Boron bioremoval by isolated Chlorella from culture media Essay


Sing good documented toxicity of B ( B ) at certain degrees, some parametric quantities for the best B remotion efficiency, including different median composings, pH, B and biomass concentrations, exerted by a freshly discovered and purified species of Chlorella sp. have been studied.

Different median composings were investigated by inoculating the microalgae into 100 milliliters BG 11 media ( pH 8.0 ) and incubating for 20 yearss ( 25 ± 2 oC, uninterrupted light 12.5 wm-2 ( 2400 sixty ) ) . The effects of add-on of triacontanol endocrine as a growing stimulation, or Na hydrogen carbonate as a C beginning and their combined effects on 5-10 mg L-1 B incorporating samples were investigated. The maximal removal output was 26.58 % at 10 mg L-1 B degree in media incorporating both of the stimulations ; the maximal specific consumption values ( qm ) was 0.49 mg g-1.

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The consequence of different pH values on the highest removal output found was investigated at pH 5, 6, 7, 8 and 9, and the maximal value was at pH 8 once more. The relation between biomass concentration and B remotion was besides investigated in the largely effectual conditions and at the 4.0-5.6 g L-1 biomass scope. The highest outputs were found as 19.59-38.03 % , and the related qm values were 0.

31-0.62 mg g-1 at these biomass concentrations, severally. These consequences indicate that at least the stray Chlorella sp. can be easy and economically stimulated to turn by adding triacontanol plus bicarbonate and used in B remotion in contaminated Waterss.

Keywords: Chlorella sp. , B remotion, triacontanol, Na hydrogen carbonate, biomass concentration, effluent intervention

1. Introduction

As known really good B ( B ) is one of the indispensable hint elements ( Waggot, 1969 ) and its biological function is still been studied by a figure of research workers ( Lee et al. , 2009 ; Sheng et al.

, 2009 ) . In malice of its biologically of import function in metamorphosis at its low concentrations ( Frick, 1985 ; Wojcik et al. , 2008 ; Lee et al. , 2009 ) , inordinate sums are deleterious and cause toxic effects ( Davis et al. , 2002 ; Gunes et al.

, 2006 ; Del-Campo MarA±n and Oron, 2007 ; Sasmaz and Obek, 2009 ) . As expected B is released to the environment chiefly in dismissed industrial effluents ( Coughlin, 1998 ) . Manufacturing installations of heat immune stuffs ( Morioka et al. , 2007 ) , storage and distribution of solar energy systems ( Abu-Hamed et al. , 2007 ) , accelerators ( Xu et al. , 2009 ) , ceramics ( Christogerou et al. , 2009 ) and glass ( Crawford et al.

, 2007 ) can be presented as the illustrations of such point pollution beginnings.Turkey on the other manus, has the largest Bacillus militias ( 60 % ) in the universe ( Okay et al. , 1985 ) . As Cervilla et Al.

( 2009 ) attracted attending to the fact that B toxicity had become of import particularly in countries near to the Mediterranean Sea, where intensive agribusiness had been developed. They evidented that surplus B in the cultivated dirts lead to B toxicity which caused suppression of nitrate decrease and attendant addition in ammonium assimilation in tomato workss, accompanied with the loss of leaf biomass and upsets in organic N metamorphosis. Nable et Al ( 1997 ) in fact, antecedently stated that B-rich dirts were of import, doing B toxicity in the field and which decreased harvest outputs. They added that assorted anthropogenetic beginnings of surplus B might increase dirt B to toxic degrees for workss, such as wastes from surface excavation, fly ash, and industrial chemicals, and the most of import beginning was irrigation H2O. They besides mentioned that bettering high-B dirts was highly hard, a normally used method was to leach extensively with low B H2O, which was non a lasting solution causation besides difficulties with the disposal of the leachates.Surely taking B from industrial effluents besides receives involvement, in the recent surveies methods including adsorption-flocculation ( Chong et al. , 2009 ; Kavak, 2009 ) , electrocoagulation ( Yilmaz et al.

, 2008 ) , rearward osmosis ( -zt & A ; uuml ; rk et al. , 2008 ) , precipitation ( Itakura et al. , 2005 ) , ion-exchange ( Okay et al.

, 1985 ) , usage of B-selective rosins ( Simonnot et al. , 2000 ) and some biological stuffs, such as duckweeds ( Del-Campo Marin and Oron, 2007 ; Sasmaz and Obek, 2009 ) have been tried.In recent old ages, on the other manus, a figure of surveies have focused on the usage of microalgae in remotion of several pollutants from the civilization media or effluents ( De-Bashan and Bashan, 2010 ; Karacakaya et al. , 2009 ; El-Sheekh et al. , 2005 ) .

