Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 741906 9 pageshttpdxdoiorg1011552013741906

Research ArticleHPLC-DAD-ESIMS Identification of Light Harvesting and LightScreening Pigments in the Lake Sediments at Edmonson Point

Rita Giovannetti Leila Alibabaei Marco Zannotti Stefano Ferraro and Laura Petetta

Chemistry Section School of Science and Technology University of Camerino Via S Agostino 1 62032 Camerino Italy

Correspondence should be addressed to Rita Giovannetti ritagiovannettiunicamit

Received 29 August 2013 Accepted 12 October 2013

Academic Editors J Gebler and A Welle

Copyright copy 2013 Rita Giovannetti 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 composition of sedimentary pigments in the Antarctic lake at Edmonson Point has been investigated and compared withthe aim to provide a useful analytical method for pigments separation and identification providing reference data for futureassessment of possible changes in environmental conditions Reversed phase high performance liquid chromatography (HPLC)with electrospray-mass spectrometry (ESI-MS) detection and diode array detection (DAD) has been used to identify light screeningand light harvesting pigments The results are discussed in terms of local environmental conditions

1 Introduction

Depletion of stratospheric ozone since the mid-1970s has ledto significant increases in ultraviolet B (UVB) irradiation overAntarctica There is currently much interest in the survivalstrategies of microorganisms in hostile environments In theremoteAntarctic continent ice-free desert regions are subjectto extreme environmental stress that provides conditions forthe growth of lichens endoliths and cyanobacteria [1 2]The increasingly intense flux of UVB radiation with shortwavelength reaching the Antarctic terrestrial surface wherecyanobacteria grow undermetabolically constrained thermaland hydrological conditions has prompted the attention ofgeologists biologists biochemists and structural chemists todraw analogies with conditions on ancient planetary surfaces[3] such as that of Mars

During summer in the areas of Antarctica small streamsand lakes fed by glacial or snow melt water are presentThis region provides many chemical data for understandingglobal processes such as climate change and dispersion ofpersistent anthropogenic pollutants because it is a symbol fora pristine environment [4] Antarctic soils were formed inan environment described as a cold desert and characterizedby extremely low temperatures which rise above 0∘C for onlybrief periods during the short Antarctic summer so chemicalweathering processes proceed very slowly Terra Nova Bay

is part of the Ross Sea and is relatively ice-free in summerand there are more than a hundred small lakes and pondsin the area Most of these small lakes are located along thecoast and receive their sediments andwater supply during thespring and summer warmer periods as melted snow becauseice acts as sediment trap [5] In the area as in the rest ofAntarctica the soils develop and vary according to the natureof the bedrock time climate and other ecological factors thatcontrol weathering

Photosynthesis is the base of life on the earth and thedevelopment of the vast range of simple organisms existingtoday can often be traced back to the earliest geological timesMost life forms depend directly or indirectly on the syntheticprocesses which harvest the sunrsquos energy utilising a range ofpigments such as chlorophylls and carotenoids which notonly determine the colour of each organism but often alsoserve a protective role against the adverse effects of ultravioletradiation other photoprotective compounds are synthesizedas reply to high solar irradiance

The preservation of pigments in the sediments dependson their water solubility and physicochemical and biologicalcharacteristics as well as on the chemical and physicalqualities of the sites In comparison to other organic materialpigments are labile compounds and the individual stabilityof the pigments varies Pigments are degraded in the aquatic

2 The Scientific World Journal

environment by chemical photochemical and biologicalprocesses Chlorophylls contain nitrogen and are thereforemore prone to being salvaged during senescence and biologi-cal breakdown than the carotenoids Chlorophyll degradationpathways include allomerization (oxidation) demetallation(loss of the Mg) and dephytylation (loss of phytyl chain)with the five-ring phorbin being relatively stable [6] Most ofthese breakdown products are detectable by regular pigmentanalysis Carotenoids are found mostly in transform and areinherently more stable than chlorophylls However unlikechlorophylls they are often broken down to colorless com-pounds by destruction of the long chain of alternating doublebonds that cannot be detected by regular pigment analysismethods [7 8] Knowledge of the type and variability inphotosynthetic pigment composition versus lake conditionsis very important for the detailed information about thehistory of this with reference to biotic communities [9] and ofenvironmental factors (eg UV radiation or anoxia) involvedin pigment transformations [10 11]

High performance liquid chromatography (HPLC) is areliable method for analysing algal pigments in sediments[10]

In order to better understand the environmental pro-cesses occurring in the Antarctic ecosystem sediment sam-ples were collected from the lake at Edmonson Point inthe Victoria Land region during an Italian expedition inthe period 2004ndash2007 In this study these sediment sampleshave been analyzed to determine the light harvesting andlight screening pigments that are very important in Antarcticecosystem because protect the life permitting the growthand survival of any organisms under low temperature andcontinuous light regime of summer

2 Material and Methods

21 Site Description The climate of central Victoria Land isa rigid Antarctic climate the monthly mean air temperatureranges between minus259∘C (August) and minus01∘C (January)There is complete darkness betweenMay 5th andAugust 10thand 24 h of light between October 10th and February 7thwhile December and January show the maximum monthlymean radiation (320 and 300Wmminus2 resp)

The Italian Base is situated in Northern Foothills and isa zone of rounded off hills and covered from local little onesglacier and accumulates of snow are present near obstacles Inthe outskirtses of the Base find little ones lakes or pools thatin summer are opened while in the rest of the area NorthernFoothills the present lakes always remain covered from theice

Edmonson Point is located in Wood Bay Victoria LandRoss Sea at the foot of the eastern slopes of Mount Mel-bourne about 50 km NE of Mario Zucchelli Terra Nova BayStation (Italy) (Figure 1)

At almost two km2 Edmonson Point is one of thelargest of the few low-lying coastal ice-free areas in NorthernVictoria Land Edmonson Point was first identified in the1980s as a site that could merit special protection principallybecause of its rich vegetation Italy established a station in

close proximity at Terra Nova Bay in 1986-87 with increasedresearch interest A variety of scientific projects have beeninitiated many of which are long-term with internationalinvolvement Edmonson Point comprises hills knolls andmoraines of volcanic materials separated by small valleyswith streams ponds and larger lakes The presence of acolony of Adelie penguins nesting skuas and some aban-doned penguin rookeries determines a patchy distributionof nutrients in terrestrial and freshwater ecosystems Theground is generally dark in colour which encourages rapidsnow-melt in spring In summer most of the area is dry andthe ground is covered by salt encrustations except below latesnow beds near stream and pool margins and in flood flatsMost streams and pools are transient However some long-lying snowdeposits and the inland glaciermargins can supplywater to the larger streams and lakes from the beginningof December to the end of January Some shallow lakes arefree of ice cover for two or three months in summer whiledeeper lakes are permanently frozen and are supplied bygroundwater or by surface waters

22 Sampling Sediment samples were collected from Lake15A at Edmondson Point during an Italian Expedition in2004ndash2007 All samples were collected with plastic toolsand were maintained at minus24∘C during all stages of storageand transport to the Italian laboratory The samples werepreserved frozen and in the dark and prior to analysis theywere freeze-dried (lyophilized) and well mixed under highvacuum (lt01 Pa)

