Research Article Biosorption of Microelements by...

Research ArticleBiosorption of Microelements by SpirulinaTowards Technology of Mineral Feed Supplements

Agnieszka Dmytryk Agnieszka Saeid and Katarzyna Chojnacka

Institute of Inorganic Technology and Mineral Fertilizers Wrocław University of Technology Smoluchowskiego 2550-372 Wrocław Poland

Correspondence should be addressed to Agnieszka Saeid agnieszkasaeidpwredupl

Received 4 April 2014 Revised 21 July 2014 Accepted 21 July 2014 Published 23 September 2014

Academic Editor Maurıcio L Torem

Copyright copy 2014 Agnieszka Dmytryk et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Surface characterization and metal ion adsorption properties of Spirulina sp and Spirulina maxima were verified by variousinstrumental techniques FTIR spectroscopy and potentiometric titration were used for qualitative and quantitative determinationof metal ion-binding groups Comparative FTIR spectra of natural and Cu(II)-treated biomass proved involvement of bothphosphoryl and sulfone groups in metal ions sorption The potentiometric titration data analysis provided the best fit with themodel assuming the presence of three types of surface functional groups and the carboxyl group as the major binding site Themechanism of metal ions biosorption was investigated by comparing the results from multielemental analyses by ICP-OES andSEM-EDX Biosorption of Cu(II) Mn(II) Zn(II) and Co(II) ions by lyophilized Spirulina sp was performed to determine themetal affinity relationships for single- and multicomponent systems Obtained results showed the replacement of naturally boundions Na(I) K(I) or Ca(II) with sorbed metal ions in a descending order of Mn(II)gtCu(II)gtZn(II)gtCo(II) for single- andCu(II)gtMn(II)gtCo(II)gtZn(II) for multicomponent systems respectively Surface elemental composition of natural and metal-loaded material was determined both by ICP-OES and SEM-EDX analysis showing relatively high value of correlation coefficientbetween the concentration of Na(I) ions in algal biomass

1 Introduction

Biosorption properties of Spirulina have been adapted inmany techniques related with metal pollution control [1ndash3]Industrial wastewater treatment or generally bioremediationof aquatic systems was the subject of several investigations[4ndash9] including smelter and refinery effluents processing[10] Besides heavy metal ions removal of inorganic con-taminants [11 12] and toxic organics was investigated [13] aswell Despite proven efficacy of Spirulina biomass Chlorellaspecies were reported as the most commonly used in currenttreatment technologies [14] Nevertheless other potentialapplications of Spirulina adsorptive capacity such as livestockfeeding and human dietary supplementation have beenalready discussed in the literature [15ndash18] Further studieson using this microalga as versatile biosorbent are thereforejustified since theymayprovide insights into both the processpathways and the mechanism of sorption

Strong molecular interactions in particular electrostaticattractions determine the quality of biosorbents and dependon chemical state and composition (type and quantity) ofsurface functional groups The state of surface moietiestheir ionization or protonation results directly from disso-ciation constant (pK

119886or pK

119887 in fact) and medium acidity

(pH) Usually value of this constant for chemical moiety inbiomolecules closely corresponds to pK value of appropriateinorganic acid or base The presence of different types offunctional groups commonly carboxyl phosphoryl sulfonehydroxyl and amine groups on biomass surfaces causes thatthe biomass has amphoteric properties Depending on pHacid or base character may be more or less apparent Proto-nated amines are positively charged while deprotonated areneutral In the case of carboxyl phosphoryl or sulfone groupsprotonation neutralizes those groups while removal of pro-tons forms negatively charged conjugate bases [19ndash21] It isespecially important when concerning biomass interaction

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 356328 15 pageshttpdxdoiorg1011552014356328

2 The Scientific World Journal

0pH

14

pKNH2

In pH 5∙

∙ COOminus

∙

pKCOOminuspKPO48minuspKSO4

2minus

SO42minus

NH3+

∙ PO43minus

Figure 1The state of ionization of functional groups on the surfaceof the microalgal cell wall depending on pH

with different ions and the fact that electrical charge ofbiomass surface governs sorbates adsorption For examplethe isoelectric point (pI) of microalgae Chlorella is about3 while Spirulina pI = 28ndash35 [19] In lower pH positivelycharged groups (eg amine group) attract anions and repulsecations while in higher pH negatively charged groups (egcarboxyl) act conversely Typical changes of ionization stateof functional groups on the surface of microalgae cell walldepending on pH are shown in Figure 1 along with the listingof functional groups (under the red arrow) and their formsin pH = 5 for which the highest model parameters fromquantitative and kinetic examination have been obtained[20]

In present work dried biomass of commercially avail-able Spirulina sp (lyophilisate) and cultivated under lab-oratory conditions Spirulina maxima were investigated forsupplementation with microelement ions Cu(II) Co(II)Mn(II) and Zn(II) which are important in livestock feedingBiosorption experiments were performed in both single-and multicomponent systems under previously evaluatedconditions Four analytical methods were used to assess thecomposition of natural and loaded biomass surface as wellas to verify the biosorption mechanism in exchange positedon the basis of earlier studies The major binding sites wereidentified along with their concentrations and thus totalcapacity of both examined biosorbents might be estimatedBased on changes observed in elemental composition of theSpirulina surface after biosorption experiments the affin-ity sequence of microelement cations was determined forsingle- andmultimetal systems separatelyThe applicability ofmicroalgal biomaterials to feed supplementationwas assesseddue to resultant of all conducted tests

2 Materials and Methods

21 Microorganisms and Media Composition Two kindsof algal biomasses were selected for biosorption researchCommercially available Spirulina sp in form of lyophilizedcells was purchased from SIGMA (USA) Spirulina maximawas obtained from Culture Collection of Algal Laboratory(CCALA) Institute of Botany Academy of Sciences of the

00 02 04 06 08 10 12 14 1600

02

04

06

08

10

12

A560

y = minus0058 + 0739 lowast x R2 = 0955

Cd

w(

gL)

Figure 2 Standard curve represents the relationship between con-centration of Spirulina maxima dry mass (119862dw) and correspondingabsorbance (A

560) results

Czech Republic Microalga grew in the Schlosser liquidmedium [22] composition of which is presented in Table 1

Schlosser culture medium was prepared with analyticalgrade reagents and purchased from POCh SA (GliwicePoland) After cultivation the biomass was separated byfiltration using the aquarium algae vacuum cleaner (ActizooZolux France) dried at 60∘C (laboratory oven WAMEDWarsaw Poland) for three days and subjected to furtherinvestigation as a biosorbent

22 Cultivation of Spirulina maxima Spirulina maxima wascultivated in laboratory-scale reactors at 35∘C [23] for 4months The cultures were carried out under 12 12 pho-toperiod conditions (12 hr light 12 hr dark cycles) providedwith artificial illumination of 19Wmminus2 intensity In orderto improve growth algal scalability tests were performed inthree consecutive systems of increasing capacity Microalgacultivation started with a working capacity of 1 L and due tocell passaging 5 L and 40 L reactor systems were examinedas well

The efficiency of the Spirulina maxima cultivation ineach of tested capacities was determined using three basicparameters the concentration of microalgal culture growthrate and relative (exponential) growth rate The first of three(119862119878) was estimated by the straight-line equation showing the

correlation between concentration ofmicroalga drymass andabsorbance measurements at 560 nm (Figure 2)

23 Semibatch Mode The experiment was conducted onSpirulina maxima culture and grown in Schlosser medium at35∘C using 40 L-working capacity reactor equipped with twolight sources (12 12 photoperiod) (Figure 3) The cultivationwas carried out for 4 months

During research the systematic partial reception ofmicroalgal biomass was performed to diminish the self-shading effect which was verified by cell concentrationmeasurements The biomass was separated by vacuum fil-tration (Actizoo Zolux France) seven times overall Thelast separation stage gathered whole population of grown

The Scientific World Journal 3

Figure 3 Spirulina maxima cultivation carried out in laboratoryscale system of 40 L

Spirulina maxima in which cells after 3-day drying at 60∘C(WAMED Warsaw Poland) were examined on biosorptionproperties in adequate tests described below

24 Metal Ions Biosorption Experiments Biosorption exper-iments were performed for copper(II) cobalt(II) man-ganese(II) and zinc(II) ions in both single- and multi-metal systems according to the procedure used in previouswork [15] Tests were carried out in a batch mode atequal initial ion concentrations 300mg Lminus1 at 35∘C (Spir-ulina cultivation temperature) and pH 5 The microalgalbiosorbent was examined at a concentration of 1 g Lminus1The solutions of metal ions were prepared in deionizedwater with appropriate amounts of inorganic salt (fromPOCh SA Gliwice Poland) Co(NO

