Considering well documented toxicity of boron (B) at certain levels, some parameters for the best boron removal efficiency, including different medial compositions, pH, boron and biomass concentrations, exerted by a newly discovered and purified species of Chlorella sp. have been studied. Different medial compositions were investigated by inoculating the microalgae into 100 mL BG 11 media (pH 8.0) and incubating for 20 days (25 ± 2 oC, continuous illumination 12.5 wm-2 (2400 lx)). The effects of addition of triacontanol hormone as a growth stimulant, or sodium bicarbonate as a carbon source and their combined effects on 5-10 mg L-1 boron containing samples were investigated. The maximum removal yield was 26.58 % at 10 mg L-1 boron level in media containing both of the stimulants; the maximum specific uptake values (qm) was 0.49 mg g-1. The effect of different pH values on the highest removal yield found was investigated at pH 5, 6, 7, 8 and 9, and the maximum value was at pH 8 again. The relation between biomass concentration and boron removal was also investigated in the mostly effective conditions and at the 4.0-5.6 g L-1 biomass range. The highest yields were found as 19.59-38.03 %, and the related qm values were 0.31-0.62 mg g-1 at these biomass concentrations, respectively. These results indicate that at least the isolated Chlorella sp. can be easily and economically stimulated to grow by adding triacontanol plus bicarbonate and used in boron removal in polluted waters.
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Keywords: Chlorella sp., boron removal, triacontanol, sodium bicarbonate, biomass concentration, wastewater treatment
As known very well boron (B) is one of the essential trace elements (Waggot, 1969) and its biological role is still been studied by a number of researchers (Lee et al., 2009; Sheng et al., 2009). In spite of its biologically important role in metabolism at its low concentrations (Frick, 1985; Wojcik et al., 2008; Lee et al., 2009), excessive amounts are injurious and cause toxic effects (Davis et al., 2002; Gunes et al., 2006; Del-Campo MarÄ±n and Oron, 2007; Sasmaz and Obek, 2009). As expected B is released to the environment mainly in discharged industrial wastewaters (Coughlin, 1998). Manufacturing facilities of heat resistant materials (Morioka et al., 2007), storage and distribution of solar energy systems (Abu-Hamed et al., 2007), catalysts (Xu et al., 2009), ceramics (Christogerou et al., 2009) and glass (Crawford et al., 2007) can be presented as the examples of such point pollution sources.
Turkey on the other hand, has the largest B reserves (60 %) in the world (Okay et al., 1985). As Cervilla et al.(2009) attracted attention to the fact that B toxicity had become important especially in areas close to the Mediterranean Sea, where intensive agriculture had been developed. They evidented that excess B in the cultivated soils lead to B toxicity which caused inhibition of nitrate reduction and consequent increase in ammonium assimilation in tomato plants, accompanied with the loss of leaf biomass and disorders in organic nitrogen metabolism. Nable et al (1997) in fact, previously stated that B-rich soils were important, causing B toxicity in the field and which decreased crop yields. They added that various anthropogenic sources of excess B might increase soil B to toxic levels for plants, such as wastes from surface mining, fly ash, and industrial chemicals, and the most important source was irrigation water. They also mentioned that ameliorating high-B soils was extremely difficult, a commonly used method was to leach extensively with low B water, which was not a permanent solution causing also difficulties with the disposal of the leachates.
Certainly removing B from industrial wastewaters also receives interest, in the recent studies methods including adsorption-flocculation (Chong et al., 2009; Kavak, 2009), electrocoagulation (Yilmaz et al., 2008), reverse osmosis (-ztürk et al., 2008), precipitation (Itakura et al., 2005), ion-exchange (Okay et al., 1985), use of B-selective resins (Simonnot et al., 2000) and some biological materials, such as duckweeds (Del-Campo Marin and Oron, 2007; Sasmaz and Obek, 2009) have been tried.
In recent years, on the other hand, a number of studies have focused on the use of microalgae in removal of several pollutants from the culture media or wastewaters (De-Bashan and Bashan, 2010; Karacakaya et al., 2009; El-Sheekh et al., 2005). Chlorella sp. is known as one of the most useful microalgae for different purposes. This genus is preferred in numerous studies considering its high growth rates under effect of many medial ambiences (Lee et al., 2002; Valderrama et al., 2002; Sung et al., 1999). Although it was shown that Chlorella sp. could remove pollutants with a high capacity and in an efficient way as a less sensitive microalgae compared to many other aquatic organisms (Ruangsomboon and Wongrat, 2006; Hanagata et al., 1992), its capacity to remove B has not been investigated previously, therefore this study aims to fill this gap.