23 Chemicals All solvents used were HPLC grade (Sigma-Aldrich) NH

4Ac and MgCO

3were analytical reagent grade

(Sigma-Aldrich) Milli-Q water was used for solution prepa-ration

24 Pigment Extraction All the laboratory procedures werecarried out in subdued light to minimize pigments degra-dation The extraction of 20 g freeze-dried of sediment wasperformed in darkened flasks to prevent photo-oxidation andthe breakdown of labile pigments extraction was carriedout with MeOHacetone (10 90 v v) and a small amountof magnesium carbonate in order to prevent the accidentalformation of chlorophyll metabolites this procedure wasrepeated until the supernatant was colourless The extractswere filtered and concentrated using a low pressure rotaryvapour at 25∘C and a dry N

2flow The concentrated extracts

were filtered (045 120583m) before HPLC analysis The driedextract were stored at minus4∘C in dark

25 Instrumentation Visible spectrum of total extracts wasobtained with a Hewlett-Packard 8452A diode array spec-trophotometer with a 1 cm quartz cell well-stoppered scan-ning from 200 to 800 nm

An HPLC Agilent 1200 Series chromatograph equippedwith autoinjector and photodiode array detector (190ndash950 nm) was used to separate and identify individual com-pounds

The Scientific World Journal 3

EdmonsonPoint

WoodBay

CoastlineIce-free groundProtected area boundaryRestricted zoneHelicopter approach zoneHelicopter landing site

A

BB

C

Map 1EdmonsonPoint

CapeWashington

Mount Melbourne (2732m)

ASPA No 118Summit of Mount Melbourne

ASPANo118Summit of Mount Melbourne

Aviator GlacierTongue

Mario ZucchelliStation (Italy)

Mario Zucchellistation (Italy)

GondwanaStation (Germany)GondwanaStation (Germany)

Wood

Bay

Wood

Bay

ASPAno161Terra Nova Bay

74degS

Inset 2

MAP 3

PERMIT

ASPA no 165entry by

permit

60degS

70deg S

80degS

90degW

0deg

RossSea

Rosssea

WeddellSea

WeddellSea

LOCATION OFWOOD BAY (INSET 2)

180deg

Colline Ippolito(Ippolito Hills)CollineIppolito(IppolitoHills)

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

74∘22

9984000998400

998400S

74∘22

9984000998400

998400S

74∘21

9984000998400

998400S

74∘21

9984000998400

998400S

74∘20

9984000998400

998400S

74∘20

9984000998400

998400S

74∘19

9984000998400

998400S

74∘19

9984000998400

998400S

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

Baia Siena(Siena bay)

MAP 2 H

H

Lower glacier slopes of Mount Melbourne

Inset 1 location of wood Bay in Antarctica

Inset 2 location of map 1 and Edmonson Point

Victor

ia La

nd

Aviator GlacierTongue

Map 1EdmonsonPoint

Mount Melbourne (2732 m)

60 ∘S

70 ∘S

80 ∘S

180∘

90∘W 90∘E

location ofwood bay (inset 2)

0∘

164∘E 165∘E 165∘E 75 ∘S

0 500 1000

July 2006PNRADSAERA

N

Map 1 Edmonson Point ASPA no 165Wood Bay Victoria Land Ross sea

Ross

sea

Terra N

ova Bay

HC

74∘S

H

N

0 10 20

(m)

(km)

Leptonychotes weddellii

Figure 1 Location map of Edmonson Point in the Victoria Land

Separation was performed from sample aliquots of 25120583Lthat were injected into reversed-phase columns LiChroCART(250 times 4mm id packed with LiChrospher 100 RP-18 (5 120583mspherical particles)) and an ODS-hypersil (C

18) precolumn

(5 120583m 20 times 40mm Hewlett-Packard)ForMSmeasurements the chromatographwas coupled to

a 1100MSDHewlett PackardMSD used under ESI conditionswith both positive and negative ion monitoring

26 HPLC Separation and Analysis The best conditions forHPLC were obtained using gradient elution with MeOH (A)and MeCN (B) as follows 0min 70 A 15min 80 A20min 90 A flow rate 1mLmin HPLC was carried outat a controlled temperature of 28∘C

In order to obtain the best conditions for ESI-MSthe chromatograms were measured with three fragmentorvoltage values (30 70 and 150 eV) in negative and positive

4 The Scientific World Journal

00

02

04

06

08

10

12

14

320 440 560 680 800Wavelength (nm)

Abso

rban

ce

Figure 2 The absorbance spectra of the MeOHacetone (10 90 v v) extract of sediment lake at Edmonson Point

2 4 6 8 10 12 14 16 18 200

20406080

100120

5

1

2

3

4

768

10981

2(a)

(b)

mAU

(min)

Figure 3 DAD chromatograms recorded at 660 nm (a) and 476 (b) of sediment extract fromEdmondson Point lake obtained using gradientelution with MeOH-MeCN (0min 70 30 15min 80 20 20min 90 10) peak numbers correspond to pigments listed in Table 1

ion mode All measurements demonstrated that the responsewas better at 70 eV in the negative ion mode and at 150 eV inthe positive ion mode

Peaks were identified by their absorption spectra at theirmaximumwavelength and characteristicMS fragmentationsTheDAD detector was set at the wavelength of 476 or 650 nmfor analysis of carotenoids and chlorophylls respectivelyPigment quantification was performed by comparing theHPLC peak areas with those of standards (chlorophyll-achlorophyll-b) from DHI Water amp Environment (HorsholmDenmark) and (canthaxanthin fucoxanthin) from the Inter-national Agency for 14C Determination VKI in Hoersholmand using published extinction coefficients [12ndash14] whenstandardswere unavailableThe specific extinction coefficientused for scytonemins was 1126 L gminus1 cmminus1 [15]

3 Results and Discussion

The absorbance spectra of the extract of sediment is reportedin Figure 2 from which the presence of chlorophyll andphaeopigments for the absorption in the range between 380and 440 nm and at about 660 nm can be deduced Theshoulders at 450ndash500 nm indicate instead the presence ofcarotenoids

The results of HPLC analysis of sediments of Lake 15A atEdmonson Point are reported in Figure 3The chromatogramobtained at 650 nm (Figure 3(a)) showed the presence of fivepigments while in that obtained at 476 nm (Figure 3(b))

eight pigmentswere observed ESI-MS analysis of themixturecombined with on-line DAD electronic spectra allowedidentification of the major components and their percentagecomposition (Table 1)

The two components eluting at 47 and 31min wereassigned as scytonemin and its reduced form [10] (2 1) from[M+H]+ at 119898119911 5455 and 5477 [M+Na]+ at 119898119911 5675 and5695 and [MminusH]minus at 119898119911 5435 and 5455 respectively andfrom the UV-vis spectra (Figure 4) that show the absorptionover relatively wide range of wavelength from 325 to 425 nm

Peaks 5 and 7 were attributed to pheophytin b and afrom [MminusH]minus at119898119911 884 and 870 respectively and from therelative UV-vis absorption bands (Figure 5)

Peaks 8 9 and 10 showed [MminusH]minus acetone adducts at119898119911 965 870 and 951 these ions together with spectral data(Figure 6) and literature information allowed us to recognizethe parent pigments chlorophyll b bacteriochlorophylls band chlorophyll a respectively