3)2sdot5H2OCuSO

4sdot5H2O

MnSO4sdot5H2O ZnSO

4sdot7H2O pH of the metal ion solution

was measured using Mettler Toledo Seven Multi-pH-meter(Greifensee Switzerland) equipped with a temperature-compensating electrode InLab413 pH adjustment to theendpoint was performed with standardized NaOH or HClsolution (01mol Lminus1 POCh SA Gliwice Poland) Thebiosorption experiments were conducted in Erlenmeyerflasks containing 100mL of Cu(II) Co(II)Mn(II) and Zn(II)synthetic solutions shaken in thermostatedwater bath shakerat 150 rpm speed The contact time of 60 minutes wasevaluated for Spirulina biomass based on previous kineticresearch [15] After completion of each test the microalgalsuspension was separated by filtration through the filterpapers oven dried (WAMED Warsaw Poland) and furtheranalyzed

25 Fourier Transform Infrared (FTIR) Spectroscopy FTIRspectroscopy was used to identify microalgal cell wall func-tional groups participating in biosorption by detection ofvibration wave number changes in the sorbent surfaceoccurred after metal ion binding FTIR measurements werethus performed for natural S maxima dried cells and thesame biomass enriched in Cu(II) (model single-metal sys-tem) In order to verify diversity of surface functional groupsfor different microalgal biosorbent pristine Spirulina splyophilisate was examined as well Before analyzing a fewmilligrams of each alga including Cu(II)-loaded S maximawere ground and mixed with potassium bromide disks inan amount provided the biomaterial concentration of 2 by

Table 1 The composition of the Schlosser medium

Component Concentration g Lminus1

NaHCO3 1361Na2CO3 403K2HPO4 05NaNO3 25K2SO4 10NaCl 10MgSO4sdot7H2O 02CaCl2 003026FeSO4sdot7H2O 001EDTA 008

Microelement solution g Lminus1

ZnSO4sdot7H2O 10MnSO4sdot5H2O 20H3BO3 50Co(NO3)2sdot6H2O 50Na2MoO4sdot2H2O 50CuSO4sdot5H2O 10FeSO4sdot7H2O 07EDTA 08Deionized water 981

massThe FTIR spectra of prepared samples were collected bythe use of a Perkin-Elmer System 2000 (WalthamMA USA)equipped with deuterated triglycine sulfate (DTGS) andmercury cadmium telluride (MCT) detector The assay wasconducted within the wave number range of 400ndash4000 cmminus1(midinfrared region) using a potassium bromide windowat room temperature (26 plusmn 1

∘C) [24] FTIR spectra wereelaborated with a System 2000-compatible software (Perkin-Elmer Waltham MA USA) by use of which backgroundcalculation was performed to set necessary entries for resultassessment Obtained background corresponding to pureKBr was automatically subtracted from each sample spec-trum All spectra were plotted using the same scale on thetransmittance axis

26 Potentiometric Titration of the Biomass Potentiomet-ric titration was performed to more specifically identifyion-binding groups compared to FTIR spectroscopy andto determine their concentrations onto microalgal cellwall Measurements were conducted with both microalgalbiomasses according to the method elaborated in previousworks [20 24] Biomass samples of 02 g suspended in deion-ized water (200mL) were titrated with 01M NaOH till pH115 and reversely with 01M HCl till pH 25 both solutionswere standardized (POCh SA Gliwice Poland) The pHchange was monitored after single dose of titrant when therecord stabilization was achieved As control (blank) sampledeionized water was used Before each titration test the waterwas purged of dissolved CO

2by 3-hour bubbling with argon

27 Scanning Electron Microscopy with Energy Dispersive X-Ray (SEM-EDX) Analytical System SEM-EDX analysis was


carried out at Wrocław University of Environmental andLife Sciences (ElectronMicroscope Laboratory) to investigatethe biosorption effect on element content of biomass cellwall including both micro- and macroelements and mapthe distribution of ions bound to its surface as it wasreported by Michalak and coworkers [25] Tests were con-ducted using pristine and ion-enriched cells of lyophilizedSpirulina sp loaded with Co(II) Cu(II) Mn(II) and Zn(II)cations in both single- and multimetal system Microalgalsamples a total of six were fixed in 25 of glutaraldehyde(Sigma httpwwwsigmaaldrichcom) and next dehydratedby ethanol (from 30 till 100 concentration) Treatedbiomass were sampled and prepared in two planes enablingto observe cross-section and its surface All samples weremounted on proper stub and gold sputtered thereafter usingHHV Scancoat Six equipment (Crawley Oxfordshire UK)Observation and photographs of microalgae surface compo-sition were taken with a Leo Zeiss 435VP SEM scanningelectron microscope (Oberkochen Germany) operating at20 kV An additional equipment of the microscope was aRONTEC GmbH energy dispersive X-ray system (BerlinGermany) by which the concentration of particular cell wallconstituent was recorded As a result the X-ray spectra ofelemental composition specific for natural and metal ion-enriched Spirulina sp surface were obtained

28 Inductively Coupled Plasma-Optical Emission Spectrom-etry (ICP-OES) ICP-OES technique was used to determineand compare total content of the elements in microalgalbiosorbent before and after metal ion binding This analysiswas proven to be precise and appropriate in evaluationof biosorption efficacy considering applicability to feedsupplementation in particular [26] In conducted researchICP-OES was performed as a complementary analysis toSEM-EDX system intended for verification of the scan-ning electron microscopy usefulness to record and elab-orate data from biosorption experiments with SpirulinaMeasurements were carried out on Spirulina sp sampleswith and without metal ion enrichment using VISTA-MPXspectrometer (Varian Victoria Australia) with ultrasonicnebulizer in the Chemical Laboratory of MultielementalAnalyses at Wrocław University of Technology accredited byILAC-MRA (Varian VISTA-MPX ICP-OES) and the PolishCentre for Accreditation (number AB 696) According toformerly elaborated procedure [15]microalgal samples of ca05 g suspended in 5mL of 65 nitric acid Suprapur fromEMD Millipore (Merck KGaA Darmstadt Germany) weresubjected to pressure microwave digestion (mineralization)in Teflon vessels using microwave oven Milestone MLS-1200(Sorisole Bergamo Italy) After mineralization all sampleswere diluted to 50mL and the concentrations of metal ionswere detected spectrometrically (VISTA-MPX Australia)in triplicate The apparatus was calibrated with standardsolutions (10 10 50 and 100mg Lminus1) prepared based onthe 100mg Lminus1 multielemental standard Astasol (PragueCzech Republic) Optimization of the test parameters wasperformed as described before [26] The analytical process ofdigested biomass samples was controlled by the use of Polish

0 2 4 6 8 10 12 14 16 18 20 22Time (days)

minus50

minus45

minus40

minus35

minus30

minus25

minus20

minus15

minus10

Ln C

s

Reactor 1LReactor 5LReactor 40L

Figure 4 Correlation between Spirulina maxima concentrationexpressed as natural logarithm and cultivation time for three testedsystems

Certified Reference Material for multielement trace analysisOriental Tobacco Leaves (CTAOTL-1) obtained from theInstitute of Nuclear Chemistry and Technology (Polandhttpwwwichtjwawpldrupal) [26]

3 Results and Discussion

31 Reactor Capacity Selection for Spirulina maxima Cul-tivation Three comparative experiments in 1 L- 5 L- and40 L-capacity reactor were performed to select the workingscale in which Spirulina maxima would be cultivated mosteffectively The biomass yield was verified by comparison ofgrowth rate and relative growth rate results obtained for eachcultivation

Relative growth rate (120583) is an experimental value specificto particular microbial system 120583 is proportional to biomassconcentration increase during the logarithmic growth phaseover a certain time period which is expressed by

120583 =

ln119862119905119878minus ln1198620

119878

119905

[120583]= [dayminus1] (1)

where 119905 time period after which the culture concentrationwas measured (assuming 1199050 = 0) 119862119905

119878 the culture concen-

tration after time 119905 and 1198620119878 the initial concentration of the

culture Relative growth rate is usually determined from thegraphically depicted correlation of ln119862

119878= 119891(119905) (Figure 4)

The linear regression is described by

ln119862119905119878= 120583 sdot 119905 + ln1198620

119878 (2)

and parameter 120583 is the slope


Table 2 Relative growth rate of continuous cultures in the reactorsof different volumes

Capacity L 120583 dayminus1 1198772

1 0145 09735 0145 097840 0285 0987

Table 3 Relative growth rates of Spirulina maxima cells in culturewith biomass reception

Time interval 120583 dayminus1 1198772

I 0285 0987II 00439 0990III 00503 0985IV 00464 0976V 00331 0949VI 00275 0876VII 00281 0923

Results of relative growth rate obtained for Spirulinamaxima cultures that grew in reactors with all selectedcapacities were presented in Table 2

