Evaluating the Impact of Atmospheric Depositions on Springtime Dinitrogen Fixation in the Cretan

13 Pages·2016·1.64 MB·English

by  Eyal Rahav

ORIGINALRESEARCH
published:23September2016
doi:10.3389/fmars.2016.00180
Evaluating the Impact of
Atmospheric Depositions on
Springtime Dinitrogen Fixation in the
Cretan Sea (Eastern
Mediterranean)—A Mesocosm
Approach
EyalRahav1,2*,CheungShun-Yan3,GuoCui3,HongbinLiu3,TatianaM.Tsagaraki4,5,
AntoniaGiannakourou5,AnastasiaTsiola5,StellaPsarra5,AnnaLagaria5,
MargaretR.Mulholland6,EleniStathopoulou7,PittaParaskevi5,BarakHerut1and
IlanaBerman-Frank2
1IsraelOceanographicandLimnologicalResearch,NationalInstituteofOceanography,Haifa,Israel,2MinaandEverard
GoodmanFacultyofLifeSciences,Bar-IlanUniversity,Ramat-Gan,Israel,3DivisionofLifeScience,HongKongUniversityof
ScienceandTechnology,HongKong,HongKong,4DepartmentofMicrobiology,UniversityofBergen,Bergen,Norway,
5HellenicCentreforMarineResearch,Heraklion,Greece,6DepartmentofOcean,EarthandAtmosphericSciences,Old
DominionUniversity,Norfolk,VA,USA,7LaboratoryofEnvironmentalChemistry,DepartmentofChemistry,Universityof
Editedby: Athens,Athens,Greece
AlbertoBasset,
UniversityofSalento,Italy
Large amounts of dust and atmospheric aerosols, originating from surrounding desert
Reviewedby:
areas(e.g.,SaharaandMiddleEast)aredepositedannuallyonthesurfaceoftheEastern
RobinsonW.(Wally)Fulweiler,
BostonUniversity,USA MediterraneanSea.Thesedepositionscanprovidehighamountsofmicro(suchasFe,
PatrickGeorgesGillet, Zn, Co) and macro nutrients (such as P and N) to supplement nutrient-poor surface
UCOAngers,France
waters- that typically limit primary productivity and also dinitrogen (N ) fixation in many
2
*Correspondence:
marine environments. Here, we studied the impact of the atmospheric deposition of
EyalRahav
[email protected] dustandaerosolsonN fixationintheCretanSea(EasternMediterraneanSea).Mixed
2
polluted aerosols (hereafter A) and Saharan dust (hereafter SD) were added to nine
Specialtysection:
mesocosms (3-m3 each) containing surface mixed layer seawater (∼10m), and N
Thisarticlewassubmittedto 2
MarineEcosystemEcology, fixation was evaluated for 6 days during May 2012 (springtime). The addition of SD
asectionofthejournal
triggeredarapid(30h)androbust(2–4-fold)increaseinN fixationratesthatremained
FrontiersinMarineScience 2
high for 6 days and contributed 3–8% of the primary productivity. The A addition also
Received:15May2016
Accepted:06September2016 resulted in higher N2 fixation rates compared to the unamended control mesocosms,
Published:23September2016 although the responses were less profound (1.5–2-fold) and accounted for only 2–4%
Citation: of the primary productivity. The microbial community responded differently to the two
RahavE,Shun-YanC,CuiG,LiuH,
additions.HeterotrophicbacterialN fixersdominatedthediazotrophcommunityinAand
TsagarakiTM,GiannakourouA, 2
TsiolaA,PsarraS,LagariaA, thecontrolmesocosms,whilethenon-filamentouscyanobacterialgroupTrichodesmium
MulhollandMR,StathopoulouE,
prevailed in the SD treatment (68% of all the operational taxonomic units, verified by
ParaskeviP,HerutBand
Berman-FrankI(2016)Evaluatingthe qPCRanalyses).Ourresultsindicatethattheaerosolsource,itsroutepriortodeposition,
ImpactofAtmosphericDepositionson anditsspecificchemicalcomposition,canalterthediazotrophicdiversityandactivityin
SpringtimeDinitrogenFixationinthe
theEasternMediterraneanSeaandmaythusimpactboththeNandCdynamicsinthis
CretanSea(Eastern
Mediterranean)—AMesocosm impoverishedenvironment.
Approach.Front.Mar.Sci.3:180.
doi:10.3389/fmars.2016.00180 Keywords:N2fixation,primaryproductivity,bacterialproductivity,Saharandust,aerosols
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Rahavetal. ImpactofAerosolsonN2-Fixation
INTRODUCTION Onepossiblereasonforthevariableresultsfromthenutrient
amendment experiments above could be the relatively small
Dinitrogen(N2)fixationisrecognizedasanimportantpathway volumes of the microcosm experiments (each a few liters
for bioavailable nitrogen inputs in many of the world’s oceans in total). The ultra-oligotrophic conditions in the study area
(Gruber and Galloway, 2008; Sohm et al., 2011). This process coupled with the short duration of the experiments (usually
canalterphytoplanktonpopulationsandprimaryproductionin less than 48h) may have been insufficient to detect active
nitrogen(N)limitedmarineenvironments(e.g.,Falkowski,1997; diazotrophy. In addition, the low population density may
Carpenter et al., 2004; Sohm et al., 2011) and thus fuels the have excluded the representation of the entire microbial food
biologicalpump.Manyphysical,chemical,andbiologicalfactors web in the small incubation experiments. In ultraoligotrophic
can affect the magnitude of N2 fixation in a given ecosystem. areas such as the Eastern Mediterranean Sea, larger-scale
WhereN2fixers(diazotrophs)occur,theyaremostoftenlimited experiments conducted over longer timescales may be required
by phosphorus (P) (Wu et al., 2000; Sanudo-Wilhelmy et al., to determine N fixation rates and responses to additions of
2
2001; Dyhrman and Haley, 2006), iron (Fe) (Paerl et al., 1987; potentially rate-limiting elements. Mesocosms (52 m3 bags)
Berman-Frank et al., 2001, 2007; Moore et al., 2009), or both were recently employed to examine diazotrophic responses
(Millsetal.,2004). to dust additions in the western Mediterranean Sea (2008,
Wet and dry deposition of atmospheric dust can increase 2010) (Ridame et al., 2013). During this experiment, addition
the availability of Fe and P in the surface ocean (Herut et al., of a dust analog (manipulated top-soils collected from the
1999, 2005; Bonnet and Guieu, 2006; Guieu et al., 2014) Sahara desert) induced a rapid (24–48h), robust (2–5.3-fold),
thereby supplementing the requirements of primary producers and lengthy (4–6 days duration) increase in N fixation
2
(mostly N and P, Zohary et al., 2005; Rahav et al., 2016a; (Table1),suggestingthatdustinputsmaystimulatediazotrophic
Tsiola et al., 2016) and N2-fixers (Gruber and Sarmiento, 1997; activity over longer timescales (>48h) than typical microcosm
Mills et al., 2004; Marañón et al., 2010). Dust enrichment experiments.
