NitrogenNitrogen FixationFixation inin thethe EasternEastern MediterraneanMediterranean SeaSea

Tali Yogev, Dikla Aharonovich, Edo Bar Zeev, Oded Béjà, Ilana Berman Frank.

December 2006 Marine cycle

(Arrigo, 2005) N inventory: *Exogenous – Terrestrial - rivrine input,

Atmospheric –N2 fixation, dust & rains *Endogenous – Lateral advection Biological (BNF) The assimilation of dinitrogen by microbial reduction to ammonia

+ - N2 + 16MgATP + 8H +8e

Nitrogenase

2NH3 + H2 + 16MgADP + 16 Pi Diversity of N2 fixing microorganisms : Prokaryotic including & Archaea

Trichodesmium spp

I. Berman

O. Levitan Cyanothece spp. Synechocystis spp.

Richelia Rhizosoleni a jpg.synechocystis/cyanoperso.orange.fr jpg.rhizosolenia/picswww.soes.soton.ac.uk www.uni-kl.de Global estimates of N Fixation are ~ 240 Tg N y-1 Marine global contributes ~ 100-200 Tg N y-1

Trichodesmium spp.

Richlia intracellularis symbiotic Rhizosolenia

Photo by : P. Lundgren Richelia Eastern

www.unipv.it Semi-enclosed ecosystem Oligotrophic Western & eastern basin Lake of denitrification Unique circulation

Depleted in P, NO3 Anthropogenic pressures One of unique characteristics of the Mediterranean is the extremely high N:P ratios found (Herut et al. 1999)

~ 28:1 Levantine basin ~ 24:1 Eastern basin ~ 22:1 Western basin

High N-fixation has been proposed as a MECHANISM resulting in these unusual N:P

(Bethoux et al. 1986, 2002; Ribera D’Alcala et al., 2003, Sarmiento et al., 1988) Stable-isotopic signatures as indicators of N- source

e

14N 15N

N fixation generates new 14N bioavailable nitrogen

15N:14N (δ15N ) = 4.8-5.0 ‰ in the worlds oceans

Low δ15N - 2.4 ‰ suggests N fixation process The case against N-fixation

1. All N:P inputs to the system are significantly in excess of 16:1(54:1) Does not require N fixation or P removal by Saharan dust

2. Lack of denitrification in the sediment or pelagic water Oxygen ~ 70% saturation (Kress et al. 2001)

3. N budget (input vs. output at the straits of Sicily) balances In – 182*109 mol y-1 Out - 179*109 mol y-1

Thus - N fixation is an insignificant process in this system

(Krom et al. 2004) The case for high N-fixation in the Mediterranean

1. N:P >>> Redfield for deep waters, and also for DIN/DIP, PON/POP, DON/DOP in nutrient-poor surface waters

2. Isotopic signatures indicate N-fixation contributes ~ 46% – 70% (Sachs and Repeta 1999) or up to 90% (Pantoja 2002) of new nitrogen to the eastern basin

3.Nutrient budgets do not add up – need N-fixation in basin to balance net loss of N at Gibraltar Straits (Bethoux 1986,1992)

4. High Fe input through dust events would alleviate any Fe limitation and increase potential for N-fixation Research Objectives

Measure and quantify spatial and temporal contribution of N2 fixation to the eastern Mediterranean sea (Tali).

Identify the pelagic organisms responsible for fixation of atmospheric N2 in the eastern Mediterranean sea and determine their spatial and temporal abundance.

a. Molecular identification – expression of nif (Dikla)

b. Characterization of symbiotic N2 fixing associations (Edo). 34030 E 35O00 E 32030 N

Water depth : 900-1000m 400 -500m 200m

Measured Parameters: * CTD (Temp, Salinity, Depth, Fluorometer) (Tali)

* Nutrients concentration - PO4, NO2, NO3, NH4 and Si(OH)4. (Nurit & Barak) * Chlorophyll a concentrations (Tali) * Photophysiological characteristics of PP - Fast repetition rate fluorometry (Edo) 15 * N2 Fixed (AR , N2) (Tali & Margie Mullholland) * C:N (Tali) * δ15N, δ13C (Tali & Margie Mullholland) * Flow cytometry (FACS) identification of Pico-phytoplankton (Tali) * Molecular identification (Dikla & Edo) * Microscopical identification (Edo & Tali) * Symbiotic N-fixing associations (Edo) CTD data