Full characterization of themicrobial cultivation requiredevaluation of biomass growth rate (119903

119909) as well This parame-

ter is expressed by proportional relationship between relativegrowth rate and the culture concentration in exact time ofmeasurement as follows

119903119909= 120583 sdot 119862

119905

119878

[119903119909] = [g sdot Lminus1 sdot dayminus1]

(3)

Changes in values of growth rate during two first weeks ofthe research including each examined system are depictedin Figure 5

Considering obtained results of growth rate and relativegrowth rate the explicit correlation between reactor capacityand cultivation yield was noted The lower the capacitysystem was used the lower the rate of microalga growth wasachieved Such disparities were likely caused by self-shadingeffect The highest of tested capacities was confirmed to besimultaneously the most effective Thus it was decided tocultivate Spirulina maxima cultures destined for biosorptionexperiments in 40 L reactor working volume in semibatchmode (Section 32)

32 SemibatchMode in 40 LReactor System Semibatchmodewas used to optimize Spirulina maxima growth during 4-month cultivation Except the biomass separation at the endof test microalga cells were filtered out in the meantimewhen stabilization of their concentrationwas noted (lag phasebeginning) The next day after each of six partial receptionsperformed a renewed increase in the biomass concentrationwas recorded (Figure 6)

Due to performing cultivation in semibatch mode sevendistinctive relative growth rates were evaluated summary ofwhich is presented in Table 3

0 2 4 6 8 10 12Time (days)

000

002

004

006

008

010

r x(g

L d

ay)


Figure 5Comparison of Spirulinamaxima growth rates for culturesin different capacities during two first weeks of cultivation

0 20 40 60 80 100Time (days)

0

1

I VIIVIVIVIIIII

Ln C

s

minus5

minus4

minus3

minus2

minus1

Figure 6Kinetics of Spirulinamaxima growth in semibatch culture

The subsequent biomass separations resulted in 120583

decrease It was noticed that average of the relative rate valuesIIndashVII (00382 dayminus1) was 87 lower compared with therate achieved at the beginning of the culture (0285 dayminus1)Limiting the relative growth rate was likely related tomediumalkalization caused by the microbial consumption of CO

2

released as a product of medium constituent decomposition(HCO

3

minus

999448999471 CO2+OHminus)

Due to similarity of the rate results obtained after fifthand sixth exceeded biomass filtration (120583 numbers VI andVII) further cultivation was discontinued and followed withbiosorption research using previously dried microalga cells

33 Fourier Transform Infrared Spectroscopy FTIR analysiswas confirmed as efficacy method for identification of both


Amine group Amide group Carboxylic group Phosphoryl and sulfone group

337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)

Figure 7 Normalized FTIR absorption spectrum of selected functional groups exposed on cell wall surface of microalgae biomass A (greenline) pristine Spirulina sp B (pink line) pristine Spirulina maxima and C (blue line) Cu(II)-enriched Spirulina maxima

total and metal ion-binding groups occurred on the biosor-bent surface [27] Evaluation of the adsorptive properties ofbiological materials including algae with FTIR spectra wasinvestigated in a number of reports Many studies focusedon applicability of seaweeds to aqueous media treatment byremoving heavy metal ions such as Pb(II) Cd(II) Ni(II)U(VI) and Th(IV) [28ndash30] There are also some works inwhich algal biomass is subjected to interactions with cationsof nutritional value [24 31] However no literature datawas found to provide information on usability of Fouriertransform infrared spectroscopy in evaluation of enrichmentSpirulina species with microelements

Results of FTIR analysis (Figure 7) proved the variety offunctional groups found on the cell wall surface of examinedbiosorbents Each group in its free form that is withoutmetalion loaded was detected at strictly defined wave numbergiving a specific signal (band) A set of all bands spectrumwhich depicts the full surface composition of particularbiomass is named as IR ldquofingerprintrdquo [32]

Figure 7 shows the bands representing the specific groupsdetected on the cell wall of pristine and Cu(II)-loadedSpirulina maxima (Figures 7(B) and 7(C) resp) as wellas lyophilized Spirulina sp (Figure 7(A)) Spectra of both

natural microalgal biomasses proved their great similarity inthe composition of surface groups At the same time smalldifferences in the wave number at which particular band wasnoted confirmed the efficacy of FTIR analysis in identifyingsamples of biological material Run sequence plot of ion-enriched microalga differed at some points from pristinemicroalgae results due to interactions of cell wall groups withmetal cations which caused visible signal changes

Considering spectra of natural biosorbent surface afew functional groups might be identified At the high-est wave number of 3300ndash3310 cmminus1 amine bands weredetected Stretching bands at 1650 cmminus1 (asymmetrical) and1400 cmminus1 (symmetrical) corresponded to carbonyl groups(C=O) of protein primary amides and carboxylate ionsrespectively The band at 1540 cmminus1 represented the stretch-ing vibrations of protein secondary amides (ndashNH) The bandat 1240 cmminus1 was given by free ndashCO bound while stretchingvibrations at 1150 and 1050 cmminus1 corresponded to CCCOmode of polysaccharides ethers Within a wave numberrange of 1240ndash1050 cmminus1 hydroxyl bands were detected aswell The detailed summary of all bands observed on themicroalgae spectra was shown in Table 4 [33 34]


Table 4 Identified functional groups on surface of natural and Cu(II)-loaded Spirulina biomass

Bond Spirulina sp Spirulina maxima Cu(II)-Spirulinamaxima Component

Wave number cmminus1

lPndashOndash ndashSO3ndash 86148 83289 mdash Polysaccharides

120574 CCCO 120574 OH 104944115086

104754114959

104980115421 Polysaccharides Polysaccharides ethers

120575 CndashOndashC 120575 OH 124196 124345 123552 Amides Polysaccharides Esters120574S COOminus 140008 140107 140745 Carboxyl group120575 CHCH2 145464 145226 145212 Alkyl chain120575 NH 154150 154470 153632 Secondary amides120574 C=O 120574 CN 165416 165715 165616 Protein primary amides120574S CH3 287417 287450 287413 Alkyl chain120574AS CH3 292779 292772 292740 Alkyl chain120574S CH2 296011 296013 295939 Alkyl chain120574 NH3 330249 331008 337202 Amines

The group(s) primarily concerned withmetal ion interac-tionmight be indicated with differences in vibration intensity(transmittance) or band wave number noted between treatedand nontreated biomass spectra FTIR records for Cu(II)-loaded biomaterial showed the characteristic peak shiftingof amine group which occurred at 60 units higher wavenumber compared to nontreated microalgal cells A smallchange in peak positions of secondary amide (lower wavenumber) and carboxyl (higher wave number) groups wasalso observed however obtained differences were not asevident as in the case of amine group Thus verificationof significance of both amide and carboxyl groups in ionsorption was required Nevertheless the band intensities ofamine amide and carboxyl groups present on Cu(II)-Smaxima surfacewere higher than for pristinemicroalga Suchresults confirmed ionic interaction between these groups andmetal cations which was in accordance with the publishedliterature [21]

On the other hand affecting ion sorption by hydroxylgroup commonly considered as one of themajor binding siteon the biomass surface [21] could not be evaluated sincenone of the obtained records represented this group onlyThere was a peak shift observed for all bands of Cu(II) loadedmicroalga which were recognized as ndashOH bound vibrationpositions (123552 115421 and 104980 cmminus1) Thereforeit was assumed that hydroxyl group interacted with metalions and further investigation was performed to prove thisstatement (Section 34)

In case of Cu(II)-Spirulina maxima spectrum no bandcorresponding to neither phosphoryl (lPndashOndash) nor sulfone(in deprotonated form of sulfonate ndashSO

3ndash) groups was

detected as well On the other hand these valleys weredetected for pristine Spirulina sp and S maxima at 862 and830 cmminus1 respectively Since occurring of particular banddepended on whether biomass was subjected to biosorptionor not surface group represented by this band should beinvolved in ion binding Thus it was concluded that metalcations were likely bound to Spirulina maxima biomass with

phosphoryl and deprotonated sulfone occurring mainly insulfonated polysaccharides group [35]

FTIR studies revealed that the surface compositionof Spirulina sp is similar to Spirulina maxima and thuscomparable adsorptive properties would be expected As aconsequence the same functional groups were considered tobe affected by the presence of metal ions and responsible fortheir sorption

The spectrophotometric records were confronted withpotentiometric titration in order to complement the surfacecharacteristics of Spirulina biomass concerning both typeand capacity of the major binding sites