enhanced N2 fixation rates in microcosm experiments from In this study, we employed mesocosms to evaluate the
the tropical Atlantic (Mills et al., 2004; Marañón et al., 2010), impact of atmospheric deposition on diazotrophy and
the North Atlantic (Gruber and Sarmiento, 1997), the Red primary productivity during springtime in the Cretan Sea,
Sea (Foster et al., 2009), and the tropical and subtropical an oligotrophic region in the Eastern Mediterranean Sea.
western North Pacific (Kitajima et al., 2009). Furthermore, a Treatments included additions of: (1) “pure” Saharan dust
100-fold increase in the abundance of Trichodesmium, a non- (1.6mg L−1), and (2) mixed polluted and desert origin
filamentous cyanobacterial diazotroph, and a ∼2-fold increase aerosols (1mg L−1). Our main objectives were to study
in dissolved organic nitrogen were reported following dust how different aerosols affect diazotrophic diversity and N
2
stimulation in the West Florida Shelf (Lenes et al., 2001). fixation in the oligotrophic Cretan Sea during the spring
However,N2 fixationratesandhighabundancesofdiazotrophs and study whether these atmospheric inputs can relieve
are not always observed in areas of the ocean where dust the nutrient limitations for diazotrophs. We hypothesized
depositionishigh.Forexample,theoligotrophicMediterranean that the aerosol source and the atmospheric route prior to
Sea receives high annual inputs of dust (20 × 106 to 50 × deposition might trigger different diazotrophic responses that
106 tons y−1, Guerzoni et al., 1999) characterized by relatively would be reflected in both diversity and activity. Specifically,
high dissolved Fe concentrations (∼42 µmol m−2 y−1, Bonnet the two deposition types, and their consequent leached
and Guieu, 2006). Yet N2 fixation rates are typically low nutrients (N, P and the subsequent N:P ratio), might result in
in surface waters and the upper mixed layer (usually <0.2 different responses of the diazotrophic community that could
nmol N L−1 d−1, Sandroni et al., 2007; Ibello et al., 2010; subsequentlyimpactthemicrobialfoodwebintheoligotrophic
Bonnet et al., 2011; Rahav et al., 2013a; Benavides et al., 2016) CretanSea.
relative to other marine systems (reviewed in Sohm et al.,
2011).
Several studies have examined the possibility of low P MATERIALS AND METHODS
availability limiting diazotrophy in the Mediterranean Sea but
results have been inconclusive (see literature compilation in Experimental Design
Table1). While N fixation was enhanced by ∼50% after the Waterwascollectedusingarotarysubmersiblepumpfromthe
2
additionofPtosurfacewatersinanultraoligotrophic,P-limited surface mixed layer (∼10m) on May 9–10, 2012, from an area
anticyclonic eddy in the Eastern Mediterranean Sea during the located5nauticalmilesnorthofHeraklion,Crete(35◦24.957N,
summerof2002(Reesetal.,2006),nosignificantincreasesinN 25◦ 14.441E). The collected seawater was brought within ∼2h
2
fixationrateswereobservedseveralyearslaterduringanidentical to the Hellenic Centre for Marine Research mesocosm facility
experiment at the same location and for the same incubation (www.cretacosmos.eu), where it was homogenously distributed
period (24h) (Ridame et al., 2011). Similarly, N fixation rates between pre-cleaned (10% HCl) mesocosms (see Figure 1 in
2
werenotenhancedbyPadditionsintheLevantineBasin(Eastern Herutetal.,thisissue,Pittaetal.,2016).Nine3-m3 mesocosms
Mediterranean) during the summer of 2009, including within werefilledandchemicalandbiologicalmeasurementsweremade
the cores of cyclonic and anticyclonic eddies (Rahav et al., tofullycharacterizetheinitialpropertiesofthewater(Table2).
2013b). The mesocosms were prepared from transparent polyethylene
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Rahavetal. ImpactofAerosolsonN2-Fixation
TABLE1|Summaryofthemicrocosm/mesocosmexperimentsperformedintheMediterraneanSeaaimedstudyingthelimitingnutrientsfordiazotrophy.