Chlorella sp. is known as one of the most utile microalgae for different intents. This genus is preferred in legion surveies sing its high growing rates under consequence of many median atmospheres ( Lee et al. , 2002 ; Valderrama et al. , 2002 ; Sung et al. , 1999 ) . Although it was shown that Chlorella sp.

could take pollutants with a high capacity and in an efficient manner as a less sensitive microalgae compared to many other aquatic beings ( Ruangsomboon and Wongrat, 2006 ; Hanagata et al. , 1992 ) , its capacity to take B has non been investigated antecedently, hence this survey aims to make full this spread.Another intent of the present survey is look intoing the effects of add-on of some growing stimulators into the media to see if they would heighten growing of Chlorella sp. and increase efficiency of bioremoval procedure. One of the growing stimulator selected is Triacontanol ( TRIA ) and the other is sodium hydrogen carbonate ( NaHCO3 ) . TRIA, a long concatenation 30-carbon primary intoxicant, ( C30H61OH ) is a good known works endocrine and growing regulator ( Ries et al.

, 1977 ; Ries and Houtz, 1983 ) . Stimulatory effects of TRIA on the photosynthesis, growing and net bioproductivity of some green algae and blue-green algae species have besides been reported ( Karacakaya et al. 2009 ; Houtz et Al. 1985a ; 1985b ) . On the other manus, as Wang et al. , ( 2008 ) reviewed that some of microalgal species could use carbonates such as NaHCO3 and Na2CO3 for cell growing with high extracellular carboanhydrase activities, which are responsible for the transition of carbonate to liberate CO2 easing higher C assimilation degrees ( Emma Huertas et al. , 2000 ; Merrett et al. , 1996 ; Ginzburg 1993 )The chief ends of the current hunt were ( I ) to uncover the use of Chlorella sp.

as an effectual biomaterial for B remotion from the civilization media, ( two ) finding of the best remotion conditions in item, by utilizing different parametric quantities, and ( three ) to look into if there was a possible offered by Chlorella sp. to develop a cost effectual and eco-friendly B remotion process. Furthermore, the effects of different median composings pH, B and biomass concentrations on the remotion procedure were studied. To our cognition this is the first study about B remotion by a new stray microalgae Chlorella sp.

, with proposed mark comprehensively. Ultimately, we hope the survey will impact the manner in which the people perceive the danger of pollutants in H2O use by the populace.

2. Materials and Methods

2.1. Isolation

The microalgal civilization was observed in the H2O samples taken from the pool in the Garden of Ankara University, Faculty of Science, Ankara, Turkey.

The samples taken were spread on the Petri plates incorporating BG 11 medium ( Rippka, 1988 ) and were incubated at 25 ± 2 oC under uninterrupted light ( blue-white fluorescent, 12.5 wm-2 ( 2400 sixty ) ) . The pH of the growing medium was adjusted to 8 by adding dilute ( 0.01 M ) and concentrated ( 1 M ) sulphuric acid or Na hydroxide solutions. Cells from microcolonies on these home bases were isolated bymicromanipulation. The microalgal cells were purified at sterile conditions by streaking the cells repeatedly on the BG 11 medium agar home base. Then, the purified microalgal cells were transferred to liquid media.

In order to formalize the axecinity, these liquid civilizations were tested for bacterial taint by plating on bacteriological media.

2.2. TRIA, NaHCO3 and Boron solutions

Stock TRIA ( 96 % , w/v ; Aldrich ) solution was prepared by fade outing 0.5 g of the chemical in trichloromethane. Sodium bicarbonate solution ( Merck ) was prepared by fade outing 17.

2 g L-1 of the NaHCO3 in distilled H2O. Stock solution of B was prepared by dilution of boracic acid ( H3BO3 ) ( Carlo Erba ) to a concluding 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 civilization experiments in undaunted flasks illuminated by cool white fluorescent lamps were carried out at 12.

5 wm-2 ( 2400 sixty ) visible radiation strength. The microalgal civilization was transferred into 100 milliliters BG 11 media or the media incorporating known concentrations of B and/ or TRIA and/or NaHCO3 solutions in 250 milliliters Erlenmeyer flasks and all were incubated at 25 ± 2 oC under uninterrupted light for 20 yearss.