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Another purpose of the present study is investigating the effects of addition of some growth stimulators into the media to see if they would enhance growth of Chlorella sp. and increase efficiency of bioremoval process. One of the growth stimulator selected is Triacontanol (TRIA) and the other is sodium bicarbonate (NaHCO3). TRIA, a long chain 30-carbon primary alcohol, (C30H61OH) is a well known plant hormone and growth regulator (Ries et al., 1977; Ries and Houtz, 1983). Stimulatory effects of TRIA on the photosynthesis, growth and net bioproductivity of some green algae and cyanobacteria species have also been reported (Karacakaya et al. 2009; Houtz et al. 1985a; 1985b). On the other hand, as Wang et al., (2008) reviewed that some of microalgal species could utilize carbonates such as NaHCO3 and Na2CO3 for cell growth with high extracellular carboanhydrase activities, which are responsible for the conversion of carbonate to free CO2 facilitating higher C assimilation levels (Emma Huertas et al., 2000; Merrett et al., 1996; Ginzburg 1993)
The main goals of the current search were (i) to reveal the usage of Chlorella sp. as an effective biomaterial for B removal from the culture media, (ii) determination of the best removal conditions in detail, by using different parameters, and (iii) to check if there was a potential offered by Chlorella sp. to develop a cost effective and eco-friendly B removal procedure. Furthermore, the effects of different medial compositions pH, B and biomass concentrations on the removal process were studied. To our knowledge this is the first report about B removal by a new isolated microalgae Chlorella sp., with proposed target comprehensively. Ultimately, we hope the study will impact the way in which the people perceive the danger of pollutants in water usage by the public.
2. Materials and Methods
The microalgal culture was observed in the water samples taken from the pond in the Garden of Ankara University, Faculty of Science, Ankara, Turkey. The samples taken were spread on the Petri plates containing BG 11 medium (Rippka, 1988) and were incubated at 25 ± 2 oC under continuous illumination (cool-white fluorescent, 12.5 wm-2 (2400 lx)). The pH of the growth medium was adjusted to 8 by adding dilute (0.01 M) and concentrated (1 M) sulfuric acid or sodium hydroxide solutions. Cells from microcolonies on these plates were isolated by
micromanipulation. The microalgal cells were purified at aseptic conditions by streaking the cells repeatedly on the BG 11 medium agar plate. Then, the purified microalgal cells were transferred to liquid media. In order to validate the axecinity, these liquid cultures were tested for bacterial contamination by plating on bacteriological media.
2.2. TRIA, NaHCO3 and Boron solutions
Stock TRIA (96 %, w/v; Aldrich) solution was prepared by dissolving 0.5 g of the chemical in chloroform. Sodium bicarbonate solution (Merck) was prepared by dissolving 17.2 g L-1 of the NaHCO3 in distilled water. Stock solution of B was prepared by dilution of boric acid (H3BO3) (Carlo Erba) to a final concentration of 10 g L-1 of B. Appropriate volumes of the stock solutions were added to the BG 11 media.
2.3. Culture conditions
A series of batch culture experiments in unshaken flasks illuminated by cool white fluorescent lamps were carried out at 12.5 wm-2 (2400 lx) light intensity. The microalgal culture was transferred into 100 mL BG 11 media or the media containing known concentrations of B and/ or TRIA and/or NaHCO3 solutions in 250 mL Erlenmeyer flasks and all were incubated at 25 ± 2 oC under continuous illumination for 20 days.
2.4. Effects of different media compositions on boron removal
To determine the effect of the different media compositions on B removal, the microalgae were cultivated in media containing increasing concentrations of B (5, 7.5 and 10 mg L-1) in; (1) BG 11 control medium without any contents; (2) BG 11 medium with 1 mg L-1 TRIA; (3) BG 11 medium with 34 mg L-1 NaHCO3 and (4) BG 11 medium with 1 mg L-1 TRIA and 34 mg L-1 NaHCO3 solutions at pH 8 (unpublished results) for 20 days incubation period. For these experiments, Erlenmeyer flasks containing 100 mL medium, to give an initial concentration of about 4.8 g dry wt. biomass L-1 inoculated in culture media.