34 Potentiometric Titration of Biosorbent Surface The valid-ity of using the potentiometric titration to investigate thenature of biomass surface is based on a postulate that ionexchange between biomass binding sites and metal cationsolution is the dominant mechanism of biosorption Themajor functional groups involved in cation binding in thepH range 2ndash12 were identified as well [20 24] In conductedresearch potentiometric titrationwas performed to verify theresults of FTIR analysis and provide detailed information onion binding sites of Spirulina sp and Spirulina maxima cellwalls

The experimental data frompotentiometric titrationwereanalyzed with three models assuming the presence of onetwo and three types of functional groups on biomaterialsurface respectively [20] Since the algal biomass has beenshown to act as weakly acidic ion exchanger the model ofmultiprotic acid might be used to characterize its titrationcurve In order to improve model conformance with exper-imental conditions modification considering different con-centration of each surface functional group was introduced[20 24] Elaborated model was expressed by a nonlinearcorrelation between molar ratio of added titrant (119909add) andpH value described with one of three equations presentedin Michalak and Chojnacka report [24] Evaluation of fittingparticular model to the experimental results required esti-mating acidic dissociation constant (119870

119886) values and heights


Table 5 Surface functional groups and their concentration evaluated with potentiometric titrations

Functional groupMicroalga(e)

Spirulina maxima Spirulina sp

pK119886

Concentrationmeq gminus1 pK

119886

Concentrationmeq gminus1

Carboxyl 266 299 277 0998Phosphoryl 668 173 696 0573Amine or hydroxyl 109 327 108 0889Total 799 246

of jumps (drop in 119909add) observed in the titration curvewhich were determined based on nonlinear regression andthe first and the second derivative course using Mathematicav 30 software (Wolfram Hanborough Oxfordshire UnitedKingdom) [20 24] In accordance with previous studies [1519 20 24 25] the model assuming three different types ofion binding groups was the best fitted to the results fromSpirulina sp and Spirulina maxima potentiometric titrationThus modified model for triprotic acid was used to describeexperimental data (see (4)) [20]

119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1

(4)Model parameters a b and c equate to heights of sub-

sequent jumps in the regression curve and represent molarratios of 1st 2nd and 3rd identified functional group re-spectively According to the definition the sum of molarratios determined for individual components of the solutionis always 1 thus in proposed model above 119886 + 119887 + 119888 = 1At the same time the sum of jumps heights is equal to molarratio of added titrant (119886 + 119887 + 119888 = 119909add) Considering bothcorrelations the solution of (4) is 119909add = 1

The use of accepted model for a triprotic acid to describepotentiometric titration records gave a curve with threeflex points (equivalence points of titration) [24] which wasshown in Figure 8 along with the first and the secondderivative course

Acidity constants of binding sites on microalgae surfacewere evaluated from logarithmic measures that is pK

119886

which corresponded to zeros of the second derivative curvecalculated from the consecutive titration equivalence points[24]Therefore pK

119886might be considered as specific pH value

In fact pK119886determines such pH above which functional

group of particular type are in the ionized form mostly andhence capable of exchanging H+ with metal cations from thesolution [20] On the basis of determined pK

119886values (black

curve in Figure 8 Table 5) carboxyl phosphoryl (phosphatein particular) and either amine or hydroxyl groups [36ndash39] were identified as ion binding sites present on both

Spirulina sp and S maxima cell walls Obtained resultswere partially consentient with those from Fourier transforminfrared analysis besides ndashOH significance of which wasnot evident on IR spectra (Figure 7) The disparity betweenthese two evaluations might be caused by conducting ionexchange at different pH values Biosorption experimentspreceding spectrophotometric assay were carried out at pH 5at which hydroxyl groups were not involved in cation bindingdue to their protonated form [20] During potentiometrictitration solution pH exceeded pK

119886enabling ndashOH to metal

ion sorptionOn the other hand potentiometric titration did not

confirm involvement of sulfone group (as ndashSO3ndash) in cation

exchange while FTIR spectrometry proved otherwise It waslikely resulted from low sulfonate pK

119886value determined in

the range of 10ndash25 [40] which was below the lowest pK119886

estimated from the titration curvemdashpK119886= 266 assigned

to carboxyl group Since interaction of metal ions withdeprotonated sulfone groups was evident on FTIR spectra itmight be concluded that ndashSO

3ndash occurred on the microalgal

surface at insufficiently high concentration to be consideredas the major binding site

Based on FTIR records capability of amide group to sorbcations was taken into consideration as well However asopposed to ndashSO

3ndash neither peak shifting nor band inten-

sity increase noted after biosorption experiments providedexplicit information on interaction between surface amidsgroups and metal cations Thus involvement of ndashNH (2∘amides) in ion exchange was verified with potentiometrictitration by comparing amide pK

119886to those evaluated based on

model (4) None of obtained results corresponded to amides(pK119886

= 100) [41] and the highest value of determinedexperimentally pK

119886was assigned to either amine or hydroxyl

group [15 20] Hence it was claimed that amide groups didnot act as binding sites or their capability to bind cations wasnot significant enough to be confirmed

The concentrations of surface functional groups of eachtype were estimated from adequate molar ratio (heightof jump) and cation exchange capacity (119902cat exch) of theSpirulina biomass at the investigated pH range as follows

119902cat exch =mEqtitrantbiosorb susp minusmEqtitrantblank

119898biosorbent

[119902cat exch] = [meq sdot gminus1]

(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)

Figure 8 Potentiometric titration results of the Spirulina maxima (a) and Spirulina sp (b) biomass samples Green curve represents modelcurve describing the experimental data red curve the first derivative of the model curve and black curve the second derivative of the modelcurve

Cation exchange capacity is an experimentally evaluatedparameter which defines capability of solid phase (sorbent)treated with an ion solution to replace surface-bound ionby another [42] In practice 119902cat exch is proportional to thequantity of standardized solution (acid or base) added perunit of biosorbent mass during entire titration [24] The useof titrant was calculated from a difference between amountsconsumed by biomass suspension and blank sample (purewater) expressed in units of milliequivalance ([mEqtitrantsample] =

[meq])There were three jumps noted in both potentiometric

titration curves obtained in the test (green lines in Figure 8)Each of jumps corresponded to specific functional group pK

119886

value of which equated to flex point occurred in the jumpline [24] Hence the concentration of particular bindingsite 119862carboxyl 119862phosphoryl 119862aminehydroxyl exposed on Spirulinacell surface was determined with equations (6) (7) and (8)respectively

119862carboxyl = 119886 sdot 119902cat exch (6)

119862phosphoryl = 119887 sdot 119902cat exch (7)

119862aminehydroxyl = 119888 sdot 119902cat exch (8)

[119862functional group] = [meq sdot gminus1] (9)

The results of the functional group concentrations as wellas total content of identified binding sites for both Spirulinabiomasses were shown in Table 5

Despite the same type of groups capable of cationexchange microalgae biosorbents differed in the incidenceof binding sites on the cell wall Spirulina maxima surfacewas characterized by 30 and 37 times higher content of bothndashCOO and lPndashOndash and ndashNH

3ndashOH respectively compared

to Spirulina sp Literature reported that surface compositionand thus adsorptive properties of biomass depend on itsspecies and condition of growth [20] Previous studies onSpirulina sp in four differentmorphological types proved thatenvironmental condition manipulating affected the qualityof biosorbent and as a result the efficacy of ion exchange

[20] Comparing two biosorbents in terms of surface com-position only identical treatment during whole investigationis required Since no detailed information was known onSpirulina sp cultivation conducted tests could not be consid-ered as comparable The aim of our own experiments was toconfirm the applicability of bothmicroalgae to microelementsorption as well as to verify whether proposed conditions ofculture growth provided material which is equally good withcommercial product Obtained results showed that Spirulinamaxima grown under laboratory conditions would be moreefficient biosorbent than lyophilized Spirulina sp purchasedon a market due to a higher number of binding sites

Evident disparities in the content of surface functionalgroups were proved however a few similarities betweenSpirulina sp and S maxima records were observed as wellIn case of both microalgae the determined concentrations ofcarboxyl and either amine or hydroxyl groups were 15ndash19times higher compared to the content of phosphoryl groupSuch results might be caused by a different amount of com-pounds from which these groups were derived Since algalcell wall is built with polysaccharides at most [43] diversityof these components influences the structure of binding sitesexposed on the biomass surface Hence obtained compo-sition of surface functional groups is specific for particularbiosorbent species which is in accordance with ldquofingerprintrdquotheory of FTIR spectra Similarity of the concentration ratiosbetween each pair of identified groups ([ndashCOO] [lPndashOndash][ndashCOO] [ndashNH

3] or [ndashOH] [lPndashOndash] [ndashNH

3] or [ndashOH])

was likely resulted from close morphological resemblancebetween examined algae

Although on S maxima surface either amine or hydroxylgroups were mostly exposed the carboxyl groups wereconsidered to act as themajor binding sites of bothmicroalgaAs carboxyl groups had the pK

