azotobacter vinelandii siderophore can provide nitrogen to support the culture of the green algae...

R E S EA RCH L E T T E R

Azotobacter vinelandii siderophore can provide nitrogen tosupport the culture of the green algae Neochloris oleoabundans

and Scenedesmus sp. BA032

Juan A. Villa1, Erin E. Ray2 & Brett M. Barney1,2

1Biotechnology Institute, University of Minnesota, St. Paul, MN, USA; and 2Department of Bioproducts and Biosystems Engineering, University of

Minnesota, St. Paul, MN, USA

Correspondence: Brett M. Barney,

Department of Bioproducts and Biosystems

Engineering, University of Minnesota, 1390

Eckles Avenue, St. Paul, MN 55108-6130,

USA. Tel.: +1 612 626 8751;

fax: +1 612 625 6286;

e-mail: [email protected]

Received 21 September 2013; revised 24

November 2013; accepted 24 November

2013. Final version published online 9

January 2014.

DOI: 10.1111/1574-6968.12347

Editor : Yaacov Okon

Keywords

Siderophore; Azotobacter vinelandii;

Neochloris oleoabundans; Scenedesmus;

nitrogen.

Abstract

Microalgae are viewed as a potential future agricultural and biofuel feedstock and

also provide an ideal biological means of carbon sequestration based on rapid

growth rates and high biomass yields. Any potential improvement using high-

yield microalgae to fix carbon will require additional fertilizer inputs to provide

the necessary nitrogen required for protein and nucleotide biosynthesis. The free-

living diazotroph Azotobacter vinelandii can fix nitrogen under aerobic conditions

in the presence of reduced carbon sources such as sucrose or glycerol and is also

known to produce a variety of siderophores to scavenge different metals from the

environment. In this study, we identified two strains of green algae, Neochloris

oleoabundans and Scenedesmus sp. BA032, that are able to utilize the A. vinelandii

siderophore azotobactin as a source of nitrogen to support growth. When grown

in a co-culture, S. sp. BA032 and N. oleoabundans obtained the nitrogen required

for growth through the association with A. vinelandii. These results, indicating a

commensalistic relationship, provide a proof of concept for developing a mutual-

istic or symbiotic relationship between these two species using siderophores as a

nitrogen shuttle and might further indicate an additional fate of siderophores in

the environment.

Introduction

Nitrogen is an important component of living systems. In

agriculture, conventional crops such as soybeans, alfalfa,

and clover meet their nitrogen requirements by forming

symbiotic relationships with diazotrophic soil bacteria

(Jones et al., 2007; Ikeda et al., 2010), while many other

agricultural crops such as corn require substantial nitro-

gen inputs from Haber-Bosch-based industrial processes.

Potential next-generation crops such as single-celled mic-

roalgae provide possible benefits in terms of total biomass

yield per square area, high density, and rapid growth vs.

current conventional agriculture crops, but would also

require substantial increases in specific nutrient inputs

such as nitrogen. Thus, a key concern in pursuing sus-

tainable next-generation oil crops should include specific

consideration of the sustainability of the nitrogen inputs.

Azotobacter vinelandii is a common Gram-negative soil

bacterium that can fix atmospheric nitrogen under

aerobic conditions. This characteristic differentiates it

from many other nitrogen-fixing bacteria that require

anaerobic or microaerobic conditions to protect the

oxygen-sensitive nitrogenase (Setubal et al., 2009). This

feature also makes it an ideal candidate strain for poten-

tial co-culture with oxygen-producing phototrophs such

as microalgae (Ortiz-Marquez et al., 2012). Additionally,

A. vinelandii is considered as an ideal strain in potential

biotechnology applications for the production of higher-

value bioproducts such as polyhydroxyalkanoates, which

could serve as potential bioplastics (Setubal et al., 2009).

Azotobacter strains produce a range of nitrogen com-

pounds which may be released into the extracellular space

under certain conditions. Extensive ammonia release was

reported two decades ago from A. vinelandii based on a

modification resulting in differential regulation of nitrogen

fixation genes, resulting in high concentrations of ammonia

accumulating in the growth media (Bali et al., 1992;

Brewin et al., 1999). This feature was recently utilized to

FEMS Microbiol Lett 351 (2014) 70–77ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

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demonstrate the potential to apply ammonia production to

the co-culture of various algae strains (Ortiz-Marquez

et al., 2012). A. vinelandii has also been reported to excrete

a range of additional nitrogen compounds to serve various

functions, including proteins involved in the production of

alginate (Gimmestad et al., 2006).