The other peaks (3 4 and 6) showed typical carotenoidUV-vis spectra (Figure 7) [MminusH]minus at119898119911 657 and [M+Na]+at119898119911 681 for peak 3 were attributable to fucoxanthin while[MminusH]minus at119898119911 567 and [M+H]+ at119898119911 569were attributableto lutein for peak 4 The [MminusH]minus ion at 119898119911 563 togetherwith [M+H]+ and [M+Na]+ ions at 119898119911 565 and 587wasassigned to canthaxanthin (peak 6)

Scytonemin is an ultraviolet screening photostablesheath pigment produced under conditions of high solarirradiance by cyanobacteria (blue-green algae) and accumu-lates in extra-cellular sheaths for photoprotection against

The Scientific World Journal 5

Table 1 Pigments in Edmonson Point lake sediments identified using DAD and ESI mass spectra

Peak Time (min) Pigment Main UV-vis bands (nm) ESI+ (119898119911) ESIminus (119898119911) Composition

1 31 Reduced scytonemin 385ndash450ndash590 [M+H]+ 5475[M+Na]+ 5695 [MminusH]minus 5455 1190

2 47 Scytonemin 385 [M+H]+ 5455[M+Na]+ 5675 [MminusH]minus 5435 867

3 51 Fucoxanthin 440ndash470[M+H]+ 681 [MminusH]minus 657 992

4 59 Lutein 420ndash450ndash480[M+H]+ 591 [MminusH]minus 567 6459

5 61 Pheophytin b (420) 450ndash475ndash665 mdash [MminusH]minus 884 030

6 78 Canthaxanthin 478 [M+H]+ 565[M+Na]+ 587 [MminusH]minus 563 207

7 93 Pheophytin a 475ndash665 mdash [MminusH]minus 870 0708 121 Chlorophyll b 465ndash600ndash650 mdash [MminusH]minus 965a 0659 151 Bacteriochlorophyll-c 370ndash420ndash610ndash660 mdash [MminusH]minus 870a 02410 192 Chlorophyll a 370ndash415ndash435ndash610ndash660 mdash [MminusH]minus 951a 030aAcetone adduct

0

400

800

1200

1600

2000(m

AU)

0

400

800

1200

1600

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(1)(2)

OH

N

O

HO

HNNH

(mAU

)

O HO

O OH

N

O

Figure 4 UV-vis spectra and formulae of scytonemin (2) and its reduced form (1)

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(mAU

)

0

30

60

90

120

(mAU

)

(5) (7)

Figure 5 UV-vis spectra of phaeophytin a (5) and phaeophytin b (7)

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 The Scientific World Journal

environment by chemical photochemical and biologicalprocesses Chlorophylls contain nitrogen and are thereforemore prone to being salvaged during senescence and biologi-cal breakdown than the carotenoids Chlorophyll degradationpathways include allomerization (oxidation) demetallation(loss of the Mg) and dephytylation (loss of phytyl chain)with the five-ring phorbin being relatively stable [6] Most ofthese breakdown products are detectable by regular pigmentanalysis Carotenoids are found mostly in transform and areinherently more stable than chlorophylls However unlikechlorophylls they are often broken down to colorless com-pounds by destruction of the long chain of alternating doublebonds that cannot be detected by regular pigment analysismethods [7 8] Knowledge of the type and variability inphotosynthetic pigment composition versus lake conditionsis very important for the detailed information about thehistory of this with reference to biotic communities [9] and ofenvironmental factors (eg UV radiation or anoxia) involvedin pigment transformations [10 11]

High performance liquid chromatography (HPLC) is areliable method for analysing algal pigments in sediments[10]

In order to better understand the environmental pro-cesses occurring in the Antarctic ecosystem sediment sam-ples were collected from the lake at Edmonson Point inthe Victoria Land region during an Italian expedition inthe period 2004ndash2007 In this study these sediment sampleshave been analyzed to determine the light harvesting andlight screening pigments that are very important in Antarcticecosystem because protect the life permitting the growthand survival of any organisms under low temperature andcontinuous light regime of summer

2 Material and Methods

21 Site Description The climate of central Victoria Land isa rigid Antarctic climate the monthly mean air temperatureranges between minus259∘C (August) and minus01∘C (January)There is complete darkness betweenMay 5th andAugust 10thand 24 h of light between October 10th and February 7thwhile December and January show the maximum monthlymean radiation (320 and 300Wmminus2 resp)

The Italian Base is situated in Northern Foothills and isa zone of rounded off hills and covered from local little onesglacier and accumulates of snow are present near obstacles Inthe outskirtses of the Base find little ones lakes or pools thatin summer are opened while in the rest of the area NorthernFoothills the present lakes always remain covered from theice

Edmonson Point is located in Wood Bay Victoria LandRoss Sea at the foot of the eastern slopes of Mount Mel-bourne about 50 km NE of Mario Zucchelli Terra Nova BayStation (Italy) (Figure 1)

At almost two km2 Edmonson Point is one of thelargest of the few low-lying coastal ice-free areas in NorthernVictoria Land Edmonson Point was first identified in the1980s as a site that could merit special protection principallybecause of its rich vegetation Italy established a station in

close proximity at Terra Nova Bay in 1986-87 with increasedresearch interest A variety of scientific projects have beeninitiated many of which are long-term with internationalinvolvement Edmonson Point comprises hills knolls andmoraines of volcanic materials separated by small valleyswith streams ponds and larger lakes The presence of acolony of Adelie penguins nesting skuas and some aban-doned penguin rookeries determines a patchy distributionof nutrients in terrestrial and freshwater ecosystems Theground is generally dark in colour which encourages rapidsnow-melt in spring In summer most of the area is dry andthe ground is covered by salt encrustations except below latesnow beds near stream and pool margins and in flood flatsMost streams and pools are transient However some long-lying snowdeposits and the inland glaciermargins can supplywater to the larger streams and lakes from the beginningof December to the end of January Some shallow lakes arefree of ice cover for two or three months in summer whiledeeper lakes are permanently frozen and are supplied bygroundwater or by surface waters

22 Sampling Sediment samples were collected from Lake15A at Edmondson Point during an Italian Expedition in2004ndash2007 All samples were collected with plastic toolsand were maintained at minus24∘C during all stages of storageand transport to the Italian laboratory The samples werepreserved frozen and in the dark and prior to analysis theywere freeze-dried (lyophilized) and well mixed under highvacuum (lt01 Pa)

23 Chemicals All solvents used were HPLC grade (Sigma-Aldrich) NH

4Ac and MgCO

3were analytical reagent grade

(Sigma-Aldrich) Milli-Q water was used for solution prepa-ration

24 Pigment Extraction All the laboratory procedures werecarried out in subdued light to minimize pigments degra-dation The extraction of 20 g freeze-dried of sediment wasperformed in darkened flasks to prevent photo-oxidation andthe breakdown of labile pigments extraction was carriedout with MeOHacetone (10 90 v v) and a small amountof magnesium carbonate in order to prevent the accidentalformation of chlorophyll metabolites this procedure wasrepeated until the supernatant was colourless The extractswere filtered and concentrated using a low pressure rotaryvapour at 25∘C and a dry N

2flow The concentrated extracts

were filtered (045 120583m) before HPLC analysis The driedextract were stored at minus4∘C in dark

25 Instrumentation Visible spectrum of total extracts wasobtained with a Hewlett-Packard 8452A diode array spec-trophotometer with a 1 cm quartz cell well-stoppered scan-ning from 200 to 800 nm