119886value of a little over 25 (the

endpoint of titration) they were in the form of carboxylateions capable of cation exchange within almost whole rangeof pH at which potentiometric titration was performed [20]Moreover deprotonation of binding sites which made themavailable tometal ions was enhanced alongwith pH increaseIn case of titration curve it might be concluded that higherpH improved the effectiveness of cation exchange


(a) (b)

Figure 9 Images of lyophilized Spirulina sp obtained with scanning electron microscope (SEM)

35 SEM-EDX Analysis of Algal Biomass Surface Enrichedin Microelements Previously conducted analysis enabledcharacterization of both type and capacity of binding sitespresent on Spirulina cell wall However neither FTIR spec-troscopy nor potentiometric titration provided informationon changes in elemental composition of microalgae surfaceresulted from metal ion biosorption Therefore scanningelectron microscopy with an energy dispersive X-ray analyti-cal system technique was carried out to compare the concen-tration of elements on natural and cation-loaded microalgaecell wall Since the same functional groups involved in ionexchange were proved to occur on both Spirulina sp andS maxima surface it was decided to perform SEM-EDXassay with one of them only For further multi-elementalanalysis lyophilized Spirulina sp was selected Although ionexchange capacity of S maximawas proved to be over 3 timeshigher commercial microalgae had certain quality whichprovided reliable records Moreover Spirulina sp was usedas biosorbent in previous tests for exploring the biosorptionmechanism [15 20] and thus comparison of the results couldbe made

SEM-EDX analysis was used in several studies to evaluatesorption of microelement cations onto seaweeds biomass[25 27] Since studies onmacroalgae proved this technique assuitable for investigating feed supplementation via biosorp-tion it was decided to apply SEM-EDX analytical system inpresent work

The assay was preceded by biosorption experimentsperformed in both single- and multimetal (Co2+ Cu2+Mn2+ Zn2+) system and under the specified conditions(Section 24) The surface composition of the microalgaesamples loaded with each metal ion in total of five and allcations at once were analyzed and compared to the results fornatural Spirulina spThe photographs of the surface structureof pristine Spirulina sp lyophilisate were taken with scanningelectron microscopy and shown in Figure 9

The samples of treated and nontreated biomass weresubjected to elemental analysis using EDX system and sixdistinct X-ray spectra were obtained (Figure 10)

The results obtained from EDX analysis showed that eachof examined Spirulina samples gave different set of bands dueto changes in the surface composition caused by biomass

treatment with cation(s) in single- or multimetal system Onthe spectra of microalgae enriched in Co(II) Cu(II) Mn(II)Zn(II) ions and all of them together the bands representingoverbalanced concentration of these cations were observedin Figures 10(b)ndash10(f) respectively while there were no cor-responding bands recorded on the natural biomass spectrum(Figure 10(a)) Diversity of the cell wall composition amongloaded Spirulina samples was noted as well Previous studies[20] proved that efficiency with which surface functionalgroups bound ions varied depending on particular element(metal) The affinity of biosorbent for sorbate is specific andattributable to both the type and valence of ion and pK

119886

value of the binding functional group Thus this parameterneeds to be separately verified for each system In presentresearch a relative affinity between Spirulina sp and Co(II)Cu(II) Mn(II) and Zn(II) ions was determined based onSEM-EDX results by comparing the concentration of the onetype of bound ion to other records The summary of elementconcentration evaluated for pristine Spirulina and samplesafter biosorption experiments was presented in Table 6

The results obtained during X-ray exams on cation-loaded Spirulina sp showed increased content of allmicroele-ment ions in which biomass was enriched via biosorptionFor single-metal system the concentration of Co(II) Cu(II)Mn(II) and Zn(II) was about 12 715 1380 and over 1760times higher respectively compared to natural microalgaewhile experiment inmulticomponent systemgave an increaseof the same cations at 145 22 87 and over 180 times (Theincrease of zinc ion concentrationwas determined as the ratioof the corresponding result for Zn(II)-enriched Spirulina spin either single-or multimetal system to the lower detectionlimit of EDX system) Although performing biosorptionin multimetal system was less effective it provided supple-mentation with all examined ions at once Thus making adecision concerning more preferable method of enrichmentSpirulinawith cation(s) depends on the application to whichthe loaded algal biomass is intended

Analysis of the surface composition of various elementsjustified the relevance to perform parallel biosorption exper-iments in single- and multimetal system since the presenceof competing cations significantly affected the binding pref-erences of biomass Disparities in absolute concentration of


(a) (b)

(c) (d)

(e) (f)

Figure 10 X-ray spectrummineral trace analysis of Spirulina sp surface (a) pristinemicroalgae biomass Spirulina sp enriched in (b) Co(II)ions (c) Cu(II) ions (d) Mn(II) ions and (e) Zn(II) ions (f) Spirulina sp enriched in all Co(II) Cu(II) Mn(II) and Zn(II) ions

each microelement and the efficacy of its interacting withsurface functional groups compared to other elements werenoted The Spirulina sp affinity for examined metal ionsin single- and multicomponent system fallowed Mn(II) gtCu(II) gt Zn(II) gt Co(II) and Cu(II) gt Mn(II) gt Co(II) gtZn(II) respectively The concentration difference betweenthemost and the least easily bound cationwas 10 times higher

in case of samples prepared in single-metal system than inthe presence of competing ions As all cations were equallycharged and biosorption experiments were carried out usingthe same biosorbent species it was concluded that interac-tion and hence affinity between biomass of Spirulina spand each microelement cation depended on sorbate type(s)only


Table 6 Atomic concentration of elements present on the surface of pristine and metal cation-loaded Spirulina sp assessed according to theanalysis of X-ray spectrum

Element Atomic concentration of elements of all detected ions Detection limits Natural biomass Co-Spirulina sp Cu-Spirulina sp Mn-Spirulina

sp Zn-Spirulina sp Co Cu MnZn-Spirulina sp

lowast

C 179 plusmn 486 548 plusmn 899 447 plusmn 794 256 plusmn 223 479 plusmn 889 337 plusmn 681 0277ndash100Cl 180 plusmn 012 071 plusmn 008 042 plusmn 007 160 plusmn 016 20 plusmn 013 020 plusmn 005 0001ndash2621O 5931 plusmn 112 394 plusmn 676 429 plusmn 767 383 plusmn 331 428 plusmn 824 549 plusmn 1014 0525ndash100P 026 plusmn 006 069 plusmn 006 137 plusmn 009 173 plusmn 013 069 plusmn 007 029 plusmn 005 0001ndash2013S 049 plusmn 007 000 plusmn 000 035 plusmn 007 137 plusmn 016 086 plusmn 009 136 plusmn 009 0001ndash2307

lowastlowast

Co 008 plusmn 008 095 plusmn 013 ltLLD ltLLD ltLLD 116 plusmn 015 0076ndash6924Cu 013 plusmn 011 ltLLD 927 plusmn 069 ltLLD ltLLD 285 plusmn 030 0083ndash8040Fe 005 plusmn 006 015 plusmn 009 015 plusmn 009 ltLLD 041 plusmn 010 036 plusmn 010 0060ndash6398Mn 002 plusmn 000 ltLLD ltLLD 276 plusmn 127 ltLLD 174 plusmn 015 0063ndash5894Zn ltLLD ltLLD ltLLD ltLLD 176 plusmn 031 018 plusmn 026 0001ndash8630

lowast lowast lowast

Ca 012 plusmn 006 ltLLD ltLLD 017 plusmn 011 019 plusmn 007 ltLLD 0341ndash3690K 258 plusmn 018 022 plusmn 006 ltLLD 008 plusmn 009 000 plusmn 001 028 plusmn 007 0001ndash3312Na 125 plusmn 085 042 plusmn 005 ltLLD 109 plusmn 019 101 plusmn 011 160 plusmn 013 0001ndash1041

lowastmacroelements lowastlowastmicroelements lowastlowastlowastalkali and alkaline earth metalsltLLD lower limit of detection

Apart from increased concentration of cobalt coppermanganese andor zinc the surface composition of loadedmicroalgae showed significantly lower content of alkali metalcompared to natural cells Such results might be referred toas ion exchange mechanism via which biosorption primarilyoccurs [35 44] The difference of sodium concentrationbefore and after biomass enrichment was more evident thanin the case of potassium thus Na(I) was claimed to be mostinvolved in ion exchanging among cations naturally boundwith surface functional groups Both sodium and potassiumions interacted with all examined sorbates According to theliterature alkaline earth metal ions might also participate inbiosorption [35 44] which was confirmed in present worksince the concentration of calcium cations decreased dueto loading Spirulina sp with microelement(s) However asopposed to Na(I) and K(I) exchanging Ca(II) with othermetal ions was noted in the presence of Co(II) and Cu(II)cations and in multicomponent system only

