Organophosphonates Fuel Cyanobacterial Blooms in Florida Bay Rafael Guevara1, Amber Enns1, and Joseph N. Boyer1,2

1Southeast Environmental Research Center, OE-148, 2Dept. Earth & Environment, School of Environment, Arts, and Society, Florida International University, Miami, FL 33199 email: rguevara@fiu.edu

Introduction

The increase in cyanobacterial blooms worldwide has generally been attributed to increased nutrient inputs. In the oligotrophic, P-limited Florida Bay, episodic cyanobacterial blooms have been dominated by members of the genus Synechococcus, often without any observed increase in nutrients. It is well known that cyanobacteria can access dissolved organic P (DOP) while eukaryotic phytoplankton cannot. DOP can be present in many forms; the most common are compounds based on phosphate ester bonds (P-O) such as DNA, RNA, and ADP. Another is the phosphonate bond (P-C) as found in plant and animal membranes, as synthetic chelators, peroxide bleach stabilizers, and organophosphate pesticides such as glyphosate (Roundup™). Only bacteria can metabolize phosphonates however many synthetic forms are not readily bioavailable. Inositol-P (phytate) is a special case of ester-P based on 6 carbon ring used by plants as primary P storage compound.

Objective

Our aim was to determine if the cyanobacterial community structure might be responding to differential P sources.

Materials and Methods

Bloom samples were collected from three sites located in the internal-central region of Florida Bay while a cyanobacterial bloom was occurring. The non-bloom samples were taken from three FCE-LTER sites in Florida Bay which are dominated by seagrass [TS/Ph-9 (FB9), TS/Ph-10 (FB21) and TS/Ph-11 (FB27)] (Fig. 1). Three one-liter samples from surface water were collected in each site. Once in the laboratory, three microcosms of 20 ml by each treatment were done (Fig. 2). Treatments were performed with two different concentrations (0.1 mM and 1 mM) of the following compounds: KH2PO4, KNO3, NH4Cl, urea, glycine, guanine, an organophosphonate-P source (2-aminoethylphosphonic acid; AEP) an ester-P (ADP), and inositol-P (phytate). A control with no nutrient addition was added. Samples were incubated in the light for 28 days.

Chl-a concentrations were determined in each microcosm using PAM fluorescence. In those microcosms where a significant increase of Chl-a was observed (p<0.05; one-way ANOVA), cyanobacteria counts and molecular analyses were performed. For both analyses, 20 milliliter of water were collected on a 0.22 µm (pore size) filter. Cyanobacteria counts were done by autofluorescence microscopy. For molecular analyses three samples from bloom areas as well as replicates for each treatment were pooled. DNA was extracted directly from the filter using the MoBio Ultraclean soil DNA extraction kit. Amplification of the cyanobacterial 16S rRNA gene was performed using specific primers (CYA106F and CYA781R). A clone library was generated from the bloom sample and three clones libraries were constructed from FB27 (control, 0.1 mM ADP and 1 mM AEP). For this purpose, PCR products were ligated into pGem-T-easy and transforming them into E. coli JM109 competent cells (Promega) according to the manufacturer’s protocol (Fig 2). The sequences were searched against the GenBank database by BLAST-N. Only sequences with a percentage of sequence identities higher than 90% were included in the phylogenetic analysis.

DNA extraction

PCR Cyano 16S rRNA gen

specific primers (Cya106F and Cya781R)

Microcosm

Water sample filtration

Chl-a (PAM)

Bacterial counts (autofluorescence

microscopy) Molecular techniques

Fig 2. Experimental design.

Cloning

Sequence analysis

bloom sample

non-bloom sample

FB 9 TS/Ph-9

FB 27 TS/Ph-11

FB 21 TS/Ph-10

Fig 1. FCE-LTER sampling ( ), and bloom sampling sites ( ).

Results

1. Chl-a concentrations were significantly higher in samples treated with ADP and AEP (Fig. 3). Also, an increase in Synechococcus-type cells number was observed in these treatments (Fig. 4).

2. Clone libraries showed an increase in number of DNA sequences related to Phormidium sp. and Limnococcus limneticus in microcosms supplemented with ADP. While sequences related to Synechococcus sp. and Limnothrix redekei increased in microcosms amended with ADP as well as in AEP-amended microcosms (Fig 5).

3. Clones related to Synechococcus sp. made up virtually 100% of clones library in bloom sample (n=42) and 71% of AEP treatment (n=31) but only 15% and 16% of clones were Synechococcus sp. in the control (n=34) and ADP treatment (n=31), respectively (Fig 5).

