Research ArticleUsing SEM-EDX and ICP-OES to Investigate the ElementalComposition of Green Macroalga Vaucheria sessilis
Izabela Michalak1 Krzysztof Marycz2 Katarzyna BasiNska2 and Katarzyna Chojnacka1
1 Department of Chemistry Institute of Inorganic Technology and Mineral Fertilizers Wrocław University of TechnologySmoluchowskiego 25 50-372 Wrocław Poland
2Department of Environment Hygiene and Animal Welfare Electron Microscope LaboratoryEnvironmental and Life Sciences University Chełmonskiego 38c 50-630 Wrocław Poland
Correspondence should be addressed to Krzysztof Marycz krzysztofmaryczinteriapl
Received 7 April 2014 Accepted 17 June 2014 Published 11 August 2014
Academic Editor Mehmet Yakup Arica
Copyright copy 2014 Izabela Michalak 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
The biomass of Vaucheria sessilis forms algal mats in many freshwaters There is a need to find the method of algal biomass utiliza-tionVaucheria sessilis is a rich source ofmicro- andmacronutrients and can be used as a soil amendment In the paper the elementalcomposition of enriched via bioaccumulation process macroalga was investigated For this purpose two independent techniqueswere used scanning electron microscopy with an energy dispersive X-ray analytical system (SEMEDX) and inductively coupledplasma optical emission spectroscopy (ICP-OES) The biomass was exposed to two microelemental solutions with Cu(II) andZn(II) ions After two weeks of the experiment macroalga accumulated 985mg of Zn(II) ions in 1 g of dry biomass and 689mg gminus1of Cu(II) ions Micrographs performed by SEM proved that bioaccumulation occurred Metal ions were bound on the surfaceand in the interior of cells Mappings of all cations showed that in the case of the surface of biomass (biosorption) the elementsconstituted aggregations and in the case of the cross section (bioaccumulation) they were evenly distributedThe algal biomass withpermanently bound microelements can find an application in many branches of the industry (feed natural fertilizers etc)
1 Introduction
Algae can be used as fertilizers soil conditioners and bios-timulants and are a source of livestock feed [1] The interestin this category of bioproducts in modern agriculture resultsfrom the trend to search for new preparations based onnatural substances that replace (eliminate) the application ofchemicals in agriculture Macroalgae can be applied on soilsin hydroponic solutions or in the form of foliar applicationsfor plants [2ndash5] The multiplicity of applications of algalbiomass results from their natural properties Macroalgaecontain a large number of organic and mineral compounds(micro- and macronutrients) They are particularly rich inphytohormones (indoleacetic acids (IAA) commonly knownas auxins gibberellic acids cytokinins abscisic acids (ABA)and ethylene) complex organic compounds vitamins simpleand complex sugars (polysaccharides like alginates laminar-ian and carrageenans) enzymes N-containing compoundslike betaines proteins and amino acids sterols [4 6]
Additionally the biomass possesses a high ability tobioaccumulate metal ions from the aqueous solution Thiscauses the biomass to also be able to serve as a mineral feedsupplement for livestock [7 8] and bioaccumulator in bio-logical wastewater treatment processes and in bioremediationtechnologies [9 10] or as a bioindicator since it provides atime-integrated picture of the bioavailable pollutants [11]
The ability of the biomass to bind metal ions is stronglydependent on the surface The surface of the biomass waswidely examined for the presence of functional groups withthe use of various analytical techniques such as titration XPSFTIR and SEM-EDX [12] Some of the active groups (car-boxylates sulfhydryls phosphates sulfates and hydroxyls)present on the surface are negatively charged and able to bindmetal cations while amine and imidazole groups (positivelycharged) can bind negatively charged metal complexes [13]
The aim of the current paper was to evaluate the bioac-cumulation properties of macroalga to present how metalions were accumulated on the surface and in the cross
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 891928 8 pageshttpdxdoiorg1011552014891928
2 The Scientific World Journal
section of the biomass and to find the potential applicationfor the biomass enriched with metal ions Therefore twomethods were chosen scanning electron microscopy with anenergy dispersive X-ray analytical system (SEM-EDX) andinductively coupled plasma optical emission spectroscopy(ICP-OES) Scanning electron microscopy is a powerfultechnique which can be used to investigate the binding ofmetal ions to seaweed SEM allows evaluating morphologicalchanges on the surface for example changes in the cellwall composition after the metal ions binding When SEMis combined with EDX technique it can provide valuableinput in determining the distribution of various elements onthe seaweed surface [14] It should be emphasized that SEMprovides only a qualitative estimation of the surface structure