As a diazotroph, A. vinelandii requires substantial

quantities of iron to grow under optimal conditions.

Iron is important for central metabolism and is also

essential as a component of the two proteins that make

up the nitrogenase complex (Barney et al., 2006;

Wichard et al., 2009). A. vinelandii contains multiple sid-

erophore biosynthetic pathways and diverts a portion of

the nitrogen obtained through biological nitrogen fixa-

tion to the specialized nitrogen-rich pyoverdine and cate-

chol siderophores (Fig. 1), which are excreted into the

extracellular space, to assist in the uptake of required

metal ions (Tindale et al., 2000; Yoneyama et al., 2011).

Siderophore systems enable this bacterium to adapt to

metal limitation in the environment (Wichard et al.,

2009). The siderophores bind various metals with a high

affinity and are then taken up by the cell through mem-

brane-bound transport systems (Cornish & Page, 1995;

Palanch�e et al., 2004; Wichard et al., 2009). It is this lat-

ter class of siderophore compounds that were of particu-

lar interest in this study to determine the potential of

these compounds to serve as a nitrogen source to various

algal strains.

Materials and methods

Reagents, strains and cell counting

Azotobacter vinelandii DJ (trans) was obtained from Den-

nis Dean (Virginia Tech), while Scenedesmus sp. BA032 is

an environmental isolate collected from the Cache Valley

in northern Utah. Neochloris oleoabundans was obtained

from the UTEX culture collection of algae. All chemicals

and reagents were obtained from Sigma Aldrich (St.

Louis, MO) or Thermo Fisher Scientific (Pittsburgh, PA).

Cell counts of S. sp. BA032 were measured using a hemo-

cytometer following the directions of the manufacturer

(Hausser Scientific, Horsham, PA).

Bacterial strains and growth conditions

Cultures of A. vinelandii DJ (trans) and gene substitution

strains described below were grown aerobically in

modified Burk’s media for siderophore production (SPB

media), which lacks added iron and includes zinc

(58 mM sucrose, 0.34 mM CaCl∙2H2O, 0.41 mM

MgSO4∙7H2O, 40 lM Na2MoO4∙2H2O, 40 lM ZnCl,

Fig. 1. Examples of nitrogen-containing siderophores produced by Azotobacter vinelandii. Shown are the chemical structures of several nitrogen-

containing siderophores of the catechol class (aminochelin, azotochelin, and protochelin) and the pyoverdine siderophore azotobactin that

contain various percentages of nitrogen.

FEMS Microbiol Lett 351 (2014) 70–77 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Siderophores support algal growth 71

0.73 mM K2HPO4, and 2.32 mM KH2PO4) at 30 °C with

agitation at 200 r.p.m. (Page & Sadoff, 1975; Huyer &

Page, 1988; Page et al., 2003).

Construction of A. vinelandii gene substitution

strains

The csbC gene of A. vinelandii codes for a key enzyme in

the biosynthesis of catechol-based siderophores (Tindale

et al., 2000). The csbC gene was cloned along with flank-

ing regions of c. 500 bp into a pUC19-derived plasmid

using primers BBP1301 (5′-CAGATAAG CTTGTCTG

GTCGATCA GGATCGCC ATG-3′) and BBP1302

(5′-GACAGGTA CCACGCTG TAGAAATA GTCGTCGT

G-3′) with HindIII and KpnI sites used to insert the gene

underlined. The csbC gene was then removed from the

plasmid using PCR with primers BBP1331 (5′-GACAGGAT CCTCTAGA TATGCATA TGGCCTCC TTACGGCT

AGAGGACG AG-3′) and BBP1332 (5′-CTGAGGAT CCG

AGCAC CTCGACCC CGACCTCT TC-3′) with BamHI

sites underlined. The spectinomycin resistance cassette

from pHP45Ω (Prentki & Krisch, 1984) was introduced

into the BamHI site to produce pPCRSIDK3. The strain

A. vinelandii AZBB040 was constructed by transforming

A. vinelandii DJ (trans) with pPCRSIDK3. Following a

double homologous recombination event, colonies were

selected for antibiotic resistance resulting in a strain

A. vinelandii AZBB040, which contains the DcsbC::spectr

substitution. Strains were confirmed to contain the geno-

mic modification by PCR with primers BBP1419 (5′-GAACAG CACGAA GCTCAG CATCAG C-3′) and BBP1420

(5′-CGAACA CCTGTT GCAGCT TGCAGC-3′) lying

outside of the region modified.

