`="0200`="003DN2 fixation in the Gulf of Aqaba

35 Pages·2013·1.71 MB·English

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BiogeosciencesDiscuss.,10,10327–10361,2013 D
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TPhleisasdeisrceufessritoonthpeapceorrrise/shpaosnbdeinegnfiCunnlaidmlepraartpeeev rOpenieinwBfoGrtifhaevjaoiularnbalel.BiogeosciencesC(lBimGa).te Open per N2 fixation in the Gulf
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p Abstract Introduction
E. Rahav1,*, B. Herut2, M.GRe.oMsucilehnotilfilcanOpd3, B. Voß4, D. Stazic4,GCe.oSscteiegnltiificchOp4, er
4 Instrumentat1ion en Instrumentation en Conclusions References
W. R. Hess , and I. Berman-Frank A A |
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Received:29May2013–Accepted:5JuneO2013–Published:26June2013 O s
Correspondenceto:I.BerOmcaena-Fnr aSnckie(inlacnea.pen [email protected])cean SDcisiceusnsiconespen Acce ionPa InteractiveDiscussion
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Abstract D
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We evaluated the seasonal contribution of heterotrophic and autotrophic diazotrophy s
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to the total dinitrogen (N ) fixation in a representative pelagic station in the northern io 10,10327–10361,2013
2 n
Gulf of Aqaba in early spring when the water column was mixed and during sum- P
a
5 mer under full thermal stratification. N2 fixation rates were low during the mixed pe- per N2 fixation in the Gulf
−1 −1
riod (∼0.1nmolNL d ) and were significantly coupled with both primary and bac- of Aqaba
|
terial productivity. During the stratified period N fixation rates were four-fold higher
2
−1 −1 D E.Rahavetal.
(∼0.4nmolNL d ) and were significantly correlated solely with bacterial productiv-
is
ity. Furthermore, while experimental enrichment of seawater by phosphorus (P) en- cu
s
hanced bacterial productivity and N fixation rates during both seasons primary pro- s
10 2 io
ductivity was stimulated by P only in the early spring. Metatranscriptomic analyses n TitlePage
P
from the stratified period identified the major diazotrophic contributors as related to a
p Abstract Introduction
heterotrophic prokaryotes from the Euryarchaeota and Desulfobacterales (Deltapro- er
teobacteria) or Chlorobiales (Chlorobia). Moreover, during this season, experimental Conclusions References
|
amendmentstoseawaterapplyingacombinationofthephotosyntheticinhibitor3-(3,4-
15 D Tables Figures
dichlorophenyl)-1,1-dimethylurea(DCMU)andamixtureofaminoacidsincreasedboth is
c
bacterialproductivityandN fixationrates.OurfindingsfromthenorthernGulfofAqaba u
2 s J I
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indicateashiftinthediazotrophiccommunityfromphototrophicandheterotrophicpop- io
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ulations, including small blooms of the cyanobacterium Trichodesmium, in winter/early J I
P
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20 spring, to predominantly heterotrophic diazotrophs in summer that may be both P and p
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carbon limited as the additions of P and amino acids illustrated. r
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1 Introduction is
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The Gulf of Aqaba, located at the tip of the Red Sea, is surrounded by land on three s
io InteractiveDiscussion
sidesandcharacterizedbyathermohalinecirculationpatterncausedbyhighevapora- n
−1 P
25 tion rates (1cmd , Wolf-Vecht et al., 1992; Biton and Gildor, 2011). The Gulf hydrol- ap
ogy is characterized by a strong seasonal variability mainly due to deep winter mixing e
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(>300m,LabiosaandArrigo,2003)andstrongsummerstratification(Manasrahetal., D
is
2007). Stratification enhances oligotrophy with surface inorganic nutrients concentra- c BGD
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tions depleted during summer, with nitrogen (N) and phosphorus (P) levels usually s
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close to their detection limits (Fuller et al., 2005; MacKey et al., 2009; Meeder et al., n
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5 2012). During winter, deep vertical mixing advects inorganic nutrients from depth to a
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thesurface,withinorganicPandNreaching∼0.1µMand∼2µM,respectively(Lindell e N fixation in the Gulf
r 2
and Post, 1995). of Aqaba
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The picophytoplankton fraction (<2µm) predominates the phytoplankton popula-
D E.Rahavetal.