To obtain quantitative information regarding changes inelemental composition of the used biomass andor solutionan additional technique is required For solutions whereconcentration of many elements is very low highly sensitivemethods should be usedmdashfor example inductively coupledplasma optical emission spectroscopy (ICP-OES) VarianVista-MPX equipment used in the present work offers a hostof benefits which include the productivity of simultaneousmeasurement of all elements from parts-per-billion to per-cent levels simple ldquoone steprdquo analysis fast optimization andhands-free operation from full PC control of all instrumentparameters [15]
In literature both methods SEM-EDX and ICP-OES areused to investigate the bioaccumulation of metal ions bybiomass but the data is insufficient and general It is indicatedthat changes in surfacemorphology are usually related to dis-ruption of the cross-linking between the metal ion and nega-tively charged chemical groups for example carboxyl groupsin the cell wall polymers Raw seaweed usually contains highcontents of calcium and magnesium (naturally present fromseawater) in the cell wall creating a net of cross-linking[16] When the seaweed is exposed to metal ions solutionsfor example cadmium these cations replace some of thecalcium andmagnesium ions thus changing the nature of thecross-linking on the surface and resulting in morphologicalchanges Yang and Chen [17] observed surface protuberanceandmicrostructures in the raw seaweed of Sargassum sp afterbiosorption of hexavalent chromium and suggested that thismay be due to calciumand another salt crystalloid depositionThis was in agreement with their EDX analysis which showedthat calcium was a major component of the seaweed surface
In the current paper bioaccumulation properties ofmacroalga Vaucheria sessilis (Xanthophyceae) towards Cu(II)and Zn(II) ions are presented Additionally the morphologi-cal changes of the surface of the biomass after bioaccumula-tion and the arrangement of metal cations on the surface andin the cross section of the biomass were investigated Perma-nently boundmicroelement ionswith the algal biomass couldconstitute a highly bioavailable source of micronutrients forplants as well as for animals
2 Materials and Methods
21 Biomass of Vaucheria Sessilis The starting culture of ma-croalga Vaucheria sessilis was obtained from the Sammlung
von Algenkulturen Albrecht-von-Haller-Institute for PlantScience University of Gottingen and was cultivated in thelaboratory according to the procedure described by Samm-lung von Algenkulturen Gottingen (SAG) [18]
22 Bioaccumulation Process Two solutions of algalmediumwhich contained Cu(II) and Zn(II) ions were preparedin deionized water by dissolving appropriate amounts ofCuSO
4
sdot5H2
O and ZnSO4
sdot7H2
O (from POCh SA GliwicePoland) The concentration of each metal ion in everymedium solution was 125mg Lminus1 pH of the solutionswas adjusted to 7 with 01mol Lminus1 standardized solutionNaOHHCl (from POCh SA Gliwice Poland) pHmeasure-ments were conducted with a pH-meter Mettler-ToledomdashSevenMulti (Greifensee Switzerland) equipped with an elec-trode InLab413 with the compensation of temperature About25 g of wet biomass of Vaucheria sessilis was added (about014 g Lminus1 of dry biomass) into each solutionThe bioaccumu-lation process lasted for two weeks at room temperature anddaylight After this process the solution was filtered throughfilter paper The composition of the solution was analyzed byICP-OES whereas the biomass was examined by SEM-EDX
23 Analytical Methods
231 Multielemental Analysis by ICP-OES The solutionsbefore and after bioaccumulation process were analysed byinductively coupled plasma optical emission spectrometermdashVarian VISTA-MPX ICP-OES (Victoria Australia) withultrasonic nebulizer in the Chemical Laboratory of Multi-elemental Analyses at Wrocław University of Technologywhich is accredited by ILAC-MRA and Polish Centre forAccreditation (number AB 696) according to EN-ISO 17025[19] For the calibration of the apparatus the multielementalstandard (100mg Lminus1 Astasol Czech Republic) was used Inorder to prepare the calibration curve the following workingdilutions of the analytical standard were prepared 10 1050mg Lminus1 As a ldquocheck standardrdquo the standard solutionmdash10mg Lminus1 was usedThe acceptable result was assessed as 10The analytical process was controlled by the use of CertifiedReferenceMaterial HardDrinkingWater (UK)mdashmetals fromLGC Standards (LGC6010) for the analysis of solutionsValues of the measurements of the CRMs were within thecertified range The examined samples were measured inthree repeats The final result was an arithmetic mean whichdiffered less than 5
