In situ Colonization of Marine Biofilms on UNS S32760 Duplex Stainless Steel Coupons in Areas with Different Water Qualities: Implications for Corrosion Potential Behavior

Luciana V. R. de Messano, Barbara L. Ignacio, Maria H. C. B. Neves and Ricardo Coutinho

1. Funda??o Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Av. Erasmo Braga, 118/6o andar. Centro. CEP: 20020-000. Rio de Janeiro. RJ. Brazil.

2. Present address: Instituto do Mar, Universidade Federal de S?o Paulo. Av. Almirante Saldanha da Gama, 89. Ponta da Praia. CEP 11030-400. Santos. SP. Brazil.

3. Marine Biotechnology Division, Instituto de Estudos do Mar Almirante Paulo Moreira, Marinha do Brasil. Rua Kioto, 253. Praia dos Anjos. CEP: 28930-000. Arraial do Cabo. RJ. Brazil.

1 Introduction1

Biofouling is a common problem affecting marine technology. Any structure placed in the marine environment is susceptible to the accumulation of microorganisms (i.e.,biofilm) and invertebrates, such as barnacles, macroalgae,mussels, bryozoans and tube worms (Hellio and Yebra,2009). One of the consequences of micro- and macrofouling growth is the intensification of corrosion on metallic materials and structures exposed to marine environments(Videla, 2003). The microfouling, typically composed of aerobic and anaerobic bacteria, protozoa and microalgae(Zobell and Allen 1935; Marszaleket al.,1979; Mieszkinet al.,2013), influences corrosion through the metabolic activity of the microorganisms (e.g. production of organic and inorganic acids, enzymes, ammonia and sulfides), as well as through the introduction of reduction reactions and the production of oxygen or chemical concentration cells caused by the biofilm’s growth (Beech and Gaylarde, 1999).Microbiologically influenced corrosion (MIC) in marine environments has been reported for several metallic materials, including stainless steels (Little and Lee, 2007). It has been well documented that in the presence of biofilms,stainless steels (SS) reveal a different corrosion potential(Ecorr) behavior, exhibiting an accented elevation ofEcorras a function of immersion time (Scottoet al., 1985; Videla,1994; Mansfeld, 2007; Littleet al.,2008). This process is called Ennoblement and although it was widely discussed,there is no unifying explanation yet (Littleet al.,2013). Its causes in marine waters have been attributed to organometallic catalysis, acidification of the electrode surface and the production of passivating siderophores. The most common hypothesis is the bioproduction of H2O2through enzymatic catalysis mainly by bacterial biofilms.This bioproduction can accelerate the cathodic reaction of oxygen and thus increase theEcorr(Scotto and Lai, 1998;Beech and Sunner, 2004; Little and Lee, 2007).

Since the increase of theEcorrvalues in the presence of marine biofilms was first described by Mollica and Travis(1976), the main concern for the consequences of ennoblement for engineering decisions is the increase of pitting and the propagation of crevice corrosion on SS(Dexter and Gao, 1988; Scotto and Lai, 1998; Acu?aet al.,2006; Larchéet al., 2011). However, this is a topic of debate when considering high alloyed SS since studies have shown that the increase of susceptibility of these SS to localized corrosion is not a certainty (Littleet al.,2008; Landoulsiet al.,2011; Machucaet al.,2014). In spite of its deleterious effect, ennoblement caused by the marine bacterial biofilm growth has been recently used to generate a biological power source in microbial fuel cells (MFC) (Littleet al.,2013), representing a promising and practical application.Some designs of MFCs consist of employing marine biofilm growth on high alloyed SS as electrodes in a benthic MFC(Dumaset al.,2007) and as a biocathode in the floating MFC, a novel prototype (Erableet al.,2013).

MIC and ennoblement are recognized as multifactorial phenomena both influenced by biotic and abiotic factors.Most studies have tested the effects of biofilms (single-and multi-species biofilms) onEcorrbehavior of SS at complete or partial laboratory conditions (e.g. Dexter and Gao, 1988;Scotto and Lai, 1998; Acu?aet al.,2006; Eashwaret al.,2011; Machucaet al.,2013). Field experimental designs for marine MIC investigations on SS are scarce, probably due to methodological limitations and/or restrictions. Apart from the valuable information provided by controlled studies, the conditions applied restrict the source of colonizers(artificially selecting species composition and altering ecological complexity) and alter important variables to the physiology and survival of the microbiota (e.g. light wavelength and diurnal oscillation, flow rate, temperature variation and nutrients availability). These field-laboratory based discrepancies may be critical for an accurate evaluation of ennoblement and its occurrence. As in addition to the mentioned biofilms interactions, it is known that abiotic factors (such as those cited below) also influence the ennoblement extent (Littleet al.,2008).

