Removal of heavy metals from mine water by cyanobacterial calcification

Available online at www.sciencedirect.comMININGScienceDirectSCIENCE ANDTECHNOLOGYELSEVIERMining Science and Technology 20 (2010) 0566 0570www.elsevier.comlocate/jcumtRemoval of heavy metals from mine water bycyanobacterial calcificationDONG Donglin , LI Hongjiang, ZHANG Jie, SUN LukeState Key Laboratory of Coal Resources and Safe Mining, China University of Mining & Technology, Bejing 100083, ChinaAbstract: The influence of different ilumination intensities on cyanobacterial calcification induced removal of heavy metals fromcontaminated mine water was studied. Cyanobacterial calcification experiments were performed using a growth medium intendedto simulate contaminated mine water. The results indicate that calcification can promote the removal of heavy metal ions. As theillumination intensity became stronger calcification rates increased and the removal of Zn*+ and Cd'+ became more obvious. Whenthe ilumination intensity was 10000 lux the removal of Pb*+ was the largest observed: stronger or weaker illumination reduced theamount of lead removed. The removal of three different heavy metals complies with an index function. For identical iluminationmintensities different ions were removed to different degres.Keywords: coal mine; cyanobacteria calcification; heavy metal; ilumination intensity; removal1 Introductionin Ca2+ ions cyanobacteria not only cause physicalThe development of large scale coal mining hascalcification but also carry out biological calcificationunder appropriate conditions.resulted in the production of a large amount of solidCyanobacterial calcification is. a process wherewaste. This has caused heavy metal pollution in thecalcareous algae transform CO3~ and Ca* intocoal mining area and in the surrounding soil and wa-CaCO3 that subsequently forms a physical part of theters. Persistent toxic contaminants, heavy metalsalgae cell. It is also a process that turns one form ofare difficult to biodegrade after entering environment.inorganic carbon turn into another form of inorganicThey usually contaminate the surface and groundcarbon'!. There are many factors that influencewater through leaching and run off'*. These elementscyanobacterial calcification such as temperature,can also enter the human body through food chain orsoluble inorganic carbon, pH and photosynthesisdirect contact and slowly accumulate resulting inDespite some achievements involving the absorp-harm to the health'5!. Pb levels have reached 1.0x10 band Cd and Zn are at 0.4x10° and 0.6x10”respec-tion of heavy metals during cyanobacterial calcifica-tion there is lttle reported on the relationship betweentively, in mine waters of a coal mine of the Pingding-it and the removal of heavy metals. Nor is there muchshan Coal Group, China. Mine water pollution causesdescribing the corresponding effects of the metal ionfurther soil contamination, which not only harms theremoval on cell growth. The complexity and diversityenvironment but also may affect the health of inhabi-of mine water makes heavy metal sequestration dif-tants. Consequently, understanding the mine waterficult so more research should be done on the subject.pollution by heavy metals is significant.Cyanobacteria are the world's most widely distrib-We have experimentally simulated removal of heavymetals from mine water. The influence of ilumina-uted organism and can be seen in fresh water, in thetion intensity during cyanobacterial calcification tosea and on continental land masses. Many species areremove heavy metals from mine water was studied.able to grow in extreme circumstances and they areThis will be of great significance for removal ofstrongly resistant to adverse conditions. Cyanobacte-heavy metals from mine water.ria are the only nuclear algae and are obviously dif-ferent from the other eukaryotic algael'. In an aque-2 Materials and methodsous environment cyanobacteria play an important rolein the calcification process'. If the area is abundant2.1 Algae cultivationeceived 02 November 2009; accepted 20 February 2010中国煤化工experiment were .