232 Scanning Electron Microscopy (SEM-EDX) NaturalVaucheria sessilis andVaucheria sessilis loaded withmicroele-ments biomass were also examined by scanning electronmicroscopy The elemental analysis and mapping wereperformed at Wrocław University of Environmental andLife Sciences (Electron Microscope Laboratory) Samples ofmacroalga were fixed in 25 of glutaraldehyde (Sigma)Then all the samples were dehydrated by ethanol (from 30till 100 concentration) In the next step macroalga wasprepared in two planes for the observation of cross sectionand its surface Samples of themacroalgaweremounted on an
The Scientific World Journal 3
Table 1 The concentration of elements in the solution before and after bioaccumulation of Zn(II) and Cu(II) ions by Vaucheria sessilis
Element
Concentration (mg Lminus1) of elements in the solutionBefore After Before After
Zn(II) ions Cu(II) ionsBioaccumulationlowast by Vaucheria sessilis
125mg Lminus1 pH of the initial solution 70 119862119878
014 g Lminus1 119879 23∘C bioaccumulation time 2 weeks
Table 2 Atomic concentration of the elements () on the surface (b) and in the cross section (b1015840) of macroalga Vaucheria sessilis afterbioaccumulation
appropriate stub and thereafter gold-sputtered (using Scan-Coat six equipmentmdashOxford) and were observed and pho-tographed with a scanning electron microscopemdashEVO LS 15(Oberkochen Germany) operating at 20 kVThemicroscopewas equipped with a BRUCKER energy dispersive X-raysystem in order to obtain a distribution of elemental compo-sition of the surface of macroalgal cell wall according to pre-viously published data [20ndash22] The X-ray spectrum of eachmacroalga loaded with a given microelement was obtained
3 Results and Discussion
31 Multielemental Analysis of the Solution before and afterBioaccumulation Process by ICP-OES Bioaccumulation is
defined as intracellular accumulation of sorbate which occursin two stages the first identical with biosorption which isquick and the subsequent which is slower and includes thetransport of sorbate inside the cells by an active transport sys-tem [9] In our previous work it was shown that the rate con-stants for biosorption of Zn(II) and Cu(II) ions by Vaucheriasessilis were much higher than for bioaccumulation [7]
Table 1 presents the composition of the solution beforeand after bioaccumulation of Zn(II) and Cu(II) ions byVaucheria sessilis The bioaccumulation capacity was deter-mined from the mass balance and by direct analysis Aftertwo weeks of the experiment the biomass of macroalgaaccumulated 985mg of Zn(II) ions in 1 g of dry biomass and689mg gminus1 of Cu(II) ions
4 The Scientific World Journal
(a) (a)
(b)
(c)
(b998400)
(c998400)
Figure 1 (a) A picture of the surface of natural biomassmdashVaucheria sessilis bottom picture of the surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) (Cmdashgreen Omdashdark blue Namdashbright blue AlmdashyellowSimdashpurple and Pmdashpink)
During bioaccumulation alkali and alkaline earth metalions were released by the biomass of Vaucheria sessilis Theorder for both macroalgae was as follows (where MAmdashmeans macroalga) MA-Zn K (466mg gminus1 dry mass) gtCa (226mg gminus1) gt Mg (749mg gminus1) gt Na (616mg gminus1)gt Ba (0129mg gminus1) MA-Cu K (622mg gminus1dry mass) gtCa (328mg gminus1) gt Mg (149mg gminus1) gt Na (804mg gminus1) gtBa (0433mg gminus1) This proves that during the first stepof bioaccumulationmdashbiosorptionmdashalkali and alkaline earthmetal ions were replaced by metal ions from the solution Itcan be assumed that in the case of Vaucheria sessilis K(I) andCa(II) cations played a dominating role in cation exchange inbiosorption process
32 Analysis of the Biomass after Bioaccumulation by ScanningElectron Microscopy (SEM-EDX) SEM-EDX pictures of livemacroalga Vaucheria sessilis were performed after two weeksof bioaccumulation of Zn(II) and Cu(II) ions This experi-ment was conducted in order to prove that bioaccumulationoccurred and metal ions were bound in the interior of thebiomass When the biomass is treated with metal ions thereis a possibility of transportation of these ions through the cellmembrane in living cells
The SEMmicrographs revealed significant changes in themorphology of examined alga Figures 1 2 and 3(a) presentthe morphological differences between natural biomass andbiomasses after accumulation Vaucheria sessilis enrichedwith Zn(II) ions exhibited medium disintegration of a
The Scientific World Journal 5
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 2 (a) A picture of the surface of Vaucheria sessilis enriched with Zn(II) ions bottom picture of the surface (b) and cross section (b1015840)of macroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Zn(II) ions on the surface (d) and in thecross section (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Znmdashred and Simdashpurple)
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
2 The Scientific World Journal