Location Watertype Typeofaddition Sampling MaximalchangeinN2 Reference Comments
depth fixationrates(%fromcontrol)
Levantine Anticyclonic P(∼100nM) Surface(16m) +50 Reesetal.,2006
Basin eddy Fe(6nM) Nochange
Western Anticyclonic P(30nM) Surface(8m) +200 Ridameetal.,2011 StationA
Basin eddy P(30nM)+Fe(2nM) +400
Dust(1.1mgL−1) +130
IonianSea Anticyclonic P(30nM) Surface(8m) Nochange Ridameetal.,2011 StationB
eddy P(30nM)+Fe(2nM) Nochange
Dust(1.1mgL−1) +210
Levantine Anticyclonic P(30nM) Surface(8m) +200 Ridameetal.,2011 StationC
Basin eddy P(30nM)+Fe(2nM) +150
Dust(1.1mgL−1) +430
Levantine Coastal P(100nM) Surface(∼2m) Nochange Rahavetal.,2016a
Basin N(1600nM) Nochange
C(1000nM) +170 Monosaccharide
NP Nochange
CP +320
CN +140
CNP +600
XanthanGum +1000 Polysaccharide
(300µgL−1)
Levantine Opensea P(100nM) Surface(5–20m) Nochange Rahavetal.,2013b
Basin Anticyclonic P(100nM) Nochange
eddy
Cyclonicgyre P(100nM) Nochange
Levantine Opensea XanthanGum Aphoticwater +400 Rahavetal.,2013c
Basin (100–4500µgL−1) (250m)
Western Coastal Dust(10gm−2) Surface(0–5m) +700 Ridameetal.,2013 Mesocosms
Basin
CretanSea Opensea Saharandust Surface(∼10m) +400 Thisstudy Mesocosms
(1.6mgL−1) Mixedpolluted +200
aerosol(1mg
L−1)
bagsandweresupportedbyaluminumframes.Themesocosms period. For a detailed description of the mesocosms’ setup and
weredeployedwithinacontinuouslycirculatingseawater350m3 analyses, see Tsagaraki et al. (this issue) and Herut et al. (this
pooltomaintainambienttemperature.Eachmesocosmwas3m issue).
deep and the seawater inside it was gently mixed using an air
pump. Two experimental amendments were made (triplicate Nutrient Concentrations
mesocosms per treatment): (1) the addition of “pure” Saharan Water samples were collected and analyzed daily to measure
dust (SD), collected from Heraklion and Tel-Shikmona (Haifa, phosphate concentrations using the MAGIC method that
Israel)duringSaharandustevents,and(2)theadditionofmixed concentrate the phosphate in the samples, thus allowing a
aerosols (A), collected in Crete and Israel, which contained a more accurately measures in oligotrophic environments such
naturalmixtureofdesertdustandpollutedEuropeanparticles. as the surface waters of the Mediterranean Sea (Rimmelin and
The leaching nutrient values of the SD and A particles are Moutin, 2005), nitrite and nitrate concentrations (Strickland
described in details in Herut et al., (this issue). In short, the and Parsons, 1972), and ammonium concentrations (Ivancic
SD leached ∼23.5nmol N per mg of dust and ∼2.3nmol P and Deggobis, 1984). The detection limits were 0.8nM for
per mg of dust. The A particles leached ∼53.0nmol N per phosphate, 0.017µM for nitrate+nitrite, and 0.019µM for
mg of dust and ∼2.8nmol P per mg of dust. These leached ammonium.
nutrients resulted in significantly different N:P ratio of ∼10
(mol:mol) in the SD and ∼19 (mol:mol) in the A particles. Chlorophyll a
Three mesocosms were used as controls, with no addition. Seawater samples (500mL) were passed through a Whatman
All mesocosms were sampled daily at 08:30 over a 6 days GF/F filter, and extracted overnight in 10mL of 90% acetone
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Rahavetal. ImpactofAerosolsonN2-Fixation
TABLE2|TheinitialchemicalandbiologicalpropertiesoftheCretanSea (Perkin Elmer, Boston, MA, USA), and incubated for 4 h
sub-surfacewaterduringMay9th2012,beforeamendmentswere under natural illumination and temperature. To determine the
performed. quantity of the added tracer, 50µl samples were immediately
Parameter Units Average±Stdev taken out from each of the polycarbonate bottles and stored
with 50µL of ethanolamine for later analysis. Incubations
NO2+NO3 nM 132±27 were terminated by filtering the samples through a GF/F
NH4 nM 131±60 filter using a low vacuum pressure (<50mmHg). The filters
PO4 nM 13±4 were then placed in scintillation vials (5mL) and 50µl of
Chlorophylla(Chla) µgL−1 0.06±0.01 32% hydrochloric acid solution were added to each vial in
Synechococcus µgCL−1 5.8±0.2 order to remove excess 14C-bicarbonate overnight. After the
Pico-eukaryotes µgCL−1 0.6±0 addition of 3mL of scintillation cocktail (ULTIMA-GOLD),
Heterotrophicbacteria µgCL−1 8.5±0.2 the samples were counted using a TRI-CARB 2100 TR
Primaryproductivity(PP) ngCL−1h−1 393±46 (PACKARD)scintillationcounter.ThehourlyPPrateswerealso
Bacterialproductivity(BP) ngCL−1h−1 13.7±5.5 compared to daily measurements taken using the NaH13CO3
N2fixation nmolNL−1d−1 0.22±0.05 method as described in Mulholland and Bernhardt (2005). The
ContributionofN2fixationtoPP % 5.4±1.7 positive (R = 0.62) and significant (P < 0.001) relationship
Trichodesmium nifHgenecopiesL−1 81264±7265 between the two measuring techniques lends credibility to
our results and suggest that the use of “potential” PP (4h
n=3forallanalysesexceptfortheTrichodesmiumgenecopieswhere1samplewas incubation and not daily) was accurate enough to characterize
analyzed.
the Cretan Sea autotrophic production during the study
period.
solution in the dark. Chlorophyll a concentrations were For the 13C uptake rate measurements, water samples
determined by the non-acidification method (Welschmeyer, were placed in triplicate clear 4.5 L polycarbonate Nalgene
1994) using a TD700 fluorometer equipped with 436nm bottles and amended with (99%) NaH13CO (Sigma) to
3
excitationand680nmemissionfilters. obtain 1% of the ambient dissolved inorganic carbon and
incubated under the same conditions and bottles as for
Picophytoplankton and Heterotrophic
the N fixation measurements (see below more details).
