Calcification of charaEffect of calcium encrustation on hevay metal ...

Charophytes as a tool forphytoremediation <strong>of</strong> heavy metalsT. Asaeda, P. I. A. Gomes, and Md.M.BaharInstitute for Environmental Scienceand Technology,Saitama Universityasaeda@mail.saitama-u.ac.jp1

Charophytes• They are one <strong>of</strong> the closestrelatives <strong>of</strong> terrestrial plants• They attach on the bottom byrhizoids• They reproduce both asexuallyand sexually. (bulbils & oosporesmainly)• Propagule bank consists <strong>of</strong>oospores (perennial) and bulbils• There are six genera; Chara,Nitella, Lamprothamnium,Tolypella, Lychnothamnus andNitellopsis.Charophytes with sexualorgans

Ecological significance <strong>of</strong> CharophytesNutrientslow bioturbationvelocity distributionsettling <strong>of</strong> SSCharophytesdead plantsrefuge for zooplanktonmechanically stablegyttjastabley stockedlow re-suspension• They do not allow materials to besuspended easily.• They are slowly decomposed andstably accumulated on the bottom• They provide habitats for variousorganisms particularly for zooplankton

Charophytes are intensively calcified in Ca rich water• CaCO 3 incrustation occurs in stems,branchlets, surface <strong>of</strong> oogonia• CaCO 3 amounts up to 50-60% dry wt(Van den Berg et al 2002)• Generally Chara spp is more calcifiedthan Nitella spp (Imahori 1954)Calcified chara. spp

Calcite Encrustation (based on McConnaughey’s Model)photosynthesisMcConnaughey (1991 ). Limnol. Oceanogr. 36:619–628.

Highly calcified Chara6

Objective and HypothesisGeneral objective:• to reduce heavy metals in water using the calcification process <strong>of</strong> charophytes.Background:• Phytochelatins (PCs) and metallothioneins (MTs) are the processes by whichplant cell detoxify heavy metals (Cobbett and Goldsbrough 2002)• Phosphorus is co-precipitated with charophyte calcite even in very lowphosphorus (Siong and Asaeda 2006)Hypothesis• Charophytes are likely relatively tolerant to heavy metals..• The co-precipitation process is not selective, thus, heavy metals can also beco-precipitated (or adsorbed) with the calcite.

Hypothesis <strong>of</strong> Co-precipitation <strong>of</strong> heavy metal with Calcitephotosynthesisheavy metalheavy metalMcConnaughey (1991 ). Limnol. Oceanogr. 36:619–628.8

We questioned:• Are charophytes lively in Ca rich water?• How tolerant are charophytes to heavy metals?• Are heavy metals stably co-precipitated?9

How lively are charophytes in Ca rich water?10

Growth measurementsa 1a 3• Lengths for primary, and sub divisions were recorded separately• Total length was used for analysis (L t =a 1 +a 2 +a 3 +b 1 )Growth rate ~ elongation rate = (L t -L 0 ) x 100/ L 0 (%)a 2b 1Flurocam images were used forCorrection <strong>of</strong> measurements• At the end <strong>of</strong> experiments relevant plants werephotographed using Flurocamlength measurement correction11

elongation ratewith respect to the initial length (%)elongation ratewith respecto to the initial length (%)1009080706050403020100Ca 4mg/lCa 40mg/lCa 80mg/lCa 120mg/l1 2 3 4 5 6weeksNitella pseud<strong>of</strong>rabellataThe growth rate was even largerin the higher Ca concentration450400350300250200150100500Effect <strong>of</strong> Ca concentrationon the growth <strong>of</strong> CharophytesChara fibrosaCa 4mg/lCa 40mg/lCa 80mg/l0 1 2 3 4 5 6 7 8 9weeks

How are charophytes tolerant to heavy metals?

Materials and methodsMaterials: Nitella pseud<strong>of</strong>labellataIncubated at 12hr:12hr (light: dark) , 24 o C.Ca concentration (CaCl 2 .2H 2 0 ):4mg/l, 40mg/l, 80mg/lAfter 4 weeks,C 412 11 10 9Low Cr (VI) High Cr (VI)C 408 7 6 54 3 2 1C 800.0 0.1 0.2 0.4 0.844080Ca concentration mg/LCr(VI) was added by K 2 Cr 2 0 7with concentrations,control, 0.1mg/l, 0.2mg/l, 0.4mg/l, 0.8mg/lThe elongation was measured every week, andthe maximum quantum efficiency <strong>of</strong> PSIIphotochemistry was measured.Cr (VI) concentration mg/L