In the present study, a field experiment was performed to characterize natural marine biofilms colonization and registerin situtheEcorrbehavior of a high-alloyed SS in areas with different water qualities in Arraial do Cabo,Southeastern Brazil. The hypothesis predicts that these biofilm communities should be distinct and would differentially influence the responsein situof theEcorrof the UNS S32760 duplex stainless steel, resulting in a site-dependent behavior as verified by Martinet al.(2007)for the Ni-Cr-Mo alloys. To achieve these goals, theEcorrbehavior of the duplex SS UNS S32760 was recorded simultaneously within situbiofilm formation in the two areas. Subsequently, a laboratory based experiment was performed to investigate the temporal effects of biofilm removal on theEcorrvalues of those SS coupons.

2 Methods and materials

Arraial do Cabo is located on the coast of the state of Rio de Janeiro, Brazil (22°57′to 23°00′S, 41°59′ to 42°01′W). Enseada dos Anjos is a bottle-shaped bay in this area, limited by the islands of Cabo Frio and Porcos.The eastern part of the bay, near Cabo Frio Island, can be considered as an undisturbed area. Whereas the northern region, where Praia dos Anjos is located, is influenced by anthropogenic activities, including a harbor (for commercial and fishery uses, named Forno Harbor) and stochastic domestic-sewage discharges (Curyet al.,2011).

The field experiment was carried out simultaneously over 18 days at two sites located on Cabo Frio Island and at Forno Harbor. During the study, abiotic parameters (salinity,pH, superficial water temperature and levels of dissolved oxygen and nutrients), chlorophyll a and polycyclic aromatic hydrocarbons (PAHs) were measured to corroborate the previous water quality characterizations of both sites.

Twelve coupons of UNS S32760 steel (70 mm×50 mm×2 mm; composition: 25% Cr, 7.11% Ni, 3.59% Mo, 0.58%Cu, 0.54% W, 0.49% Mn, 0.21% N, 0.28% Si, 0.019% P and 0.015% C) were submerged at a 1 m depth at each site.This SS was selected due to its known excellent resistance to MIC and localized corrosion in seawater (Franciset al.,2011), given that the goal of the present study was to register theEcorrvariation without pitting or crevice propagation (Littleet al.,2008). The coupons were placed inside cages made of 100 μm nylon mesh to avoid settlement by macrofoulers. These cages were scraped daily to avoid clogging.

For the biofilm analysis, the coupons were brought to the laboratory on days 2, 10 and 18. The biofilm on each coupon was scraped with sterile brushes and suspended in 50 mL of 2 % sterile formaldehyde. The standardized final volumes (100 mL) were stained with 4', 6-diamidino-2-phenylindol (DAPI) at a final concentration of 2 μg/mL.The biofilm suspension was filtered onto black polycarbonate filters with 0.22 μm pores (25 mm diameter)that were subsequently fixed on glass slides with immersion oil and cover slides. Bacteria, Cyanobacteria, diatoms and dinoflagellates were identified and counted using epifluorescence microscopy.

The microbial community was taxonomically identified following morphological criteria. For this, the taxonomic nomenclature of the diatoms followed Hasle and Syvertsen(1997); dinoflagellates taxonomy followed Steidinger and Tangen (1997) and the cyanobacterial nomenclature followed Komárek and Anagnostidis (1986) and Anagnostidis and Komárek (1988, 1990). As there was no intention to investigate the biochemical basis of the process,Bacteria was also morphologically classified considering the morphotypesCoccus-like,Bacillus-like and filamentous.