tionsinthissystemandiscomprisedmainlyofSynechococcus,Prochlorococcus,and
is
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10 picoeukaryotes(Sommer,2000;MacKeyetal.,2007,2009;Iluzetal.,2009).Thewin- u
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ter deep mixing as well as sporadic events of nutrient inputs during summer (i.e., Sa- s
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haran dust events, Paytan et al., 2009) often induce blooms of larger phytoplankton n TitlePage
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suchasdiatoms(LindellandPost,1995;Mackeyetal.,2007)andTrichodesmiumspp. a
p Abstract Introduction
e
(Post et al., 2002). r
Pelagic dinitrogen (N ) fixation by diazotrophs is an important source of new N in Conclusions References
15 2 |
oligotrophic marine systems, converting nitrogen from the otherwise unavailable pool
D Tables Figures
ofatmosphericN2 toammonia(Falkowski,1997).N2 fixationoccursinnitratedepleted isc
surface photic layers of tropical oceans where diazotrophic phototrophs such as Tri- u
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chodesmium spp. (Capone et al., 2005), unicellular cyanobacteria (Zehr et al., 2001; io
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Montoya et al., 2004), and, also non-photosynthetic diazotrophic bacterioplankton (re- J I
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viewed in Riemann et al., 2010) are predominantly responsible for this process. p
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In the Gulf of Aqaba, several groups of diazotrophs have been identified based on r
theiraminoacidsequences,includingαandγ proteobacteria,aswellasT.erythraeum | FullScreen/Esc
andtheunicellularcyanobacterialgroupA(Fosteretal.,2009).Toourknowledge,only
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threestudieswerepublishedonN fixationfromtheGulfofAqaba:oneshowingactual is
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ratesusingthe N assimilationtechnique(Fosteretal.,2009);anotherbasedonindi- s
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rect measurements of acetylene reduction rates for concentrated Trichodesmium spp. io InteractiveDiscussion
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colonies (Post et al., 2002); and the third based on isotopic δ15N estimations (Aberle P
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et al., 2010). During the stratified summer (September), Foster et al. (2009) measured pe
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−1 −1 D
N fixationratesrangingfromundetectableto1.9nmolNL d .Althoughestimations
2 is
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based on isotopic analyses suggested that N fixation plays a minor role during win- u
2 s
ter mixing (Aberle et al., 2010), the highest rates measured (1.9nmolNL−1d−1) were sio 10,10327–10361,2013
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during March, when the water column was mixed (Foster et al., 2009).
P
Inthisstudy,weevaluatedtheseasonalcontributionofheterotrophicandautotrophic a
5 p
e N fixation in the Gulf
diazotrophy to the total dinitrogen fixation in a representative pelagic station in the r 2
of Aqaba
northern Gulf of Aqaba in early spring the water column was mixed and during sum-
|
mer under full thermal stratification. We also experimentally examined the role of het-
D E.Rahavetal.
erotrophic diazotrophy and the potential of P-limitation on diazotrophs in this system. is
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2 Materials and methods n TitlePage
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◦ 0 p Abstract Introduction
WatersampleswerecollectedfromtheR.V.RotenbergatsamplingStationA(29 28 N, e
r
◦ 0
34 55 E) located at the northern tip of the Gulf of Aqaba (Fig. 1) during the mixed win- Conclusions References
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ter (March 2010) and the stratified summer periods (September 2010 and July 2012).