The gene coding for Avin_25580 of A. vinelandii encodes

a key enzyme in the biosynthesis of azotobactin sidero-

phore (Yoneyama et al., 2011). The gene for Avin_25580

was cloned along with flanking regions of c. 500 bp into a

pUC19-derived plasmid using primers BBP1502 (5′-GACTAAGC TTGAAGCG TTCCCGGC TGAAGGTC-3′) and

BBP1503 (5′-GTGACC CTGTTCAT GCTGCTGC TG-3′)with HindIII and EcoRI (downstream of primer) sites used

to insert the gene. The gene for Avin_25580 was then

removed from the plasmid using PCR with primers

BBP1562 (5′-NNNGGATC CACCCAGG TCAACGAC

CTGCTGCT G-3′) and BBP1563 (5′-NNNGGATC CG

TCCTCG CCACCTGC TCCTCGAT CAACTG-3′) with

BamHI sites. A tetracycline resistance cassette derived from

pRK415 (Mather et al., 1995) was introduced into the

BamHI site to produce pPCRSIDK9. A. vinelandii was

transformed as described above. Colonies were selected for

antibiotic resistance resulting in the strain A. vinelandii

AZBB041, which contains the substitution to the gene cod-

ing for Avin_25580. Strains were confirmed to contain the

genomic modification by PCR with primers BBP1623

(5′-CACTTG CTGGAC AAAGAG ACGGTC C-3′) and

BBP1624 (5′-CTGCGG TTCAGC TCTCCG TAGCTC

ATTTC-3′) lying outside of the region modified.

Optical characterization to confirm phenotype

Azotobacter vinelandii wild-type and gene substitution

strains were grown for 72 h in SPB media, and cell density

was determined by measuring the optical density (OD) at

600 nm to confirm similar rates of growth. Specific optical

features of the siderophores were used to monitor the

culture supernatant (Page & Huyer, 1984; Page et al.,

1991; Rodr�ıguez-L�opez et al., 1991). The absorbance spec-

tra were measured using a Varian Bio 50 UV/Vis spectro-

photometer. Levels of catechols were determined using an

approach similar to that used by Page and Huyer (Page &

Huyer, 1984) and the reported extinction coefficient for

dihydroxybenzoic acid (DHBA) at pH 3.0 (Rodr�ıguez-

L�opez et al., 1991). The absorbance at 380 nm was used

to estimate the levels of azotobactin in culture superna-

tants using the reported extinction coefficients

[kmax = 380 nm at pH 4.0, e = 23 500 M�1 cm�1 (Page

et al., 1991)] and catechols [kmax = 310 nm at pH 3.0,

e = 9200 M�1 cm�1 (Rodr�ıguez-L�opez et al., 1991)].

Algae growth medium and conditions

Algae were maintained on a derivative of Bold’s basal med-

ium (Bold, 1949) containing 25 mg L�1 NaCl, 75 mg L�1

MgSO4�7H2O, 25 mg L�1 CaCl2�2H2O, 100 mg L�1

Na2SO4, 300 mg L�1 K2HPO4, 600 mg L�1 NaNO3, and

5 mg L�1 FeSO4�7H2O, adjusted to pH 7.6. The medium

was further supplemented with 1 mL L�1 of trace metals

solution containing 1.0 g L�1 boric acid, 1 g L�1 sodium

EDTA, 200 mg L�1 MnCl2�4H2O, 20 mg L�1 ZnCl2,

15 mg L�1 CuCl2�2H2O, 15 mg L�1 Na2MoO4�2H2O, and

15 mg L�1 CoCl2�6H2O. Cultures of S. sp. BA032 and

N. oleoabundans were maintained on agar plates of the

modified medium listed above.

