Seasonal variation in the biological succession of marine diatoms over 316L stainless steel in a coastal environment of Chile

Abstract Characterizing seasonal changes in diatom community profiles in coastal environments is scarce worldwide. Despite diatoms being prevalent in microfouling, their role in microbially influenced corrosion of metallic materials remains poorly understood. This study reports the effect of seasonal variations on the settlement of marine diatoms and corrosion of 316 L stainless steel surfaces exposed to Chilean coastal seawater. Electron microscopy imaging revealed a diverse assembly of diatoms, exhibiting pronounced differences at genus level between summer and winter seasons, with a significant delay in diatom settlement during winter. Electrochemical measurements indicated an active role of diatoms in increasing corrosion current during biofilm development. While the final diatom composition was similar irrespective of the season, the analyses of diatom assemblages over time differed, showing faster colonization when silicate and nitrate were available. This study lays the foundation for future research on the dominant season-specific genera of diatoms to unveil the microbial interactions that could contribute to corrosion and to evaluate their potential as bioindicators for alternative surveillance strategies.


Introduction
Microbially influenced corrosion (MIC) contributes to metallic failures in marine environments and represents roughly 20% of the trillions in annual corrosion costs for seawater structures (Koch et al. 2002;Little et al. 2020).By adhering to the metal surface and forming a biofilm, microorganisms drive the generation of MIC through their metabolic alteration of the metal's chemistry (Videla 1994;Al-Abbas et al. 2013;Sowards et al. 2014).
Biofilms develop when planktonic microorganisms interact with organic and inorganic particles that have settled on a surface, such as metals found in seawater (Comp� ere et al. 2001).Recent reviews of the biofilm concept highlight the complexity of this process (Flemming et al. 2021;Flemming et al. 2023).A historical analysis of metallic surfaces immersed in marine environments is associated with the presence of bacteria, the first colonizers that induce MIC (Videla 1994;Dang and Lovell 2000;Dang et al. 2008;Rampadarath et al. 2017).Nevertheless, previous studies have also shown the involvement of photosynthetic eukaryotes, such as diatoms in the MIC of metallic materials (Videla 1994;Qian and Xu 2012).Further analysis of marine steel structures affected by MIC has revealed a milieu of bacteria, archaea, and photosynthetic eukaryotes at the metal/biofilm interface after months of exposure, supporting the hypothesis of combined community participation in MIC processes during the early colonization (Celikkol-Aydin et al. 2016).It has been suggested that diatoms could play a role in MIC by producing reactive oxygen species, such as hydrogen peroxide, or by generating oxygen within the biofilm, thus increasing oxygen availability to aerobic or facultatively anaerobic bacteria involved in MIC (Messano et al. 2014;Eashwar et al. 2014).
Exposure to stainless steel (SS) in marine environments leads to an ennobling effect on the metal's electrochemical behavior, which involves displacement of the surface potential, such as open circuit potential (OCP) and corrosion potential (Ecorr), towards more positive (noble) values after immersion in natural seawater (Characklis and Cooksey 1983;Videla et al. 1989;F� eron and Dupont 1998).While diatoms alone may not cause ennoblement, their interaction with other microorganisms in marine biofilms appears to contribute to the ennoblement of the metallic surface (Dexter and Zhang 1991;reviewed in Landoulsi et al. 2011).Thus, the role of diatoms and their interactions in contributing to marine MIC mechanisms remains a topic of discussion and investigation.
The settlement of diatoms is considered the first notable increase in biomass throughout the biofilm succession (Little et al. 1991).These microorganisms are essential and abundant in coastal areas that are rich in nutrients (Malviya et al. 2016).However, anthropogenic industrial and commercial activities have had a significant impact on these dynamic areas (Halpern et al. 2008;Malone et al. 2010).For example, modifications in temperature, light, water velocity, nutrient concentration, and turbidity contribute to a decrease in the diatom community's ecological richness, abundance, and diversity (Cochero et al. 2014).Likewise, heavy metals and varying nutrient loads have a negative effect on community profiles (Belando et al. 2017), as observed in the coast of Chile (Glasner et al. 2021).Additionally, all the above, combined with immersion or intermittent exposure to steel structures, have resulted in distinct seasonal patterns of diatom colonization on the coast of England (Edyvean 1986).
While differences in the marine diatom community resulting from environmental changes have been reported, how this modification influences the development of MIC is missing.This study proposes that seasonality in the Chilean coasts determines the recruitment of marine diatoms and influences the temporal development of corrosion in SS.To evaluate this hypothesis, the diatom succession and the corrosion of 316 L grade SS during winter and summer were determined.To achieve this, a microcosm system was used to expose SS coupons to a continuous flow of natural Chilean coastal seawater for approximately fifteen weeks.Visual observations, environmental scanning electron microscopy (ESEM) imaging, and electrochemical measurements were conducted to determine the effect of temporal changes in the diatom community on SS corrosion.