2
Bacterial Abundance Triplicate parallel dark bottles were also incubated and
Samples for determining picophytoplankton and heterotrophic subtracted from the light bottles to correct for dark carbon
bacterial abundance were collected every day during the fixation.Incubationswereterminatedandanalyzedasdescribed
experimental period. The samples were fixed with 0.2µm for the N2 fixation measurements. The contribution of
filtered glutaraldehyde (0.5% final concentration) and held N2 fixation to the N demand for primary productivity
at 4◦C for approximately 45 min, flash-frozen in liquid was estimated using the measured particulate C: N ratio
nitrogen, and then transferred to a −80◦C refrigerator until obtained for each sample. A comparison between the rates
further processing. Frozen samples were thawed at room measured using the 13C and 14C methods can be seen in
temperature and sub-samples were stained with SYBR Green FigureS1.
I and incubated for 10 min in the dark, according to
Bacterial Productivity (BP)
Marie et al. (1997) and Vaulot and Marie (1999). Samples
for picophytoplankton abundance were analyzed based on Rates of bacterial productivity (BP) were estimated using the
their auto-fluorescence signals, without pre-staining using a [4,5-3H]-leucine incorporation method (Simon et al., 1990).
FACSCalibur(BectonDickinson)flowcytometerequippedwith Triplicate (1.7mL) samples were incubated with 100 nmol L−1
an air-cooled laser at 488nm and a standard filter set-up [4,5-3H]-leucine (Perkin Elmer, Boston, MA, USA) for 4h at
(Marie et al., 1997; Vaulot and Marie, 1999). Flow cytometry in situ temperatures in the dark, with triplicate trichloroacetic
data were acquired and processed with the Cell Quest Pro acid(TCA)killedsamplesservingascontrols.Incubationswere
software(BectonDickinson).Anaverageestimatedflowrateof terminatedbyadding100µLofcoldTCA(100%)tothevialsand
58µLmin−1 wasused.Thepicophytoplanktoncarbonbiomass treated according to the micro-centrifugation protocol (Simon
was calculated from cell counts, assuming 175fg C cell−1 for et al., 1990). After the addition of 1mL of scintillation cocktail
Synechococcuscells,53fgCcell−1 forProchlorococcuscells,and (ULTIMA-GOLD), the samples were counted using a TRI-
2100fg C cell−1 for picoeukaryotes (Campbell and Yentsch, CARB2100TR(PACKARD)scintillationcounter.Aconversion
1989). factor of 3.1kg C mol−1 and an isotope dilution factor of
2.0 were used to estimate bacterial productivity (Simon et al.,
Primary Productivity (PP) 1989).
Photosynthetic carbon fixation rates were estimated using the
14C incorporation method (Nielsen, 1952). Water samples Dinitrogen (N2) Fixation Rates
were analyzed in triplicate with dark and zero time controls. 15N uptake measurements were performed using the
2
Polycarbonate (Nalgene) bottles were filled with 50mL of 15N-enriched seawater protocol described by Mohr et al.
sample water, inoculated with 5µCi of NaH14CO tracer (2010),withminormodificationsfortheEasternMediterranean
3
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Rahavetal. ImpactofAerosolsonN2-Fixation
Sea (Rahav et al., 2013a,c). Enriched seawater was prepared Nested PCR and 454-Pyrosequencing
daily by degassing filtered (0.2-µm) natural seawater using a Samples collected onto 0.2µm filters were sequenced using a
degassingmembrane(Liqui-Cel,MiniModule(cid:13)R G542)for∼1h. 454-pyrosequencingat−48,48,and,144haftertheatmospheric
Then 1 ml of 15N gas (99%) was added for every 100ml of additionswerecarriedout.TheGenomicDNAsamplescollected
2
the degassed seawater and shaken vigorously until the bubbles from each of the different treatments were pooled into one
disappeared (∼30min). Aliquots of this 15N - sea enriched sample prior to pyrosequencing, granting a representative view
2
water were then added to the incubation bottles, with the of the microbial microorganisms of each mesocosm type.
enriched water constituting 5% of the total volume of the nifH gene fragments were amplified from the genomic DNA
sample (i.e., 225mL). Similar enriched seawater additions samples following the nested polymerase chain reaction (PCR)
collected from the oligotrophic North Pacific Subtropical Gyre protocol(ZehrandTurner,2001).ThenestedPCRreactionwas
(NPSG) resulted in a final 15N enrichment of 1.5 atom%, performed in triplicate using a Platinum Taq DNA polymerase
2
following the addition of 50ml of 15N -enriched water to a PCR system in a volume of 12.5µL (Invitrogen, Carlsbad,
2
4.5L bottle (Wilson et al., 2012). The bottles were then shaken CA), which contained a 1X rxn PCR buffer, 4 mM MgCl ,
2
and incubated under ambient surface seawater temperatures. 400µM dNTPs, 1µM of primers (nifH 3 and nifH 4 for the
Incubation bottles were either covered with neutral density first round and nifH 1 and nifH 2 for the second round
screening to simulate ambient light or were kept under of the nest PCR), 1 unit of Platinum Taq polymerase and
complete darkness for 24h (see Rahav et al., 2013b). The 1µL of total genomic DNA. Thirty cycles were performed
incubations under ambient irradiance (representative of a for each of the nested PCR rounds. After the nested PCR
full diel cycle having both light and dark cycles) recorded the cycles ended, 1µL of the PCR products were used to run
activities of both autotrophic and heterotrophic diazotrophs, 10 cycles of PCR with sample-specific multiplex identifiers
whereas the dark incubations reflected the activity of mainly (MIDs) and adaptor-attached primers (Farnelid et al., 2011).
heterotrophic diazotrophs who do not require light energy The PCR condition was the same as that used for the
for fixing N (Rahav et al., 2013b). We estimated the nested PCR. The PCR products were then gel-purified with a
2
heterotrophic contribution to N fixation by comparing the Quick gel purification kit (Invitrogen, Carlsbad, CA). For 454-
2
dark incubations to the bottles incubated under ambient diel pyrosequencing, the MID-adaptor-labeled PCR products were
irradiance. mixed in the same concentrations to construct an amplicon
The incubations were terminated by filtering water through library following the Rapid Library construction protocols
pre-combusted 25mm GF/F filters (with a nominal pore size (Roche,454LifeScience).TheDNAlibraryattachedbeadswere
of 0.7µm). The filters were then dried in an oven at 60◦C and loaded onto a Pico TiterPlate and sequenced with a GS Junior
stored in a desiccator until analysis. In the laboratory, samples System.