*Corresponding author. Tel: 86 10 62331293Syneto Cyanophyta,E-mail address: ddI@ cumtb.edu.cnCyanYHC N M H G Chrococcaceae,do: 10.1016/S1674-5264(09)60245-3Synechocystis. Algae seed was purchased from theDONG Donglinet alRemoval of heavy metals from mine water by cyanobacterial calcification567National Laboratory of Freshwater Algae, Institute oftration reflects the amount of metal removal.Hydrobiology, Chinese Academy of Sciences. Culti-vation conditions were: 25 C, 3000 lux, pH 8 in a3 Results and discussionculture medium of BG11"". The medium contains, in3.1 Change in pHone liter: NaNO3, 1500 mg; K2HPO4:3H2O, 40 mg;MgSO47H2O, 75 mg; CaCl2:2H2O, 36 mg; citric acid,The pH changes observed with the three different6 mg; ferric ammonium citrate, 6 mg; Na2 EDTA, 1heavy metal ion doped substrates are shown in Fig.1.mg; Na2CO3, 20 mg; H;BO3, 0.061 mg; MnSO4:H2O,As time passes the pH changes significantly. There0.169 mg; ZnSO47H2O, 0.287 mg; CuSO4:5H2O,are two types of change and the trends are the same.0.0025 mg; and, (NH)6MoO244H2O, 0.0125 mg.The general trend is that for the first six days the pHThe ilumnination cycle was 12 h light followed by 12of the substrates iluminated with 20000, 10000, 5000h dark. Cultivation lasted for 15 days during whichand 2000 lux rapidly increased. Then during the sixthtime the algae grew well.to tenth days the pH remains near a steady value. II-2.2 Preparation of Ca+, Pb2+, zn2+ and Cd+lumination levels of 20000 or 10000 lux resulted in apH of 9.8 for media containing Pb and Zn, see Figs.containing substratesla and lb. The pH of the substrates containing Cd isThe experiment was intended to simulated the ionlower, 9.6, see Fig. Ic. Ilumination levels of 5000 orcontent of mine water. The BG11 medium was first2000 lux resulted in pH of 9.1 for all three substrates.prepared and disinfected. Then Ca^*(CaCl2) andBetween the tenth to fourteenth days pH values of allHCO3 (NaHCO;) were added to it giving a Ca2+ con-three substrates appeared to decline. The low 50 luxcentration of 100 mg/L and a HCO3~ concentration ofillumination level caused lower pH and the extent of152 mg/L. The added HCO3 is to provide CO32- ionspH increase was small and occurred for only a shortfor precipitation and to promote calcification.time. Soon the pH begins to decline and then becomeThen solutions containing Pb*+, Zn'+ and Cd2+steady. The main reason for the pH rise is that photo-were prepared and added to the BG11 medium.synthesis by the algae produces OH. In later periodsAdded Pb'+, Zn'+ and Cd4+ concentrations were al-the algae die off because the substrate is relativelymost 10 μmol/L. ThePb-*, Zn*+ and Cd * containingpoor in nutrients. As the nutrient starved algal cellssubstrate was then inoculated using the previouslybegan catabolism the pH fall while enzymes in theprepared, vigorously growing algae culture. Thecell break down various macromolecules. When thedoped substrate and the growing culture were mixedlight level was very low photosynthesis was slow orin the proportion of 1:1, 600 mL of each. The mixturenon-existent and the pH rise was not very obvious. Awas gently stirred with a disinfected glass bar untilthe same time algae cells soon die because of effectshomogeneous. The ion concentrations were then:from the heavy metals. The pH changes are in accor-Ca2+, 50 mg/L; Pb2*, 5 μmol/L; Zn2+, 5 μmol/L; and,dance with the principles of photosynthesis and calci-Cd*, 5 umol/L. This corresponds to a typical ionfication. Change in pH can indirectly reveal thecontent of mine water. Finally, 200 mL samples weregrowth state of the algae cells.put into 250 mL beakers. Five beakers were prepared3.2 Calcificationfor each kind of heavy metal to give three groups anda total of 15 beakers. These beakers were placed inThe disappearance of Ca2+ from the medium waslab boxes provided with different levels of ilumina-established through analysis of the remaining iontion. The levels were 50, 2000, 5000, 10000 or 20000content. The curves shown in Fig. 2 illustrate thelux of light with a color temperature of 4000 K. Thetrends. The content of Ca+ clearly.decreased.temperature and pH were the same as during thPrevious research has attributed the decline in Coriginal cultivation of the algae.to the fact that cyanobacteria are prokaryotic micro-organisms that are able to produce oxygen via photo-2.3 pH measurementsynthesis. During the growth phase photosynthesisSample pH was checked daily with a modelcauses the balance of CO2 to shift. The CO2 in thePHS -25B pH meter.water from biological species also causes the local pH2.4 Measurement of Ca2+, ,Pb2+, Zn2+ and Cd2+to rise. The transformation of HCO3~ into CO35- re-sults in the deposition of CaCO3. This reduces theAfter measuring the pH the samples were extractedCat + concentration in the substrate medium'2and centrifuged at 3000 r/min for three minutes. TheThe