An HPLC Agilent 1200 Series chromatograph equippedwith autoinjector and photodiode array detector (190ndash950 nm) was used to separate and identify individual com-pounds

The Scientific World Journal 3

EdmonsonPoint

WoodBay

CoastlineIce-free groundProtected area boundaryRestricted zoneHelicopter approach zoneHelicopter landing site

A

BB

C

Map 1EdmonsonPoint

CapeWashington

Mount Melbourne (2732m)

ASPA No 118Summit of Mount Melbourne

ASPANo118Summit of Mount Melbourne

Aviator GlacierTongue

Mario ZucchelliStation (Italy)

Mario Zucchellistation (Italy)

GondwanaStation (Germany)GondwanaStation (Germany)

Wood

Bay

Wood

Bay

ASPAno161Terra Nova Bay

74degS

Inset 2

MAP 3

PERMIT

ASPA no 165entry by

permit

60degS

70deg S

80degS

90degW

0deg

RossSea

Rosssea

WeddellSea

WeddellSea

LOCATION OFWOOD BAY (INSET 2)

180deg

Colline Ippolito(Ippolito Hills)CollineIppolito(IppolitoHills)

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

74∘22

9984000998400

998400S

74∘22

9984000998400

998400S

74∘21

9984000998400

998400S

74∘21

9984000998400

998400S

74∘20

9984000998400

998400S

74∘20

9984000998400

998400S

74∘19

9984000998400

998400S

74∘19

9984000998400

998400S

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

Baia Siena(Siena bay)

MAP 2 H

H

Lower glacier slopes of Mount Melbourne

Inset 1 location of wood Bay in Antarctica

Inset 2 location of map 1 and Edmonson Point

Victor

ia La

nd

Aviator GlacierTongue

Map 1EdmonsonPoint

Mount Melbourne (2732 m)

60 ∘S

70 ∘S

80 ∘S

180∘

90∘W 90∘E

location ofwood bay (inset 2)

0∘

164∘E 165∘E 165∘E 75 ∘S

0 500 1000

July 2006PNRADSAERA

N

Map 1 Edmonson Point ASPA no 165Wood Bay Victoria Land Ross sea

Ross

sea

Terra N

ova Bay

HC

74∘S

H

N

0 10 20

(m)

(km)

Leptonychotes weddellii

Figure 1 Location map of Edmonson Point in the Victoria Land

Separation was performed from sample aliquots of 25120583Lthat were injected into reversed-phase columns LiChroCART(250 times 4mm id packed with LiChrospher 100 RP-18 (5 120583mspherical particles)) and an ODS-hypersil (C

18) precolumn

(5 120583m 20 times 40mm Hewlett-Packard)ForMSmeasurements the chromatographwas coupled to

a 1100MSDHewlett PackardMSD used under ESI conditionswith both positive and negative ion monitoring

26 HPLC Separation and Analysis The best conditions forHPLC were obtained using gradient elution with MeOH (A)and MeCN (B) as follows 0min 70 A 15min 80 A20min 90 A flow rate 1mLmin HPLC was carried outat a controlled temperature of 28∘C

In order to obtain the best conditions for ESI-MSthe chromatograms were measured with three fragmentorvoltage values (30 70 and 150 eV) in negative and positive

4 The Scientific World Journal

00

02

04

06

08

10

12

14

320 440 560 680 800Wavelength (nm)

Abso

rban

ce

Figure 2 The absorbance spectra of the MeOHacetone (10 90 v v) extract of sediment lake at Edmonson Point

2 4 6 8 10 12 14 16 18 200

20406080

100120

5

1

2

3

4

768

10981

2(a)

(b)

mAU

(min)

Figure 3 DAD chromatograms recorded at 660 nm (a) and 476 (b) of sediment extract fromEdmondson Point lake obtained using gradientelution with MeOH-MeCN (0min 70 30 15min 80 20 20min 90 10) peak numbers correspond to pigments listed in Table 1

ion mode All measurements demonstrated that the responsewas better at 70 eV in the negative ion mode and at 150 eV inthe positive ion mode

Peaks were identified by their absorption spectra at theirmaximumwavelength and characteristicMS fragmentationsTheDAD detector was set at the wavelength of 476 or 650 nmfor analysis of carotenoids and chlorophylls respectivelyPigment quantification was performed by comparing theHPLC peak areas with those of standards (chlorophyll-achlorophyll-b) from DHI Water amp Environment (HorsholmDenmark) and (canthaxanthin fucoxanthin) from the Inter-national Agency for 14C Determination VKI in Hoersholmand using published extinction coefficients [12ndash14] whenstandardswere unavailableThe specific extinction coefficientused for scytonemins was 1126 L gminus1 cmminus1 [15]

3 Results and Discussion

The absorbance spectra of the extract of sediment is reportedin Figure 2 from which the presence of chlorophyll andphaeopigments for the absorption in the range between 380and 440 nm and at about 660 nm can be deduced Theshoulders at 450ndash500 nm indicate instead the presence ofcarotenoids

The results of HPLC analysis of sediments of Lake 15A atEdmonson Point are reported in Figure 3The chromatogramobtained at 650 nm (Figure 3(a)) showed the presence of fivepigments while in that obtained at 476 nm (Figure 3(b))

eight pigmentswere observed ESI-MS analysis of themixturecombined with on-line DAD electronic spectra allowedidentification of the major components and their percentagecomposition (Table 1)

The two components eluting at 47 and 31min wereassigned as scytonemin and its reduced form [10] (2 1) from[M+H]+ at 119898119911 5455 and 5477 [M+Na]+ at 119898119911 5675 and5695 and [MminusH]minus at 119898119911 5435 and 5455 respectively andfrom the UV-vis spectra (Figure 4) that show the absorptionover relatively wide range of wavelength from 325 to 425 nm

Peaks 5 and 7 were attributed to pheophytin b and afrom [MminusH]minus at119898119911 884 and 870 respectively and from therelative UV-vis absorption bands (Figure 5)

Peaks 8 9 and 10 showed [MminusH]minus acetone adducts at119898119911 965 870 and 951 these ions together with spectral data(Figure 6) and literature information allowed us to recognizethe parent pigments chlorophyll b bacteriochlorophylls band chlorophyll a respectively

The other peaks (3 4 and 6) showed typical carotenoidUV-vis spectra (Figure 7) [MminusH]minus at119898119911 657 and [M+Na]+at119898119911 681 for peak 3 were attributable to fucoxanthin while[MminusH]minus at119898119911 567 and [M+H]+ at119898119911 569were attributableto lutein for peak 4 The [MminusH]minus ion at 119898119911 563 togetherwith [M+H]+ and [M+Na]+ ions at 119898119911 565 and 587wasassigned to canthaxanthin (peak 6)

Scytonemin is an ultraviolet screening photostablesheath pigment produced under conditions of high solarirradiance by cyanobacteria (blue-green algae) and accumu-lates in extra-cellular sheaths for photoprotection against

The Scientific World Journal 5

Table 1 Pigments in Edmonson Point lake sediments identified using DAD and ESI mass spectra

Peak Time (min) Pigment Main UV-vis bands (nm) ESI+ (119898119911) ESIminus (119898119911) Composition

1 31 Reduced scytonemin 385ndash450ndash590 [M+H]+ 5475[M+Na]+ 5695 [MminusH]minus 5455 1190