were pelletized in tin disks and analyzed with a Europa 20/20
mass spectrometer equipped with an automated carbon and Quantitative PCR (qPCR)
nitrogenanalyzer.Forisotoperatiomassspectrometry,standard Trichodesmium sp. nifH gene copies were quantified using a
curves were performed with each sample run to determine N TaqMan probe qPCR analysis as described in Moisander et al.
mass.Samples were run onlywhenthestandardcurves hadR2 (2012).Noothercyanobacterialdiazotrophsweredetectedinany
values >0.99. At masses >4.7µg N, the precision for the atom ofthemesocosmbags.TheqPCRreactionwas10µLinvolume,
%15Nmeasurementwas<0.0001%,basedondailycalibrations contained 1 X Premix Ex Taq (Takara), 1µL genomic DNA or
made in association with sample runs and with calibrations RNA,0.5µMreverseandforwardprimersand0.25µMprobes
averagedmadethroughoutseveralyears.Formostoftheresults (labledwith5′-FAMand3′-TAMRA).TheqPCRreactionswere
reported here, the masses were >4.7µg N. However, samples run in triplicate using the Applied Biosystems’ 7500 Fast Real-
with <4.7µg N were only used if the precision was 0.0001% Time PCR System (Applied Biosystems, CA, USA) (Moisander
for that sample run. Standard masses ranged between 1.2 and et al., 2012). The linear regression r2 value of the standard
100µg N. In addition to the daily standard curves, reference curve was 0.99 for all the reactions and the efficiency of the
standardsandstandardsprocessedassampleswererunevery6 qPCRreactionwas101%.Thedetectionlimitwas10nifH gene
to8samples. copiesL−1.
Sequences Quality Control and Analysis
DNA Sample Collection and Extraction The sequencing quality control and analysis were conducted
Seawater (3–8L) was filtered through GF/D glass microfiber with Mothur (Schloss et al., 2009). Low quality sequences were
filters (2.7µm, Whatman International Ltd.) and 0.2µm pore- removed, including short sequences (<300 bases in length),
sizedpolyethersulfonemembranefilters(Supor-200,PallCorp.) ambiguous base-containing sequences and chimeric sequences.
usingaperistalticpumpforadurationof40min.Totalgenomic The trimmed sequences were de-noised with 0.01 sigma value
DNA was recovered from biomass left on the membrane filters in order to reduce the effects of the PCR bias and were
using PureLink Genomic DNA Kits(Invitrogen, Carlsbad,CA) then aligned with the nifH DNA database of the Ribosomal
(Kongetal.(2013).ExtractedDNAwaselutedinto50–60µlof Database Project (Wang et al., 2013). The distances between
TE buffer (Tris and EDTA) and stored at −20◦C until further these high quality DNA sequences were then calculated and
analysis. clustered at 95% similarity. Based on this similarity clustering,
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Rahavetal. ImpactofAerosolsonN2-Fixation
operational taxonomic units (OTUs), representative sequences within the range previously reported for the Cretan Sea
of each OTU, the Shannon diversity index, and the Chao (Ignatiades et al., 2002; Tsiola et al., 2016), with Synechococcus
richness estimator were generated using Mothur. The OTUs the predominant autotrophic picoplankton (5.8 ± 0.2µg C
that contained more than 10 sequences were selected for the L−1), followed by pico-eukaryotes (0.6 ± 0.2µg C L−1). The
above analysis, whereas the rest of the OTUs were grouped heterotrophic bacterial biomass was higher than that of the
as “other” species. Note that the rarefaction curves of our autotrophic bacterioplankton (8.5 ± 0.2µg C L−1), although
samples did not reach a plateau (not shown), suggesting that it was still at the lower end of that reported for oligotrophic
the sequencing depth of our study was insufficient to look oceans (Cho and Azam, 1988 and see Figure 3 in Herut
at rare diazotrophic species. We therefore set a cutoff of 10 etal.,thisissue).Thesurfaceprimaryandbacterialproductivity
sequences to filter the less abundant OTUs (e.g., Farnelid rates (PP and BP) were also low (393 ± 46ng C L−1 h−1
et al., 2013). The OTU representative DNA sequences were for PP and 13.7 ± 5.5 ng C L−1 h−1 for BP), and were
translatedintoaminoacid(aa)sequencesusingtheFrameBOT consistent with previous studies performed in the Cretan Sea
pipeline in which the frameshift errors were corrected (Wang (Gotsis-Skretas et al., 1999; Psarra et al., 2000; Tsiola et al.,
et al., 2013). The OTU representative sequences were used 2016).