change of [Ca*] in the medium containing Pbsupernatant was taken and the Ca2*, Pb*, Zn2+ ands shown in Fig. 2a. The largest calcification rate isCd4+ levels were determined by flame atomic absorp-fouriring a linear drop intion spectrometry using a PE5 100PC atomic absorp-diss中国煤化工aer and enualyion spectrometer. The reduced Ca-+ level in the sub-[CaiYHC N M H Gder in calcifcationstrate denotes the amount of calcification that hasrate Tungwu sualn,; as a luncuon oi ilumination fall inoccurred and a reduction in heavy metal ion concen-the sequence 10000, 5000, 20000, 2000 and 50 lux.568Mining Science and TechnologyVol.20 No.410.2r10.2 [9.8-9.4).4 t- 10000 lux一- 500ux.0 t”2000 lux3.63.6 t8.6+ S0lux12 16Time (day)(a) Pb(b)Zn(C)CdFig. 1 pH changes observed with the three dfferent heavy metal ion doped substrates522[of Ii 48548_ 10000 luxE 44。44色- 500 lux台44-2000 lux4C4+ 50lux4204812162-(2)Pb(e)CdFig.2 Changes in Ca2+ observed with the three different heavy metal ionFig. 2b shows the changes in. Ca2+ when Zn is5000 lux. The probable reason is that strong light mayadded to the substrate. Again, Ca2+ declines as timedestroy the photosynthetic reaction center and therebypasses. Stronger ilumination levels speed the declinerestrain photosynthesis and cut down on the rate ofin [Ca2*]. The rate of calcification is higher.calcification'5. When Zn or Cd were added to theFig. 2c shows the changes of Ca2+ for Cd contain-substrate this limit to photosynthesis was not ob-ing substrate. During the first six days the decline inserved, an interesting point for further study.[Cat*] is the fastest of all samples. Later the calcifica-3.3 Removal of heavy metal ionstion rate drops and [Ca2*] becomes constant. In aword, the changes in Ca2+ ion concentration are asso-The drop in solution heavy metal ion concentra-ciated with illumination levels. The stronger the lighttions is shown in Fig. 3. The heavy metal ion concen-the greater the decline. The reason may be that in thetrations do show a decreasing trend. In the Pb con-darker conditions the level of photosynthesis is low.taining substrate, see Fig. 3a, the first six daysThis validates the connection between calcificationshowed the fastest rate of removal. The rate then de-and photosynthesis. But in the Pb containing sub-creased in a stepwise manner until, finally, the con-strate the fastest calcification rate occurs at an illu-tent of Pb stabilized. Removal was also affected bymination level of 10000 lux. A level of 20000 luxillumination levels. The rate, from big to small, fell inshowed lower calcification rates than either 10000 orthe order: 10000, 5000, 20000, 2000 and 50 lux.5.2r.o050.5.8-.0.6-2000ux .上3.0f与3.5t.2|.s.0002048古.162504.81216(a) Pb”+(b) Zn2*(c) Cd* .Fig. 3 Trend in solution heavy metal ion concentrationsFig. 3b shows the respective changes for Zn. Again,dark light levels the changes are modest.the stronger the light the more Zn ions disappearedFig I中国煤化工->ntaining medium.from solution over a given time period: the more ob-Rennination levels. Atvious the removal effect. The drop here is neither atheeYHC N M H GCd concentrationsstepwise trend nor stationary but smoothly decreasing.become constant. rigs. 2 ana 5 snow that the varia-For strong light levels removal rates are high and fortion in heavy metal ions follows the variation in Ca2+DONG Donglin et alRemoval of heavy metals from mine water by cyanobacterial calcificationlevels. This implies the metal ion removal has a closesamples showed a drop in Zn of 47.2%, 38.6%,relationship to cyanobacterial induced calcification.23.3%, 18.3% or 8.6%. Here 20000 lux provided thePerhaps during sequestration of calcium by thelargest drop in [Zn] and that condition was chosen ascyanobacterium the heavy metal ion is also taken upthe baseline for the Zn model, Fig. 4b.becoming part of the cell structure. It was also prob-After 14 days 20000, 10000, 5000, 2000 or 50 lux,able that the shell of the algae cell wall was able togave 21.3%, 16.9%, 15.6%, 11.2% or 4.6% drops inexcrete mucilage glue that collects heavy, metal ions[Cd]. The 20000 lux condition ilustrated the greatestand fixes them to the bacterial flamentdrop in [Cd]. But this condition gave results onlyslightly higher than did the 10000 and 5000 lux con-3.4 A model for heavy metal sequestrationditions. From a cost perspective 5000 lux is a betterThe disappearance of heavy metal ions from solu-illumination level and so it was used as the baselinetion was fit to an exponential