2.4. Effectss of different media composings on B remotion

To find the consequence of the different media composings on B remotion, the microalgae were cultivated in media incorporating 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 milligrams L-1 TRIA ; ( 3 ) BG 11 medium with 34 milligrams L-1 NaHCO3 and ( 4 ) BG 11 medium with 1 milligrams L-1 TRIA and 34 mg L-1 NaHCO3 solutions at pH 8 ( unpublished consequences ) for 20 yearss incubation period. For these experiments, Erlenmeyer flasks incorporating 100 mL medium, to give an initial concentration of about 4.8 g dry wt.

biomass L-1 inoculated in civilization media.

2.5. Consequence of initial pH on B remotion

As the preliminary experiments indicated that alterations in pH values affected B remotion by Chlorella sp. , tests 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 milligrams L-1 TRIA and 34 mg L-1 NaHCO3 for 15 yearss incubation period in order to happen a suited pH. For pH experiments, Erlenmeyer flasks incorporating 100 mL medium, to give an initial concentration of about 4.8 g dry wt.

biomass L-1 inoculated in civilization media.

2.6. Effectss of the biomass concentrations on B remotion

The influence of microalgal biomass on B remotion was investigated utilizing four different biomass concentrations. The biomass concentrations were estimated utilizing the dry weight method. The experiments were performed in BG 11 + TRIA + NaHCO3 media at pH 8 for 20 yearss incubation period and Erlenmeyer flasks incorporating 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 civilization media.

2.6. Analytic methods

During the incubation period, 3 milliliter samples were taken at 5, 10, 15 and 20 yearss from each of the flasks. The B concentration was determined by mensurating the optical density at 585 nanometer with a Shimadzu UV 2001 theoretical account spectrophotometer ( Adams, 1990 ) .For the measuring of microalgal growing at the terminal of 10, 15 and 20 yearss incubation periods the biomass concentration was determined by mensurating both of ocular denseness and dry weight parametric quantities for any set of growing conditions. Ocular denseness was measured at 600 nanometer with the spectrophotometer.Dry weight was determined by centrifugation on the samples at 5000 revolutions per minute for 10 min ( Hettich EBA 12 theoretical account extractor ) and drying at 80 & A ; deg ; C ( N & A ; uuml ; ve FN 400 theoretical account sterilizator ) overnight.


7. Statistical analysis

The experiments were set in a wholly randomized design up with three replicates. The informations were subjected to analysis of discrepancy utilizing important differences among intervention agencies were compared by descriptive statistics ( ±S.E. ) .

3. Consequences and Discussion

In the survey, the stray and purified microalgal strain which was identified as Chlorella sp. by measuring the morphological belongingss was used ( Fig 1 ) .

Chlorella sp. is a one-celled fresh-water, microscopic green algae with spherical cells about 2-8 ?m in diameter, about the same size as human ruddy blood cells ( Bewicke and Potter 1984 ) .

& A ; lt ; Here Fig. 1 & A ; gt ;

3.1. Effectss of different media composings and B concentrations on the B remotion

Previous surveies showed that the initial concentration of B was an of import parametric quantity in B bioremoval efficiency that was measured in the civilization media ( Del-Campo Mar & A ; iacute ; n and Oron, 2007 ) . Therefore, we foremost tested this parametric quantity with three B concentrations, i.e.

about 5, 7.5 and 10 milligram L-1, in the first series of experiments which were designed besides for measuring of the consequence of different media composings. The purpose was to see whether B remotions were besides affected by TRIA and / or NaHCO3. Four different BG 11 media composings were investigated for this proposes: ( I ) BG 11 control samples, ( two ) BG 11 + TRIA, ( three ) BG 11 + NaHCO3, ( four ) BG 11 + TRIA + NaHCO3. The consequences which are presented in Fig 2. showed that the removal output decreased when initial B concentrations were increased up to 10 milligrams L-1.

& A ; lt ; Here Fig. 2 & A ; gt ;

As presented in the Figure 2 a-b and with Fig 2 c-d, the remotion outputs were close to each other at initial B concentrations of 5-10 milligram L-1, and all of the remotion outputs found showed important additions merely after 10 yearss of the experiments. The maximal B remotion was observed at 5.08 mg L-1, i.e. , 23.

36 % in BG 11 control samples in 20 yearss of incubation, and there was no important difference between the controls and the BG 11 + TRIA samples. For illustration, the highest B removal output was 24.13 % , in the presence of 5.

66 mg L-1 B in the BG 11 + TRIA samples. The remotion outputs were besides near to each other at initial concentrations of about 7.5 and 10 milligram L-1 B containing samples. These consequences can be interpreted as a mark of ineffectualness of TRIA in B remotion when applied entirely. NaHCO3, on the other manus, showed its highest B remotion 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 effectual factor on B remotion by Chlorella sp.