section of the biomass and to find the potential applicationfor the biomass enriched with metal ions Therefore twomethods were chosen scanning electron microscopy with anenergy dispersive X-ray analytical system (SEM-EDX) andinductively coupled plasma optical emission spectroscopy(ICP-OES) Scanning electron microscopy is a powerfultechnique which can be used to investigate the binding ofmetal ions to seaweed SEM allows evaluating morphologicalchanges on the surface for example changes in the cellwall composition after the metal ions binding When SEMis combined with EDX technique it can provide valuableinput in determining the distribution of various elements onthe seaweed surface [14] It should be emphasized that SEMprovides only a qualitative estimation of the surface structure
To obtain quantitative information regarding changes inelemental composition of the used biomass andor solutionan additional technique is required For solutions whereconcentration of many elements is very low highly sensitivemethods should be usedmdashfor example inductively coupledplasma optical emission spectroscopy (ICP-OES) VarianVista-MPX equipment used in the present work offers a hostof benefits which include the productivity of simultaneousmeasurement of all elements from parts-per-billion to per-cent levels simple ldquoone steprdquo analysis fast optimization andhands-free operation from full PC control of all instrumentparameters [15]
In literature both methods SEM-EDX and ICP-OES areused to investigate the bioaccumulation of metal ions bybiomass but the data is insufficient and general It is indicatedthat changes in surfacemorphology are usually related to dis-ruption of the cross-linking between the metal ion and nega-tively charged chemical groups for example carboxyl groupsin the cell wall polymers Raw seaweed usually contains highcontents of calcium and magnesium (naturally present fromseawater) in the cell wall creating a net of cross-linking[16] When the seaweed is exposed to metal ions solutionsfor example cadmium these cations replace some of thecalcium andmagnesium ions thus changing the nature of thecross-linking on the surface and resulting in morphologicalchanges Yang and Chen [17] observed surface protuberanceandmicrostructures in the raw seaweed of Sargassum sp afterbiosorption of hexavalent chromium and suggested that thismay be due to calciumand another salt crystalloid depositionThis was in agreement with their EDX analysis which showedthat calcium was a major component of the seaweed surface
In the current paper bioaccumulation properties ofmacroalga Vaucheria sessilis (Xanthophyceae) towards Cu(II)and Zn(II) ions are presented Additionally the morphologi-cal changes of the surface of the biomass after bioaccumula-tion and the arrangement of metal cations on the surface andin the cross section of the biomass were investigated Perma-nently boundmicroelement ionswith the algal biomass couldconstitute a highly bioavailable source of micronutrients forplants as well as for animals
2 Materials and Methods
21 Biomass of Vaucheria Sessilis The starting culture of ma-croalga Vaucheria sessilis was obtained from the Sammlung
von Algenkulturen Albrecht-von-Haller-Institute for PlantScience University of Gottingen and was cultivated in thelaboratory according to the procedure described by Samm-lung von Algenkulturen Gottingen (SAG) [18]
22 Bioaccumulation Process Two solutions of algalmediumwhich contained Cu(II) and Zn(II) ions were preparedin deionized water by dissolving appropriate amounts ofCuSO
4
sdot5H2
O and ZnSO4
sdot7H2
O (from POCh SA GliwicePoland) The concentration of each metal ion in everymedium solution was 125mg Lminus1 pH of the solutionswas adjusted to 7 with 01mol Lminus1 standardized solutionNaOHHCl (from POCh SA Gliwice Poland) pHmeasure-ments were conducted with a pH-meter Mettler-ToledomdashSevenMulti (Greifensee Switzerland) equipped with an elec-trode InLab413 with the compensation of temperature About25 g of wet biomass of Vaucheria sessilis was added (about014 g Lminus1 of dry biomass) into each solutionThe bioaccumu-lation process lasted for two weeks at room temperature anddaylight After this process the solution was filtered throughfilter paper The composition of the solution was analyzed byICP-OES whereas the biomass was examined by SEM-EDX
23 Analytical Methods
231 Multielemental Analysis by ICP-OES The solutionsbefore and after bioaccumulation process were analysed byinductively coupled plasma optical emission spectrometermdashVarian VISTA-MPX ICP-OES (Victoria Australia) withultrasonic nebulizer in the Chemical Laboratory of Multi-elemental Analyses at Wrocław University of Technologywhich is accredited by ILAC-MRA and Polish Centre forAccreditation (number AB 696) according to EN-ISO 17025[19] For the calibration of the apparatus the multielementalstandard (100mg Lminus1 Astasol Czech Republic) was used Inorder to prepare the calibration curve the following workingdilutions of the analytical standard were prepared 10 1050mg Lminus1 As a ldquocheck standardrdquo the standard solutionmdash10mg Lminus1 was usedThe acceptable result was assessed as 10The analytical process was controlled by the use of CertifiedReferenceMaterial HardDrinkingWater (UK)mdashmetals fromLGC Standards (LGC6010) for the analysis of solutionsValues of the measurements of the CRMs were within thecertified range The examined samples were measured inthree repeats The final result was an arithmetic mean whichdiffered less than 5