2 47 Scytonemin 385 [M+H]+ 5455[M+Na]+ 5675 [MminusH]minus 5435 867

3 51 Fucoxanthin 440ndash470[M+H]+ 681 [MminusH]minus 657 992

4 59 Lutein 420ndash450ndash480[M+H]+ 591 [MminusH]minus 567 6459

5 61 Pheophytin b (420) 450ndash475ndash665 mdash [MminusH]minus 884 030

6 78 Canthaxanthin 478 [M+H]+ 565[M+Na]+ 587 [MminusH]minus 563 207

7 93 Pheophytin a 475ndash665 mdash [MminusH]minus 870 0708 121 Chlorophyll b 465ndash600ndash650 mdash [MminusH]minus 965a 0659 151 Bacteriochlorophyll-c 370ndash420ndash610ndash660 mdash [MminusH]minus 870a 02410 192 Chlorophyll a 370ndash415ndash435ndash610ndash660 mdash [MminusH]minus 951a 030aAcetone adduct

0

400

800

1200

1600

2000(m

AU)

0

400

800

1200

1600

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(1)(2)

OH

N

O

HO

HNNH

(mAU

)

O HO

O OH

N

O

Figure 4 UV-vis spectra and formulae of scytonemin (2) and its reduced form (1)

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(mAU

)

0

30

60

90

120

(mAU

)

(5) (7)

Figure 5 UV-vis spectra of phaeophytin a (5) and phaeophytin b (7)

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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International Journal ofPhotoenergy

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Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

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Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

The Scientific World Journal 3

EdmonsonPoint

WoodBay

CoastlineIce-free groundProtected area boundaryRestricted zoneHelicopter approach zoneHelicopter landing site

A

BB

C

Map 1EdmonsonPoint

CapeWashington

Mount Melbourne (2732m)

ASPA No 118Summit of Mount Melbourne

ASPANo118Summit of Mount Melbourne

Aviator GlacierTongue

Mario ZucchelliStation (Italy)

Mario Zucchellistation (Italy)

GondwanaStation (Germany)GondwanaStation (Germany)

Wood

Bay

Wood

Bay

ASPAno161Terra Nova Bay

74degS

Inset 2

MAP 3

PERMIT

ASPA no 165entry by

permit

60degS

70deg S

80degS

90degW

0deg

RossSea

Rosssea

WeddellSea

WeddellSea

LOCATION OFWOOD BAY (INSET 2)

180deg

Colline Ippolito(Ippolito Hills)CollineIppolito(IppolitoHills)

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

74∘22

9984000998400

998400S

74∘22

9984000998400

998400S

74∘21

9984000998400

998400S

74∘21

9984000998400

998400S

74∘20

9984000998400

998400S

74∘20

9984000998400

998400S

74∘19

9984000998400

998400S

74∘19

9984000998400

998400S

165 ∘09984000998400 998400E 165 ∘49984000998400 998400E 165 ∘89984000998400 998400E

Baia Siena(Siena bay)

MAP 2 H

H

Lower glacier slopes of Mount Melbourne

Inset 1 location of wood Bay in Antarctica

Inset 2 location of map 1 and Edmonson Point

Victor

ia La

nd

Aviator GlacierTongue

Map 1EdmonsonPoint

Mount Melbourne (2732 m)

60 ∘S

70 ∘S

80 ∘S

180∘

90∘W 90∘E

location ofwood bay (inset 2)

0∘

164∘E 165∘E 165∘E 75 ∘S

0 500 1000

July 2006PNRADSAERA

N

Map 1 Edmonson Point ASPA no 165Wood Bay Victoria Land Ross sea

Ross

sea

Terra N

ova Bay

HC

74∘S

H

N

0 10 20

(m)

(km)

Leptonychotes weddellii

Figure 1 Location map of Edmonson Point in the Victoria Land

Separation was performed from sample aliquots of 25120583Lthat were injected into reversed-phase columns LiChroCART(250 times 4mm id packed with LiChrospher 100 RP-18 (5 120583mspherical particles)) and an ODS-hypersil (C

18) precolumn

(5 120583m 20 times 40mm Hewlett-Packard)ForMSmeasurements the chromatographwas coupled to

a 1100MSDHewlett PackardMSD used under ESI conditionswith both positive and negative ion monitoring

26 HPLC Separation and Analysis The best conditions forHPLC were obtained using gradient elution with MeOH (A)and MeCN (B) as follows 0min 70 A 15min 80 A20min 90 A flow rate 1mLmin HPLC was carried outat a controlled temperature of 28∘C

In order to obtain the best conditions for ESI-MSthe chromatograms were measured with three fragmentorvoltage values (30 70 and 150 eV) in negative and positive

4 The Scientific World Journal

00

02

04

06

08

10

12

14

320 440 560 680 800Wavelength (nm)

Abso

rban

ce

Figure 2 The absorbance spectra of the MeOHacetone (10 90 v v) extract of sediment lake at Edmonson Point

2 4 6 8 10 12 14 16 18 200

20406080

100120

5

1

2

3

4

768

10981

2(a)

(b)

mAU

(min)

Figure 3 DAD chromatograms recorded at 660 nm (a) and 476 (b) of sediment extract fromEdmondson Point lake obtained using gradientelution with MeOH-MeCN (0min 70 30 15min 80 20 20min 90 10) peak numbers correspond to pigments listed in Table 1

ion mode All measurements demonstrated that the responsewas better at 70 eV in the negative ion mode and at 150 eV inthe positive ion mode

Peaks were identified by their absorption spectra at theirmaximumwavelength and characteristicMS fragmentationsTheDAD detector was set at the wavelength of 476 or 650 nmfor analysis of carotenoids and chlorophylls respectivelyPigment quantification was performed by comparing theHPLC peak areas with those of standards (chlorophyll-achlorophyll-b) from DHI Water amp Environment (HorsholmDenmark) and (canthaxanthin fucoxanthin) from the Inter-national Agency for 14C Determination VKI in Hoersholmand using published extinction coefficients [12ndash14] whenstandardswere unavailableThe specific extinction coefficientused for scytonemins was 1126 L gminus1 cmminus1 [15]

3 Results and Discussion

The absorbance spectra of the extract of sediment is reportedin Figure 2 from which the presence of chlorophyll andphaeopigments for the absorption in the range between 380and 440 nm and at about 660 nm can be deduced Theshoulders at 450ndash500 nm indicate instead the presence ofcarotenoids

The results of HPLC analysis of sediments of Lake 15A atEdmonson Point are reported in Figure 3The chromatogramobtained at 650 nm (Figure 3(a)) showed the presence of fivepigments while in that obtained at 476 nm (Figure 3(b))

eight pigmentswere observed ESI-MS analysis of themixturecombined with on-line DAD electronic spectra allowedidentification of the major components and their percentagecomposition (Table 1)

The two components eluting at 47 and 31min wereassigned as scytonemin and its reduced form [10] (2 1) from[M+H]+ at 119898119911 5455 and 5477 [M+Na]+ at 119898119911 5675 and5695 and [MminusH]minus at 119898119911 5435 and 5455 respectively andfrom the UV-vis spectra (Figure 4) that show the absorptionover relatively wide range of wavelength from 325 to 425 nm

Peaks 5 and 7 were attributed to pheophytin b and afrom [MminusH]minus at119898119911 884 and 870 respectively and from therelative UV-vis absorption bands (Figure 5)