2.5. Effect of initial pH on boron removal
As the preliminary experiments indicated that changes in pH values affected B removal by Chlorella sp., trials were performed at five different initial pH values 5, 6, 7, 8 and 9 at 10 mg L-1 B concentration in media with 1 mg L-1 TRIA and 34 mg L-1 NaHCO3 for 15 days incubation period in order to find a suitable pH. For pH experiments, Erlenmeyer flasks containing 100 mL medium, to give an initial concentration of about 4.8 g dry wt. biomass L-1 inoculated in culture media.
2.6. Effects of the biomass concentrations on boron removal
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The influence of microalgal biomass on B removal was investigated using four different biomass concentrations. The biomass concentrations were estimated using the dry weight method. The experiments were performed in BG 11 + TRIA + NaHCO3 media at pH 8 for 20 days incubation period and Erlenmeyer flasks containing 100 mL medium, to give an initial concentration of about 4.0, 4.8, 5.2 and 5.6 g dry wt. biomass L-1 were inoculated in culture media.
2.6. Analytical methods
During the incubation period, 3 mL samples were taken at 5, 10, 15 and 20 days from each of the flasks. The B concentration was determined by measuring the absorbance at 585 nm with a Shimadzu UV 2001 model spectrophotometer (Adams, 1990).
For the measurement of microalgal growth at the end of 10, 15 and 20 days incubation periods the biomass concentration was determined by measuring both of optic density and dry weight parameters for any set of growth conditions. Optic density was measured at 600 nm with the spectrophotometer.
Dry weight was determined by centrifugation on the samples at 5000 rpm for 10 min (Hettich EBA 12 model centrifuge) and drying at 80 °C (Nüve FN 400 model sterilizator) overnight.
2.7. Statistical analysis
The experiments were set in a completely randomized design up with three replicates. The data were subjected to analysis of variance using significant differences among treatment means were compared by descriptive statistics (±S.E.).
3. Results and Discussion
In the study, the isolated and purified microalgal strain which was identified as Chlorella sp. by evaluating the morphological properties was used (Fig 1). Chlorella sp. is a single-celled fresh-water, microscopic green algae with spherical cells about 2-8 μm in diameter, almost the same size as human red blood cells (Bewicke and Potter 1984).
< Here Fig. 1 >
3.1. Effects of different media compositions and boron concentrations on the boron removal
Previous studies showed that the initial concentration of B was an important parameter in B bioremoval efficiency that was measured in the culture media (Del-Campo Marín and Oron, 2007). Therefore, we first tested this parameter with three B concentrations, i.e. about 5, 7.5 and 10 mg L-1, in the first series of experiments which were designed also for measurement of the effect of different media compositions. The aim was to see whether B removals were also affected by TRIA and / or NaHCO3. Four different BG 11 media compositions were investigated for this proposes: (i) BG 11 control samples, (ii) BG 11 + TRIA, (iii) BG 11 + NaHCO3, (iv) BG 11 + TRIA + NaHCO3. The results which are presented in Fig 2. showed that the removal yield decreased when initial B concentrations were increased up to 10 mg L-1.
< Here Fig. 2 >
As presented in the Figure 2 a-b and with Fig 2 c-d, the removal yields were close to each other at initial B concentrations of 5-10 mg L-1, and all of the removal yields found showed significant increases only after 10 days of the experiments. The maximum B removal was observed at 5.08 mg L-1, i.e., 23.36 % in BG 11 control samples in 20 days of incubation, and there was no significant difference between the controls and the BG 11 + TRIA samples. For example, the highest B removal yield was 24.13 %, in the presence of 5.66 mg L-1 B in the BG 11 + TRIA samples. The removal yields were also close to each other at initial concentrations of about 7.5 and 10 mg L-1 B containing samples. These results can be interpreted as a sign of ineffectiveness of TRIA in B removal when applied alone. NaHCO3, on the other hand, showed its highest B removal efficiency at 5.55 mg L-1 B (29.31 %), its lowest value was found at 9.32 mg L-1 B concentration (23.55 %), higher than both of the controls and TRIA samples. The presence of NaHCO3 was an effective factor on B removal by Chlorella sp.