Siderophores isolation from A. vinelandii

Cultures of A. vinelandii wild-type cells were cultured on

SPB medium. Following growth, cells were centrifuged at

7000 g for 10 min. Supernatant was separated from the

cells, then filtered (0.22 lm pore size, Nalgene Filtration,

Thermo Scientific), and collected. The filtered supernatant

was added to a column containing Q-Sepharose (GE

Healthcare). The material retained by the column was

rinsed with distilled water and eluted with 500 mM NaCl

in distilled water. The sample of crude concentrated

siderophores was substituted for sodium nitrate in the


72 J.A. Villa et al.

medium described above based on Bold’s basal medium

(Bold, 1949) to screen algae strains for potential growth

on agar plates.

Algae growth with siderophores

To determine the ability of specific algae strains to grow

on A. vinelandii siderophores, A. vinelandii wild-type and

gene substitution strains were first grown on SPB med-

ium for 3 days, then cells were removed by centrifuga-

tion, and the obtained supernatant was filtered as

described above into an autoclaved container. The super-

natant was transferred aseptically to a clean Erlenmeyer

flask, and an equivalent quantity of algal cells was inocu-

lated into each flask. Cells were grown under a bank of

fluorescent lights (c. 200 µmols s�1 m�2) with constant

agitation for c. 8 days while counting the algal cells using

a hemocytometer.

Results and discussion

Identification of algae capable of growing on

siderophores as a nitrogen source

A primary objective of this work was to determine

whether A. vinelandii-derived siderophores could serve as

a suitable shuttle of nitrogen from A. vinelandii to nondi-

azotrophic photosynthetic species such as algae. It has

been reported previously that certain species are known

to take advantage of siderophore-producing bacteria by

utilizing foreign siderophores for metal uptake (Amin

et al., 2009; D’Onofrio et al., 2010). To determine

whether this feature could also satisfy fixed nitrogen

requirements for a phototroph, we first isolated sidero-

phores from media of A. vinelandii using a simple ion-

exchange resin to collect the siderophores from spent

media as described in the methods. Then, 18 green algae

strains were screened on a simple Bold’s basal media

where sodium nitrate was replaced with the isolated sid-

erophores. Two strains were able to replicate on the sid-

erophore-containing media. The first was a strain of

Scenedesmus sp. BA032 isolated from the Cache Valley in

northern Utah, and the second was Neochloris oleoabun-

dans, a strain of interest for potential neutral lipid pro-

duction (Li et al., 2008; Gouveia et al., 2009). These two

strains were selected for further siderophore utilization

studies as described below.

Identification of specific class of siderophores

supporting algae growth

The siderophores produced by A. vinelandii fall into two

different classes called the catechol and pyoverdine

siderophores (Fig. 1). Others have recently reported the

successful disruption of siderophore biosynthetic genes in

A. vinelandii (Yoneyama et al., 2011). Here, we utilized a

similar approach to what was taken previously by target-

ing a key gene in each of the two pathways that produce

either class of siderophore (Tindale et al., 2000; Yoney-

ama et al., 2011). Gene substitution strains were con-

structed and the phenotype (deficient in the production

of either class of siderophore) was confirmed by measur-

ing the absorbance of spent culture media (Fig. 2), con-

firming the results reported previously by others

(Yoneyama et al., 2011). These gene substitution strains

are important for several reasons. First, there are two

classes of siderophores produced by A. vinelandii, and

disruption of either class should allow us to determine

whether algae are growing on either class specifically. Sec-

ond, siderophores are not the only extracellular nitrogen

compound produced by A. vinelandii. Azotobacter vine-

landii also produces other compounds, including ammo-

nia, urea, or extracellular proteins (Bali et al., 1992;

Brewin et al., 1999; Gimmestad et al., 2006), each of

which could serve as sources of nitrogen to support algae

growth. Thus, if the algae strain were able to grow on

both substitution strains, this might indicate that another

compound was responsible for providing the nitrogen, as

has been shown for alternative algae strains (Ortiz-Mar-

quez et al., 2012).

Fig. 2. Production of siderophores in Azotobacter vinelandii wild-type

and gene substitution strains AZBB040 and AZBB041. Bacterial cells

were grown for 72 h in SPB medium and the cultural supernatants

were collected. Specific absorbance of the siderophores at 310 and

380 nm is derived from the catechol and azotobactin, respectively.

Absorbance spectra were measured at pH 4.0 for AZBB041 and wild-

type strains and at pH 3.0 for AZBB040.



