Azotobacter Siderophore as a Source of Nitrogen from Azotobacter vinelandii to
Support the Growth of the Green Algae Scendesmus sp. BA032
Juan Antonio Villa Torrecilla and Brett M. Barney
Department of Bioproducts and Biosystems Engineering and BioTechnology Institute
University of Minnesota
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. Any potential improved biological route to fix carbon
will require addition fertilizer inputs to provide the necessary nitrogen required for protein and nucleotide biosynthesis. Conventional crops such as soybeans and clover are able to meet their nitrogen requirements by forming
symbiotic relationships with diazotrophic soil bacteria. 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 for different metals. In this study, we have identified a strain of the green algae Scenedesmus that is able to utilize the A. vinelandii siderophore azotobactin as a source of
nitrogen. When grown in a co-culture, the algae obtained all nitrogen required for growth through the association with A. vinelandii. These results provide a proof of concept for the first step toward developing a mutualistic or
symbiotic relationship between these two species.
Materials and methods
Bacterial strains and growth conditions
Cultures of Azotobacter vinelandii DJ were grown aerobically for siderophore induction in modified Burk’s medium (B media), containing 58 mM sucrose, 0.34 mM CaCl·2H2O, 0.4 mM MgSO4·7H2O, 40 µM Na2MoO4 ·2H2O, 0.73 mM K2HPO4 and 2.32 mM KH2PO4 buffer at 30 °C with agitation at 200 rpm (Page and Sadoff,
1975), but no source of iron. Each gene deletion strain (SIDK3 and SIDK9) was cultured in the same way as the parental A. vinelandii strain. The cultures of Scenedesmus sp. BA032 were aerobically grown in MB medium for inducing siderophores production after 3 days of growth with A. vinelandii modified with 0.42 mM of
NaCl and 0.11 µM of CuCl2. Daily samples of BA0032 (10 µl) were collected to measure cell density (determined by hemacytometer counting). All A. vinelandii and S. sp. BA032 cells were grown under nitrogen-free conditions, except for the presence of atmospheric nitrogen gas. S. sp. BA032 cells were maintained on plates of
modified Bristol’s medium.
Azotobacter vinelandii mutant strains
A. vinelandii SIDK3 was constructed by transforming A. vinelandii DJ (trans) with the plasmid pPCRSIDK3, which contains approximately 500 bp DNA segments flanking the gene for csbC and an antibiotic marker (spectinomycin) in place of csbC. Following double homologous recombination and selection on B plates
containing the specified antibiotic, strains were confirmed to contain the modification by PCR. The csbC gene codes for a key enzyme in the biosynthesis of catechol based siderophores. A. vinelandii SIDK9 was constructed by transforming A. vinelandii DJ (trans) with the plasmid pPCRSIDK9, which contains approximately 500
bp DNA segments flanking a region inside the large gene Avin_25580 and an antibiotic marker (tetracycline) in place of the internal region of Avin_25580 as above. The Avin_25580 gene codes for a key enzyme in the biosynthesis of the nonribosomal peptide synthase for azotobactin siderophore.
Optical characterization
A. vinelandii wild type and gene deletion strains were grown for 48 h, and cell growth was determined by measuring the optical density (OD) at 600 nm. The specific optical features of the siderophores were used to monitor culture supernatant after 48 h. The absorbance spectrum of the siderophores was measured using a Varian
50 Bio visible spectrophotometer. Catechols quantitation was determined using the extinction coefficient at 310 nm (Page et al, 1988) and azotobactin was determined using the extinction coefficient at 380 nm for culture supernatants.