Another result, perhaps more important than above, was obtained by adding TRIA and NaHCO3 to the culture media together enhanced the growth of Chlorella sp. Consequently the B removal values reached to the highest of all held in this study. Enhancement of growth by TRIA and NaHCO3 could be the reason of the increase of growth and the maximum specific B uptake values (qm) presented below (Fig. 2d). It is clearly seen that TRIA, stimulated all of the parameters measured in the presence of NaHCO3. Oxidation of the HCO3 to CO2 and its assimilation increased the primary production in terms of dry weight. In the medium with 1 mg L-1 TRIA and 34 mg L-1 NaHCO3 the B removal reached its highest value on day 20 of incubation. The removal yield of B was 18.9 % at 5.45 mg L-1 B concentration on 10th day, and increased to 32.95 % on the 20th day. In TRIA + NaHCO3 samples the microalgae removed the B efficiently up to 8.68 mg L-1 level, with the highest yield of 26.58 %.
The findings reported by Del-Campo Marín and Oron (2007), who used Lemna gibba in their study, bioremoval of B by L. gibba decreased when its initial concentrations in the media were increased, and the most efficient removal measured was below 2 mg L-1 B concentration. They added that there was no removal at about 10 mg L-1 B concentration even on 12th day of cultivation. In the present study however, Chlorella sp. showed significant removal efficiency at 10 mg L-1 B concentration at the four different media compositions tested. Removal capacity could be increased further through stimulation of the biomass production by the addition of TRIA and NaHCO3 to the BG 11 medium. There are some of the studies in the literature, describing removal of industrial pollutants in an effective way by including TRIA hormone in BG 11 culture media by freshwater cyanobacterium Synechocystis sp. (Karacakaya et al., 2009). Thus, we can conclude that further studies on the use of Chlorella sp. in bioremoval of some pollutants may give more meaningful and promising results in terms of higher level of removal yields for several pollutants.
The comparison of the maximum specific B removal values (qm) found in four different media compositions used in the present study are summarized in Table 1. In an effort to test the validity of the above presented results, the maximum specific B removals in TRIA + NaHCO3 samples were also measured and found higher values at all tested initial B concentrations, parallel to the growth rates which were also higher than the controls. In the samples with higher amounts of biomass the removal of B per one gram of dry weight was also higher and the lowest biomass was in the BG 11 control samples. Comparison of qm values for B removal in media with and without TRIA showed that there were slight difference, indicating the presence hormonal stimulation. As presented in the table, addition of TRIA to the media increased qm values from 0.28 mg g-1 to 0.31 mg g-1 at about 5 mg L-1 B concentration, and from 0.26 mg g-1 to 0.34 mg g-1 at about 7.5 mg L-1 B level. The highest qm values in NaHCO3 samples were found as 0.34, 0.38 and 0.35 mg g-1 at 5.55, 6.71 and 9.32 mg L-1 B concentrations, respectively. Stimulation by NaHCO3 in the media was clearly evident, as shown by the maximum specific qm values at all tested initial B concentrations. On the other hand, Chlorella sp. showed its highest qm values in TRIA + NaHCO3 medium as 0.39, 0.40 and 0.49 mg g-1 at 5.45, 6.84, and 8.68 mg L-1 B concentrations. In general, the maximum specific B removal values were increased by the higher initial B concentrations up to a certain level. qm values decreased above the 7.5 mg L-1 B concentrations without any exception, but the qm values measured in the samples containing BG 11 + TRIA + NaHCO3 increased when boron concentrations increased from 5 to 10 mg L-1 . This pattern of changes was due to the high growth rate of Chlorella sp. in TRIA + NaHCO3 samples.
< Here Tab. 1 >
3.2. Effect of initial pH on boron removal
In line with the above informations, selected optimum media compositions we tested for optimum pH value for the maximum B removal. The effect of initial pH on the removal by Chlorella sp. was summarized in Fig 3. As shown in the figure, B removal was 13.64 %, 15.24 %, and 18.71 % at pH 5, 6 and 7 respectively. Chlorella sp. removed B with the highest yield of 22.4 % at pH 8. It was observed that the removal yield of B increased with an increase in pH values up to pH 8, but decreased sharply to 13.50 % when pH increased to 9. As these experiments demonstrated that Chlorella sp. showed its highest B removal yields at pH 8, the following experiments were conducted at this pH value. Bursali et al. (2009) and Yilmaz et al. (2008) also marked basic pH values for efficient B removal by the macroalgae Caulerpa racemosa var. cylindracea and by the electrocoagulation in a batch reactor.
