin Lagoon polluted water using wild and mutant strains of microalgae

In this study, an attempt was made to reduce the pollutants of Al-Sayyadin Lagoon water, which is still the main site of open fishing in Ismailia, Egypt, using the wild and mutant strains of two green microalgae called Chlamydomonas reinhardtii CC1021 and Parachlorella kessleri PC. Four mutant strains were obtained from UV mutagenesis of the two wild types. One mutant strain was from C. reinhardtii CC1021 (CC1021Mut1) and the 3 other strains ((PCMut2, PCMut3 and PCMut4) were from P. kessleri PC. Reduction of nutrients like phosphate, ammonia, chemical oxygen demand (COD), biological oxygen demand (BOD) as well as some heavy metals like Co, Zn by the six microalgal strains was studied. The results obtained showed that the treated wild and mutant strains with mixture of TAP medium and polluted water showed highest growth rate and were more efficient in improving water quality than that treated with polluted water only in phycoremediation. The dose of UV radiation used in this study had no negative impact on the efficiency of bio-remediation potentials of the tested mutant strains but in some results it enhanced their growth rate and removal efficiency. The statistical analysis indicated that there was significant differences (p<0.05) found in bioremediation of water parameters between the selected algae. The wild and mutant strains of P. kessleri had higher efficiency in phycoremediation than the wild and mutant strains of C. reinhardtii CC1021. The mutant algae of P. kessleri PC exhibited significant higher growth rate and removal efficiency than the wild type.


INTRODUCTION
Pollution of surface water has become one of the most important environmental problems.Two types of large and long-lasting pollution threats can be recognized at the global level: organic pollution leading to high organic content in aquatic ecosystems and eutrophication.It is well-known that polluted water can reduce water quality, thus restricting the use of water bodies for many purposes.Organic pollution occurs when large quantities of organic compounds from many sources are released into the receiving running waters, lakes and also seas.Organic pollutants originate from domestic sewage farm water and could negatively affect water quality in many ways.During the decomposition process of organic water, dissolved oxygen in the water may be used up at greater rate than it can be replenished, thus giving rise to oxygen depletion which has severe consequences on aquatic biota.Organic effluents also frequently contain large quantities of suspended solid which reduces the light available to photosynthetic organisms, mainly algae.In addition to organic wastes from people and animals, these may also be rich in disease causing (pathogenic) organisms (Xu and Nirmalakhandan, 1998;Altenburger et al., 2000).Numerous natural and chemical substances have been used and released without knowledge of the possible impact on the structure and function of aquatic ecosystems (Häder et al., 2006).There are many physico-chemical methods available in treating polluted waters, but recent progress in bioremediation suggests that algae can play dual role in increasing biomass by utilizing waste as nutrients and can help in solving problems of pollution created by effluents (Mehta and Gaur, 2005).Efforts have been made to apply intensive microalgal cultures to remediate different wastes that have been described by de-Bashan et al. (2002), Rao et al. (2011) and Kshirsagar (2013).
The aim of the present investigation is to examine the efficiency of microalgae strains in the removal of inorganic nutrient and some heavy metals to prevent further deterioration of water quality of El Sayyadin Lagoon.It also aims to boost published literatures on mutagenesis in this area, which is far more limited.

Collection of wastewater
The polluted water samples used in this study were collected from Al Sayyadin Lagoon located in Ismailia City, Egypt (Figures 1 and  2).This lagoon is polluted by sanitary and agricultural wastewater from El Bahtini and Mahsama drains.

Microalgal strains
The microalgal strains of Chlamydomonas reinhardtii (CC1021mt+) and Parachlorella kessleri PC (the wild type strain SAG 211-11h) were formerly referred to as Chlorella vulgaris but were later reassigned into Chlorella kessleri (Fott and Nováková, 1969).Today, these species are referred to as P. kessleri (Krienitz et al., 2004), obtained from the Biotechnology Department, Kazakh National University-Al-Farabi culture collection.Both of these strains were cultured in TAP medium in 250 ml Erlenmeyer flask at 28°C and were exposed to continuous illumination at a light intensity of 120 µE m -2 s -1 .TAP medium composed of 25 ml from the first stock solution (NH 4 Cl 15 g/l, MgSO 4 .7H 2 O 4 g/l, CaCl 2 .2H 2 O 2 g/l) and 1 ml from the second stock solution (Phosphate buffer; K 2 HPO 4 28.8 g/100 ml and KH 2 PO 4 14.4 g/100 ml) in addition to Tris-base (2.42 g), 1 ml of glacial acetic acid, 1 ml of microelements and pH = 6-7.