to search the protein sequence database in the National Dinitrogen (N ) fixation rates, measured using the enriched
2
Center of Biotechnology Information (NCBI) via the protein seawater method (Mohr et al., 2010), were low (0.22 ±
BLAST webpage (McGinnis and Madden, 2004). The OTU 0.05nmol N L−1 d−1), and similar to the rates measured
representativesequencesandtheaffiliatedreferenceswereused the previous spring in surface waters in the Levantine Basin
to construct a Neighbor-joining phylogenetic tree (p-distance) using identical methodology (Rahav et al., 2013a). Two days
with MEGA 6.0 (Tamura et al., 2013). The OTUs affiliated prior to the experiment, N fixation contributed 5.4 ± 1.7%
2
with the same reference sequences were grouped and these to PP (Table2), a slightly higher contribution than those
groupswerethennamedwiththespeciesnameofthereference estimated during the previous spring (∼2%, Rahav et al.,
sequences. To display the diazotrophic community structures, 2013a) and summer (∼0.5–2%, Yogev et al., 2011; Rahav
therelativeabundancesofthesegroupswerecalculated.Forthe et al., 2013b) in the Eastern Mediterranean Sea. We postulate
relationships between the samples, the samples were clustered that this may have been due to the relatively high number
with a Thetayc calculator using an Unweighted Pair Group of nifH gene copies observed from the non-filamentous
Method with an Arithmetic Mean (UPGMA) algorithm using photoautotrophic cyanobacteria Trichodesmium (>80,000 gene
Mothur.SequencedatawasdepositedinNCBIunderaccession copies L−1, Table2) found in the Cretan water prior to the
numberSRP075730. experiment.Todate,onlyonesporadicbloomofTrichodesmium
has been recorded in the EMS (Spatharis et al., 2012), and the
Statistical Analysis
reason for its rare occurrence in this warm, N-impoverished
Data are displayed as averages; error bars signify one standard environmentisunknown(Berman-FrankandRahav,2012).Our
deviation (n = 3). A repeated measures analysis of variance findings here combined with the published literature of N in
2
(ANOVA) was used to compare differences between the the Eastern Mediterranean Sea (e.g., Ibello et al., 2010; Bonnet
control and the SD or A mesocosm treatments. Prior analyses, et al., 2011; Yogev et al., 2011; Rahav et al., 2013a,b,c; Raveh
the ANOVA assumptions, namely the normally and the et al., 2015; Rahav et al., 2016a) suggest patchy spatial and
heterogeneityofvariancesofthedata,wereexamined.Atselected temporal occurrence of cyanobacterial (and other) diazotrophs
time-points throughout the experiment, a one-way ANOVA withvariablecontributiontoPPintheCretanSea.
followed by Tukey’s post-hoc test was applied (P < 0.05).
The relationship between the N fixation and the autotrophic
2
The Response of Diazotrophy to Saharan
and heterotrophic variables was determined with a Pearson
correlationtest(n=3,P<0.05).Alltestswereperformedusing Dust and Mixed Aerosol Amendments
theXLSTATsoftware. TheadditionofSaharandust(SD)triggeredarapid(30hafter
enrichment) response, with a ∼2-fold increase in N fixation
2
RESULTS AND DISCUSSION compared to the unamended controls (0.42 ± 0.08 and 0.24 ±
0.04nmolNL−1 d−1,respectively,one-wayANOVA,P=0.02)
Characteristics of Cretan Sea Surface
(Figure1).AtT48,N fixationratesdeclinedinallmesocosms,
2
Water yet remained higher in the SD and A mesocosms than in the
The surface Cretan Sea water used for the mesocosm study controls (Figure1). This decline in all mesocosms may suggest
exhibited typical low-nutrient, low-chlorophyll, oligotrophic a weak, yet notable, bottle effect imposed by the mesocosm
Eastern Mediterranean Sea characteristics (Table2; Pitta et al., bags (Calvo-Díaz et al., 2011), low % enrichment of the
2016).Thedissolvedinorganicnitrite+nitrate,ammonium,and enrichedseawaterusedfortheN fixationmeasurementsatthis
2
phosphorus (P) concentrations were close to their detection specifictimepoint,orcompetitionbetweentheambientsurface
limits (132 ± 27, 131± 60, and 13 ± 4nM, respectively), microbialpopulationsanddiazotrophsthatprecludediazotrophs
resulting in a high N:P ratio (∼20:1). Correspondingly, low from thriving. The marked and significant differences between
surface chlorophyll-a was measured prior to any addition thetreatmentsthroughoutmostsamplingssuggestthatevenifan
(0.06 ± 0.01µg L−1). The picophytoplankton biomass was experimentalartifactoccurredatT48,itsimpactonthemeasured
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Rahavetal. ImpactofAerosolsonN2-Fixation
The short-term enhancement of N fixation rates measured
2
after the addition of SD or A were similar to those observed
previously in mesocosm experiments amended with a Saharan
dust analog performed in the Western Mediterranean Sea
(WMS) during the summer of 2008 and 2010 (2–5.3-fold and
4–6 days, Ridame et al., 2013). This may suggest common
limitingfactorsfordiazotrophyinthesetwocontrastingsystems.
Elementsleachedoffdustcanintroduceothernutrientsandtrace
elements to the surface water in different quantities, chemical
forms, concentrations and stoichiometry, depending on the
dust/aerosolorigin(Léonetal.,2015).Inaddition,atmospheric
deposition can be a source of viable diazotrophic microbes as
demonstratedfromaerosolsthatoriginatedintheSaharadesert,
collected in Israel, and dissolved in seawater from the east
Mediterranean Sea (Rahav et al., 2016b). N fixation by these
2
airborne diazotrophs delivered with atmospheric deposition
comprised ∼10–15% (∼0.03nmol N L−1 d−1) of the “typical-
FIGURE1|TemporalchangesinvolumetricN2fixationratesduring average” rates measured in the EMS (0.10–0.20nmol N L−1
themesocosmenrichmentexperiments.Dataareaveragesof3replicate d−1),suggestingthatatmosphericdepositioncansupplynotonly
bottlespertreatmentofthecontrol(gray),SD(white)andA(black) nutrients and trace metals to stimulate in situ N fixation but
2
mesocosmsduringMay2012.
may potentially deliver diazotrophs (Rahav et al., 2016b). Yet,
theroleofairbornediazotrophsshouldbeextensivelystudiedin
theMediterraneanSeaaswellasinothermarineenvironments
rateswasweakoverall.After78h,N fixationdeclinedinbothSD exposed to high atmospheric depositions such as the North
2
treatmentsandcontrolincubations.Yet,N fixationremained4 Atlantic Ocean and the China Sea. In the present study, we
2
timeshigherintheSDtreatmentsrelativetothecontrols(∼0.25 did not determine the contribution of diazotrophs or other
± 0.04 and ∼0.07 ± 0.01nmol N L−1 d−1, respectively, one- bacteriaoriginatingfromourSDandAadditions(i.e.,measure
way ANOVA, P = 0.004). By day 5 (120h), N fixation rates N fixation and BP, PP in SD or A added to sterile seawater).