elongation ratewith respect to the initial length (%)elongation ratewith respect to the initial length (%)elongation ratewith respect to the initial length (%)350300250200Ca: 4mg/lcontrol0.1mgCr(IV)/l0.2mgCr(VI)/l0.4mgCr(IV)/l0.8mgCr(IV)/l350300250200Ca:40mg/lcontrol0.1mgCr(VI)/l0.2mgCr(VI)/l0.4mgCr(VI)/l0.8mgCr(VI)/l15015010010050Cr(VI) added50Cr(VI) added00 1 2 3 4 5 6 7 8 9weeks00 1 2 3 4 5 6 7 8 9weeksElongation rate <strong>of</strong> charophytesin different Cr (VI) concentration mediaCr (VI) is added after 4 th weekTolerance to heavy metal (Cr)toxicity has the positivecorrelation with Ca inwater/calcification.45040035030025020015010050Ca:80mg/lcontrol0.1mgCr(VI)/l0.2mgtCr(VI)/l0.4mgCr(VI)/l0.8mgCr(VI)/lCr(VI) added00 1 2 3 4 5 6 7 8 9weeks

Maximum quantum efficiency <strong>of</strong> PSII photochemistry (F v /F m )Cr(VI) concentrationF v /F m0.8 0.39-0.440.4 0.40-0.630.2 0.72-0.760.1 0.77-0.79Fv/Fm values before Cr (VI) addition did not showsignificant difference (t-test, P< 0.05)Stress free plants show Fv/Fm greater than 0.8 (DeEll and Toivonen, 2003; Maxwelland Johnson 2000). ・・・more affected with increasing Cr concentrationCharophytes have satisfactory tolerance to Cr(VI).

How stably are heavy metals co-precipitated?

Materials:Cara fibrosa Agrardh ex Bruzelius vs CdIncubation condition:2h:12h (light (40mmol/m 2 s -1 ): dark) for 1.5 monthpH:7.0±0.1; Ca: 17±2 mg/L; Mg: 4±1 mg/L; SRP 5±1 mgP/LNO 3- : 1.1±0.2 mgN/L; SO 42-: 35±3 mg/L;Total alkalinity: 33±3 mg CaCO3/LCd concentration:Control (mg Cd/L), 0.001 mg Cd/L, 0.01mg Cd/L, 0.1mg Cd/LFor comparison,Attached algae (Oscillatoria spp.) were incubated with 0.01 mg Cd/L

Chemical analysessample• Dissolved in 1.0mol/L HNO 3• Cd and Ca were measurer by double-beam atomic absorption spectrophotometerSequential fractionation:• extraction exchangeable fraction with magnesium chloride (MgCl 2 , pH 7.0)• carbonate-bound fraction with sodium acetate (NaOAc, pH 5.0)• organic fraction with H 2 O 2 and HNO 3

Chemical AnalysisSequentialFractionation20-40 mg sample10 mL <strong>of</strong> 1.0 mol L -1MgCl 2(pH 7.0): 30 minSample0.1 g sample,dry-ashing 400 o C, 20 hTotal Ca and CdExtractExchangeable -FractionResidue10 mL NaOAc 1.0mol L -1(pH 5.0); 5 h50 mL HNO 31 mol L -1 filteredResidueExtractExtractCarbonate -bound FractionextractResidue5 mL HNO 335% + 5 mLH 2O 230%, 85 o C, 5mL + 20 distilled water 25 mL, filteredResidue(discharged)Atomic AbsorptionSpectrophotometer-Graphite furnace(GFAA)Total MetalsOrganic-boundFraction

Cd in media (mg/L) : 0.001 Cd: 0.01 mg/L Cd: 0.1 mg/LTotal Cd in plant: 0.3 mg/kg dryweight basis (d.w.),BCF (bio-concentration factor) :300Total Cd: 125-134 mg/kg d.w.BCF: 12500 - 13400Total Cd: 734 mg/kg d.w.BCF: 7340

fraction (%)Total Ca (mg/g dry weight)30025020015010050Ca content was not muchaffected by Cd concentration,more affected by age.(alkaline zone extends with age)0controlmature0.001mg/L Cdyoung0.01mg/L Cdmature0.1mg/L Cdmaureepiphytes0.01mg/L Cd1009080706050403020100controlmature0.001mg/L Cdyoung0.01mg/L Cdmature0.1mg/L Cdmaureepiphytes0.01mg/L CdWith increasing Cd concentration,the fraction <strong>of</strong> carbonate-bound Ca increases.Most <strong>of</strong> Ca was exchangeable for epiphytic algae.Organic-bound Ca Carbonate-bound Ca exchangeable Ca (%)