Experimental set-up
AISI 316 L grade SS plates of dimensions 2 cm x 10 cm x 0.3 cm were exposed in 118 � 36 x 20 cm 3 acrylic aquaria (Figure 1).Four independent aquariums were employed as experimental units during each exposure season, i.e. summer and winter.The aquaria were installed in an open laboratory at the Estaci� on Costera de Investigaciones Marinas (ECIM) of the Pontificia Universidad Cat� olica de Chile, located in Las Cruces, Valpara� ıso, Chile (33 30 '16 S; 71 38 0 23 W).To replicate coastal water dynamics for exposure of the SS plates per season, a constant flux of 6-7 L min −1 of natural seawater taken from 5 m depth near the coast was used.Upon entry, the seawater was filtered by an 800 lm membrane and transported using an enclosed distribution system made of PVC without exposure to sunlight until it reached the aquariums.Surfaces of the SS plates were manually polished to obtain 310 grade finish (according to the UK scale) using silicon carbide paper (sandpaper), sonicated in distilled water to remove any remaining particles, rinsed with distilled water, degreased with acetone (Merck KGaA, Darmstadt, Germany), and dried at room temperature (�23 � C) according to standard protocol ASTM G1 − 03 (2011).The experimental design included 72 polished plates (per season) assembled in groups, each comprising three plates.Such groups of plates were placed in 24 acrylic holders, 6 per experimental unit (Figure 1), and kept at room temperature (�23 � C) in 50-mL plastic sterile conical tubes (Falcon by Corning Lab Sciences, Tewksbury, MA, USA) until exposure.
Biofilm development was evaluated using a time series of six selected points for each season based on previously reported marine corrosion studies conducted in the central zone of Chile (Fischer et al. 2016;Daille et al. 2020).The sampling times were 1, 3, 5, 8, 12, and 15 weeks of exposure.A plate holder was randomly removed from each aquarium.Hence, four holders were obtained at each sampling time.From each removed plate holder: one plate was used for microscopy imaging, and two for electrochemical analysis (e.g.eight plates were used for electrochemical analysis per week per season).Plates for ESEM observations were placed in 50-mL sterile conical tubes with aqueous 2% (wt/vol) glutaraldehyde solution (Merck KGaA, Darmstadt, Germany).While plates for electrochemical analysis were deposited in 50-mL plastic conical tubes with filter-sterilized (0.22 mm pore size, hydrophilic polyethersulfone membrane filter (Merck Millipore, Darmstadt, Germany) seawater.Additionally, water quality parameters were monitored at the beginning of the experiment and discretely at each sampling time using a multi-parameter digital meter (Hach HQ40dTM by Hach, CO, Ames, Iowa, USA).Main nutrients were measured according to Hansen and Grasshoff (1983).Values are listed in Table S1.

Morphological characterization of biofilms
The biofilms on SS plates were morphologically characterized using microscopic imaging to determine the surface's colonization pattern, i.e. a homogeneous or patchy distribution, and to taxonomically classify diatoms characterized by morphological features.Two SS plates from different holders were used for each sampling time to carry out ESEM characterization of the selected biofilms.The plates were aseptically cut with a 420 DremelV R cutting disc (Dremel, WI, USA).
Coupons of 2 cm x 2 cm x 0.3 cm were obtained from the bottom section of each plate.The cutting was carried out at short and intermittent intervals (< 30 s) to avoid heating and drying out the sample.The coupons were stored in separate containers filled with aqueous 2% (wt/vol) glutaraldehyde solution at 4 � C until analysis.For ESEM observations, the coupons were dried at room temperature (�23 � C) for 5 min, mounted with conductive carbon adhesive tape (Nisshin EM Co., Tokyo, Japan) on the sample stage, and observed in a Hitachi TM-1000 environmental scanning electron microscope (Tokyo, Japan) with magnifications of 100x, 500x, 1000x, 2500x, 5000x and 8000x at the Colorado School of Mines, CO, USA.Three to five randomly selected areas on each coupon were analyzed to obtain representative images.