Ultra violet (UV) mutagenesis
According to Harris (1989), 5 ml of the liquid culture with a density of 1x10 6 / ml of algal cells was placed in 9 cm Petri dish, forming a thin layer covering the bottom.The dish was placed on shaker with 20 rpm and exposed to UVC lamp of 254 nm and 5 W/m 2 at a distance equal to 15 cm for 0, 1, 3, 5, 7 and 10 min, respectively.After UV irradiation, the cells were inoculated in solid TAP medium and incubated in the dark for 24 h to prevent photo-reactivation.After 24 h, some dishes were incubated in light under photoautotrophic condition and the others incubated in the dark under heterotrophic condition for a period of 15 days.

Selection of the mutant strains
After the incubation period, four mutant strains were selected based on the phenotypic characteristics which appeared in the color and size of colonies that differ from the wild type colonies.These mutant strains were CC1021Mut1 obtained from exposure of C. reinhardtii (CC1021mt+) to UV irradiation for 1 min.The three others (PCMut2, PCMut3 and PCMut4) were obtained from exposure of P. kessleri to UV irradiation for 3, 7 and 10 min, respectively.These mutants were transferred from solid to liquid medium and kept under phototrophic growth conditions to be tested for their efficiency in the bioremediation of polluted water (Figures 3 and 4).

Design of the bioremediation experiment
To study the efficiency of microalgae in bioremediation of polluted water, the following methods were employed: (i) Cultural media inoculated with algal strain (negative control), (ii) polluted water inoculated with algal strain (positive control), (iii) series of diluted polluted water and cultural media inoculated with algal strain as follows: Sample number 1: Negative control (1000 ml of the culturing medium) inoculated with the algal strain.
The total number of samples for the six algal strains was 36 and these samples were incubated for one week at the same growth condition of light and temperature.

Investigation of algal growth strains in the samples
Examination of the algal cells was done by using light inverted microscope of the OLYMPUS series.The initial cell density was about 1×10 5 cells/ml.The number of cells was counted everyday for one week by using UTERMÖHL's technique (1958) under light microscope (Wetzel and Likens, 1979).The growth of algal strains was plotted for the six samples for each strain.The algal growth percentage of the six samples for each algal strain was calculated (Figure 5).

Analytical method
Water samples were filtered onto Whatman GF/C glass fiber filters to get rid of the algal cells.The physical and chemical parameters and some heavy metals of polluted water were analyzed before (Table 1) and after using the selected algae (Tables 2 and 3) according to the Standard Methods for Examination of Water and Waste Water (APHA, 1989).The parameters under study were ammonia, phosphate, biological oxygen demand (BOD), chemical oxygen demand (COD) and cobalt and zinc.After one week, these parameters were analyzed in samples numbers 2, 4 and 6 for each of the algal strain.

Algal growth rate
It is clear from Figure 5 that the algal growth rate of the two wild types and their mutants in both selected dilutions are lower in the positive controls which contain the polluted water only as compared to other dilution samples and the negative control.On the other hand, the negative control of C. reinhardtii CC1021, P. kessleri and CC1021 Mut1 strains, respectively achieved a higher percentage of cell numbers as compared to their dilutions and decreased with the increase of polluted water content.In contrast, there is a significant increase of the growth rate recorded in sample numbers 2, 3, 4 for PCMut2 and in samples 3 and 4 for PCMut3 as compared to the negative control.PCMut4 strain growth rate percentage was high in samples 2, 3, 4, 5 as compared to the control and showed the best growth rate of 152.2% in sample number 3 (dilution 3:2).

Water analysis
The results obtained from water analysis (Tables 2 and 3; Figures 6,7,8) showed that, the initial pH of the waste water was 6.02, and after being treated with the algal strains, it increased from 7 to 7.