232 Scanning Electron Microscopy (SEM-EDX) NaturalVaucheria sessilis andVaucheria sessilis loaded withmicroele-ments biomass were also examined by scanning electronmicroscopy The elemental analysis and mapping wereperformed at Wrocław University of Environmental andLife Sciences (Electron Microscope Laboratory) Samples ofmacroalga were fixed in 25 of glutaraldehyde (Sigma)Then all the samples were dehydrated by ethanol (from 30till 100 concentration) In the next step macroalga wasprepared in two planes for the observation of cross sectionand its surface Samples of themacroalgaweremounted on an
The Scientific World Journal 3
Table 1 The concentration of elements in the solution before and after bioaccumulation of Zn(II) and Cu(II) ions by Vaucheria sessilis
Element
Concentration (mg Lminus1) of elements in the solutionBefore After Before After
Zn(II) ions Cu(II) ionsBioaccumulationlowast by Vaucheria sessilis
125mg Lminus1 pH of the initial solution 70 119862119878
014 g Lminus1 119879 23∘C bioaccumulation time 2 weeks
Table 2 Atomic concentration of the elements () on the surface (b) and in the cross section (b1015840) of macroalga Vaucheria sessilis afterbioaccumulation
appropriate stub and thereafter gold-sputtered (using Scan-Coat six equipmentmdashOxford) and were observed and pho-tographed with a scanning electron microscopemdashEVO LS 15(Oberkochen Germany) operating at 20 kVThemicroscopewas equipped with a BRUCKER energy dispersive X-raysystem in order to obtain a distribution of elemental compo-sition of the surface of macroalgal cell wall according to pre-viously published data [20ndash22] The X-ray spectrum of eachmacroalga loaded with a given microelement was obtained
3 Results and Discussion
31 Multielemental Analysis of the Solution before and afterBioaccumulation Process by ICP-OES Bioaccumulation is
defined as intracellular accumulation of sorbate which occursin two stages the first identical with biosorption which isquick and the subsequent which is slower and includes thetransport of sorbate inside the cells by an active transport sys-tem [9] In our previous work it was shown that the rate con-stants for biosorption of Zn(II) and Cu(II) ions by Vaucheriasessilis were much higher than for bioaccumulation [7]
Table 1 presents the composition of the solution beforeand after bioaccumulation of Zn(II) and Cu(II) ions byVaucheria sessilis The bioaccumulation capacity was deter-mined from the mass balance and by direct analysis Aftertwo weeks of the experiment the biomass of macroalgaaccumulated 985mg of Zn(II) ions in 1 g of dry biomass and689mg gminus1 of Cu(II) ions
4 The Scientific World Journal
(a) (a)
(b)
(c)
(b998400)
(c998400)
Figure 1 (a) A picture of the surface of natural biomassmdashVaucheria sessilis bottom picture of the surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) (Cmdashgreen Omdashdark blue Namdashbright blue AlmdashyellowSimdashpurple and Pmdashpink)
During bioaccumulation alkali and alkaline earth metalions were released by the biomass of Vaucheria sessilis Theorder for both macroalgae was as follows (where MAmdashmeans macroalga) MA-Zn K (466mg gminus1 dry mass) gtCa (226mg gminus1) gt Mg (749mg gminus1) gt Na (616mg gminus1)gt Ba (0129mg gminus1) MA-Cu K (622mg gminus1dry mass) gtCa (328mg gminus1) gt Mg (149mg gminus1) gt Na (804mg gminus1) gtBa (0433mg gminus1) This proves that during the first stepof bioaccumulationmdashbiosorptionmdashalkali and alkaline earthmetal ions were replaced by metal ions from the solution Itcan be assumed that in the case of Vaucheria sessilis K(I) andCa(II) cations played a dominating role in cation exchange inbiosorption process
32 Analysis of the Biomass after Bioaccumulation by ScanningElectron Microscopy (SEM-EDX) SEM-EDX pictures of livemacroalga Vaucheria sessilis were performed after two weeksof bioaccumulation of Zn(II) and Cu(II) ions This experi-ment was conducted in order to prove that bioaccumulationoccurred and metal ions were bound in the interior of thebiomass When the biomass is treated with metal ions thereis a possibility of transportation of these ions through the cellmembrane in living cells
The SEMmicrographs revealed significant changes in themorphology of examined alga Figures 1 2 and 3(a) presentthe morphological differences between natural biomass andbiomasses after accumulation Vaucheria sessilis enrichedwith Zn(II) ions exhibited medium disintegration of a
The Scientific World Journal 5
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 2 (a) A picture of the surface of Vaucheria sessilis enriched with Zn(II) ions bottom picture of the surface (b) and cross section (b1015840)of macroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Zn(II) ions on the surface (d) and in thecross section (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Znmdashred and Simdashpurple)