4. Of the Synechococcus sp. recovered from bloom samples, 88% were Synechococcus sp. KORDI-63, 10% Synechococcus sp. WH8101, and 2% Synechococcus sp. KORDI-65. Only a 20% of the sequences from non-bloom sample were related to Synechococcus sp. KORDI-63. The AEP treatment showed a shift in cyanobacterial community structure towards 100% Synechococcus sp. KORDI-63, while ADP treatment changed to 80% Synechococcus sp. KORDI-63 and 20% Synechococcus sp. KORDI-65 (Fig 6 and table 1).

Fig 4. Number of Synechococcus-type cells per ml in Florida Bay water samples under different treatments.

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

Control 0.1mM ADP 1 mM AEP

log

(num

ber

of c

ells

/ml)

Treatments

0

100

200

300

400

500

600

700

800

900

1000

Chl-a

(ug

/L)

treatment

FB9

FB21

FB27

Fig 3. Chl-a concentrations measured from microcosms supplemented with different N and P compounds. Inorganic and organic supplemental substrates were added to provide final concentrations of 0.1 mM. (***) p<0.001, (**) p<0.01, (*) p< 0.05 according to one-way ANOVA.

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*

Fig 6. Relative abundance of clones related to different Synechococcus strains (Genbank accession number)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Control (n=5) 0.1 mM ADP (n=5)

1 mM AEP (n=22)

bloom (n=42)

Synechococcus sp. str. WH8101 (AF001480)

Synechococcus sp. str. KORDI-65 (FJ497728)

S. elongatus str. PCC 7942 (AF132930)

Synechococcus sp. str. KORDI-63 (FJ497740)

Synechococcus sp. str. KORDI-28 (FJ497720)

Table 1. Cyanobacterial clones related to Synechococcus strains. Clone name

Number of clones

Closest cultivable strain using Blast-N analysis Accession no. Similarity

(%) Source

C-03 2 Synechococcus sp. str. KORDI-28 FJ497720 99 Seawater of the East Sea (China) C-07 1 Synechococcus elongatus str. PCC 7942 AF132930 92 Freshwater C-10 1 Synechococcus sp. str. KORDI-28 FJ497720 98 Seawater of the East Sea (China) C-13 1 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea

ADP-04 1 Synechococcus sp. str. KORDI-28 FJ497720 99 Seawater of the East Sea (China) ADP-10 1 Synechococcus sp. str. KORDI-63 FJ497740 92 Seawater of the East China Sea ADP-11 2 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea ADP-12 1 Synechococcus sp. str. KORDI-63 FJ497740 93 Seawater of the East China Sea AEP-03 21 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea AEP-04 1 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea

CyaBloom-01 30 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea CyaBloom-02 1 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea CyaBloom-03 1 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea CyaBloom-04 1 Synechococcus sp. str. KORDI-65 FJ497728 99 Seawater of the East China Sea CyaBloom-05 5 Synechococcus sp. str. KORDI-63 FJ497740 99 Seawater of the East China Sea CyaBloom-06 4 Synechococcus sp. str. WH8101 AF001480 100 Woods Hole Harbor

Fig 7. Phylogenetic tree based on cyanobacterial 16S rRNA gene sequences. .

Discussion

These preliminary findings suggest that, under P-limitation, growth of cyanobacteria is favored by organophosphonate additions, especially some Synechococcus strains which have also been observed as a dominant strains in cyanobacterial blooms occurred in Florida Bay. It is possible that natural or anthropogenic inputs of organophosphonate compounds estuaries and marine waters may be the trigger which shifts phytoplankton community structure to that of cyanobacteria and promotes bloom formation.

Acknowledgements

We wish to thank Sandro Stumpf, Jeff Absten and the Microbial Ecology field crew. This research was supported by the National Science Foundation through the FCE LTER program.

Fig 5. Relative abundance of clones related to different cyanobacterial taxa (>90% of sequence identity).

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10%

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30%

40%

50%

60%

70%

80%

90%

100%

Control (n=34) 0.1 mM ADP (n=31) 1 mM AEP (n=31) bloom (n=42)

Uncultured cyano/chloroplast

Gloeothece sp.

Xenococcus sp.

Phormidium sp.

Symploca atlantica

Limnothrix redekei

Limnococcus limneticus

Geitlerinema sp.

Leptolyngbya sp.

Synechocystis aquatilis

Synechococcus sp.