Azotobactin
Molecular Formula: C44H59N13O23
Molecular Weight: 1138
N 16%
Protochelin
Molecular Formula: C30H36N3O9
Molecular Weight: 546
N 7.6%
Azotochelin
Molecular Formula:C20H22N2O8
Molecular Weight: 418.3
N 6.6%
Figure 2. Production of siderophores in Azotobacter vinelandii wild type and knock outs construction PCRSIDK3 and
PCRSIDK9. Bacterial cells were grown 48 hours in B medium in the presence of molybdenum (40 µmol-1) and the
cultural supernatants were collected. Specific absorbance of the siderophores were detected and compared for wild type
and knock outs. The peaks at 380 and 310 nm are derived from the azotobactin and catechol siderophores, respectively.
Aminochelin
Molecular Formula: C11H16N2O3
Molecular Weight: 219
N 12.7%
Figure 1. Structure of the siderophores produced by Azotobacter that contain
nitrogen and their percentage. Azotobactin is a pyoverdine-like siderophore;
Protochelin, Azotochelin and Aminochelin are catechol siderophores.
Figure 4. Light microscopy images of cultures of A. vinelandii WT (White arrow) and Scenedesmus sp. BA032
(Black arrow). A. vinelandii grows around the cells of BA032 forming coaggregates..
Figure 3. Growth of different consortiums using only atmospheric nitrogen as a nitrogen source and sucrose as a
carbon source for A. vinelandii. A. vinelandii strains were grown with S. sp. BA032 in co-culture (panel A).
Supernatant from growths of A. vinelandii were filtered through a 0.2 µm filter and used to grow S. sp. BA032 (panel
B). The microalgae grew in modified Burk’s medium lacking any source of nitrogen other than air (black squares),
with 0.7 mM NaNO3 (open squares) with A. vinelandii WT strain (black triangles), A. vinelandii PCRSIDK3 (grey
circles) and A. vinelandii PCRSIDK9 (open diamonds). Each data point represents the overage and standard
deviation from three independent samples.
Discussion
The carbon sequestration from microorganisms, plants, or other feedstock for the production of sugars or biofuels may demand unsustainable inputs of nitrogen fertilizers. A. vinelandii is of interest in the biotechnological, biomedical and pharmaceutical fields and is one of the best candidates for the production of biological
fertilizers (Kizilkaya, 2009) A. vinelandii is equipped with a siderophore production system and uses part of this nitrogen fixed to produce compounds as azotobactin or catechol siderophores for the uptake of metal ions (Yoneyama et al.) Azotobactin, a specific siderophore, has a short peptide chain, contains a significant
percentage of nitrogen and can constitute a source of nitrogen for the proliferation of nondiazotrophic microalgae organisms. In a classic mutualistic relationship, these compounds and the metals bounded are used by free-living diazotrophs to fix nitrogen that is utilized by the plants (Kraepiel et al., 2009). This approach makes
use of organisms specialized for aerobic nitrogen fixation as an alternative of diazotrophic cultivation with the microalgae Scenedesmus sp. BA032 for the assembling of microbial synthetic communities. In this study we demonstrated that azotobatin siderophore can be used as a source of nitrogen by the microalgae, in mass
weight, the azotobactin contains 16% nitrogen which can be a potential nitrogen source. We are able to growth S. sp. BA032 with azotobactin as the primary nitrogen source. This results is a strong support that algae have the ability to utilize the short peptides chain in the structure of azotobactin. In addition, genetic and
biochemical information on this bacteria is available to address other complementary mutations to boost ammonium excretion by altering ammonium assimilation into azotobatin or other stable compounds and might also allow the study of less characterized bacteria naturally associated with microalgae. On the other hand, sugars
and several trace elements are enough to support the growth of A. vinelandii. The idea of this approach is that the nitrogen can be provided by the bacterium as stable compounds such as azotabactin and the energy required for nitrogen fixation may be provided by microalgae exudates.
Acknowledgements
References
We would like to thank Erin Ray, Janet Ohlert and Jiashi Wei for their efforts in strain
construction and preliminary data collection, the University of Minnesota Institute on the
Environment and the Synthetic Ecology Initiative of the BioTechnology Institue for funding.
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