< Here Fig. 3 >
3.3. Effect of the biomass concentrations on boron removal
Influence of the initial amount of Chlorella sp. biomass on B removal was investigated at four different biomass concentrations (4.0, 4.8, 5.2 and 5.6 g L-1). Figure 4 presents the results obtained by incubating the samples for 20 days which evidenced the presence of crucial effects of initial level on the bioremoval, which increased from 19.59 % to 26.58 % at the end of 20 days when biomass concentration increased from 4.0 g L-1 to 4.8 g L-1. Removal value was 29.34 % at 5.2 g L-1 biomass concentration on 15th day of incubation, and increased to 33.48 % at the end of 20 days. The best result for B removal value was 38.03 % at 5.6 g L-1 biomass concentration on the day 20. It is clearly seen in Fig 4 that the relation between these variables was roughly linear. This linearity can be interpreted as the indication of the relation between higher growth rates and tolerance to B in Chlorella sp. used for the experiments.
< Here Fig. 4 >
The relation between the maximum specific B uptake values and different biomass concentrations has also been determined and presented in Table 2. The effect of biomass concentrations on qm values after the incubation period showed that the microalgal uptake capacity and removal yield of B at 5.6 g L-1 biomass concentration was higher than 4.0 g L-1.
< Here Tab. 2 >
The maximum specific boron removal levels were higher at higher biomass concentrations, as the qm values and growth rates of microalgae were higher at higher biomass concentrations. At 4.0 g L-1 biomass concentration, the qm value was 0.31 mg g-1, and the removal yield was 19.59 %. The qm values increased from 0.49 mg g-1 to 0.58 mg g-1 as the increase in biomass concentration from 4.8 to 5.2 g L-1. The highest qm value was 0.62 mg g-1 at 5.6 g L-1 biomass concentration and the highest B removal percentage reached to 38.03 %.
The data given in Table 2 also shows the pattern of the changes in chlorophyll (a+b) concentrations and growth rates (µ) of Chlorella sp. within the 20 days incubation period. As shown in Table, chlorophyll values increased from 0.21 µg mL-1 to 1.254 µg mL-1 in parallel to the increasing biomass concentrations from 4.0- 5.6 g L-1. Chlorophyll (a+b) concentration reached to its maximum value in the samples containing 5.6 g L-1 initial biomass level. There was noticeable difference between the growth rates at the samples containing different biomass concentrations, and the highest µ value of 0.177 d-1 was found at the sample containing 5.2 g L-1 biomass concentration. As expected, growth rate decreased to 0.139 d-1 in the 5.6 g L-1 samples, when the initial biomass concentration exceeded this optimum level. The reason was due to already high biomass amounts of microalgae.
If the removal (%), qm, chlorophyll and µ values discussed, correspond to the very high amounts of biomass would also higher removal and chlorophyll values. But, due to the high microalgal biomass in the culture media, the growth rate was less and hence the maximum specific B uptake was even too. In the light of these results, appropriate biomass concentration to B removal was 5.2 g L-1. Finally, correlate with the removal (%), qm, chlorophyll (a+b) and µ results between the effect of media compositions and the effect of biomass, showed that higher biomass concentrations and addition of TRIA and NaHCO3 in the culture media were enhance effects of growth and the removal yield did so.
The ultimate aim of this study was to investigate if the presently isolated, which is an easy to find, isolate and cultivate Chlorella sp. could serve as biomaterial for B removal from water and if its potential could be increased by the stimulatory effect of some growth stimulating agents. We performed various experiments for this purpose and brought to light that this Chlorella sp., was capable of removing B from the BG 11 culture media initially containing B up to 10 mg L-1 concentration.
Some investigators in fact, have used Chlorella spp. as a biomaterial for removal of some pollutants (Lim et al., 2010; Valderrama et al., 2002), but no Chlorella sp. has been used in such studies for B bioremoval. The stimulants used in the present study have not been tested for increasing the growth and bioremoval capacity of the subject green alga. Thus, it can be concluded that the present study has revealed the exploitable potential of B removal capacity of Chlorella sp. as a well defined, cost-effective and eco-friendly treatment organism.
Financial support was gratefully acknowledged by the Scientific and Technological Research Council of Turkey (TÜB°TAK-B°DEB).