The composition of microalgae surface besides ionsexchanged during biosorption contained elements such ascell wall building blocks (eg carbon) or functional groupconstituents (eg phosphorus) the concentration of whichshould not have varied after microelement ion bindingwhile it did Disparities in records for natural and enrichedSpirulina sp were likely observed due to conformationalchanges of neighboring functional groups as they interactedwith different cations According to posited mechanism ofbiosorption-ion exchange cation binding onto biosorbentoccurs through electron donor-acceptor interactions result-ing in a replacement of naturally bound ions Na(I) K(I)andor Ca(II) Both alkali and alkaline earth metal ions are

larger in size compared to microelement cations used forbiosorption experimentsThe ionic radius forNa(I) K(I) andCa(II) is 95 pm 133 pm and 99 pm respectively in contrastionic radius for Co(II) Cu(II) Mn(II) and Zn(II) is in therange of 73ndash80 pm [45 46] The exchange of larger cationsto smaller cations affects the spatial distribution of ions ontothe biosorbent surface and either revealing or concealingthe particular elements on Spirulina sp surface might beobserved as increase or decrease of the corresponding bandsin the X-ray spectra

36 Comparison of SEM-EDX and ICP-OES TechniquesInductively coupled plasma-optical emission spectrometrywas used in several studies as the suitable method for inves-tigating the elemental composition of biosorbent surfacethus assessing themechanism of the process and applicabilityof the biomass to binding particular ions [25 26] SinceICP-OES gave reliable and explicit results from previousexperiments with Spirulina [15ndash17 20] it was decided toemploy this technique to verify the records from SEM-EDXanalysis in present work However there was no cationexchanged during biosorption the atomic concentration ofwhich was evaluated in all examined samples Thus thecorrelation coefficient between ICP-OES and SEM-EDX ana-lytical techniques was assessed on the example of the resultsobtained for sodium ion as it was most frequently replacedby microelement cations regardless of the system testedThe ratios of Na(I) concentration on natural and loadedSpirulina surface determined by both compared analysiswere depicted graphically and linear regression equation wasdetermined (Figure 11) Records for microalga loaded with


0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna

y = minus09287 + 902 lowast 10minus5 lowast x P = 0010 R2 = 09801

Na(I) content in the biomass ICP-OES (mgkg)

Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()

Figure 11 Correlation between the concentration of Na(I) ions onSpirulina sp surface assessed by ICP-OES and SEM-EDX analysisNaturalnamdashpristine microalgae Co Mn Znmdashbiomass enrichedin Co(II) Mn(II) and Zn(II) cations respectively MIXmdashbiomassenriched inmetal cations inmulti-component system Values on the119909 and 119910 axis indicate Na(I) concentration determined by ICP-OESand EDX and analytical system respectively

Cu(II) were not involved in estimating the ICP-OECSEM-EDX correlation coefficient since the content of sodium ionswas under detection limit of X-ray system

Due to comparison between ICP-OES and SEM-EDXanalysis relatively high value of the correlation coefficientwas achieved (119877

2

= 09891) R-squared value close to 1confirmed both techniques to give the similar results ofsurface elemental composition and thus to be equally usefulin biosorption investigations

4 Conclusions

Biosorption of microelement ions copper cobalt zinc andmanganese from aqueous solutions by Spirulina biomasswas investigated to assess the applicability of enriched algaeas a feed supplement for livestock Parallel experimentsin single- and multicomponent system were performedIn present work adsorptive properties of cultivated underlaboratory conditions Spirulina maxima were verified bycomparing to commercially available lyophilized Spirulinasp Both examined biosorbents were proved to increaseThe results obtained from potentiometric titration showedthat higher pH value favored metal ions biosorption sincemore binding sites were deprotonatedThereby ion exchangewas confirmed as the dominant mechanism of the processAlthough biosorption capacity was over 3 times higher for Smaxima than commercial lyophilisate loading Spirulina spwithmicroelement ions increased their surface concentrationfrom 12 up to 1760 times The affinity of biomass formicroelement cations determined on the example of pristineand loaded Spirulina sp in single- and multicomponentsystem was Mn(II) gt Cu(II) gt Zn(II) gt Co(II) and Cu(II) gtMn(II) gt Co(II) gt Zn(II) respectively FTIR analysis showed

similar chelating characteristics ofmetal coordination groupsto results evaluated with titration The binding sites presenton Spirulina cell wall included carboxyl phosphoryl andeither hydroxyl or amine groups Sulfonate groups were alsoproved to participate in ion sorption (FTIR spectra) Theconcentration of elements on the surface of natural andmetalion-loaded biomass was assessed by both ICP-OES and SEM-EDX techniques Since relatively high value of correlationcoefficient was shown between these systems usefulnessof SEM-EDX analysis in biosorption studies on Spirulinabiomass was confirmed as well

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theworkwas financially supported by PolishNational Centrefor Research and DevelopmentmdashProject no N R05 001410 and Grant entitled ldquoInnovative technology of seaweedextracts components of fertilizers feeds and cosmeticsrdquomdashProject no PBS1A122012 and National Science CentremdashProject no 201205EST803055 The work was financiallysupported by Polish National Science CentremdashProject noUMO-201205EST803055

References

[1] R Gupta P Ahuja S Khan R K Saxena and H MohapatraldquoMicrobial biosorbents meeting challenges of heavy metalpollution in aqueous solutionsrdquo Current Science vol 78 no 8pp 967ndash973 2000

[2] Y Liu and Y-J Liu ldquoBiosorption isotherms kinetics andthermodynamicsrdquo Separation and Purification Technology vol61 no 3 pp 229ndash242 2008

[3] R Vannela and S K Verma ldquoCu2+ removal and recoveryby SpiSORB batch stirred and up-flow packed bed columnarreactor systemsrdquo Bioprocess and Biosystems Engineering vol 29no 1 pp 7ndash17 2006

[4] H Chen and S-S Pan ldquoBioremediation potential of spirulinatoxicity and biosorption studies of leadrdquo Journal of ZhejiangUniversity Science vol 6 no 3 pp 171ndash174 2005

[5] A G Murugesan S Maheswari and G Bagirath ldquoBiosorptionof cadmiumby live and immobilized cells of Spirulina platensisrdquoInternational Journal of Environmental Research vol 2 no 3 pp307ndash312 2008

[6] E Finocchio A Lodi C Solisio and A Converti ldquoChromium(VI) removal by methylated biomass of Spirulina platensis theeffect of methylation processrdquo Chemical Engineering Journalvol 156 no 2 pp 264ndash269 2010

[7] C D Magro M C Deon A D Rossi C O Reinehr MHemkemeier and L M Colla ldquoChromium (VI) biosorptionand removal of chemical oxygen demand by Spirulina platensisfrom waste water-supplemented culture mediumrdquo Journal ofEnvironmental Science and Health A ToxicHazardous Sub-stances and Environmental Engineering vol 47 no 12 pp 1818ndash1824 2012


[8] C Solisio A Lodi and E Finocchio ldquoEffects of pH onchromate(VI) adsorption by Spirulina platensis biomass batchtests and FT-IR studiesrdquo Water Science and Technology vol 67no 9 pp 1916ndash1922 2013

[9] O Murali and S K Mehar ldquoBioremediation of heavy metalsusing Spirulinardquo International Journal of Geology Earth andEnvironmental Sciences vol 4 no 1 pp 244ndash249 2014

[10] K Chojnacka A Chojnacki and H Gorecka ldquoTrace elementremoval by Spirulina sp from copper smelter and refineryeffluentsrdquo Hydrometallurgy vol 73 no 1-2 pp 147ndash153 2004

[11] P G Hiremath and P Binnal ldquoFirst report on biosorption offluoride on the microalga Spirulina platensis batch studiesrdquoAsian-American Journal of Chemistry vol 1 no 1 pp 1ndash10 2013

[12] H Doshi A Ray and I L Kothari ldquoLive and dead Spirulinasp to remove arsenic (v) from waterrdquo International Journal ofPhytoremediation vol 11 no 1 pp 53ndash64 2009

[13] G L Dotto V M Esquerdo M L G Vieira and L A A PintoldquoOptimization and kinetic analysis of food dyes biosorption bySpirulina platensisrdquo Colloids and Surfaces B Biointerfaces vol91 no 1 pp 234ndash241 2012

[14] I Priyadarshani D Sahu and B Rath ldquoMicroalgal bioremedia-tion current practices and perspectivesrdquo Journal of BiochemicalTechnology vol 3 no 3 pp 299ndash304 2011

