Open Access Highly Accessed Research article

On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study

Alberto J Martín-Rodríguez15*, Alejandro González-Orive2, Alberto Hernández-Creus26, Araceli Morales3, Roberto Dorta-Guerra4, Manuel Norte1, Víctor S Martín1 and José J Fernández1*

Author Affiliations

1 Institute for Bio-Organic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, Avenida Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain

2 Department of Physical Chemistry, University of La Laguna, Avenida Astrofísico Francisco Sánchez 1, La Laguna, Tenerife 38206, Spain

3 Department of Physiology, Institute of Biomedical Technologies, CIBICAN, University of La Laguna, Campus de Ofra, s/n, La Laguna, Tenerife 38071, Spain

4 Department of Statistics, Operations Research and Computation, University of La Laguna, Avenida Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain

5 Oceanic Platform of the Canary Islands, Carretera de Taliarte s/n, Telde, Gran Canaria 35214, Spain

6 Institute of Materials and Nanotechnology, University of La Laguna, Avenida Astrofísico Francisco Sánchez s/n, La Laguna, Tenerife 38206, Spain

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BMC Microbiology 2014, 14:102  doi:10.1186/1471-2180-14-102

Published: 23 April 2014



A variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical properties of the biofilms.

The main objectives were:

1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm morphology, thickness, roughness, surface coverage, elasticity and adhesion forces.

2) To establish and characterise in detail a biofilm model with a relevant marine strain.


Both the medium and the temperature significantly influenced the total cell densities and biofilm biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2 (0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in MH2 (0.73 ± 0.29 nN).


All the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the assay conditions.

Biofilm; Biofouling; Antifouling; Shewanella algae; CLSM analysis; Atomic Force Microscopy; Young modulus; Adhesion forces