Electrochemical measurements were performedin situto avoid any disturbance in the biofilm community and in the interface biofilm–SS. For that, electric contacts were prepared on the coupons by welding an SS wire and covering the contacts with epoxy and the edge with non-conductive resin. The experimental unit was carefully suspended at the subsurface for theEcorrmeasurements of the coupons and, subsequently, placed back. TheEcorrvalues were registered daily using a multimeter and an Ag/AgCl electrode. At day 18, the last coupons were retrieved from both of the sites. Biofilms were removed and the coupons were kept in filtered and sterile seawater under UV light for 10 days to monitor theEcorrcurve in the absence of a biofilm.

A paired t-test was performed to evaluate the variation of theEcorrbetween the sites using InStat v.3.10? (GraphPad Software Inc., La Jolla, CA, USA). Multivariate analyses were conducted to compare the distribution oftaxain the biofilm communities. The communities were graphically presented in two-dimensional ordination plots using non-metric multidimensional scaling (nMDS) based on the Bray-Curtis measure of similarity. Major group comparisons(within the groups of Bacteria,Cyanobacteria,dinoflagellates and diatoms) were conducted based ontaxadensities and an overall comparison was conducted based on transformed data (presence and absence). A one-way analysis of similarities (ANOSIM) test was conducted to formally test the factor site. The similarity percentage procedure (SIMPER) was used to identify the similarities and dissimilarities within and between groups (i.e. sites). All of the multivariate analyses were performed using PRIMER(Plymouth Routines In Multivariate Ecological Research)v5? software.

3 Results and discussion

The water quality differences between Forno Harbor and Cabo Frio Island recorded during this experiment (Table 1)are in total agreement with the previous characterizations of Forno Harbor as a disturbed (polluted) site and Cabo Frio Island as an undisturbed site (Curyet al.,2011).

Items Cabo Frio Island Forno Harbor VMin VMax VMin VMax Temperature / oC 22.00 23.20 22.74 23.10 Salinity / (p.s.u.) 35.25 35.36 35.41 35.44 Diss. Oxygen / (ml?L-1) 5.51 5.69 5.16 5.47 pH 8.51 8.68 8.68 8.70 PAHs / (μg?L-1) 1.46 6.14 3.10 11.09 Nitrate /(mmol?L-1) 0.22 0.72 0.27 3.33 Phosphate / (mmol?L-1) 0.15 0.21 0.21 0.48 Cl a / (mg?m-3) 0.36 0.63 0.46 3.03

The biofilm communities were comprised of Bacteria,Cyanobacteria, diatoms and dinoflagellates. Higher richness values were recorded at Cabo Frio Island (23taxain total: 9taxaat 2 days, 20taxaat 10 days and 11taxaat 18 days of biofilm development) than at Forno Harbor (15taxain total:3taxaat 2 days, 11taxaat 10 days and 11taxaat 18 days of biofilm development). In general, Bacteria were more abundant at Forno Harbor. At this site,Coccus-like bacteria showed high densities in the period between day 2 and day 10 of the biofilm development (Fig. 1). Scarce information regarding the microbial composition of natural environments complicates the understanding of microbial communities’ dynamics (Mc Dougaldet al.,2012). Arraial do Cabo is not an exception to the range of poorly studied natural environments and no information is available in the literature to compare the results.

Fig. 1 Cell density of the Bacteria in biofilm communities on UNS S32760 stainless steel coupons at Cabo Frio Island (undisturbed) and Forno Harbor(anthropogenically disturbed); and the times of the coupons’ immersion, which were sampled on the 2nd,10th and 18th days

In relation to diatoms, genera commonly found in fouling communities were recorded in this study (Cookseyet al.,1984; Brandiniet al.,2001; Patil and Anil, 2005; Zargielet al.,2011). Despite the similar total abundance between sites,Cabo Frio Island showed highertaxarichness (Fig. 2). At Forno Harbor,Naviculawas the most abundant diatom genus. This genus showed a marked local imbalance in its distribution.Naviculais mostly a facultative heterotroph that is frequently considered a superior competitor in conditions of abundant ammonium and nitrates as well as in limited light (e.g. Werner, 1977; Underwood and Barnett,2006). These features may explain its prevalence. In addition, at this anthropogenically disturbed site, only four of the thirteentaxaof diatoms were recorded, and two of them (Chaetocerossp. andPseudonitzchiasp.) were exclusively recorded.