Another consequence, possibly more of import than above, was obtained by adding TRIA and NaHCO3 to the civilization media together enhanced the growing of Chlorella sp. Consequently the B remotion values reached to the highest of all held in this survey. Enhancement of growing by TRIA and NaHCO3 could be the ground of the addition of growing and the maximal specific B consumption values ( qm ) presented below ( Fig. 2d ) . It is clearly seen that TRIA, stimulated all of the parametric quantities measured in the presence of NaHCO3. Oxidation of the HCO3 to CO2 and its assimilation increased the primary production in footings of dry weight. In the medium with 1 milligrams L-1 TRIA and 34 mg L-1 NaHCO3 the B remotion reached its highest value on twenty-four hours 20 of incubation.

The removal output of B was 18.9 % at 5.45 mg L-1 B concentration on 10th twenty-four hours, and increased to 32.95 % on the twentieth twenty-four hours. In TRIA + NaHCO3 samples the microalgae removed the B expeditiously up to 8.68 milligrams L-1 degree, with the highest output of 26.

58 % .The findings reported by Del-Campo Mar & A ; iacute ; n and Oron ( 2007 ) , who used Lemna gibba in their survey, bioremoval of B by L. gibba decreased when its initial concentrations in the media were increased, and the most efficient remotion measured was below 2 mg L-1 B concentration. They added that there was no remotion at about 10 mg L-1 B concentration even on 12th twenty-four hours of cultivation. In the present survey nevertheless, Chlorella sp.

showed important removal efficiency at 10 mg L-1 B concentration at the four different media composings tested. Removal capacity could be increased farther through stimulation of the biomass production by the add-on of TRIA and NaHCO3 to the BG 11 medium. There are some of the surveies in the literature, depicting remotion of industrial pollutants in an effectual manner by including TRIA endocrine in BG 11 civilization media by fresh water cyanobacterium Synechocystis sp.

( Karacakaya et al. , 2009 ) . Therefore, we can reason that farther surveies on the usage of Chlorella sp. in bioremoval of some pollutants may give more meaningful and promising consequences in footings of higher degree of removal outputs for several pollutants.

The comparing of the maximal specific B remotion values ( qm ) found in four different media composings used in the present survey are summarized in Table 1. In an attempt to prove the cogency of the above presented consequences, the maximal specific B remotions in TRIA + NaHCO3 samples were besides measured and found higher values at all tested initial B concentrations, analogue to the growing rates which were besides higher than the controls. In the samples with higher sums of biomass the remotion of B per one gm of dry weight was besides higher and the lowest biomass was in the BG 11 control samples. Comparison of qm values for B remotion in media with and without TRIA showed that there were little difference, bespeaking the presence hormonal stimulation. As presented in the tabular array, add-on of TRIA to the media increased qm values from 0.28 milligrams g-1 to 0.31 milligrams g-1 at about 5 mg L-1 B concentration, and from 0.

26 milligrams g-1 to 0.34 milligrams g-1 at about 7.5 mg L-1 B degree. 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 milligram L-1 B concentrations, severally. Stimulation by NaHCO3 in the media was clearly apparent, as shown by the maximal specific qm values at all tested initial B concentrations.

On the other manus, 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 milligram L-1 B concentrations. In general, the maximal specific B remotion values were increased by the higher initial B concentrations up to a certain degree. qm values decreased above the 7.5 milligram L-1 B concentrations without any exclusion, but the qm values measured in the samples incorporating BG 11 + TRIA + NaHCO3 increased when B concentrations increased from 5 to 10 milligrams L-1. This form of alterations was due to the high growing rate of Chlorella sp. in TRIA + NaHCO3 samples.

& A ; lt ; Here Tab.

1 & A ; gt ;

3.2. Consequence of initial pH on B remotion

In line with the above informations, selected optimal media composings we tested for optimal pH value for the maximal B remotion. The consequence of initial pH on the remotion by Chlorella sp. was summarized in Fig 3. As shown in the figure, B remotion was 13.

64 % , 15.24 % , and 18.71 % at pH 5, 6 and 7 severally. Chlorella sp. removed B with the highest output of 22.

4 % at pH 8. It was observed that the removal output of B increased with an addition in pH values up to pH 8, but decreased aggressively to 13.50 % when pH increased to 9.

As these experiments demonstrated that Chlorella sp. showed its highest B remotion outputs at pH 8, the undermentioned experiments were conducted at this pH value. Bursali et Al. ( 2009 ) and Yilmaz et Al.