Growth of S. sp. BA032 in liquid culture

containing siderophore

To test whether a specific siderophore class was responsible

for the growth of S. sp. BA032, in the first experiment,

A. vinelandiiwild-type strain and the two single-gene substi-

tution strains were first grown in SPB media, which results

in the production of siderophores (Fekete et al., 1983; Huyer

& Page, 1988). Culture supernatant was separated from

A. vinelandii cells as described in the methods, and an equiv-

alent quantity of S. sp. BA032 cells was inoculated into each

supernatant sample for culture. Figure 3 (top) shows the

results of this experiment along with a positive control (SPB

media supplemented with nitrate, but not subjected to

A. vinelandii cells) and a negative control (SPB media with-

out added nitrogen sources, not subjected to A. vinelandii

cells). These controls were compared to spent SPB media

from the specific A. vinelandii strains following several days

of growth. The experiment demonstrated that supernatants

of A. vinelandii wild-type strain and the catechol sidero-

phore gene substitution strain (A. vinelandii AZBB040)

were able to support the growth of S. sp. BA032 cells, while

the supernatant from the strain containing a substitution for

a pyoverdine (azotobactin) siderophore gene (A. vinelandii

AZBB041) had limited growth under similar conditions.

This result supports the proposal that azotobactin is provid-

ing the source of nitrogen to sustain growth in the media.

Co-culture of A. vinelandii and S. sp. BA032

In addition to using the spent media of A. vinelandii, we

also wished to study the potential to grow A. vinelandii

and S. sp. BA032 as a co-culture. In this experiment, cells

of A. vinelandii wild-type or the individual gene substitu-

tion strains were inoculated along with S. sp. BA032 into

sterile media and grown together. As shown in Fig. 3

(bottom), a similar result was found to that shown in

Fig. 3 (top), where the strain containing the substitution

for the gene involved in the biosynthetic pathway to pro-

duce azotobactin (A. vinelandii AZBB041) resulted in

very minimal growth, while A. vinelandii wild-type cells

reached a similar level of growth as was found when

using filtered supernatants. The strain containing the gene

substitution of catechol siderophores (A. vinelandii

AZBB040) was able to support S. sp. BA032, although the

levels of cells obtained in the culture were not as high as

when only isolated supernatant was utilized.

Growth of N. oleoabundans in liquid culture

containing siderophore

The second strain that grew well on plates supplemented

with A. vinelandii siderophores as the primary nitrogen

Fig. 3. Growth of various consortiums using atmospheric nitrogen as

the nitrogen source and sucrose as the reduced carbon source for

Azotobacter vinelandii. Supernatants collected from growths of

A. vinelandii strains were filtered through a 0.2-mm filter and used to

grow Scenedesmus sp. BA032 (Top). These results are compared to a

positive control of the same media (not grown with any A. vinelandii

strains) supplemented with 700 µM NaNO3 and a negative control of

the same media with no added nitrogen. Additionally, A. vinelandii

strains were grown together with S. sp. BA032 in co-culture

(Bottom). Each data point represents the average and standard

deviation from three independent samples.



source was N. oleoabundans. As was found for S. sp.

BA032, supernatants of the wild-type and catechol sidero-

phore gene substitution strains (A. vinelandii AZBB040)

were able to support the growth of N. oleoabundans to

comparable cell densities, while the supernatant of the

strain carrying the azotobactin gene substitution resulted

in a limited growth (Fig. 4). These results further support

the likelihood that azotobactin is responsible for meeting

the nitrogen requirements in N. oleoabundans as well.

Quantities of siderophore available to culture

algae

The production of siderophores in A. vinelandii has been

studied for many years and is well characterized, includ-

ing the enzymatic pathways and genes involved in their

biosynthesis (Tindale et al., 2000; Page et al., 2003;

Wichard et al., 2009; Yoneyama et al., 2011). In many

reports, the production of specific siderophores under

laboratory culture conditions has been found to be very

high [40–60 mg L�1 of various azotobactin forms

(Demange et al., 1988)]. Based on the absorbance values

obtained in these studies (See Fig. 2 as an example) and a

molecular weight of c. 1370 g mol�1, our yields of azot-

obactin were calculated to be around 25–50 mg L�1 in

various cultures, which agrees well with previous reports

(Demange et al., 1988). Based on the molecular formula

of azotobactin 87 (C55H79N14O27), this constitutes a con-

siderable number of nitrogen equivalents. From the start-

ing quantities of azotobactin in supernatants used to

grow S. sp. BA032 (c. 25 µM for A. vinelandii wild-type

strain and AZBB040 gene substitution strain, Fig. 3 top)

and the positive nitrogen control containing 700 µM of

sodium nitrate, the number of cells obtained with S. sp.