With consideration to the dinoflagellates, higher values of the total abundance of this group were found at Cabo Frio Island (Fig. 3). Four of the eight recordedtaxawere found at Forno Harbor, and all of these were also recorded at Cabo Frio Island. It is important to note that three of the species recorded at Forno Harbor belong to the same genus,Prorocentrum, which is mixotrophic and related to harmful bloomsin situations of high nutrient inputs (e.g. Zingoneet al.,2006).Protoperidinium, the other recorded genus, is also related to harmful blooms (e.g. Zingoneet al.,2006).This heterotrophic organism is a voracious eater and exhibits positive growth rates on several diatom and dinoflagellate species (e.g. Gribbleet al.,2007), which may explain the dominance of this genus after theNaviculabloom at Forno Harbor.

In relation to Cyanobacteria, 3taxawere recorded, withSpirulina subtilissimabeing recorded only at a low density in Forno Harbor (day 18) (Fig. 4).

Fig. 2 Cell density of diatoms in biofilm communities on UNS S32760 stainless steel coupons at Cabo Frio Island (undisturbed) and Forno Harbor(anthropogenically disturbed) and the times of the coupons’ immersion, which were sampled on the 2nd,10th and 18th days

Fig. 3 Cell density of dinoflagellates in biofilm communities on UNS S32760 stainless steel coupons at Cabo Frio Island (undisturbed) and Forno Harbor(anthropogenically disturbed) and the times of the coupons’ immersion, which were sampled on the 2nd,10th and 18th days

The temporal and spatial variability of the biofilm communities pointed out above were confirmed using multivariate analysis. The nMDS analysis showed scattered biofilm communities for both the overall (Fig. 5) and major-group (data not shown) approaches, a finding that was confirmed using the ANOSIM test for the factor site (p>gt;0.05 for all comparisons). Despite the high values of the average dissimilarity between Cabo Frio Island and Forno Harbor(overall biofilm communities: 51%; diatoms: 92%;dinoflagellates: 54%; Cyanobacteria: 68%; and Bacteria:73%;

Fig. 4 Cell density of Cyanobacteria in biofilm communities on UNS S32760 stainless-steel coupons at Cabo Frio Island (undisturbed) and Forno Harbor(anthropogenically disturbed) and the times of the coupons’ immersion, which were sampled on the 2nd,10th and 18th days

SIMPER analysis), the similarity within groups (i.e. sites)was lower than 50%.

The dissimilarity between groups (i.e. spatial variability of the biofilm) can be reasonably attributed to anthropogenically-mediated alterations of the water qualities’ parameters at Forno Harbor. This affirmation is supported by a water samples analysis taken during this experiment, data from the literature (Curyet al.,2011) and microbiota dispersal-related patterns, which are strongly related to natural dispersal and viability of seawater microorganisms (Greenet al.,2008; Van der Guchtet al.,2007) and consequently, related to biofilm formation. The extent of the biological differences between sites with respect to microbial colonization patterns was remarkable.The biofilm at Forno Harbor was characterized by higher relative abundances of Bacteria at day 2, followed by diatoms (especiallyNaviculasp.) on day 10 and dinoflagellates on day 18, whereas no clear trend was recorded at Cabo Frio Island when considering the same major biological groups.

Concerning theEcorrvalues of the UNS S32760 duplex coupons, temporal curves tended to show increased values over time at both of the sites; however, this pattern was significantly prominent only at the Forno Harbor site (t=5.240;p>lt;0.0001;n= 16) (Fig. 6). At this disturbed site, the averageEcorrvalues ranged from -258 mVAg/AgClto +261 mVAg/AgCl, and a shift in the averageEcorr(approximately 250 mVAg/AgCl) was recorded on the 10th day (Fig. 6)characterizing the ennoblement phenomenon. Shifts inEcorrvalues and the occurrence of the ennoblement phenomenon in the first 15 days of immersion were previously recorded in seawater conditions (e.g. Dexter, 1993; Littleet al.,2008).At Cabo Frio Island (the undisturbed site) the average values ranged from -228 mVAg/AgClto -46 mVAg/AgCland ennoblement was not clearly recorded. Nevertheless, it is important to point out that in a previous study at the Cabo Frio Island site, under different experimental conditions,ennobled OCP values of UNS S32760 were recorded on the 5thday in coupons protected with meshes of 500 μm(Messanoet al.,2009).