Peaks 8 9 and 10 showed [MminusH]minus acetone adducts at119898119911 965 870 and 951 these ions together with spectral data(Figure 6) and literature information allowed us to recognizethe parent pigments chlorophyll b bacteriochlorophylls band chlorophyll a respectively

The other peaks (3 4 and 6) showed typical carotenoidUV-vis spectra (Figure 7) [MminusH]minus at119898119911 657 and [M+Na]+at119898119911 681 for peak 3 were attributable to fucoxanthin while[MminusH]minus at119898119911 567 and [M+H]+ at119898119911 569were attributableto lutein for peak 4 The [MminusH]minus ion at 119898119911 563 togetherwith [M+H]+ and [M+Na]+ ions at 119898119911 565 and 587wasassigned to canthaxanthin (peak 6)

Scytonemin is an ultraviolet screening photostablesheath pigment produced under conditions of high solarirradiance by cyanobacteria (blue-green algae) and accumu-lates in extra-cellular sheaths for photoprotection against

The Scientific World Journal 5

Table 1 Pigments in Edmonson Point lake sediments identified using DAD and ESI mass spectra

Peak Time (min) Pigment Main UV-vis bands (nm) ESI+ (119898119911) ESIminus (119898119911) Composition

1 31 Reduced scytonemin 385ndash450ndash590 [M+H]+ 5475[M+Na]+ 5695 [MminusH]minus 5455 1190

2 47 Scytonemin 385 [M+H]+ 5455[M+Na]+ 5675 [MminusH]minus 5435 867

3 51 Fucoxanthin 440ndash470[M+H]+ 681 [MminusH]minus 657 992

4 59 Lutein 420ndash450ndash480[M+H]+ 591 [MminusH]minus 567 6459

5 61 Pheophytin b (420) 450ndash475ndash665 mdash [MminusH]minus 884 030

6 78 Canthaxanthin 478 [M+H]+ 565[M+Na]+ 587 [MminusH]minus 563 207

7 93 Pheophytin a 475ndash665 mdash [MminusH]minus 870 0708 121 Chlorophyll b 465ndash600ndash650 mdash [MminusH]minus 965a 0659 151 Bacteriochlorophyll-c 370ndash420ndash610ndash660 mdash [MminusH]minus 870a 02410 192 Chlorophyll a 370ndash415ndash435ndash610ndash660 mdash [MminusH]minus 951a 030aAcetone adduct

0

400

800

1200

1600

2000(m

AU)

0

400

800

1200

1600

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(1)(2)

OH

N

O

HO

HNNH

(mAU

)

O HO

O OH

N

O

Figure 4 UV-vis spectra and formulae of scytonemin (2) and its reduced form (1)

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(mAU

)

0

30

60

90

120

(mAU

)

(5) (7)

Figure 5 UV-vis spectra of phaeophytin a (5) and phaeophytin b (7)

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 The Scientific World Journal

00

02

04

06

08

10

12

14

320 440 560 680 800Wavelength (nm)

Abso

rban

ce

Figure 2 The absorbance spectra of the MeOHacetone (10 90 v v) extract of sediment lake at Edmonson Point

2 4 6 8 10 12 14 16 18 200

20406080

100120

5

1

2

3

4

768

10981

2(a)

(b)

mAU

(min)

Figure 3 DAD chromatograms recorded at 660 nm (a) and 476 (b) of sediment extract fromEdmondson Point lake obtained using gradientelution with MeOH-MeCN (0min 70 30 15min 80 20 20min 90 10) peak numbers correspond to pigments listed in Table 1

ion mode All measurements demonstrated that the responsewas better at 70 eV in the negative ion mode and at 150 eV inthe positive ion mode

Peaks were identified by their absorption spectra at theirmaximumwavelength and characteristicMS fragmentationsTheDAD detector was set at the wavelength of 476 or 650 nmfor analysis of carotenoids and chlorophylls respectivelyPigment quantification was performed by comparing theHPLC peak areas with those of standards (chlorophyll-achlorophyll-b) from DHI Water amp Environment (HorsholmDenmark) and (canthaxanthin fucoxanthin) from the Inter-national Agency for 14C Determination VKI in Hoersholmand using published extinction coefficients [12ndash14] whenstandardswere unavailableThe specific extinction coefficientused for scytonemins was 1126 L gminus1 cmminus1 [15]

3 Results and Discussion

The absorbance spectra of the extract of sediment is reportedin Figure 2 from which the presence of chlorophyll andphaeopigments for the absorption in the range between 380and 440 nm and at about 660 nm can be deduced Theshoulders at 450ndash500 nm indicate instead the presence ofcarotenoids

The results of HPLC analysis of sediments of Lake 15A atEdmonson Point are reported in Figure 3The chromatogramobtained at 650 nm (Figure 3(a)) showed the presence of fivepigments while in that obtained at 476 nm (Figure 3(b))

eight pigmentswere observed ESI-MS analysis of themixturecombined with on-line DAD electronic spectra allowedidentification of the major components and their percentagecomposition (Table 1)

The two components eluting at 47 and 31min wereassigned as scytonemin and its reduced form [10] (2 1) from[M+H]+ at 119898119911 5455 and 5477 [M+Na]+ at 119898119911 5675 and5695 and [MminusH]minus at 119898119911 5435 and 5455 respectively andfrom the UV-vis spectra (Figure 4) that show the absorptionover relatively wide range of wavelength from 325 to 425 nm

Peaks 5 and 7 were attributed to pheophytin b and afrom [MminusH]minus at119898119911 884 and 870 respectively and from therelative UV-vis absorption bands (Figure 5)

Peaks 8 9 and 10 showed [MminusH]minus acetone adducts at119898119911 965 870 and 951 these ions together with spectral data(Figure 6) and literature information allowed us to recognizethe parent pigments chlorophyll b bacteriochlorophylls band chlorophyll a respectively

The other peaks (3 4 and 6) showed typical carotenoidUV-vis spectra (Figure 7) [MminusH]minus at119898119911 657 and [M+Na]+at119898119911 681 for peak 3 were attributable to fucoxanthin while[MminusH]minus at119898119911 567 and [M+H]+ at119898119911 569were attributableto lutein for peak 4 The [MminusH]minus ion at 119898119911 563 togetherwith [M+H]+ and [M+Na]+ ions at 119898119911 565 and 587wasassigned to canthaxanthin (peak 6)

Scytonemin is an ultraviolet screening photostablesheath pigment produced under conditions of high solarirradiance by cyanobacteria (blue-green algae) and accumu-lates in extra-cellular sheaths for photoprotection against

The Scientific World Journal 5

Table 1 Pigments in Edmonson Point lake sediments identified using DAD and ESI mass spectra

Peak Time (min) Pigment Main UV-vis bands (nm) ESI+ (119898119911) ESIminus (119898119911) Composition

1 31 Reduced scytonemin 385ndash450ndash590 [M+H]+ 5475[M+Na]+ 5695 [MminusH]minus 5455 1190