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Samples were collected using 12L Niskin bottles mounted on a rosette equipped
is
15 with a CTD (Seabird 19 Plus) and fluorometer (Turner designs, Cyclops7 for real- cu
s J I
time chlorophyll [Chl a] fluorescence). For metatranscriptome analyses samples were s
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taken from surface and 3 depths within the photic layer during September 2010: n
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60m, the DCM (approximately 100m), and 130m. FiftyL (60m and DCM samples) a
p
or 200L (130m sample) of seawater were pre-filtered through 2µm mesh and 10L er Back Close
aliquots were vacuum filtered on Supor-400 0.45µm filters (Pall. Corp.). Filters were
20 | FullScreen/Esc
immersedinto2mLPGTXbuffer(4.2Mphenolamendedwith6.9%v/vglycerol,5mM
D
8-hydroxyquinoline, 15.6mM Na2EDTA, 0.1M sodium acetate, 0.8M guanidine thio- isc Printer-friendlyVersion
cyanateand0.48Mguanidinehydrochloride(Pintoetal.,2009)andimmediatelyfrozen u
s
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in liquid nitrogen. For all other analyses seawater was dispensed into 4.6L Nalgene io InteractiveDiscussion
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incubation bottles. The filled Nalgene bottles were placed in transparent outdoor in-
25 P
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cubators with continuously flowing seawater to maintain ambient surface-water tem- p
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peratures. The incubators were shaded with neutral density screening to mimic in situ D
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irradiance conditions. c BGD
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2.1 Inorganic nutrients io 10,10327–10361,2013
n
P
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Duplicatewatersampleswerecollectedin15mLacid-washedplasticscintillationvials p
e N fixation in the Gulf
and kept frozen. Nutrients were determined using a segmented flow Technicon Au- r 2
5 of Aqaba
toAnalyser II (AA-II) system as described by Kress and Herut (2001). The precision |
of nitrite+nitrate (NO2+NO3), phosphate (PO4), and silicic acid (Si(OH)4) measure- D E.Rahavetal.
ments were 0.02, 0.003, and 0.06µM, respectively. The limit of detection (2 times the isc
u
standard deviation of the blank) was 0.075µM for NO +NO , 0.008µM for PO , and s
2 3 4 s
10 0.03µM for Si(OH)4. ion TitlePage
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2.2 Chl a extraction p Abstract Introduction
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Conclusions References
Duplicate seawater samples were filtered onto glass fiber filters (25mm Whatman
|
◦
GF/F, ca. 0.7µm in pore size). The filters were stored at −20 C in a dark box until
D Tables Figures
analysis within 2–3 days. Samples were extracted in 5mL of 90% acetone overnight is
c
15 at4◦Cinthedark.ChlaconcentrationsweredeterminedusingaTurnerDesigns(TD- us J I
s
700) fluorometer with a 436nm excitation filter and a 680nm emission filter (Holm et io
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al.,1965).Ablankfilterwasalsostoredin90%acetoneunderthesameconditionsas P J I
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those of the samples. Pure Chl a (Sigma C6144- from Anacystis nidulans) was used p
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to calibrate the measurements. r
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2.3 Dinitrogen (N ) fixation rates
20 2 D
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N fixationratesweremeasuredonfieldsamplesusingthe15N assimilationtechnique cu Printer-friendlyVersion
2 2 s
s
described by Montoya (1996) and Mulholland et al. (2006). Water was added to 4.6L io InteractiveDiscussion
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polycarbonateNalgenebottlesthatweresealedwithseptumtopsandspikedwith9mL
P
of15N2(99%).Bottleswereincubatedfor24hunderambientsurfaceseawatertemper- ap
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aturesandcoveredwithneutraldensityscreeningasdescribedabove.Toterminatethe r
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incubations,waterwasfilteredontopre-combusted25mmGF/Ffilters(450◦Cfor4h), D
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and filtered samples were analyzed on a Europa 20/20 mass spectrometer equipped c BGD
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with an automated N and Carbon (C) analyzer preparation module. N fixation was s
2 io 10,10327–10361,2013
calculated according to Mulholland et al. (2006) using N solubility factors described by n
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5 Weiss (1970). a
p
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r 2
2.4 Primary productivity of Aqaba
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13
Photosynthetic C fixation rates were estimated by determining C uptake (Mulholland D E.Rahavetal.