fraction (%)Total Cd (mg/Kg dry weight)10009008007006005004003002001000controlmature0.001mg/L Cdyoung0.01mg/L Cdmature0.1mg/L Cdmaureepiphytes0.01mg/L CdTotal Cd content increaseswith Cd concentration1009080706050403020100controlmature0.001mg/L Cdyoung0.01mg/L Cdmature0.1mg/L Cdmaureepiphytes0.01mg/L CdOrganic-bound Cd Carbonate-bound Cd exchangeable CdThe fraction <strong>of</strong> carbonate-bound Cd increases with Cd concentration.Cd <strong>of</strong> epiphytic algae is mostly exchangeable.

Major components <strong>of</strong> <strong>calcium</strong> and cadmium on the cell surface <strong>of</strong> Charathe results <strong>of</strong> visual Minteq (Gustafson, 2007)Input data Ca: 17mg/l, Mg: 4mg/l, PO 43-: 5mgP/l, NO 3- : 1.1mgN/l, SO 42-:35mg/l,Total alkalinity: 33mgCaCO 3 /l, temperature 25 o CMedium (mgCd/l)0.0010.01-0.1under illuminationAcidic (pH 5.5-6.1) Alkaline (pH 8.0-9.6)Ca 2+ CaSO 4 (aq) Ca 2+ CaCO 3(aq) CaSO 4(aq) Ca 2+ CaSO 4 (aq)Cd 2+ CdSO 4 (aq) Cd 2+ CdSO 4(aq) CdHCO 3+CdCO 3(aq) Cd(CO 3 ) 2 2- Cd 2+ CdSO 4 (aq)Ca 2+ CaSO 4 (aq) Ca 2+ CaCO 3(aq) CaSO 4(aq) Ca 2+ CaSO 4 (aq)Cd 2+ CdSO 4 (aq) Cd 2+ CdSO 4(aq) CdHCO 3+darkness(pH 6.0-7.2)CdCO 3(aq) Cd(CO 3 ) 2 2- Cd 2+ CdSO 4 (aq)Red : oversaturated > precipitatedCa CalciteCd OtaviteCalcite was oversaturated over pH=8.3CdCO 3 was oversaturated at pH=8.5-9.4( 0.01mgCD/l), 7.9-9.9 (0.1mgCd/l)CaCO 3 and CdCO 3were precipitated simultaneously. In the alkaline zone.

Alkaline band(calcified)Acidic band(noncalcified)Calcified Otavite = alkaline layer on the calcite only slowlydissolved.Carbonate-bound Cd is not affectedby anoxic condition.The extension <strong>of</strong> alkaline bandwith age cause the precipitation <strong>of</strong>otavite encrusted with calcite.

heavy metalsNutrientslow bioturbationsettling <strong>of</strong> SSvelocity distributionCharophytesgyttjamechanically stabledead plantsstabley co-precipitatedand accumulatedrefuge for zooplanktonlow re-suspension

ConclusionsPossible usage <strong>of</strong> calcification <strong>of</strong> carophytes for the trap <strong>of</strong> heavy metals wereinvestigated.• Charophytes preferably grow in high concentration <strong>of</strong> Ca 2+ in the media.• Although the growth is more affected with increasing concentration,charophytes still have satisfactory tolerance against heavy metals andaccumulate them.• Heavy metals are co-precipitated by the <strong>calcium</strong> carbonate <strong>encrustation</strong>,are stably trapped.Therefore, charophytes become a feasible media to trap heavy metals in water.27

Thank you for your attention28

fraction (%)1009080706050403020100CaCO3-Cd CaCO3-Cdco-precipitated co-precipitatedas anion as cationCommercialCaCO 3Cd adsorbedby calciteOtavite layer on the calcite only slowlydissolved.Carbonate-bound Cd is not affectedby anoxic condition.Organic-bound CdExchangeable CdCarbonate-bound Cd29

alkaline regionacid region<strong>Calcification</strong> model (Borowitzka, 1982)medium cell wall cellHCO 3-HCO 3-OH - + CO 2HCO 3- + OH -OH -H 2 O + CO 32-Ca 2+ C a CO 330

Calcified = alkalineUncalcified =acidCalcified = alkalineUncalcified = acidCalcified = alkaline31