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
The Scientific World Journal 3
Table 1 The concentration of elements in the solution before and after bioaccumulation of Zn(II) and Cu(II) ions by Vaucheria sessilis
Element
Concentration (mg Lminus1) of elements in the solutionBefore After Before After
Zn(II) ions Cu(II) ionsBioaccumulationlowast by Vaucheria sessilis
125mg Lminus1 pH of the initial solution 70 119862119878
014 g Lminus1 119879 23∘C bioaccumulation time 2 weeks
Table 2 Atomic concentration of the elements () on the surface (b) and in the cross section (b1015840) of macroalga Vaucheria sessilis afterbioaccumulation
appropriate stub and thereafter gold-sputtered (using Scan-Coat six equipmentmdashOxford) and were observed and pho-tographed with a scanning electron microscopemdashEVO LS 15(Oberkochen Germany) operating at 20 kVThemicroscopewas equipped with a BRUCKER energy dispersive X-raysystem in order to obtain a distribution of elemental compo-sition of the surface of macroalgal cell wall according to pre-viously published data [20ndash22] The X-ray spectrum of eachmacroalga loaded with a given microelement was obtained
3 Results and Discussion
31 Multielemental Analysis of the Solution before and afterBioaccumulation Process by ICP-OES Bioaccumulation is
defined as intracellular accumulation of sorbate which occursin two stages the first identical with biosorption which isquick and the subsequent which is slower and includes thetransport of sorbate inside the cells by an active transport sys-tem [9] In our previous work it was shown that the rate con-stants for biosorption of Zn(II) and Cu(II) ions by Vaucheriasessilis were much higher than for bioaccumulation [7]
Table 1 presents the composition of the solution beforeand after bioaccumulation of Zn(II) and Cu(II) ions byVaucheria sessilis The bioaccumulation capacity was deter-mined from the mass balance and by direct analysis Aftertwo weeks of the experiment the biomass of macroalgaaccumulated 985mg of Zn(II) ions in 1 g of dry biomass and689mg gminus1 of Cu(II) ions
4 The Scientific World Journal
(a) (a)
(b)
(c)
(b998400)
(c998400)
Figure 1 (a) A picture of the surface of natural biomassmdashVaucheria sessilis bottom picture of the surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) (Cmdashgreen Omdashdark blue Namdashbright blue AlmdashyellowSimdashpurple and Pmdashpink)
During bioaccumulation alkali and alkaline earth metalions were released by the biomass of Vaucheria sessilis Theorder for both macroalgae was as follows (where MAmdashmeans macroalga) MA-Zn K (466mg gminus1 dry mass) gtCa (226mg gminus1) gt Mg (749mg gminus1) gt Na (616mg gminus1)gt Ba (0129mg gminus1) MA-Cu K (622mg gminus1dry mass) gtCa (328mg gminus1) gt Mg (149mg gminus1) gt Na (804mg gminus1) gtBa (0433mg gminus1) This proves that during the first stepof bioaccumulationmdashbiosorptionmdashalkali and alkaline earthmetal ions were replaced by metal ions from the solution Itcan be assumed that in the case of Vaucheria sessilis K(I) andCa(II) cations played a dominating role in cation exchange inbiosorption process
32 Analysis of the Biomass after Bioaccumulation by ScanningElectron Microscopy (SEM-EDX) SEM-EDX pictures of livemacroalga Vaucheria sessilis were performed after two weeksof bioaccumulation of Zn(II) and Cu(II) ions This experi-ment was conducted in order to prove that bioaccumulationoccurred and metal ions were bound in the interior of thebiomass When the biomass is treated with metal ions thereis a possibility of transportation of these ions through the cellmembrane in living cells
The SEMmicrographs revealed significant changes in themorphology of examined alga Figures 1 2 and 3(a) presentthe morphological differences between natural biomass andbiomasses after accumulation Vaucheria sessilis enrichedwith Zn(II) ions exhibited medium disintegration of a
The Scientific World Journal 5
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 2 (a) A picture of the surface of Vaucheria sessilis enriched with Zn(II) ions bottom picture of the surface (b) and cross section (b1015840)of macroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Zn(II) ions on the surface (d) and in thecross section (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Znmdashred and Simdashpurple)
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
4 The Scientific World Journal
(a) (a)
(b)
(c)
(b998400)
(c998400)
Figure 1 (a) A picture of the surface of natural biomassmdashVaucheria sessilis bottom picture of the surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) (Cmdashgreen Omdashdark blue Namdashbright blue AlmdashyellowSimdashpurple and Pmdashpink)