model: y= =Aexp(- -x/b)+C.for the Cd model, Fig. 4c.Here y is the heavy metal concentration and x is theThe results from ftting the data show that the threetime under ilumination. After 14 days, for ilumina-models comply with the index function and that alltions of 20000，10000, 5000, 2000 or 50 lux, thethe correlation coefficients are over 0.95 (Table 1).amount of Pb removed from the substrate medium isTable 1 Fitting results for the removal of heavy metals40%，53.3%, 46.6%, 33.3% or 14.3% respectively.Metal llumination (lux) Fiting equationAn ilumination of 10000 lux caused the most Pb toP10000y=- 190.17exp(-/47.42)+193.65 0.9535be sequestered. So 10000 lux was selected as thezn 2000.y-43.15exp(-x/3.91)+45.12 0.9767baseline for the model, Fig. 4a.After 14 days 20000, 10000, 5000, 2000 or 50 luxCd5000y=-15.04exp(-x/5.08)+15.940.95380r6024个。 cd 5000 lux50B 20 -Fittng function ofCd40-16-30-30, Pb 1000 lux20●Zn 2000 luxg10- Fiting function of Pb一ilting function ofZn024681012140246810124Time (day)(a) Fit model for Pb removal (0000 lux)(b) Fit model for Zn removal (0000 lux)(C) Fit model for Cd removal (5000 lux)Fig. 4 Fit curves for dfferent heavy ion removal3.5 Effect on the three heavy metals under iden-general trends are more or less the same. For identicaltical iluminationlight flux the largest drop is in the Pb doped sample:Fig. 5 shows the removal of the three heavy metalsZn, and Cd levels fall to a lesser extent.under the five different light intensities. Note that the5.0.0 t4.5.st. 4.0- Pb.5.s5- Cd0f2.581216.0(a) 2000 lux(6) 1000 lux551 4.: 4.45 4.03.4.0W3.68 2:3.3中国煤化工_481216E8 12 16:YHCN M H G;(day)(c) 5000 lux(d) 2000 lux(c) 50 luxFig. 5 Removal of the three heavy metals under the five different light intensities570Mining Science and TechnologyVol.20 No.4Fig. 5a shows the results for 20000 lux ilumina-Acknowledgementstion. In this case the drop in [Zn] is largest followedby [Pb] and [Cd]. Removal rates are the highest in theThis work was supported by the National Basicinitial six days where [Zn] and [Cd] decrease in aResearch Program of China (No.2007CB209401),linear fashion and [Pb] first falls in a stepwise mannerResearch on basic theory about the mechanism ofwater inrush and its prevention in coal mines andbefore becoming constant.Fig. 5b shows results for 10000 lux. The removal,supported by the Fundamental Research Funds for thefrom most to least, falls in the order Pb, Zn and thenCentral Universities.Cd. The most obvious change in [Zn] and [Cd] occurReferencesduring the initial six days. There is a four day sta-tionary period in the [Pb] decline. Fig. 5c is for the]Yao E Q, Gui H R. Characteristics of the main pollutingtrace elements in the water environment of mining5000 lux flux. The most removal is in the case of Pb.subsidence pools. Journal of China University of MiningAs time passes Zn and Cd levels are constant al-& Technology, 2008, 18(3): 362-367.though the Pb content shows a stepwise decrease. The2] Dong D L, Wu Q, Zhang R. Environmental charac-most prominent changes occur over the fourth toterstics of groundwater: an application of PCA to waterchemistry analysis in Yulin. Journal of China Universityeighth days although [Pb] also becomes constant atofMining & Technology. 2007, 17(1): 73-77.the end. Fig. 5d shows the 2000 lux data. The trend is[3] DongD L WuQ, QianZJ, Xie HL, WuGX, BiCC,the same as for 5000 lux ilumination. [Zn] and [Cd]Lv Z Q. 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On the tenth day the[6] Chen S J, Zheng W J, Yang F Study advances on heavymeasured value of Zn was very high, which is proba-metal bio-absorbed by cyanaobacteria, Marine Enviro-bly a sample or measurement error.nmental Science, 2006, 25(4): 103- 106. (In Chinese)The metal sequestration is different for different[7] Dittrich M, Miller B, Mavrocordatos D, Wehri B.Induced calcite precipitation by cyanobacterium Synec-metals when the ilumination is constant. This ishococcus. Acta Hydrochim Hydrobiol, 2003, 31(2): 162-probably due to the different activities of the differentmetals. Algae exposed to Pb died sooner than algae[8] Gao K s. On the calcification of coralline algae. Ocea-exposed to Zn or Cd. At lower illumination levelsnologia et Limndoogia Sirica, 1999, 30(3): 290-294.algae death occurred earlier. In addition, low light[9] Reynaud S, Leclercq N, Romaine L S, Ferrier-Pages C,levels gave concentrations that varied. This probablyJaubert J, Gattuso J. 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