2 2
in both SD treatments and control incubations were near the Other factors that regulating diazotrophs and N fixation rates
2
limit of analytical detection (∼0.10nmol N L−1 d−1, one-way includetop–downeffectsbygrazing(Stukeletal.,2014),viralcell
ANOVA, P > 0.05, Figure1). Similarly, the mixed aerosol (A) lysis (Hewson et al., 2009), competition with non-diazotrophic
additionledtoanoverallincreaseinN fixationratesintheshort primary producers such as Prochlorococcus (Moisander et al.,
2
term(30hpostaddition,Figure1),althoughtheresponseswere 2012)orbacterialheterotrophs(Thingstadetal.,2005),andother
moderate (∼1.5–2-fold higher rates in treatment incubations nutritional constraints such as carbon for heterotrophs (Rahav
relative to controls) and differences between treatments and et al., 2016a) or vitamin B12 (Bonnet et al., 2010) may further
controls were significant only at the 2 days time point. Similar limit production. Top–down grazing of diazotrophs probably
to the SD treatments, N fixation rates in the A treatments did not greatly impact the diazotrophic populations in our
2
decreased to near the limits of analytical detection and were experiments.Zooplanktonabundancedidnotvarysignificantly
not significantly different from controls after 144h (Figure1). between the mesocosms (M. Lou personal communication).
The observed changes in N fixation following the SD or A Specifically, no known diazotroph grazers such as Macrosetella
2
addition can be explained by the enhanced concentrations of gracilis that feed on Trichodesmium cells were found in our
essential (and limiting in the unamended seawater) nutrients mesocosms nor observed in the few surveys performed in the
from the additives such as P (2–3nmol P per mg dust/aerosol, EMS (Kimor and Wood, 1975; Zakaria, 2015). In contrary, we
Herutetal.,thisissue).ItisalsopossiblethatleachedFeinthe hypothesizethatairbornemicrobesorvirusescanbeimportant
SD mesocosms (net change of ∼6nM in dissolved Fe 3h after inregulatingsurfaceN fixationintheMediterraneanSea.Such
2
SDaddition),andtheinsignificantchangeintheAmesocosms interactions may occur especially in light of the high amount
(Herut et al., this issue) could explain the observed increase in of aerosols deposited to the surface waters of Mediterranean
N fixation.DespitetheincreaseinN fixationratesinthe30– Sea (Guerzoni et al., 1999), which potentially brings ∼107
2 2
48hfollowingtheSDorAadditions,absoluteN fixationrates viruses per m3 of air (Prospero et al., 2005; Womack et al.,
2
remainedlowoverallthroughouttheexperiment(Figure1).This 2010; Polymenakou, 2012). Yet, examination of these factors
couldbeduetolimitedabundancesofdiazotrophsintheinitial was beyond the scope of the present study and warrants more
conditions so that overall rates remained low. Furthermore, research.
after the initial enhanced response of the diazotrophs to the Both heterotrophic and cyanobacterial diazotrophs were
added dust- or aerosol-borne nutrients and/or trace elements, present in this mesocosm study (Figure2) with the Shannon
competition by more efficient non-diazotrophic phytoplankton diversityindexrangingbetween0.7and2.5throughouttheentire
or bacteria could have maintained low overall N fixation experiment (Figure2). Based on the relative abundance of the
2
rates. nifH OTUs, most diazotrophs present in the original Cretan
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Rahavetal. ImpactofAerosolsonN2-Fixation
FIGURE2|Aneighbor-joiningphylogentictreeconstructedwithnifHgeneaminoacidsequences.Samplesweretaken2dayspriortotheadditionofthe
SDorAaddition.Thedifferently-coloredsequencesarerepresentativesequencesoftheOTUsrevealedinthisstudy.ThesequencesfallintoClusterIproteobacteria
(blue),ClusterIII(yellow),andcyanobacteria(green).Theaccessionnumbersofeachreferencesequenceareshowninfrontofthesequencenames.
SeawatersamplesgroupedintoknownnifH clusters(Figure3; Novosphingobium, Azorhizobium in the SD treatments, and
ChienandZinder,1996;Zehretal.,2003).After2days(48h),the Burkholderia in the A amended mesocosms (Figure3). By
diazotrophcommunityinthecontroltreatmentswasdominated the end of the experiment (144h), the Shannon diversity
by Sideroxydans (30%) and Acrobacter (20%), whereas in the dropped to 0.5–1.5. One explanation for this convergence
SD and A treatments Trichodesmium (68%) and Pseudomonas in microbial populations is the potential of those OTUs to
(41%) respectively dominated the diazotroph populations. At successfully utilize heavy metal concentrations as those found
theconclusionoftheexperiment,thediazotrophiccommunities in the SD and A additions (see Table 4 in Herut et al., this
in the control mesocosms had shifted to Azorhizobium and issue).
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Rahavetal. ImpactofAerosolsonN2-Fixation
While some cyanobacteria, including Trichodesmium, We assumed that dark incubations mainly reflect the activity
were present, most nifH OTUs retrieved in this experiment of heterotrophic diazotrophs that do not require light energy
belonged to Proteobacteria. The overall predominance of for N fixation, and estimated the heterotrophic contribution
2
non-cyanobacterial heterotrophic bacteria over autotrophic to N fixation by comparing the dark incubations to light
2
diazotrophs, highlights the importance of heterotrophic bottles incubated under the ambient surface irradiance.
diazotrophy in the EMS waters as recorded in the Levantine When we compared our light vs. dark bottle incubations,
Basin (Man-Aharonovich et al., 2007; Yogev et al., 2011; we found that the light:dark N fixation ratio in the
2
Rahav et al., 2013c) and also across the Mediterranean control mesocosms was usually ∼1 (average 1.7 ± 1.6,
(Benavidesetal.,2016).Agreatercontributionofheterotrophic Table3), suggesting N was fixed by both autotrophic and
2
diazotrophs is now recognized also from many other heterotrophic diazotrophs. Enrichment by SD or A caused
oceanic environments (reviewed in Riemann et al., 2010) the light:dark ratio to decline (an average of 0.8 ± 0.7 for
including the Red Sea (Foster et al., 2009; Rahav et al., SD and 0.8 ± 0.3 for A, Table3), implying that the aerosol
2015), the western North Atlantic Ocean (Mulholland additions disproportionately stimulated heterotrophic N
2
et al., 2012) and the English Channel (Rees et al., 2009, fixation. Similar comparisons between ambient light and
2016). dark bottle N fixation rates showed the same trend in
2
To further estimate the role of heterotrophic diazotrophs the Mediterranean (also in the spring) and the Gulf of
in our system, we simultaneously incubated light and dark Aqaba/Northern Red Sea (in summertime), highlighting
4.6 L bottles (microcosms) from each treatment (Table3). the important role of heterotrophs in fixing dinitrogen in
FIGURE3|Therelativeabundance(%)ofthenifHDNAphyladuringthemesocosmenrichmentexperimentofMay2012.