[15] A Zielinska andK Chojnacka ldquoThe comparison of biosorptionof nutritionally significant minerals in single- and multi-mineral systems by the edible microalga Spirulina sprdquo Journalof the Science of Food and Agriculture vol 89 no 13 pp 2292ndash2301 2009

[16] A Saeid K Chojnacka M Korczynski D Korniewicz andZ Dobrzanski ldquoBiomass of Spirulina maxima enriched bybiosorption process as a new feed supplement for swinerdquoJournal of Applied Phycology vol 25 no 2 pp 667ndash675 2013

[17] A Saeid K Chojnacka M Korczynski D Korniewicz and ZDobrzanski ldquoEffect on supplementation of Spirulina maximaenriched with Cu on production performance metabolical andphysiological parameters in fattening pigsrdquo Journal of AppliedPhycology vol 25 no 5 pp 1607ndash1617 2013

[18] I Zinicovscaia G Duca V Rudic et al ldquoSpirulina platensisas biosorbent of zinc in waterrdquo Environmental Engineering andManagement Journal vol 12 no 5 pp 1079ndash1084 2013

[19] S Schiewer and B Volesky ldquoModeling multi-metal ionexchange in biosorptionrdquo Environmental Science and Technol-ogy vol 30 no 10 pp 2921ndash2927 1996

[20] K Chojnacka A Chojnacki and H Gorecka ldquoBiosorptionof Cr3+ Cd2+ and Cu2+ ions by blue-green algae Spirulinasp kinetics equilibrium and the mechanism of the processrdquoChemosphere vol 59 no 1 pp 75ndash84 2005

[21] K Chojnacka ldquoBiosorptionrdquo in Biosorption and Bioaccumula-tion in Practice chapter 22 pp 5ndash15 Nova Science PublishersNew York NY USA 2009

[22] U G Schlosser ldquoSammlung von algenkulturenrdquo Berichte derDeutschen Botanischen Gesellschaft vol 95 no 1 pp 181ndash2761982

[23] A Vonshak ldquoSpirulina growth physiology and biochemistryrdquoin Spirulina Platensis (Arthrospira) Physiology Cell-Biology andBiotechnology chapter 3 pp 43ndash66 Taylor amp Francis LondonUK 1997

[24] I Michalak and K Chojnacka ldquoInteractions of metal cationswith anionic groups on the cell wall of the macroalga Vaucheriasprdquo Engineering in Life Sciences vol 10 no 3 pp 209ndash217 2010

[25] IMichalak K Chojnacka andKMarycz ldquoUsing ICP-OES andSEM-EDX in biosorption studiesrdquo Microchimica Acta vol 172no 1 pp 65ndash74 2011

[26] K Chojnacka ldquoThe application of multielemental analysis inthe elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006

[27] I Michalak K Chojnacka and A Witek-Krowiak ldquoState of theart for the biosorption processmdasha reviewrdquoApplied Biochemistryand Biotechnology vol 170 no 6 pp 1389ndash1416 2013

[28] A B Dekhil Y Hannachi A Ghorbel and T BoubakerldquoRemoval of lead and cadmium ions from aqueous solutionsusing the macroalga Caulerpa racemosardquo Chemistry and Ecol-ogy vol 27 no 3 pp 221ndash234 2011

[29] W H Fan Z Z Xu and Q Li ldquoKinetics and mechanism ofnickel(II) ion biosorption by immobilized brown Laminariajaponica algaerdquo Adsorption Science and Technology vol 28 no6 pp 499ndash507 2010

[30] S S Bozkurt Z B Molu L Cavas andMMerdivan ldquoBiosorp-tion of uranium (VI) and thorium (IV) onto Ulva gigantea(Kutzing) bliding discussion of adsorption isotherms kineticsand thermodynamicrdquo Journal of Radioanalytical and NuclearChemistry vol 288 no 3 pp 867ndash874 2011

[31] J Plaza Cazon M Viera S Sala and E Donati ldquoBiochemicalcharacterization ofMacrocystis pyrifera andUndaria pinnatifida(Phaeophyceae) in relation to their potentiality as biosorbentsrdquoPhycologia vol 53 no 1 pp 100ndash108 2014

[32] R Davis and L J Mauer ldquoFourier tansform infrared (FT-IR)spectroscopy a rapid tool for detection and analysis of food-borne pathogenic bacteriardquo in Current Research Technologyand Education Topics in Applied Microbiology and MicrobialBiotechnology vol 2 of Microbiology Series pp 1582ndash1594FORMATEX Badajoz Spain 2010

[33] S Venkatesan K Pugazhendy D Sangeetha C VasantharajaS Prabakaran andMMeenambal ldquoFourier transform infrared(FT-IR) spectoroscopic analysis of Spirulinardquo InternationalJournal of Pharmaceutical amp Biological Archives vol 3 no 4 pp969ndash972 2012

[34] P Prabakaran and A D Ravindran ldquoEfficacy of differentextraction methods of phycocyanin from Spirulina platensisrdquoInternational Journal of Research in Pharmacy and Life Sciencesvol 1 no 1 pp 15ndash20 2013

[35] E Fourest C Canal and J-C Roux ldquoImprovement of heavymetal biosorption by mycelial dead biomasses (Rhizopusarrhizus Mucor miehei and Penicillium chrysogenum) pHcontrol and cationic activationrdquo FEMS Microbiology Reviewsvol 14 no 4 pp 325ndash332 1994

[36] J S Cox D S Smith L AWarren and F G Ferris ldquoCharacter-izing heterogeneous bacterial surface functional groups usingdiscrete affinity spectra for proton bindingrdquo EnvironmentalScience and Technology vol 33 no 24 pp 4514ndash4521 1999

[37] L N Johnson and R J Lewis ldquoStructural basis for control byphosphorylationrdquo Chemical Reviews vol 101 no 8 pp 2209ndash2242 2001

[38] J DeRuiter ldquoCarboxylic Acid Structure and Chemistry Part 1rdquo2005 httpwwwauburnedusimderuijapda1 acids1pdf

[39] N L Bauld ldquoAminesrdquo 2005 httpresearchcmutexasedunbauldCHAPTER2021htm

[40] P X Sheng Y-P Ting J P Chen andLHong ldquoSorption of leadcopper cadmium zinc and nickel by marine algal biomasscharacterization of biosorptive capacity and investigation of


mechanismsrdquo The Journal of Colloid and Interface Science vol275 no 1 pp 131ndash141 2004

[41] J DeRuiter ldquoAmides and related functional groupsrdquo 2005httpwwwauburnedusimderuijapda1 amidespdf

[42] G M Gadd ldquoBiosorption critical review of scientific rationaleenvironmental importance and significance for pollution treat-mentrdquo Journal of Chemical Technology and Biotechnology vol84 no 1 pp 13ndash28 2009

[43] D S Domozych ldquoAlgal Cell Wallsrdquo in eLS John Wiley amp SonsChichester UK 2011

[44] T A Davis B Volesky and A Mucci ldquoA review of thebiochemistry of heavymetal biosorption by brown algaerdquoWaterResearch vol 37 no 18 pp 4311ndash4330 2003

[45] Periodic table Lenntech 2014 httpwwwlenntechcompe-riodicperiodic-charthtm

[46] R Kayestha Sumati and K Hajela ldquoESR studies on the effectof ionic radii on displacement of Mn2+ bound to a soluble 120573-galactoside binding hepatic lectinrdquo FEBS Letters vol 368 no 2pp 285ndash288 1995

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Active and Passive Electronic Components

Control Scienceand Engineering

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Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design



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Civil EngineeringAdvances in

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of


Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Chemical EngineeringInternational Journal of Antennas and

Propagation




Navigation and Observation



DistributedSensor Networks



0pH

14

pKNH2

In pH 5∙

∙ COOminus

∙

pKCOOminuspKPO48minuspKSO4

2minus

SO42minus

NH3+

∙ PO43minus

Figure 1The state of ionization of functional groups on the surfaceof the microalgal cell wall depending on pH

with different ions and the fact that electrical charge ofbiomass surface governs sorbates adsorption For examplethe isoelectric point (pI) of microalgae Chlorella is about3 while Spirulina pI = 28ndash35 [19] In lower pH positivelycharged groups (eg amine group) attract anions and repulsecations while in higher pH negatively charged groups (egcarboxyl) act conversely Typical changes of ionization stateof functional groups on the surface of microalgae cell walldepending on pH are shown in Figure 1 along with the listingof functional groups (under the red arrow) and their formsin pH = 5 for which the highest model parameters fromquantitative and kinetic examination have been obtained[20]