( 2008 ) besides marked basic pH values for efficient B remotion by the macroalgae Caulerpa racemosa volt-ampere. cylindracea and by the electrocoagulation in a batch reactor.

& A ; lt ; Here Fig. 3 & A ; gt ;


3. Consequence of the biomass concentrations on B remotion

Influence of the initial sum of Chlorella sp. biomass on B remotion was investigated at four different biomass concentrations ( 4.0, 4.8, 5.2 and 5.6 g L-1 ) .

Figure 4 nowadayss the consequences obtained by incubating the samples for 20 yearss which evidenced the presence of important effects of initial degree on the bioremoval, which increased from 19.59 % to 26.58 % at the terminal of 20 yearss 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 twenty-four hours of incubation, and increased to 33.

48 % at the terminal of 20 yearss. The best consequence for B remotion value was 38.03 % at 5.6 g L-1 biomass concentration on the twenty-four hours 20. It is clearly seen in Fig 4 that the relation between these variables was approximately additive. This one-dimensionality can be interpreted as the indicant of the relation between higher growing rates and tolerance to B in Chlorella sp. used for the experiments.

& A ; lt ; Here Fig. 4 & A ; gt ;

The relation between the maximal specific B consumption values and different biomass concentrations has besides been determined and presented in Table 2. The consequence of biomass concentrations on qm values after the incubation period showed that the microalgal uptake capacity and removal output of B at 5.6 g L-1 biomass concentration was higher than 4.0 g L-1.

& A ; lt ; Here Tab. 2 & A ; gt ;

The maximal specific B remotion degrees were higher at higher biomass concentrations, as the qm values and growing 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 output was 19.59 % .

The qm values increased from 0.49 milligrams g-1 to 0.58 milligrams g-1 as the addition 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 remotion per centum reached to 38.03 % .

The information given in Table 2 besides shows the form of the alterations in chlorophyll ( a+b ) concentrations and growing rates ( µ ) of Chlorella sp. within the 20 yearss incubation period. As shown in Table, chlorophyll values increased from 0.21 µg mL-1 to 1.254 µg mL-1 in analogue to the increasing biomass concentrations from 4.

0- 5.6 g L-1. Chlorophyll ( a+b ) concentration reached to its maximal value in the samples incorporating 5.6 g L-1 initial biomass degree. There was noticeable difference between the growing rates at the samples incorporating different biomass concentrations, and the highest µ value of 0.177 d-1 was found at the sample incorporating 5.2 g L-1 biomass concentration.

As expected, growing rate decreased to 0.139 d-1 in the 5.6 g L-1 samples, when the initial biomass concentration exceeded this optimal degree. The ground was due to already high biomass sums of microalgae.If the remotion ( % ) , qm, chlorophyll and µ values discussed, correspond to the really high sums of biomass would besides higher remotion and chlorophyll values. But, due to the high microalgal biomass in the civilization media, the growing rate was less and hence the maximal specific B consumption was even excessively.

In the visible radiation of these consequences, appropriate biomass concentration to B remotion was 5.2 g L-1. Finally, correlative with the remotion ( % ) , qm, chlorophyll ( a+b ) and µ consequences between the consequence of media composings and the consequence of biomass, showed that higher biomass concentrations and add-on of TRIA and NaHCO3 in the civilization media were enhance effects of growing and the removal output did so.



The ultimate purpose of this survey was to look into if the soon isolated, which is an easy to happen, isolate and cultivate Chlorella sp. could function as biomaterial for B remotion from H2O and if its potency could be increased by the stimulatory consequence of some growing exciting agents. We performed assorted experiments for this intent and brought to visible radiation that this Chlorella sp. , was capable of taking B from the BG 11 civilization media ab initio incorporating B up to 10 milligrams L-1 concentration.Some research workers in fact, have used Chlorella spp. as a biomaterial for remotion of some pollutants ( Lim et al. , 2010 ; Valderrama et al.

, 2002 ) , but no Chlorella sp. has been used in such surveies for B bioremoval. The stimulations used in the present survey have non been tested for increasing the growing and bioremoval capacity of the topic green alga. Therefore, it can be concluded that the present survey has revealed the exploitable potency of B remotion capacity of Chlorella sp.

as a well defined, cost-efficient and eco-friendly intervention being.


Fiscal support was appreciatively acknowledged by the Scientific and Technological Research Council of Turkey ( T & A ; Uuml ; B & A ; deg ; TAK-B & A ; deg ; DEB ) .


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