is
and Bernhardt, 2005). Water samples were placed in clear 4.6L polycarbonate Nal- c
u
gene bottles and amended with highly enriched (99%) NaH13CO (Sigma) to obtain s
3 s
io
10 1% of the ambient dissolved inorganic C and incubated under the same conditions n TitlePage
as for 15N incubations described above. Parallel dark bottles (n=3–5) were also incu- P
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p Abstract Introduction
bated and subtracted from the light bottles to correct for dark C fixation. Incubations e
r
were terminated by immediately filtering the entire contents of incubation bottles onto Conclusions References
◦ ◦ |
pre-combusted 25mm GF/F filters (450 C for 4h). Filters were stored at −20 C and
then dried and pelleted in tin disks before their analysis using the Europa 20/20 mass D Tables Figures
15 is
spectrometer. c
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s
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2.5 Bacterial productivity rates n
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P
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Bacterial production was estimated using the 3H-leucine (Amersham, specific activity: p
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−1 r
160Cimmol ) incorporation method (Simon et al., 1992). Triplicate 1.7mL samples
wereincubatedfor4–8hatinsitutemperaturesinthedark.Killedsamplesoftriplicate | FullScreen/Esc
20
trichloroacetic acid (Sigma) served as controls. One milliliter of high 3H affinity (Ultima D
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Gold)scintillationcocktailwasaddedtosamplesthatweresubsequentlycountedusing c Printer-friendlyVersion
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a TRI-CARB 2100 TR, PACKARD scintillation counter. ss
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2.6 Addition of phosphorus D
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Amendments of orthophosphate (PO ) solution (Sigma) were added into 4.6L poly- s
4 s
carbonate Nalgene bottles when 15N2 was added, bringing the seawater to a final ion 10,10327–10361,2013
concentration of 0.5µM PO . The incubation times were identical to that of the un- P
4 a
p
5 amended controls, and incubations were terminated under the same conditions (see er N2 fixation in the Gulf
above Sect. 2.3). of Aqaba
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2.7 Additionof3-(3,4-dichlorophenyl)-1,1-dimethylureaandaminoacidmixture D E.Rahavetal.
is
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The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), dis- s
s
solved in dimethyl sulfoxide, was added to a final concentration of 50µM in 4.6L poly- ion TitlePage
carbonate Nalgene bottles along with 15N . Clavier and Boucher (1992) reported this P
10 2 a
as the most effective for photosynthetic inhibition by using a minimal concentration of p Abstract Introduction
e
r
DCMU. Furthermore, a mixture of 20 amino acids (Sigma A9906) was added bringing
Conclusions References
the seawater to a final concentration of 500 nM of dissolved organic C (DOC). We hy- |
pothesized that the combination of DCMU and amino acids would promote heterotro- D Tables Figures
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phy over autotrophy (not necessarily diazotrophs) by supplying DOC and dissolved c
15 u
inorganic nitrogen (DON) sources from the amino acids while suppressing photosyn- ss J I
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thesis as DCMU blocks the linear electron flow from PSII to the plastoquinone. n
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2.8 RNA extraction and cDNA library preparation for sequencing p
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Total cellular RNA was extracted from cells on frozen filters following the hot phenol | FullScreen/Esc
20 method (Steglich et al., 2006) and yielded 5–13µg total RNA for each sample. For D
cDNAlibrarypreparationDNAwasremovedfromtotalRNAbyTURBO™DNase(Am- isc Printer-friendlyVersion
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bion, USA) treatment and resulting RNA was depleted in ribosomal RNA using MI- s
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CROBExpressTM (Ambion, USA). cDNA was prepared following the manufacturers’ io InteractiveDiscussion
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recommended protocol. The obtained cDNA libraries of the three depths (60, DCM P
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and130m–seeabove)weremixedandanalysedonanIlluminaHiSeq2000resulting D
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in 2.3 million paired-ends reads of 100nt length for each run. c BGD
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2.9 Bioinformatic analyses io 10,10327–10361,2013
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All 6309 protein sequences with a gene name matching “nif” or with the annotation p
e N fixation in the Gulf
“nitrogenase” were extracted from RefSeq database (www.ncbi.nlm.nih.gov/refseq; r 2
5 of Aqaba
query: “(nif[Gene Name]) OR nitrogenase”; date: 28 February 2013). Using TBlastN, |
sequencing reads matching one of these with an E value ≤1×10−9 were searched D E.Rahavetal.