BA032 correlated well with the amount of nitrogen pro-

vided from either source, indicating that S. sp. BA032

was able to utilize essentially all of the provided azotobac-

tin to support growth. Thus, siderophores constitute a

source of nitrogen for the proliferation of nondiazotroph-

ic microalgae organisms when excreted into the extracel-

lular space. Azotobactin is composed of a small peptide

chain (Fig. 1) synthesized via a nonribosomal peptide

synthetase (Yoneyama et al., 2011). Due to the extensive

number of nitrogen atoms found in an azotobactin mole-

cule, even smaller molar concentrations of this compound

can account for a significant mass amount of available

nitrogen when released to the environment. Based on the

potential for diffusion and previous reports of other

organisms hijacking foreign siderophores for their own

benefit (Amin et al., 2009; D’Onofrio et al., 2010), it was

of interest to demonstrate whether siderophores from

Azotobacter would also serve to satisfy nitrogen require-

ments to support other organisms in the environment.

Azotobacter is a bacterium predominantly associated

with soils, while Scenedesmus is a ubiquitous green algae

found in freshwater environments. Thus, there are some

differences between the conditions where these strains are

found. The light utilized to culture the algae strains could

also potentially lead to photodegradation of the sidero-

phores. Even if this were the case, the final fate of the

nitrogen appears to be suitable for the culture of the algae

strains selected here, which represent only a small sam-

pling of potential strains of phototrophs from the envi-

ronment that might make use of the excreted nitrogen

siderophore compounds provided by a diazotroph.

Implications of findings

Under the current experimental design, the cultures tested

here are best defined as commensal co-cultures and not

symbiotic, as A. vinelandii is providing a nitrogen source,

but is not getting anything in return from the algal strain.

Azotobacter vinelandii grows on simple media requiring

only sugar and minor amounts of additional minerals.

Because the sugar provided to support A. vinelandii is an

external source, this relationship is currently an

open-loop system. However, many strains of algae and

cyanobacteria are known to produce extracellular reduced

Fig. 4. Growth of various consortiums using atmospheric nitrogen as

the nitrogen source and sucrose as the reduced carbon source for

Azotobacter vinelandii. Supernatants collected from growths of

A. vinelandii strains were filtered through a 0.2-µm filter and used to

grow Neochloris oleoabundans. Each data point represents the

average and standard deviation from three independent samples.



carbon compounds that could support the growth of

A. vinelandii (Brechignac & Schiller, 1992; Wolf, 1997; De

Philippis & Vincenzini, 1998; Corzo et al., 2000; Bar-Zeev

et al., 2009) and could be used as a secondary selection

pressure. Under such conditions, the two strains could be

grown in such a manner that a symbiotic relationship

might evolve. Examples of phototrophs producing extra-

cellular carbon that is released to the environment in the

form of polysaccharides and simple sugars are common,

although the exact reason for the energetically wasteful

excretion of extracellular reduced carbon is not clear

(Passow, 2002; Bar-Zeev et al., 2009). This research pro-

vides clear evidence that the nitrogen released to the envi-

ronment as siderophores can be utilized by other

organisms not only for obtaining metals from the envi-

ronment (D’Onofrio et al., 2010), but also to provide

requisite nitrogen required for growth and replication in

some strains of green algae.

Acknowledgements

This work is supported by a grant (RC-0007-12) from the

Initiative for Renewable Energy & the Environment (Insti-

tute on the Environment) to B.M.B and the Biotechnology

Institute at the University of Minnesota for fellowship

funding to J.A.V. We thank Jiashi Wei for assistance in

early isolation of siderophores and preliminary algal cul-

ture studies. We thank the kind suggestions of the anony-

mous reviewers.

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azotobacter vinelandii siderophore can provide nitrogen to support the culture of the green algae...

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