Fig.6 Average values and minimum and maximum values of the corrosion potential (Ecorr) vs. time (days) of UNS 32760 duplex stainless steel at both of the sites: Cabo Frio Island (undisturbed) and Forno Harbor(anthropogenically disturbed)

The present results confirm ennoblement site-dependence on the UNS S32760 duplex SS coupons as previously proposed by Martinet al.(2007) to Ni-Cr-Mo alloys in seawater. These authors showed that the extent of ennoblement was higher in the lower Delaware Bay(temperate and eutrophic waters) than in Key West, Florida(tropical and oligotrophic waters). In spite of the differences in the SS characteristics, ennoblement phenomenon was recorded/prevalent at sites with high nutrients input in both studies. Biofilm compositions and densities seem to be the plausible explanation for the differences and similarities between both studies, but no information concerning the biofilm is available in the study of Martinet al.(2007).

Current data suggest an important contribution of diatoms to the ennoblement of UNS S32760 at Forno Harbor. At the time in which ennobledEcorrvalues were recorded at Forno Harbor,Naviculawas the dominant genus.Naviculasecretes exopolymeric substances (EPS) with both hydrophobic and hydrophilic properties (Arceet al.,2004)and has been implicated in corrosion processes (Brankevichet al.,1990; Eashwaret al.,2011). It was proposed that the metabolites embedded in the EPS would be involved in ennoblement (Motodaet al., 1990; Machucaet al., 2014) as well as the metabolic activity via photosynthesis (Eashwaret al., 2011). However, most of the studies that showed biologically-induced corrosion and ennoblement were related to Bacteria. There is a lack of basic knowledge concerning the mechanism by which diatoms, by themselves or via their metabolites, influence the corrosion potential of SS and the whole process is not well understood(Landoulsiet al., 2011).

Also at Forno Harbor,Coccus-like bacteria showed high densities in the period marked by an ascendant curve of theEcorrvalues until day 10, when ennoblement clearly occurred. This assumption is corroborated by the low densities of this group at Cabo Frio Island, the site where ennoblement was not recorded (Fig. 1 and Fig. 2). Poor information is available forin situconditions and/or multi-species biofilm. Culturable bacteria and their effects on SS are well studied (Ismailet al.,1999; Bakeret al.,2003; Parotet al.,2011), but the lack of studies of natural microbial communities, particularly in tropical areas,hampers the understanding of biofilm-mediated ennoblement (Acu?aet al.,2006). Regarding Arraial do Cabo, the only available information about the microbial composition of this area is related to water-column seawater(Curyet al.,2011), and the authors did not mention any genus of Bacteria which had metabolic activity related to ennoblement or corrosion processes.

In consideration to dinoflagellates and Cyanobacteria, to the best of our knowledge, there is no information regarding the influence of these groups on ennoblement or corrosion processes, but particularly Cyanobacteria have several metabolic attributes that could be acting and influencing the electrochemical behavior of SS. These organisms are photoautotrophic and secret EPS in a large proportion in relation to their biomass production to form biofilms. The cyanobacterial EPS are hydrofobic, have a complex structure (they are constituted by heteropolysaccharides),serve as an energy and carbon source for heterotrophic bacteria and have an anionic nature, exhibiting capacity to chelate and sequester metal cations (Kamennayaet al.,2012). Further studies focusing on the role of Cyanobacteria in corrosion processes are essential to understand ecological and metabolic interactions and their consequences for practical applications.

Results from the present study highlight the complex nature of the biofilm-SS relationship. The complexity is shown, for example, by the findings of similarEcorrvalues with different biofilm communities (after 2 days of immersion) and the maintenance of ennobledEcorrvalues(after 10 to 18 days of immersion at Forno Harbor) whilst cell densities of some groups decrease and others increase over time. Another example of this complexity is the comparison of the current data with the results of Messanoet al.(2009). These authors described different diatom dominance on the same duplex SS immersed at Cabo Frio Island when a shift ofEcorrwas recorded. So, in spite of laboratory evidence that the presence of one target species can produce the ennoblement phenomenon, metabolic interactions of multiple microbial species, such as the consumption vs. production of O2in SS interfaces by the combined effect of microorganisms as highlighted by Landoulsiet al.(2011), would be a key-factor in natural systems. Moving toward an understanding of the whole process is particularly important for engineering decisions and especially concerning novel technologies such as the floating MFC design that depends on the ennoblement of natural biofilms to generate current more efficiently (Erableet al.,2013).