and extracted. This set of reads was compared against the NCBI nt database using is
c
BlastNatanE-valuecut-offof1×10−5 andbyBlastXatanE valuecut-offof1×10−8. us
s
10 The phylogenetic distribution was analysed with MEGAN 4.70.4 (Huson et al., 2011). ion TitlePage
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p Abstract Introduction
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3 Results r
Conclusions References
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3.1 Seasonal changes of productivity in the water column
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The upper mixed layer of the Gulf of Aqaba showed a clear seasonal pattern be- cu
s J I
tween the stratified (September 2010 and July 2012) and mixed (March 2010) periods s
io
(Fig. 2a). During the latter period, the upper 200m was relatively mixed, and the water n
15 J I
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column during summer was well stratified (Fig. 2a). The average sea surface temper- a
p
ature (SST) was 23◦C in March and 28◦C in July. At a depth of 350m, no seasonal e Back Close
r
temperature effect was observed, and the seawater temperature remained at ∼21◦C.
| FullScreen/Esc
The highest surface (5–20m) Chl a concentration was detected during the winter
−1 −1 D
20 sampling with 0.19µgL relative to a concentration of 0.14µgL during stratification is
(Fig. 2b). The deep chlorophyll maximum (DCM) value differed between periods, with cu Printer-friendlyVersion
s
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the shallowest DCM (∼50m) recorded during March 2010 increasing to 80m in mid- io InteractiveDiscussion
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July2012andreaching100mattheendofthestratifiedperiod(September2010).The
P
ChlaconcentrationinthewinterDCMwas0.25µgL−1,whileduringsummer,theChla ap
−1 e
concentration in the DCM reached 0.33µgL (Fig. 2b). r
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Inorganic nutrient concentrations in the upper 50m were low in March, averaging D
∼0.15µM and 0.01µM for NO +NO and PO , respectively (Fig. 3a, b). During sum- isc BGD
2 3 4 u
mer (stratified water column), NO2+NO3 was close to its detection limit in the upper ssio 10,10327–10361,2013
160m (<0.1µM), whereas PO was scarce in the upper 50m (∼0.01µM; Fig. 3a, b). n
4
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5 The maximal nutrient concentrations were found below 200m for all samplings (not a
p
shown). During March, the N:P ratio (mol:mol) was higher (∼20:1) than the con- e N fixation in the Gulf
r 2
ventional 16:1 “Redfield ratio” (Redfield et al., 1963), except at 40m (N:P of 12:1; of Aqaba
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Fig. 2c), while during summer, the N:P was lower than 16:1 in the upper 200m (∼2
D E.Rahavetal.
to 8:1; Fig. 2c).