In present work dried biomass of commercially avail-able Spirulina sp (lyophilisate) and cultivated under lab-oratory conditions Spirulina maxima were investigated forsupplementation with microelement ions Cu(II) Co(II)Mn(II) and Zn(II) which are important in livestock feedingBiosorption experiments were performed in both single-and multicomponent systems under previously evaluatedconditions Four analytical methods were used to assess thecomposition of natural and loaded biomass surface as wellas to verify the biosorption mechanism in exchange positedon the basis of earlier studies The major binding sites wereidentified along with their concentrations and thus totalcapacity of both examined biosorbents might be estimatedBased on changes observed in elemental composition of theSpirulina surface after biosorption experiments the affin-ity sequence of microelement cations was determined forsingle- andmultimetal systems separatelyThe applicability ofmicroalgal biomaterials to feed supplementationwas assesseddue to resultant of all conducted tests

2 Materials and Methods

21 Microorganisms and Media Composition Two kindsof algal biomasses were selected for biosorption researchCommercially available Spirulina sp in form of lyophilizedcells was purchased from SIGMA (USA) Spirulina maximawas obtained from Culture Collection of Algal Laboratory(CCALA) Institute of Botany Academy of Sciences of the

00 02 04 06 08 10 12 14 1600

02

04

06

08

10

12

A560

y = minus0058 + 0739 lowast x R2 = 0955

Cd

w(

gL)

Figure 2 Standard curve represents the relationship between con-centration of Spirulina maxima dry mass (119862dw) and correspondingabsorbance (A

560) results

Czech Republic Microalga grew in the Schlosser liquidmedium [22] composition of which is presented in Table 1

Schlosser culture medium was prepared with analyticalgrade reagents and purchased from POCh SA (GliwicePoland) After cultivation the biomass was separated byfiltration using the aquarium algae vacuum cleaner (ActizooZolux France) dried at 60∘C (laboratory oven WAMEDWarsaw Poland) for three days and subjected to furtherinvestigation as a biosorbent

22 Cultivation of Spirulina maxima Spirulina maxima wascultivated in laboratory-scale reactors at 35∘C [23] for 4months The cultures were carried out under 12 12 pho-toperiod conditions (12 hr light 12 hr dark cycles) providedwith artificial illumination of 19Wmminus2 intensity In orderto improve growth algal scalability tests were performed inthree consecutive systems of increasing capacity Microalgacultivation started with a working capacity of 1 L and due tocell passaging 5 L and 40 L reactor systems were examinedas well

The efficiency of the Spirulina maxima cultivation ineach of tested capacities was determined using three basicparameters the concentration of microalgal culture growthrate and relative (exponential) growth rate The first of three(119862119878) was estimated by the straight-line equation showing the

correlation between concentration ofmicroalga drymass andabsorbance measurements at 560 nm (Figure 2)

23 Semibatch Mode The experiment was conducted onSpirulina maxima culture and grown in Schlosser medium at35∘C using 40 L-working capacity reactor equipped with twolight sources (12 12 photoperiod) (Figure 3) The cultivationwas carried out for 4 months

During research the systematic partial reception ofmicroalgal biomass was performed to diminish the self-shading effect which was verified by cell concentrationmeasurements The biomass was separated by vacuum fil-tration (Actizoo Zolux France) seven times overall Thelast separation stage gathered whole population of grown





3)2sdot5H2OCuSO

4sdot5H2O

MnSO4sdot5H2O ZnSO

















0 2 4 6 8 10 12 14 16 18 20 22Time (days)

minus50

minus45

minus40

minus35

minus30

minus25

minus20

minus15

minus10

Ln C

s







120583 =

ln119862119905119878minus ln1198620

119878

119905

[120583]= [dayminus1] (1)





119878= 119891(119905) (Figure 4)


ln119862119905119878= 120583 sdot 119905 + ln1198620

119878 (2)





1 0145 09735 0145 097840 0285 0987



I 0285 0987II 00439 0990III 00503 0985IV 00464 0976V 00331 0949VI 00275 0876VII 00281 0923





119903119909= 120583 sdot 119862

119905

119878


(3)





0 2 4 6 8 10 12Time (days)

000

002

004

006

008

010

r x(g

L d

ay)



0 20 40 60 80 100Time (days)

0

1

I VIIVIVIVIIIII

Ln C

s

minus5

minus4

minus3

minus2

minus1




2


3

minus

999448999471 CO2+OHminus)





337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)












120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation













3)2sdot5H2OCuSO

4sdot5H2O

MnSO4sdot5H2O ZnSO

















0 2 4 6 8 10 12 14 16 18 20 22Time (days)

minus50

minus45

minus40

minus35

minus30

minus25

minus20

minus15

minus10

Ln C

s







120583 =

ln119862119905119878minus ln1198620

119878

119905

[120583]= [dayminus1] (1)





119878= 119891(119905) (Figure 4)


ln119862119905119878= 120583 sdot 119905 + ln1198620

119878 (2)





1 0145 09735 0145 097840 0285 0987



I 0285 0987II 00439 0990III 00503 0985IV 00464 0976V 00331 0949VI 00275 0876VII 00281 0923





119903119909= 120583 sdot 119862

119905

119878


(3)





0 2 4 6 8 10 12Time (days)

000

002

004

006

008

010

r x(g

L d

ay)



0 20 40 60 80 100Time (days)

0

1

I VIIVIVIVIIIII

Ln C

s

minus5

minus4

minus3

minus2

minus1




2


3

minus

999448999471 CO2+OHminus)





337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)












120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation












0 2 4 6 8 10 12 14 16 18 20 22Time (days)

minus50

minus45

minus40

minus35

minus30

minus25

minus20

minus15

minus10

Ln C

s







120583 =

ln119862119905119878minus ln1198620

119878

119905

[120583]= [dayminus1] (1)





119878= 119891(119905) (Figure 4)


ln119862119905119878= 120583 sdot 119905 + ln1198620

119878 (2)





1 0145 09735 0145 097840 0285 0987



I 0285 0987II 00439 0990III 00503 0985IV 00464 0976V 00331 0949VI 00275 0876VII 00281 0923





119903119909= 120583 sdot 119862

119905

119878


(3)





0 2 4 6 8 10 12Time (days)

000

002

004

006

008

010

r x(g

L d

ay)



0 20 40 60 80 100Time (days)

0

1

I VIIVIVIVIIIII

Ln C

s

minus5

minus4

minus3

minus2

minus1




2


3

minus

999448999471 CO2+OHminus)





337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)












120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation












1 0145 09735 0145 097840 0285 0987



I 0285 0987II 00439 0990III 00503 0985IV 00464 0976V 00331 0949VI 00275 0876VII 00281 0923





119903119909= 120583 sdot 119862

119905

119878


(3)





0 2 4 6 8 10 12Time (days)

000

002

004

006

008

010

r x(g

L d

ay)



0 20 40 60 80 100Time (days)

0

1

I VIIVIVIVIIIII

Ln C

s

minus5

minus4

minus3

minus2

minus1




2


3

minus

999448999471 CO2+OHminus)





337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)












120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation











337202

295939

267413

296013

292740

331008

292772

287417

287450

285506

296011292779

330249

165416

154150

140008

165715

145464

154470

124196

108028104944

115086

140107

145226165616

153632

145212140745

123552

124345

115421

104980

114959

93263104754

86148

8328957797

57546

43159

6993261595

57710

54550

40000 3000 2000 1500 1000 5004000

A

B

C

(cmminus1)












120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation














120574 CCCO 120574 OH 104944115086

104754114959






3ndash) groups was












pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation













pK119886


119886




119883add =119886

1 + 10minuspH

1198701198862+ 10minus2pH

(1198701198861sdot 1198701198862) + 10

pHsdot 1198701198863

+

2 sdot 119887

1 + 10minuspH

1198701198861+ 10

pHsdot 1198701198862+ 102sdotpH

sdot 1198701198862sdot 1198701198863

+ (3 sdot 119888) (1 + 10pH

sdot 1198701198861+ 102sdotpH

sdot 1198701198861sdot 1198701198862

+ 103sdotpH

sdot 1198701198861sdot 1198701198862sdot 1198701198863)

minus1





119886





119886values (black























119898biosorbent


(5)


2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(a)

2 4 6 8 10 12 14pH

02

04

06

08

1

minus02

xad

d

(b)





119886













3] or [ndashOH])






(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










(a) (b)








119886





(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










(a) (b)

(c) (d)

(e) (f)








lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation













lowast


lowastlowast










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










0 20000 40000 60000 80000 100000 120000 140000 160000

MIXZn

CoMn

0

2

4

6

8

10

12

14Naturalna



Na(

I) co

ncen

trat

ion

on th

e bio

mas

s sur

face

SEM

()




2


4 Conclusions





Acknowledgments


References




















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation





















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation









Research Article Biosorption of Microelements by...

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