In the laboratory assay (after biofilm removal), coupons retrieved from Forno Harbor showed a decrease inEcorrvalues from +234 mVAg/AgClto -67 mVAg/AgClafter 24 hours under sterile conditions. TheEcorrvalues remained constant until day 6, when theEcorrvalues increased to +50 mVAg/AgCl.After that time, theEcorrvalues showed a slight decrease to-27 mVAg/AgCl. In contrast, the coupons retrieved from Cabo Frio Island (where ennoblement was not recorded) showed increasing values (to +127 mVAg/AgCl) until day 3 under laboratory conditions. Then, theEcorrvalues slightly decreased, and after day 6 and until the end of the assay(day 10), they resembled the values found at Forno Harbor.The data showed that once the biofilm communities were removed, coupons from both sites converged to similarEcorrvalues after 6 days of immersion in sterile seawater (Fig. 7).These findings supported the previous conclusion concerning the influence of biofilm communities on theEcorrbehavior of the duplex SS UNS 32760.

Fig. 7 Corrosion potential (Ecorr) vs. time (hours/days) of UNS 32760 duplex stainless steel coupons after biofilm removal and under sterile conditions

4 Conclusions

Results showed that theEcorrvalues of the UNS S32760 duplex stainless steel responded to the spatial and temporal variability of the biofilm communities. The diatomNaviculaandCoccus-like bacteria were pointed out as the microorganisms primarily related to theEcorrbehavior and ennoblement was site-dependent. Moving toward an understanding of biofilm-SS interactions is crucial and has important implications for materials science and engineering decisions.

Acknowledgements

We are grateful to Hector A. Videla (in memoriam) for his insightful suggestions and the Corrosion Laboratory(UFRJ/COPPE/DMM) researchers for their suggestions and technical assistance. We are in debt to the IEAPM team for their assistance. We especially thank Eliane Gonzalez Rodriguez for her invaluable support and our Marine Biotechnology division colleagues: Carlos G. W. Ferreira,Bruno Masi, Wagner Costa, and Soledad Lopez. We are also thankful to the IEAPM Chemistry Group for seawater analysis. Thanks to FAPERJ for the post-doctoral fellowship of BL Ignacio. Thanks to FAPERJ/CAPES for the post-doctoral fellowship of LVR Messano.

Acu?a N, Ortega-Morales BO, Valadez-González A (2006).Biofilm colonization dynamics and its influence on the corrosion resistance of austenitic UNS S31603 stainless steel exposed to Gulf of Mexico seawater.Marine Biotechnology, 8,62-70.

Anagnostidis K, Komárek J (1988). Modern approach to the classification system of cyanophytes. 3-Oscillatoriales.

Algological Studies / Archiv for Hydrobiologie, Supplement Volumes 50-53, 327-472.

Anagnostidis K, Komárek J (1990). Modern approach to the classification system of Cyanophytes. 5 - Stigonematales.

Algological Studies/Archiv for Hydrobiologie,Supplement Volume 59, 1-73.

Arce FT, Avci R, Beech IB, Cooksey KE, Wigglesworth-Cooksey B (2004). A live bioprobe for studying diatom surface interactions.Biophysical Journal, 87, 4284-4297.

Baker PW, Kimio I, Kazuya W (2003). Marine prosthecate bacteria involved in the ennoblement of stainless steel.Environmental Microbiology,5 (10), 925-932.

Beech IB,Gaylarde CC (1999). Recent advances in the study of biocorrosion, an overview.Revista de Microbiologia, 30,170-190.

Beech IB, Sunner J (2004). Biocorrosion: towards understanding interactions between biofilms and metals.Current Opinion in Biotechnology, 15, 181-186.

Brandini FP, da Silva ET, Pellizzari FM, Fonseca ALO, Fernandes LF (2001). Production and biomass accumulation of periphytic diatoms growing on glass slides during a 1-year cycle in a subtropical estuarine environment (Bay of Paranaguá, southern Brazil).Marine Biology, 138, 163-171.