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10 Bacterialproductivity(BP)rateswereuniformlylowthroughouttheupper200mdur- cu
ing the mixed period (0.2–0.8µgCL−1d−1), whereas higher BP rates were usually ob- ss
io
tained during the summer samplings (1.2–3.7µgCL−1d−1), except for the upper 10m n TitlePage
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duringJuly2012(Table1).Primaryproductivity(PP)ofthesurfacewatersrangedfrom a
p Abstract Introduction
−1 −1 e
2.4 to 3.1µgCL d in March, whereas during the stratified period, PP was lower, r
ranging from 0.3 to 0.6µgCL−1d−1 (Table 1). PP declined with depth during both the Conclusions References
15 |
mixed and stratified periods, with non-detectable rates at 100m and 200m, respec-
D Tables Figures
tively, where <0.1% of surface irradiance was measured (Table 1). is
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The ratio between bacterial productivity (BP) and primary productivity (BP:PP) pro- u
s J I
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vides a measure of the metabolic status of the environment, i.e., whether the environ- io
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ment is predominantly heterotrophic or autotrophic (Lagaria et al., 2011; Rahav et al., J I
20 P
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2013a). A ratio <1 indicates higher autotrophic fixation of C relative to heterotrophic p
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Cfixation,and,conversely,whenthisratiois>1,thereishigherheterotrophicproduc- r
tion. In this study the BP:PP ratio was usually <1 during March, except for depths | FullScreen/Esc
below the DCM, where it was ∼1 (Table 1). In the stratified periods, the BP:PP ratio
D
was always >1 (Table 1, Fig. 4). is
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N fixation rates were uniformly low at the upper 150m during March 2010 s
2 s
(∼0.1nmolNL−1d−1); however, in the aphotic zone (>200m), slightly higher N fix- io InteractiveDiscussion
2 n
ation rates were recorded (0.2nmolNL−1d−1, Fig. 5). During both samplings of the P
a
−1 −1 p
stratified period, N fixation rates for the surface waters were ∼0.4nmolNL d in e
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−1 −1 D
July, and increased to a maximum of 0.5nmolNL d at 100m depth in September
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2010 (Fig. 5). u
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The relationship between N fixation rates from March 2010 (mixed), July 2012 s
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and September 2010 (stratified) was examined with the corresponding heterotrophic n
P
metabolism (BP) and phototrophic C fixation (PP) (Fig. 6). BP correlated significantly a
5 p
andpositivelywithN2 fixationduringboththemixed(R2=0.98,P=0.003,n=21)and er N2 fixation in the Gulf
stratifiedperiods(R2=0.52,P=0.01,n=21)(Fig.6a).NocorrelationwithPPwasap- of Aqaba
|
parent for the stratified periods (Fig. 6a). In March, PP and N fixation were positively
2 D E.Rahavetal.
(R2=0.84, n=15), although not significantly (P=0.07), correlated (Fig. 6b). is
c
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10 To identify active diazotrophs, metatranscriptome analyses were carried out dur- s
s
ing the fully stratified water column in September 2010. Samples were collected io
n TitlePage
from surface waters, 60m, the DCM, and 130m. The latter three, encompassing the P
a
depths in which maximum fixation rates were measured, were pooled and subjected p Abstract Introduction
e
to analytical metatranscriptomics. We found mRNA reads from two different groups r
Conclusions References
of prokaryotes related to N fixation, none of which were of cyanobacterial origin |
15 2
(Table S1 in the Supplement). One set of reads was closely related to sequences D Tables Figures
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from the Euryarchaeota, more specifically the Methanosarcinales, which matched the c
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nifH, nifD and nifB sequences of organisms such as Methanosarcina mazei strain s J I
s
Gö1 (Fig. 7). The second set of reads was of bacterial origin, indicating the Desul- ion
J I
fobacterales (Deltaproteobacteria) or Chlorobiales (Chlorobia). For several of these P
20 a
p
reads, the top hit in a BlastN search came from the Desulfobacterales but the sec- e Back Close
r
ond hit from the Chlorobiales, or vice versa. For example, the sequence lcl|HWI-
ST1172:64:D1FDDACXX:8:1206:19713:16690_1:N:0:ATCGCG (Table S1 in the Sup- | FullScreen/Esc
plement) has in a BlastN search as top match a stretch in the genome sequence of D
is
a marine sulfate reducer, Desulfobacterium autotrophicum HRM2 (Strittmatter et al., c Printer-friendlyVersion
25 u
2009) with an e value of 4e−14 (76/95 identical residues, 80% ID), whereas the hit ss
io InteractiveDiscussion
rankedsecondisinthegenomesequenceofChlorobiumphaeobacteroidesBS1,with n
an e value of 6e−12 (76/98 identical residues, 78% ID). Sequences (SAR406) point- Pa
p
ing to the green sulfur bacterial phylum, which includes the genus Chlorobium, were e
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