2 47 Scytonemin 385 [M+H]+ 5455[M+Na]+ 5675 [MminusH]minus 5435 867

3 51 Fucoxanthin 440ndash470[M+H]+ 681 [MminusH]minus 657 992

4 59 Lutein 420ndash450ndash480[M+H]+ 591 [MminusH]minus 567 6459

5 61 Pheophytin b (420) 450ndash475ndash665 mdash [MminusH]minus 884 030

6 78 Canthaxanthin 478 [M+H]+ 565[M+Na]+ 587 [MminusH]minus 563 207

7 93 Pheophytin a 475ndash665 mdash [MminusH]minus 870 0708 121 Chlorophyll b 465ndash600ndash650 mdash [MminusH]minus 965a 0659 151 Bacteriochlorophyll-c 370ndash420ndash610ndash660 mdash [MminusH]minus 870a 02410 192 Chlorophyll a 370ndash415ndash435ndash610ndash660 mdash [MminusH]minus 951a 030aAcetone adduct

0

400

800

1200

1600

2000(m

AU)

0

400

800

1200

1600

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(1)(2)

OH

N

O

HO

HNNH

(mAU

)

O HO

O OH

N

O

Figure 4 UV-vis spectra and formulae of scytonemin (2) and its reduced form (1)

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(mAU

)

0

30

60

90

120

(mAU

)

(5) (7)

Figure 5 UV-vis spectra of phaeophytin a (5) and phaeophytin b (7)

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

The Scientific World Journal 5

Table 1 Pigments in Edmonson Point lake sediments identified using DAD and ESI mass spectra

Peak Time (min) Pigment Main UV-vis bands (nm) ESI+ (119898119911) ESIminus (119898119911) Composition

1 31 Reduced scytonemin 385ndash450ndash590 [M+H]+ 5475[M+Na]+ 5695 [MminusH]minus 5455 1190

2 47 Scytonemin 385 [M+H]+ 5455[M+Na]+ 5675 [MminusH]minus 5435 867

3 51 Fucoxanthin 440ndash470[M+H]+ 681 [MminusH]minus 657 992

4 59 Lutein 420ndash450ndash480[M+H]+ 591 [MminusH]minus 567 6459

5 61 Pheophytin b (420) 450ndash475ndash665 mdash [MminusH]minus 884 030

6 78 Canthaxanthin 478 [M+H]+ 565[M+Na]+ 587 [MminusH]minus 563 207

7 93 Pheophytin a 475ndash665 mdash [MminusH]minus 870 0708 121 Chlorophyll b 465ndash600ndash650 mdash [MminusH]minus 965a 0659 151 Bacteriochlorophyll-c 370ndash420ndash610ndash660 mdash [MminusH]minus 870a 02410 192 Chlorophyll a 370ndash415ndash435ndash610ndash660 mdash [MminusH]minus 951a 030aAcetone adduct

0

400

800

1200

1600

2000(m

AU)

0

400

800

1200

1600

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(1)(2)

OH

N

O

HO

HNNH

(mAU

)

O HO

O OH

N

O

Figure 4 UV-vis spectra and formulae of scytonemin (2) and its reduced form (1)

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800(nm)

350 400 450 500 550 600 650 700 750 800(nm)

(mAU

)

0

30

60

90

120

(mAU

)

(5) (7)

Figure 5 UV-vis spectra of phaeophytin a (5) and phaeophytin b (7)

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 The Scientific World Journal

0

20

40

60

80

mAU

0

10

20

30

40

350 400 450 500 550 600 650 700 750 800

(nm)350 400 450 500 550 600 650 700 750 800

(nm)

350 400 450 500 550 600 650 700 750 800

(nm)

(8) (9)

(10)

(mAU

)

0

20

40

60

80

(mAU

)

Figure 6 UV-vis spectra of chlorophyll b (8) bacteriochlorophylls b (9) and chlorophyll a (10)

DNA damage [16 17] Scytonemin allows the renownedsurvival ability of these bacteria [18] and is produced inresponse to elevated levels of UV-A radiation its productionis governed by a complex mechanism in which a number ofenvironmental factors are implicatedTheUVVis absorptionprofile of scytonemin is such that it absorbs UV-A radiationstrongly while transmitting the wavelengths of light that areabsorbed by the photosynthetic apparatus of cyanophyta

In addition the photobleaching of chlorophyll a by UVAradiation was retarded in scytonemin-containing cyanobac-terial sheaths It was noted also that there seemed to be adirect relationship between the production of the scytone-min in cyanobacteria and the exposure to UV flux Thescytonemin molecule is not photodegraded and is believedto have a long-term persistence in terrestrial crusts or driedcyanobacterial cultures [16 17 19]

The reduced form of scytonemin called Red Scytoneminis another UV-absorbing compound that is stable over longtime scales and is formed by transformation of scytoneminunder anoxic reducing conditions after loss of cell integrity ofcyanobacteria [16] Also canthaxanthin [20] is UV-induciblepigments that protects organisms against the deleteriouseffects of UV radiation [16]

Because scytonemin its reduced form and the carotenoidcanthaxanthin were detected in the sediment of EdmonsonPoint it is possible to assume the presence of colonialcyanobacteria in the water column at this site

Fucoxanthin is an indicator pigment for diatoms chrys-ophytes and prymnesiophytes [21] while chlorophyll a isindicative of the presence of autotrophic benthic algae

The presence of chlorophyll b and lutein can be attributedto an input from green algae (chlorophyta) and perhapsto the vegetation present in the area Lutein is a lipophilicmolecule is the most abundant carotenoid in photosyntheticplant tissues that has distinctive light harvesting propertiesand is generally insoluble in water so may be present inthe sediment as an aggregate Because the polyenic chain issusceptible to oxidative degradation via light or heat and isunstable in acid its presence is indicative of anaerobic andnot acidic conditions during its deposition in the sediment

Phaeophytin-a and b are metal-free chlorophylls acidicconditions promoting displacement of the metals (Figure 8)so their presence is indicative of pH change at the time of theirdeposition

Bacteriochlorophyll-c is found only in green photosyn-thetic bacteria organisms that contain a special antennacomplex known as a chlorosome [22] It has a naturaltendency to form large aggregates with highly organizedhelical structures that absorb light very efficiently with fastenergy transfer to some membrane bound energy acceptorsit is highly probable that it was deposited in such an aggregateform

The quantitative analysis of pigments (Table 1) revealedthat the major components are in the order lutein scytone-mins fucoxanthin and canthaxanthin respectively and onlysmall amounts of chlorophyll and its degradation productsare present

Scytonemins is about 10 times cyanobacterial carotenoids(canthaxanthin) confirming high biological receipt of UVradiation while cyanobacterial carotenoids are about 6 times

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

The Scientific World Journal 7

0

20

40

60

80

100

(mAU

)

0

2

4

6

8

350 400 450 500 550 600

(nm)

(mAU

)

0

2

4

6

8

(mAU

)

(5)

(4)

(3)

(3)

(5)

(4)

350 400 450 500 550 600

350 400 450 500 550 600

(nm)

(nm)

OH

O

O

HO 3OCOCH

HOO

O

HO

Figure 7 UV-vis spectra of fucoxanthin (3) lutein (4) and canthaxanthin (5)

Chlorophyll Phaeophytin

+2H+

minusMgO

NMg

phytilO

O

O

O

N

NN

O

O

O

O

N

N HN

NH O

phytil

Figure 8 Reaction that occurs in the chlorophyll demetallation for the formation of phaeophytin

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 The Scientific World Journal

total chlorophyll-a showing high cyanobacterial receipt ofphotosynthetically active radiation Comparison betweenscytonemins with total chlorophyll that is about 60 confirmshigh proportion on photoprotection versus photosyntheticproduction

