Marine bacteria from the Gulf of California with antimicrofouling activity against colonizing bacteria and microalgae Diana Elizabeth Sánchez-Rodríguez1, Ismael Ortiz-Aguirre2, Ruth Noemí Aguila-Ramírez3*, Erika Guadalupe Rico-Virgen1, Bárbara González-Acosta3 & Claire Hellio4 1. Universidad de Guadalajara. CUCSur, Gómez Farías 82. San Patricio Melaque, Jalisco, México; [email protected], [email protected] 2. Universidad Autónoma de Baja California Sur, Carretera al Sur Km 5.5, La Paz, Baja California Sur, México; [email protected] 3. Instituto Politécnico Nacional- Centro Interdisciplinario de Ciencias Marinas, Av. IPN S/N Col. Playa Palo de Santa Rita, La Paz, Baja California Sur, México; [email protected], [email protected] 4. Université de Bretagne Occidentale, Biodimar/LEMAR UMR 6539, Plouzané, France; [email protected] * Correspondence Received 11-I-2018. Corrected 11-VI-2018. Accepted 06-IX-2018. Abstract: One way of reducing the input of pollutants into the marine environment is to enforce the use of non-toxic antifouling paints in marine protected areas. Thus, the purpose of this study was to detect marine microorganisms that secrete inhibitory substances against bacteria and microalgae to avoid biofouling on man- made structures in La Paz bay, B.C.S., Mexico. The inhibitory potential of 125 bacteria was evaluated against biofilm-forming bacteria. Crude extracts were obtained with methanol and ethyl acetate from 16 bacterial strains that exhibited antagonistic and antibacterial activity in a preliminary screening. Antibacterial and antimicroalgal assays were performed using crude extracts, the minimum inhibitory concentration (MIC) was determined. The highest activity against bacteria and microalgae was found in two strains, Shewanella algae and Staphylococcus sp. The results of this study suggest that extracts of bacteria from the Gulf of California with antimicrobial properties against biofilm-forming bacteria can also prevent the adhesion of microalgae, which may control the development of biofilm formation and, as a consequence, biofouling. Key words: epibionts; extracts; Shewanella; Staphylococcus; biofouling; antifouling. Sánchez-Rodríguez, D. E., Ortiz-Aguirre, I., Aguila-Ramírez, R. N., Rico-Virgen, E. G., González-Acosta, B., & Hellio, C. (2018). Marine bacteria from the Gulf of California with antimicrofouling activity against colonizing bacteria and microalgae. Revista de Biología Tropical, 66(4), 1649-1663 The biofouling process starts immediately stratified biofilm, generating chemical signals after a substrate comes into contact with sea- that act as attractants or deterrents for the water. Biochemical conditioning occurs by establishment of microalgae, spores of algae, adsorption of dissolved organic material such fungi, and protozoa; then comes the settlement as polysaccharides, proteins, lipids and amino of invertebrate larvae, known as macrofouling acids forming a film that enables organisms to (Fusetani & Clare, 2006). This process affects colonize the surface (Callow & Callow, 2011). the maritime industry in general, given that the Pioneer bacteria adhere and start forming a establishment of micro and macroorganisms on Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1649-1663, December 2018 1649 man-made structures generates biocorrosion of In this study, the potential of the Gulf surfaces and boats, reducing their useful life of California as source of bioactive-bacteria (Yebra, Kiil, & Dam-Johansen, 2004). strains was evaluated. Several environments Metal biocides, such as tributyltin (TBT) and marine organisms were considered for and copper are added to marine paints as isolation of bacteria, these included marine antifouling compounds. These biocides are sediments and bacteria associated with man- very toxic and affect the marine fauna; some grove plants. The mangrove ecosystem is a examples include gastropod imposex, mus- unique environment harboring diverse groups sel larvae mortality and oyster shell mal- of microorganisms that play key roles in nutri- formations that have all been associated to ent transformation (Thatoi, Behera, Mishra, & ecotoxicological effects caused by TBT, even Dutta, 2013). The degradation of mangrove- at extremely low concentrations (Alzieu, 2000; plant material begins with colonization by Konstantinou & Albanis, 2004). Due to the fungi and bacteria (Grossart, Kiørboe, Tang, environmental problems related to the use of & Ploug, 2003). Within this community, this toxic-antifouling biocides, natural alternatives is considered a selective force, a competi- are required to regulate organism-colonization tion for resources that promotes the biosyn- processes. Therefore, given the high toxicity of thesis of antimicrobial