DISCUSSION
Microalgae usually play an important role during the  tertiary treatment of domestic wastewaters in maturation ponds or the treatment of small/middle scale municipal wastewater in facultative or aerobic ponds (Aziz and Ng, 1993;Abeliovich, 1986;Mara and Pearson, 1986;Oswald et al., 1996).Algae utilize wastes as nutritional source and enzymatically degrade pollution.They enhance the removal of nutrients, heavy metals and pathogens and furnish O 2 to heterotrophic aerobic bacteria to mineralize organic pollutants, using in turn the CO 2 released from bacterial respiration (Munoz and Guieysse, 2006).The present work aims to describe the screening of 6 microalgal strains to select a good candidate for the removal of pollutants from waste water.In this study, two different species of green microalgae-C.reinhardtii CC1021 strain and P. kessleri were used as target organisms because they were used as model for phototrophic organisms and are green algae in particular.
It is well known that genus Chlamydomonas was considered as a significant step toward the use of algae for remediation of contaminated sites and waters (Hallmann, 2007).The mutations in this study were induced by using UV radiation.UV mutagenesis offers many advantages such as less pollution, simple operation and sterile cultivation condition (Huang et al., 2010).UV-induced physiological effects such as declining photosynthetic rates can be related not only to damaged biomolecules, but also to ultrastructural changes in organelles or membranes (Holzinger and Lütz, 2006).The two microalgae were treated with the mutagen UV and the result was 4 mutant strains and such mutagenically treated algae were compared with untreated wild types in terms of growth and remediation efficiency.The results showed that all the cultures of the selected strains showed highest growth percent as compared to that of the positive control (Figure 6).The less growth of the algal strains in the positive control is an indication of highly polluted water (Afkar et al., 2010).This may be attributed to the presence of other microorganisms and bacteria that compete with our strains and lowered their growth as compared to the others that contained mixture of polluted water and culturing medium.It is well known that the role of culture media is to make algal growth to be faster than the growth of the other microorganisms which is not adapted to this culture media.This result was confirmed by reports of Cho et al. (2011) and Olumayowa et al. (2013) that the growth of microalgae increases by 1.5 and 2.5 fold in autoclaved wastewater to get rid of other microorganisms and bacteria, even though some bacteria have been found to secrete algal growth promoters (Mazur et al., 1995).
The growth rates of the wild type strain of CC1021, CC1021 mutant and PC wild type in this study are nearly the same, that is, they attained the higher growth percentage in the negative control sample, and decreased with the increase of polluted water in the dilutions (Figure 5).In contrast, the growth rate of mutants PCMut2, PCMut3 and PCMut4 exhibited significant higher growth values in the presence of cultural media and polluted water than the wild type.This demonstrates that UV radiation had a negative potential effect on the mutant growth of CC1021 Mut1 strain.This is in line with the study of Ikehata and Ono (2011) that showed it had no negative potential effect on the growth of the three mutants of PC but it enhanced their growth and resistance to pollutants.This result contradicts that of Michler et al. (2002) who reported that UV can have a strong effect on nutrient uptake, motility, reproduction and growth.About the resistance of microalgae, it was found that, microalgae are able to survive short-term unpredictable environmental stress by means of physiological acclimatization as a result of the modification of gene expression (Bradshaw and Hardwick, 1989;Fogg, 2001;Costas et al., 2008).However, when environmental stress exceeds physiological limits, only the occurrence of mutations that confers resistance can allow adaptation (Costas et al., 2001).More and more examples accumulate on rapid adaptation of microalgae to extreme environments through the selection of rare spontaneous mutations conferring resistance which affects only one gene.
By means of these mechanisms, microalgae achieve adaptation toxenobiotics (García-Villada et al., 2002).In addition, Chlorophytes species were more capable of rapid adaptation to extreme environments than other algae phyla (de-Godos et al., 2010).The obtained results (Tables 1, 2 and  3; Figures 6, 7 and 8) demonstrate that in samples number 2 and 4 (dilution 4:1 and dilution 2:3), all the algal strains did not only attain higher growth percent, but also achieved high efficiency of removal percent; this result may be in accordance with that of Wang et al. (2009) and Choi and Lee (2012) who reported more quantitatively increasing Chlorella caused an increase of removal rate.Generally, the result showed that NH 4 -N contents were removed more effectively than phosphate and this is in agreement with Kshirsagar (2013).The above results are in agreement with many researchers with respect to phycoremediation especially Chlorella and Chlamydomonas (Cho et al., 2011;Johnson and Wen, 2010;Kong et al., 2010).The mechanism involved in algal nutrient removal was an uptake by the cells and stripping ammonia through elevated pH (Aslan and Kapdan, 2006;Kong et al., 2010).
The initial pH in this study was 6.02 and reached 7.2 after bioremediation.Microalgae were reported to be more efficient in sequestering metal species from solution than bacterial and fungal biomass (Khoshmanesh et al., 1996).
The mechanism of the effectiveness in removing heavy metals from waste water by microalgae is related to their large surface area and high binding affinity (Roy et al., 1993).Different algal species have different sizes, shapes and cell wall compositions which affect their metal binding (Wehrheim and Wettern, 1994;Cai et al., 1995).Generally in the pH (6 to 7) most of metal ions can be significantly removed (Rivas, et al., 2007;Sannasi and Salamija, 2011).It was remarkable that CC1021 showed the best removal of Zn and this was similar to that reported by Jennett et al. (1980).The effect of BOD and COD reduction in the presence of 6 algal strains is seen in Tables 2 and 3 and graphically represented in Figures 9a  and b.The percentage of BOD and COD reduction in CC1021 reached 94.8, 99.5 and 97.4,99.7%, respectively; for the mutant strain, CC1021Mut1, 91.7, 98.3 and 94.4,99.7%; for the wild type PC, 91.7, 98.3 and 94.4,99.7%; while the removal percents, 88.2, 93.1 and 98.6, 99.8%; 9 2.4, 87% and 95.8, 99.5%; 86.2, 86.2% and 96.9, 99.4% were achieved for PCMut2, PCMut3 and PCMut4.A similar result was observed by Kshirsagar (2013), Kotteswari et al. (2012) and Wang et al. (2009); C. vulgaris mostly showed that COD and BOD removal efficiency was 88 and 89.60% (Valderrama et al., 2002), which is in agreement with our results.