4 Conclusions

Detailed studies of chlorophylls carotenoids and theirderivatives in aquatic sediments and their isolation andidentification may permit to use fossil pigments to recon-struct past changes in lake production Thus the type offossil pigments can reflect the sedimentation history andsimultaneously give indirect information about the dynamicsof the abundance of algae in lakes

Because of the complexity of the pigments mixtureswhich typically occur in sediments the separation of theindividual components can be problematic in this paperwe have shown that the combination of HPLCDADESIMSin negative and positive ionization modes allowed the sep-aration and the conclusive identification of sedimentarypigments extracts from lake 15A at Edmonson Point Thismethod allowed us to determine the characteristics of sev-eral photosynthetic pigments that are present in the lakesediments because the cold climate in the area favours theirpreservation

The pigments could be classified into four families ofcompounds light screening pigments as scytonemins andlight harvesting pigments as carotenoids and chlorophyllsother than phaeophytins (its degradation pigments) Esti-mates of these pigments suggest a stronger UV stress in theLake 15A at Edmonson Point

This chemical approach may be useful in the extensivestudies of these sites in the temporal and spatial variationsin composition of sedimentary pigments as responses tochanging environmental conditions

Acknowledgments

The authors wish to thank PNRA (Progetto NazionaleRicerche in Antartide) for providing funding and the oppor-tunity to undertake the research and would like to expresstheir gratitude to Professor V Bartocci for important scien-tific suggestions

References

[1] D D Wynn-Williams ldquoPotential effects of ultraviolet radiationonAntarctic primary terrestrial colonizers cyanobacteria algaeand cryptogamsrdquo in Ultraviolet Radiation in Antarctica Mea-surements and Biological Effects C S Weiler and P A PenhaleEds vol 62 ofAntarctic Research Series pp 243ndash257 AmericanGeophysical Union Washington DC USA 1994

[2] D D Wynn-Williams N C Russell and H G M EdwardsldquoMoisture and habitat structure as regulators for microalgalcolonists in diverse Antarctic terrestrial habitatsrdquo in EcosystemProcesses in Antarctic Ice-Free Landscapes W B Lyons CHoward-Williams and IHawes Eds pp 77ndash88 BalkemaPressRotterdam The Netherlands 1997

[3] D DWynn-Williams ldquoAntarctica as a model for ancient MarsrdquoinThe Search for Life onMars Special Publication J Hiscox Edpp 49ndash57 British Interplanetary Society London UK 1999

[4] J C Farman B G Gardiner and J D Shanklin ldquoLargelosses of total ozone in Antarctica reveal seasonal ClO

119909

NO119909

interactionrdquo Nature vol 315 no 6016 pp 207ndash210 1985[5] S J Fitzsimons ldquoParaglacial redistribution of glacial sediments

in the Vestfold Hills East Antarcticardquo Geomorphology vol 15no 2 pp 93ndash108 1996

[6] J Porra E E Pfundel and N Engel ldquoMetabolism and functionof photosynthetic pigmentsrdquo in Phytoplankton Pigments inOceanography SW Jeffrey R F CMantoura and SWWrightEds pp 85ndash126 UNESCO Paris France 1997

[7] P R Leavitt ldquoA review of factors that regulate carotenoid andchlorophyll deposition and fossil pigment abundancerdquo Journalof Paleolimnology vol 9 no 2 pp 109ndash127 1993

[8] Y Z Yacobi U Pollingher Y Gonen V Gerhardt and ASukenik ldquoHPLC analysis of phytoplankton pigments from LakeKinneret with special reference to the bloom-forming dinoflag-ellate Peridinium gatunense (Dinophyceae) and chlorophylldegradation productsrdquo Journal of Plankton Research vol 18 pp1781ndash1796 1996

[9] N Cannone D Wagner H W Hubberten and M GuglielminldquoBiotic and abiotic factors influencing soil properties across alatitudinal gradient in Victoria Land Antarcticardquo Geodermavol 144 no 1-2 pp 50ndash65 2008

[10] P R Leavitt and D A Hodgson ldquoSedimentarypigmentsrdquo inTracking Environmental Change Using Lake Sediments J PSmol H J B Birks and W M Last Eds Developments inPaleoenvironmental Research pp 295ndash325 Kluwer AcademicPublishers Dordrecht The Netherlands 2001

[11] C G Oviatt G D Habiger and J E Hay ldquoVariation in thecomposition of Lake Bonneville marl a potential key to lake-level fluctuations and paleoclimaterdquo Journal of Paleolimnologyvol 11 no 1 pp 19ndash30 1994

[12] J P Hurley and C J Watras ldquoIdentification of bacteriochloro-phylls in lakes via reverse-phase HPLCrdquo Limnology amp Oceanog-raphy vol 36 no 2 pp 307ndash315 1991

[13] J Villanueva J O Grimalt R de Wit B J Keely and J RMaxwell ldquoChlorophyll and carotenoid pigments in solar salternmicrobial matsrdquo Geochimica et Cosmochimica Acta vol 58 no21 pp 4703ndash4711 1994

[14] SW Jeffrey R F CMantoura and T Bjornland PhytoplanktonPigments in Oceanography Guidelines to Modern MethodsUNESCO Paris France 1997

[15] W F Vincent D R Mueller and S Bonilla ldquoEcosystems on icethe microbial ecology of Markham Ice Shelf in the high ArcticrdquoCryobiology vol 48 no 2 pp 103ndash112 2004

[16] F Garcia-Pichel and R W Castenholz ldquoCharacterization andbiological implications of scytonemin a cyanobacterial sheathpigmentrdquo Journal of Phycology vol 27 no 3 pp 395ndash409 1991

[17] F Garcia-Pichel N D Sherry and R W Castenholz ldquoEvidencefor an ultraviolet sunscreen role of the extracellular pigmentscytonemin in the terrestrial cyanobacterium Chlorogloeopsissprdquo Photochemistry and Photobiology vol 56 no 1 pp 17ndash231992

[18] R W Castenholz and F Garcia-Pichel ldquoCyanobacterialresponses to UV-radiationrdquo in The Ecology of CyanobacteriaM Potts and B A Whitton Eds Kluwer Dordrecht TheNetherlands 1999

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

The Scientific World Journal 9

[19] P R Leavitt R D Vinebrooke D B Donald J P Smol and DW Schindler ldquoPast ultraviolet radiation environments in lakesderived from fossil pigmentsrdquo Nature vol 388 no 6641 pp457ndash459 1997

[20] F Vincent and A Quesada ldquoUltraviolet radiation effects oncyanobacteria implications for Antarctic microbial ecosys-temsrdquo inUltraviolet Radiation in Antarctica Measurements andBiological Effects C S Weiler and P A Penhalew Eds vol 62of Antarctic Research Series pp 111ndash124 American GeophysicalUnion Washington DC USA 1994

[21] M A Burford B G Long and P C Rothlisberg ldquoCopepodpredation in phagotrophic ciliates in Oregon coastal watersrdquoMarine Ecology Progress Series vol 107 no 1-2 pp 103ndash111 1994

[22] A Egawa T Fujiwara T Mizoguchi Y Kakitani YKoyama and H Akutsu ldquoStructure of the light-harvestingbacteriochlorophyll-c assembly in chlorosomes from Chloro-bium limicola determined by solid-state NMRrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 3 pp 790ndash795 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of