During bioaccumulation alkali and alkaline earth metalions were released by the biomass of Vaucheria sessilis Theorder for both macroalgae was as follows (where MAmdashmeans macroalga) MA-Zn K (466mg gminus1 dry mass) gtCa (226mg gminus1) gt Mg (749mg gminus1) gt Na (616mg gminus1)gt Ba (0129mg gminus1) MA-Cu K (622mg gminus1dry mass) gtCa (328mg gminus1) gt Mg (149mg gminus1) gt Na (804mg gminus1) gtBa (0433mg gminus1) This proves that during the first stepof bioaccumulationmdashbiosorptionmdashalkali and alkaline earthmetal ions were replaced by metal ions from the solution Itcan be assumed that in the case of Vaucheria sessilis K(I) andCa(II) cations played a dominating role in cation exchange inbiosorption process
32 Analysis of the Biomass after Bioaccumulation by ScanningElectron Microscopy (SEM-EDX) SEM-EDX pictures of livemacroalga Vaucheria sessilis were performed after two weeksof bioaccumulation of Zn(II) and Cu(II) ions This experi-ment was conducted in order to prove that bioaccumulationoccurred and metal ions were bound in the interior of thebiomass When the biomass is treated with metal ions thereis a possibility of transportation of these ions through the cellmembrane in living cells
The SEMmicrographs revealed significant changes in themorphology of examined alga Figures 1 2 and 3(a) presentthe morphological differences between natural biomass andbiomasses after accumulation Vaucheria sessilis enrichedwith Zn(II) ions exhibited medium disintegration of a
The Scientific World Journal 5
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 2 (a) A picture of the surface of Vaucheria sessilis enriched with Zn(II) ions bottom picture of the surface (b) and cross section (b1015840)of macroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Zn(II) ions on the surface (d) and in thecross section (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Znmdashred and Simdashpurple)
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
The Scientific World Journal 5
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 2 (a) A picture of the surface of Vaucheria sessilis enriched with Zn(II) ions bottom picture of the surface (b) and cross section (b1015840)of macroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Zn(II) ions on the surface (d) and in thecross section (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Znmdashred and Simdashpurple)
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
6 The Scientific World Journal
(a) (a)
(b)
(c)
(d)
(b998400)
(c998400)
(d998400)
Figure 3 (a) A picture of the surface of Vaucheria sessilis enriched with Cu(II) ions bottom picture of surface (b) and cross section (b1015840) ofmacroalga mapping of all elements on the surface (c) and in the cross section (c1015840) mapping of Cu(II) ions on the surface (d) and in the crosssection (d1015840) (Cmdashgreen Omdashdark blue Namdashbright blue Almdashyellow Pmdashpink Cumdashred and Simdashpurple)
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
The Scientific World Journal 7
meshwork structure The dysfunction of typical biomassmorphology was observed in Vaucheria sessilis enriched withCu(II) ions Scanning microscope observations indicated aloss of filaments thickness additionally a high occurrence ofreproductive structures was noticed Moreover the biomassenriched with Zn(II) and Cu(II) ions revealed visible defor-mation of the cell wall
Additionally the chemical distribution analysis of allinvestigated elements confirmed that the bioaccumulationof metal ions took place inside the cell The distribution ofCu(II) ions on the surface of the biomass was very denseand regularly arranged however cross sections revealedheterogeneous locations Analysis of Zn(II) ions distributionshowed a slide aggregation of the mentioned element onthe surface of the biomass whereas a highly aggregatedarrangement on the cross section was visible
Table 2 presents atomic concentration of the elements ()on the surface and in the cross section ofmacroalgaVaucheriasessilis after bioaccumulationThe changes in atomic concen-tration on the surface of the biomass after bioaccumulation(b) concerned mainly the increase of carbon concentration(except of MA-Cu) and decrease of oxygen (except of MA-Cu) sulphur calcium and sodium (except of MA-Cu)Theseresults stay in agreement with data obtained by ICP-OESDecrease of the content of Ca(II) and Na(I) ions on thesurface of the biomass (increase in the solution) was due toion exchange with metal ions from the solutionmdashZn(II) andCu(II) Atomic concentration of carbon in the cross sectionofMA-Cu andMA-Znwas lower than in the natural biomassA different observation concerned the atomic concentrationof oxygen It was also noticed that atomic concentration ofsulphur and calcium decreased and sodium increased
4 Conclusions
Vaucheria sessilis is a widespread alga but has not beenthoroughly studied yet The performed experiments showedthat it can act as a good bioaccumulator of metal ionsHigher bioaccumulation was more observed for Zn(II) thanfor Cu(II) ions