Conclusion
1) The dose of UV radiation used in this study had no negative impact on the efficiency of bio-remediation potentials of the tested mutant strains; however, in some results, it enhanced the resistance of the mutants to polluted water, increasing their growth rate and in turn their removal efficiency.2) NH 4 contents were removed more effectively than phosphate in all algal strains.3) Statistical analysis using a post hoc multiple test showed that the wild and mutant strains had convergent efficiency in treatment of polluted water, as there was significant difference (p<0.05)found in bioremediation of water parameters between the six algae in both dilutions of 4:1 and 2:3.4) The mutant strain, PCMut2 showed the best removal capacity for BOD, COD and phosphate, while the mutant strain, PCMut3 showed the best result for Co; the wild strain PC has the best removal of ammonia (Duncan, p< 0.05).On the other hand, the wild type CC1021 was the best efficient strain in lowering the levels of Zn.However, the mutant strain CC1021Mut1 ranked the second order in lowering the levels of COD and Zn (Duncan, p< 0.05).5) The sample number 4, dilution 2:3 (400 ml culturing medium + 600 ml of the polluted water) had better concentration than the others for effective nutrients removal.6) The wild and mutant strains of P. kessleri had higher efficiency in phycoremediation than the wild and mutant strains of C. reinhardtii CC1021.7) The mutant strains of Parachlorella PC appeared more efficient in bioremediation than the wild type as they have fast growth rates by mutagenesis.This removable efficiency exhibited by these mutants makes them excellent candidates for the removal of polluted water.
In summary, there is a possibility of producing mutants of Chlorella that have higher growth rate and removal efficiency.

Figure 5 .
Figure5.The growth percent of the six algal strains in different selected samples after 7 days.

Figure 6 .
Figure 6.Decrease % of the selected parameters in sample No. 2 after using the algal strains for one week.

Figure 7 .
Figure 7. Decrease % of the selected parameters of sample No.4 after using the algal strains.

Figure 8 .
Figure 8. Increase % of the selected parameters of sample No. 6 after using the algal strain except Cobalt.

Figure 9 .
Figure 9. Decrease % of the BOD and COD after using the algal strains (a) in sample No. 2 (b) in sample No. 4.

Table 1 .
The analysis of water before using algae.

Table 2 .
Analysis of water samples after bioremediation for one week using CC1021 and CC1021Mut1 strains.

Table 3 .
Analysis of water samples after bioremediation for one week using Pc, PCMut2, PCMut3 and PCMut4 strains.