Brankevich GJ, De Mele MLF, Videla HA (1990). Biofouling and corrosion in coastal power plant cooling water system.Marine Technological Journal, 24, 18-28.

Cooksey B, Cooksey KE, Miller C, Paul JH, Rubin RW, Webster D (1984). The attachment of microfouling diatoms.Marine Biodeterioration: an Interdisciplinary Study, Costlow JD,Tipper RC (Eds.)., Naval Institute Press, Annapolis, MD, USA,168-171.

Cury JC, Araujo FV, Coelho-Souza SA, Peixoto RS, Oliveira JAL,Santos HF, D’Avila AMR, Rosado AS (2011). Microbial diversity of a Brazilian coastal region influenced by an upwelling system and anthropogenic activity.PLoS ONE,6,e16553.

Dexter SC (1993). Role of microfouling organisms in marine corrosion.Biofouling, 7, 97-127.

Dexter SC, Gao GY (1988). Effect of seawater biofilms on corrosion potential and oxygen reduction of stainless steel.Corrosion, 44, 717-723.

Dumas C, Mollica A, Feron D, Basseguy R, Etcheverry L, Bergel A (2007). Marine microbial fuel cell: use of stainless steel electrodes as anode and cathode materials.Electrochimica Acta,53, 468-473.

Eashwar M, Subramanian G, Palanichamy S, Rajagopal G (2011).The influence of sunlight on the localized corrosion of UNS S31600 in natural seawater.Biofouling,27(8), 837-849.

Erable B, Lacroix R, Etcheverry L, Féron D, Delia ML, Bergel A(2013). Marine floating microbial fuel cell involving aerobic biofilm on stainless steel cathodes.Bioresources Technology,142, 510-516.

Francis R, Byrne G, Warburton G (2011). The corrosion of superduplex stainless stell in different types of seawater.Corrosion/2011, Houston, TX, USA, Paper No. 11351.

Green JL, Bohannan BJ, Whitaker RJ (2008). Microbial biogeography: from taxonomy to traits.Science, 320,1039-1043.

Gribble KE, Nolan G, Anderson DM (2007). Biodiversity,biogeography and potential trophic impact ofProtoperidiniumspp. (Dinophyceae) of the southwestern coast of Ireland.Journal of Plankton Research, 29, 931-947.

Hasle GR, Syvertsen EE (1997). Marine Diatoms. In:Identifying Marine Phytoplankton. Academic Press, San Diego, CA, USA,5-385.

Hellio C, Yebra DM (2009). Introduction.Advances in marine antifouling coatings and technologies. CRC Press, Boca Raton,FL, USA, 1-15.

Ismail KM, Jayaraman A, Wood TK, Earthman JC (1999). The influence of bacteria on the passive film stability of 304 stainless steel.Electrochimica Acta, 44, 4685-4692.

Kamennaya NA, Ajo-Franklin CM, Northen T, Jansson C (2012).Cyanobacteria as biocatalysts for carbonate mineralization.Minerals, 2(4), 338-364.

Komárek J, Anagnostidis K (1986). Modern approach to the classification system of cyanophytes. 2. Chroococcales.Algological Studies/Archiv for Hydrobiologie, Supplement Volume 43, 157-226.

Landoulsi J, Cooksey KE, Dupres V (2011). Review-Interactions between diatoms and stainless steel: focus on biofouling and biocorrosion.Biofouling, 27, 1105-1124.

Larché N, Thierry D, Debout V, Blanc J, Cassagne T, Peultier J,Johansson E, Taravel-Condat C (2011). Crevice corrosion of duplex stainless steels in natural and chlorinated seawater.Rev.Métallurgie, 108(7-8), 451-463.

Little BJ, Lee JS (2007).Microbiologically Influenced Corrosion.John Wiley and Sons, Hoboken, NJ, USA, 261.

Little BJ, Lee JS, Ray RI (2008). The influence of marine biofilms on corrosion: a concise review.Electrochimica Acta, 54, 2-7.

Little BJ, Lee JS, Ray RI, Austin S, Biffinger JC (2013).Ennoblement due to biofil ms: indicator for potential corrosion and source of electrical energy.Recent Patents Mater.Sci., 6, 20-28.