compounds (Slattery, antifouling paints based on chemical agents, Starmer, & Paul, 2001). Furthermore, growth environmentally-friendly approaches are now a processes and cell-to-cell interactions, which priority in the search for new methods to control are regulated by signaling molecules involved biofouling on surfaces. In addition, antifouling in population-mediated gene expression (quo- paints with toxic agents are not biodegradable, rum-sensing), play an important role in the since they include organometallic compounds population dynamics of some bacterial spe- in their matrix (Martínez-Matamoros, 2012). cies (Gomes, Grunau, Lawrence, Eberl, & The industrial requirements for novel anti- Gademann, 2003). fouling compounds are: absence of toxicity Some marine organisms such as sponges, towards non-targeted species; several years of seaweed and seahorses have a large number of antifouling and anti-biocorrosion activities; no associated bacteria, which among other func- bioaccumulation in marine food webs; and con- tions, work as protection and defense against taining active compounds that can be produced epibionts and other microorganisms (Hentschel in large quantities by green processes (Cirimin- et al., 2003; Balcázar, Loureiro, Da Silva, na, Bright, & Pagliaro, 2015). Marine bacteria Pintado, & Planas, 2010; Egan et al., 2013). are good candidates for bioactive-compound In the marine environment, this microbial production due to their ability to generate anti- association with living surfaces (symbiosis) bacterial substances, which allow ecological offers ample opportunities for bioprospection stability of multiple marine ecosystems, as well of natural products (Satheesh, Ba-akdah, & as interrelationships among microorganisms in Al-Sofyani, 2016). In some cases, bacterial epiphytic environments (Vimala, 2016). Micro- symbionts are capable of producing a wide bial communities have strong affinities towards variety of bioactive compounds that prevent living and non-living surfaces (El Bour, Ismail- colonization by opportunistic or predatory Ben, & Ktari, 2013), they can be cultivated organisms on the host’s surface. This suggests in large batches and culture conditions can that bioactive compounds act as antimicrobial be modified to enhance the yield of active agents. Whether as antibiotics or as inhibitors compounds; these characteristics comprise an of bacterial communication systems (quorum environmentally friendlier process than the sensing), it is expected that these microorgan- chemical synthesis of active compounds (Ber- isms regulate the establishment of other micro nbom, Ng, Kjelleberg, Harder, & Gram, 2011). and macroorganisms (Penesyan, Kjelleberg, & 1650 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1649-1663, December 2018 Egan, 2010) and thus protect holobionts from and incubated for 24 h at 35 °C. Plate con- epibiosis (Dobretsov, Dahms, & Qian, 2006). tents were poured off and the wells washed Strategies based on biomimetics have in triplicate with sterile distilled water. The shown strong potential in solving fouling- remaining attached bacteria were fixed with caused problems on man-made structures. Con- 200 μl of methanol in each well, and after sidering that succession within the community 15 min, the microplates were emptied and air will depend on the success of initial coloniza- dried. The microplates were stained with 200 tion (bacteria and microalgae), growth inhibi- μl of crystal violet for each well during 5 min. tion may be the key to control or prevent the Surplus stain was rinsed off by placing the entire process. Therefore, this research aimed microplate under running tap water. After the to examine the effects of marine bacteria microplates were air dried, the dye bounded to extracts against pioneer groups in the coloniza- adherent cells was resolubilized with 200 μl of tion process. acetone-isopropanol (1:3) per well. The optical density (OD) of each well was measured at 620 nm. Based on OD produced by bacterial films, MATERIAL AND METHODS strains were classified into the following cat- Biofilm sampling from paint panels: egories described by Stepanovic et al. (2000): This study was carried out during June 2016 no biofilm producers and weak, moderate or at a marine pier in La Paz, Baja California Sur, strong biofilm producers. Mexico (24º08’32” N - 110º18’39” W). Three different experimental steel panels (20 x 10 Isolation of associated bacteria: Samples cm) were used, each coated with a different of different organisms and sediments were paint type: Copper (C), silicon (S) and without collected under aseptic conditions; the meth- Copper (CF). Panels were immersed to a depth odology presented in Table 1 was followed. All of one meter