TABLE3|TheN2fixationratesofCretansub-surfacewaterinexperimentalmesocosms(controls,+SD,+A),forbottlesincubatedunderambient
lighting(L)andincompletedarkness(D)for24h.
Timeafterenrichment(h) Incubationunderambientlight Incubationundercompletedark Light:darkN2fixationratio
Control SD A Control SD A Control SD A
0 0.22±0.01 0.05±0.00 4.4
30 0.24±0.04 0.42±0.08 0.31±0.06 0.17±0.09 0.22±0.06 0.28±0.02 1.4 1.9 1.1
48 0.07±0.02 0.28±0.02 0.15±0.07 N.A N.A N.A N.A N.A N.A
72 0.07±0.01 0.25±0.04 0.16±0.03 0.05±0.01 0.60±0.02 0.34±0.08 1.3 0.4 0.5
120 0.10±0.07 0.14±0.06 0.15±0.09 0.44±0.20 0.38±0.01 0.18±0.13 0.2 0.4 0.8
144 0.07±0.00 0.15±0.05 0.02±0.00 0.07±0.00 0.29±0.03 0.05±0.01 1.0 0.5 0.7
Average 0.14±0.08 0.25±0.09 0.18±0.09 0.16±0.16 0.37±0.16 0.21±0.13 1.7±1.6 0.8±0.7 0.8±0.3
Valuesareaveragesof3independentreplicates.Thetime0measurementisanaverageof3randomlypickedmesocosms.N.A,notavailable.
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Rahavetal. ImpactofAerosolsonN2-Fixation
ultra-oligotrophic systems (Bonnet et al., 2013; Rahav et al., (Figure4). These estimations (2–8%) are in line with other
2013a,2015). estimatesfromtheMediterraneanSea(Garciaetal.,2006;Bonnet
et al., 2011; Yogev et al., 2011; Rahav et al., 2013b). However,
The Contribution of N Fixation to Primary
2 thisisnotsurprisingasheterotrophicdiazotrophscomprisedthe
Productivity in the Cretan Sea during bulk of the retrieved nifH OTUs and the dark N fixation was
2
Spring generally higher than the rates measured under ambient light
(Table3 and discussion above). The positive coupling between
To estimate the contribution of N fixation to primary
2
N fixationandbacterialabundance(BA)orbacterialproduction
productivity, we calculated the contribution of the fixed N 2
(BP),whilenodistinctpatternwasobservedwithTrichodesmium
(as particulate organic nitrogen, PON) to the total particulate
nifHgeneexpression(Figure5),furthersupportstheimportance
organic carbon (POC) from the mesocosms. An average
ofheterotrophicbacterialdiazotrophsinthissystem(Rahavetal.,
POC:PONratioof∼8concurswithpreviousmeasurementsfrom
2013a,2015).
theopen-watersoftheLevantinebasin(Yogevetal.,2011;Rahav
Highercontributionsofdiazotrophstoprimaryproductivity
etal.,2013b).Wesubsequentlyusedthisaveragetocalculatethe
are typically found where filamentous cyanobacterial N -fixers
percentcontributionofN fixationtoPP.Ourresultsshowthat 2
2
N fixation contributed 2–4% (P > 0.05) of PP following the predominate.Forexample,thecontributionofTrichodesmiumto
2
PPwas11.6%intheouterSanyaBayoftheChinaSea(Dongetal.,
mixedaerosoladditionsand3–8%(P=0.04)afterSDadditions
2008),22and44%intheequatorialAtlanticandSouthAtlantic
Gyre,respectively(Fernándezetal.,2010),and47%ofthetotal
depth-integrated PP in the North Atlantic Ocean (Carpenter
etal.,2004).
IncaseswhereEMSsurfacewaterreceivesSaharandustinputs
(as tested here), Trichodesmium-derived PP may be enhanced
if there is a seed population that can respond to the addition.
Yet, as previously observed, the diazotrophs that characterize
the EMS are predominantly small-sized bacterial phylotypes
(Figure3;Man-Aharonovichetal.,2007;Yogevetal.,2011),and
Trichodesmiumisuncommon(Yogevetal.,2011;Spatharisetal.,
2012).
Finally,ourresultsdemonstratethatintensedustandaerosol
deposition events (as frequently occur in the Mediterranean
Sea, Herut et al., 2002; Bonnet and Guieu, 2006) may
potentially fuel diazotrophy and alter the composition of
diazotrophic communities. Furthermore, these depositions to
the Mediterranean Sea can also enrich surface waters with
bioavailableNsources(Carboetal.,2005;Herutetal.,2005;and
seeTableS1inRahavetal.,2016c).ThehigherNconcentrations
FIGURE4|ThecontributionofN2fixationtoprimaryproductivity(PP) canstimulatenewproductionandinducechangesincommunity
inthecontrol(gray),SD(white),andA(black)mesocosms.
structuresuchasashiftfrompopulationsofpicophytoplankton
FIGURE5|TherelationshipbetweenN2fixationand(A)bacterialabundance;BA,(B)bacterialproduction;BP,and(C)TrichodesmiumnifHcopies,in
thefirst144hfollowingtheSaharandust(SD)andmixedaerosol(A)enrichments.
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