Machuca LL, Bailey SI, Gubner R, Watkin ELJ, Ginige MP,Kaksonen AH, Heidersbach K (2013). Effect of oxygen and biofilms on crevice corrosion of UNS S31803 and UNS N08825 in natural seawater.Corrosion Science, 67, 242-255.

Machuca LL, Jeffrey R, Bailey SI, Gubner R, Watkin EL, Ginige MP, Kaksonen AH, Heidersbach K (2014). Filtration-UV irradiation as an option for mitigating the risk of microbiologically influenced corrosion of subsea construction alloys in seawater.Corrosion Science, 79, 89-99.

Mansfeld F (2007). The interaction of bacteria and metal surfaces.Electrochimica Acta, 52, 7670-7680.

Marszalek DS, Gerchakov SM, Udey LR (1979). Influence of substrate composition on marine microfouling.Applied and Environmental Microbiology, 38, 987-995.

Martin FJ, Dexter SC, Strom M, Lemieux, EJ (2007). Relations between seawater ennoblement selectivity and passive film semiconductivity on Ni-Cr-Mo alloys.Corrosion/2007,Houston, TX, USA, Paper No. 07255.

McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S(2012). Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal.Nature Reviews Microbiology, 10, 39-50.

Messano LVR, de Sathler L, Reznik LY, Coutinho R (2009). The effect of biofouling on localized corrosion of the stainless steels N08904 and UNS S32760.International Biodeterioration and Biodegradation, 63, 607-614.

Mieszkin S, Callow ME, Callow JA (2013). Interactions between microbial biofilms and marine fouling algae: a mini review.Biofouling, 29(9), 1097-113.

Mollica A, Trevis A (1976). Correlation entre la formation de la pellicule primaire et la modification de la cathodique sur des aciers inoxydables experimentes en eau de mer aux vitesses de 0.3 A 5.2 m/s.4th Int. Cong. Marine Corrosion and Fouling,Juan-Les-Pins, Antibes, France, 351–365.

Motoda S, Suzuki Y, Shinohara T, Tsujikawa S (1990). The effect of marine fouling on the ennoblement of electrode potential for stainless steels.Corrosion science, 31, 515-520.

Parot S, Vandecandelaere I, Cournet A, Delia ML, Vandamme P,Bergé M, Roques C, Bergel A (2011). Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater.Bioresource Technology, 102, 304-311.

Patil JS, Anil AC (2005). Biofilm diatom community structure:influence of temporal and substratum variability.Biofouling, 21,189-206.

Scotto V, DiCintio R, Marcenaro G (1985). The influence of marine aerobic microbial film on stainless steel corrosion behaviour.Corrosion, 25, 185-194.

Scotto V, Lai E (1998). The ennoblement of stainless steels in seawater: a likely explanation coming from the field.Corrosion Science, 40, 1007-1018.

Steidinger KA, Tangen K (1997). Dinoflagellates. Thomas, CR(Ed.).Identifying Marine Phytoplankton, Academic Press, San Diego, CA, USA, 387-584.

Underwood GJC, Barnett M (2006). What determines species composition in microphytobenthic biofilms?Microphytobenthos Symposium, Royal Netherlands Academy of Arts and Sciences,Amsterdam, Netherland, 121-138.

Van der Gucht K, Cottenie K, Muylaert K, Vloemans N, Cousin S,Declerck S, Jeppesen E, Conde-Porcuna JM (2007). The power of species sorting: local factors drive bacterial community composition over a wide range of spatial scales.Proceedings of the National Academy of Sciences, 104, 20404-20409.

Videla HA (1994). Biofilms and corrosion interactions on stainless steel in seawater.International Biodeterioration and Biodegradation, 34, 245-257.

Videla HA (2003).Biocorros?o, Biofouling e Biodeteriora??o de materiais. Edgard Blücher, S?o Paulo, SP, Brazil, 130.

Werner D (1977). The biology of diatoms. University of California Press, Berkeley, CA, USA, 484.

Zargiel KA, Coogan JS, Swain GW (2011). Diatom community structure on commercially available ship hull coatings.Biofouling,27, 955–965.

Zingone A, Siano R, D’Alelio D, Sarno D (2006). Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterranean Sea).Harmful Algae, 5,321–337.

Zobell CE, Allen EC (1935). The significance of marine bacteria in the fouling of submerged surfaces.Journal of Bacteriology, 29,239–251.