Shallow water Station Deep water Station Depth (m) Apr -05 Apr Oct -06 Oct -05 Jul - 06 Oct -06 Jan -06 -06 Feb Mar -06 -06 Apr May -06 Jun -06 Jul - 06 -Aug 06 -06 Sep Jul - 05 -05 Sep Oct -05 Nov -05 -05 Sep May -05 -05 Aug Nov -05 Jan -06 -06 Feb Jun -05 Jul - 05 Aug -05 Aug Mar -06 -05 Apr May -06 Jan -06 -Aug 06 -06 Sep May -05 Jun -05 Dec -05 Dec -05 a ƒ Characterization of Pico-phytoplankton populations:

1. Flow cytometer identification (FACS) Discriminate different populations and quantify the abundance, on the basis of their light scatter signals and the fluorescence emitted from individual natural pigments

FACS does not identify specifically but can distinguish between 2 groups of prokaryotes on basis of photosynthetic pigments

• Prochlorococcus-like - divinyl chl.b,a., 0.5 µm • Synechococcus-like - phycoerythrin, 1-2 µm • Small eukaryotic algae (<10 µm) Phytoplankton population distribution (FACS). -1 Shallow station0 20000 40000 60000Cell no. ml Deep station 0 20000 40000 60000 80000

0 0 50 50 100 150 May-06 100 150 200 200 Depth (m)Depth 250 250 300 (m) Depth 300 0 20000 40000 60000 0 20000 40000 60000 80000

0 0 50 50 0 20000 40000 60000 80000 Jun-06 100 0 100 150 50 150 200 200

Depth (m) Depth 100 250150 250 300200 300 Depth (m) 250 0 20000 40000 60000 80000 3000 20000 40000 60000

0 0 Sep-06

50 50

100 100

150 150 200

Depth 200

250 (m) Depth 250 Eukaryotic 300 300 Synechococcus Prochlorococcus Detection of the in field populations.

The enzyme is tagged with - Alexa 488 probe.

10µ

Bright-field : e.H

Chlorophyll a. filter : g.j (ex:450nm, em:680nm) FITC ( filter ) : f.i (ex:488nm, em:520nm).

Photo by Edo Bar Zeev Integrated values of N fixation, (Stations east of Herzelia, 2006)

400 -1

d 350 -2 300 250 200 150 100 50 N fixation µmol N m 0 Nov Apr Jul Oct Mar Jun Aug Feb 0123456789101112Sep Tb01Deep Station May Time Tb04Shallow Station

SD= +/- 10% Integrated values of N fixation, (Stations east of Herzelia, 2006)

Deep water Station Shallow water Station N2 fixed Tb01 Tb04N2 fixed Tb04 -1 d -2 400 400 -1 -1 350 350 d 300 d -2

-2 300 250 250 200 200 150 150 nm m ol

100 nm m ol 100 50 50 N fixation µmol N m N fixation µmol N 0 0

234567891011

mon. mon. 234567891011Time Time

>100 100>10 10>1 Total No data SD= +/- 10% Total N2 fixed (AR) vs. month Deep station Shallow station N fixation nmol N l-1 d-1

00.511.522.533.500.511.522.533.5

5 5

No data No data

40-80 50-100

Depth (m) Depth 100-150 Apr-05 81-130 May-05 Jun-05 150-195 Feb-06 No data No data Mar-06 Apr-06 May-06 150- Jun-06 Sep-06 Oct-06 Nov-06 Correlation between AR and Synechococcus-like cells

Cell no. ml-1

0 10000 20000 30000 40000 50000 60000 70000 0 20000 40000 60000 80000 0 0

50 50

100 100

150 150

200 200 May Sep 250 250

300 0123300 0123

0 10000 20000 30000 40000 50000 60000 70000 0 20000 40000 60000 80000 0 0

Depth (m) Depth 50 50

100 100

150 150 200 200 Jun Oct 250 250

300 0123300

0123 Synechococcus nmol N L-1 d-1 Prochlorococcus Measurements values of biological nitrogen fixation

• East of Herzelia– Range 0.4 -3.05 nmol N L-1d-1 (maximal value Apr-05 & May-06)

• Eastern Atlantic surface ocean – 3.1 nmol N L-1d-1 (October) (Voos et al. 2004)

• Western Mediterranean (north western Mediterranean sea) 4 nmol N L-1d-1 (April), 7.5 nmol N L-1d-1 (August) (Sandroni et al. 2005)

• Warm-core eddy (Cyprus) – 129 nmol N L-1d-1 (May) (Rees et al. 2006) Preliminary conclusions

• N-fixation rates low but measurable range - 0.4 - 3.05 nmol N L-1d-1 Integrated values - 130-357 µmol N m-2 d-1 *Dust/nutrient enrichment experiments (P & Fe limitation) *Data processing artificially enriched in 15N • Rates not significantly different between 2 stations *Nutrient information • The values show peak in Apr 2005 & May 2006 *Summer information