The combination of two advancedmethods ICP-OES andSEM-EDX allowed to characterize the process of bioaccu-mulation of metal ions by Vaucheria sessilis The scanningelectron microscopy technique allowed us to understand theinteraction between metal ions and the biomass In the caseof the surface (biosorption) the elements revealed aggregatedistribution in contrast to the cross section (bioaccumu-lation) where even arrangements were noticed The SEManalysis also revealed significant changes in the morphologyof the investigated algae ICP-OES analysis of the solutionbefore and after bioaccumulation showed that metal ionsfrom aqueous solution were bound to the biomass Bioac-cumulation properties of algae may be used in the industryfor example in the production of natural feed supplementsAdditionally the possibility of algae enrichment in chosenelements may find an application in production of naturalfertilizers The enriched biomass can serve as a rich sourceof highly bioavailable and nontoxic forms of microelementsboth for animals as well as for plants
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project is financed in the framework of Grant enti-tled Biologically Active Compounds in Extracts from BalticSeaweeds (201205DST503379) attributed to The NationalScience Centre and Grant entitled Innovative Technology ofSeaweed ExtractsmdashComponents of Fertilizers Feed andCos-metics (PBS1A122012) attributed to The National Centrefor Research and Development in Poland
References
[1] S-K Kim Handbook of Marine Macroalgae Biotechnology andApplied Phycology John Wiley amp Sons 2012
[2] WKhanU P Rayirath S Subramanian et al ldquoSeaweed extractsas biostimulants of plant growth and developmentrdquo Journal ofPlant Growth Regulation vol 28 no 4 pp 386ndash399 2009
[3] J S Craigie ldquoSeaweed extract stimuli in plant science andagriculturerdquo Journal of Applied Phycology vol 23 no 3 pp 371ndash393 2011
[4] P du Jardin ldquoThe science of plant biostimulantsmdasha biblio-graphic analysisrdquo Final report Contract No 30-CE045551500-96 ldquoAd Hoc Study on Bio-Stimulants Productsrdquo EuropeanCommission 2012
[5] F N Verkleij ldquoSeaweed extracts in agriculture and horticulturea reviewrdquo Biological Agriculture amp Horticulture vol 8 no 4 pp309ndash324 1992
[6] B Hamza and A Suggars ldquoBiostimulants myths and realitiesrdquoTurfGrass Trends vol 8 pp 6ndash10 2001
[7] I Michalak and K Chojnacka ldquoPorownanie procesu biosor-pcji i bioakumulacji jonow mikroelementow przez makroalgęVaucheria sessilisrdquo Biotechnologia vol 1 pp 125ndash139 2010(Polish)
[8] K Chojnacka ldquoBiosorption and bioaccumulation of microele-ments by Riccia fluitans in single and multi-metal systemrdquoBioresource Technology vol 98 no 15 pp 2919ndash2925 2007
[9] K Chojnacka ldquoBiosorption and bioaccumulationmdashtheprospects for practical applicationsrdquoEnvironment Internationalvol 36 no 3 pp 299ndash307 2010
[10] A Rehman F R Shakoori and A R Shakoori ldquoUptake ofheavy metals by a ciliate Tachysoma pellionella isolated fromindustrial effluents and its potential use in bioremediation oftoxic wastewaterrdquo Bulletin of Environmental Contamination andToxicology vol 77 no 3 pp 469ndash476 2006
[11] D J H Phillips ldquoThe use of biological indicator organismsto monitor trace metal pollution in marine and estuarineenvironments a reviewrdquo Environmental Pollution vol 13 no 4pp 281ndash317 1977
[12] 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
[13] R H Crist K Oberholser N Shank and M Nguyen ldquoNatureof bonding between metallic ions and algal cell wallsrdquo Environ-mental Science and Technology vol 15 no 10 pp 1212ndash1217 1981
[14] M M Figueira B Volesky and H J Mathieu ldquoInstrumen-tal analysis study of iron species biosorption by Sargassum
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
8 The Scientific World Journal
biomassrdquo Environmental Science and Technology vol 33 no 11pp 1840ndash1846 1999
[15] H Gorecka K Chojnacka and H Gorecki ldquoThe applicationof ICP-MS and ICP-OES in determination of micronutrients inwood ashes used as soil conditionersrdquo Talanta vol 70 no 5 pp950ndash956 2006
[16] E Percival and R H McDowell Chemistry and Enzymologyof Marine Algal Polysaccharides Academic Press London UK1967
[17] L Yang and J P Chen ldquoBiosorption of hexavalent chromiumonto raw and chemically modified Sargassum sprdquo BioresourceTechnology vol 99 no 2 pp 297ndash307 2008
[18] httpwwwuni-goettingendeen184982htmlhtmlsaghtml[19] K Chojnacka ldquoThe application of multielemental analysis in
the elaboration of technology of mineral feed additives basedon Lemna minor biomassrdquo Talanta vol 70 no 5 pp 966ndash9722006
[20] A Witek-Krowiak D Podstawczyk K Chojnacka ADawiec and K Marycz ldquoModelling and optimization ofchromium(III)biosorption on soybeanmealrdquo Central EuropeanJournal of Chemistry vol 11 no 9 pp 1505ndash1517 2013
[21] M Kania D Mikołajewska K Marycz and M KobielarzldquoEffect of diet on mechanical properties of horsersquos hairrdquo Acta ofBioengineering and Biomechanics vol 11 no 3 pp 53ndash57 2009
[22] K Kalinski K Marycz J Czogala E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgenmicroanalysis (SEM-EDS) in canine urolithiasisrdquoJournal of Electron Microscopy vol 61 pp 47ndash55 2012