Determination of surface coverage
The ESEM images with a magnification of 500x were processed using ImageJ software (https://imagej.nih.gov/ij/).The threshold tool was used to define the biofilm areas in 2-9 summer images and 3-8 winter images.The measure analysis tool was employed to establish the percentage of biofilm cover.Measured values are detailed in Table S2.

Electrochemical analysis
Two SS plates per plate holder were used to determine the electrochemical behavior of the metal/biofilm interface using a Reference 600 potentiostat (Gamry, Philadelphia, PA, USA) for each sampling time.The open circuit potential (OCP) of the plates was measured for approximately 1 h until stabilization (variation < 1 mV) was achieved.Potentiodynamic curves were recorded starting from the open circuit potential value at a rate of 0.5 mV s −1 .These two SS plates per sampled holder were used to obtain the cathodic and anodic curves, each performed on a different plate, as specified by Torres- Bautista et al. (2015) and Fischer et al. (2019).One-way ANOVA was employed to determine significant differences between the corrosion current measurements (p < 0.05).

Statistical analysis
Presence-absence matrices were constructed according to the identifications based on ESEM imaging, and they were evaluated in the Primer7 software (http:// www.primer-e.com/).Bray-Curtis similarity analyses were carried out, together with cluster analyses, to determine the similarity between the community patterns.Multivariate non-metric multidimensional scaling (NMDS) analyses were performed to visualize the differences between samples.From the same matrices, the ecological indices of richness (Margalef), evenness (Pielou index, J'), and diversity (Shannon-Wiener index, H'-log e ) (Morin 2011) of each community were determined.

Seasonal variation in the development and cover of marine biofilms
ESEM images revealed deposition of material of an unknown origin on surfaces of SS plates during the initial stage of exposure (first week of exposure in summer and three weeks of exposure in winter) (Figure S1(a, d), opaque areas).A more detail observation of these unidentified materials is presented in the supplementary Figure S2.Notably, it was observed that the regions with irregularities/imperfections had accumulated more of this unknown material and that the settlement of diatoms was associated with such areas (Figure S1(b, e)).
The settlement of larger microorganisms (> 3 mm) was delayed during the winter, shifting from one week (observed in summer) to even five weeks.Due to the sizes of those cells, predominantly composed of diatoms, the overall SS surface coverage was directly correlated with diatom presence, and the quantification showed similar trends for both seasons (Figure 2 -Upper Panel).ESEM analyses demonstrated that surface coverage during the summer increased by approximately 30% during the first week of exposure.After three weeks, there was a turning point in the process of surface colonization, with an average biofilm cover of approximately 60%.Finally, from three to fifteen weeks of exposure, between 80% and 100% of plate surfaces were colonized (Figure 2 -Upper Panel).Biofilm surface coverage during winter reached similar levels to that recorded for the summer-exposed plates after 15 weeks.The main differences in biofilm spread were noted between one to three weeks, with the winter-exposed plates having lower coverage than the summer plates.

Electrochemical behavior of AISI 316 grade stainless steel during summer and winter exposures
The free corrosion potential (E corr ) showed a peak of 0.22 V vs Ag/AgCl after one week of exposure during summer that decreased to −0.04 ± 0.02 V vs Ag/AgCl (week 3), −0.21 ± 0.04 V vs Ag/AgCl (week 5), and −0.22 ± 0.06 V vs Ag/AgCl (week 8) throughout the exposure.The E corr of the first three weeks showed a significant difference (p < 0.05) to the rest of the experiment, but no significant differences were found among the values measured after five weeks.During winter exposure, E corr decreased throughout the exposure, reaching values of −0.30 ± 0.05 V vs Ag/AgCl (Figure 2 -Lower Panel).Only the values of E corr reached at fifteen weeks showed significant differences (p < 0.05) when compared to all the values measured throughout the experiment.

Seasonal variation in the genera of diatoms settled over stainless steel
The experimental design led to recording the succession of diatoms assemblage along summer and winter.The NMDS analysis demonstrated that after one week of summer exposure and five weeks of winter exposure, there was a 60% similarity between diatom assemblages.Thus, the initial diatom recruitment was similar, despite the season, albeit there was a winter delay.The trajectory of diatom community changes over the remaining period followed a pattern with a similarity of 40%.The winter community profile reached a similarity of 60% with that of the summer community over the period from eight to fifteen weeks (Figure 3).Although kinetics was different, the final degree of similarity within the winter and summer assemblages indicated comparable recruitment.
However, temporal specificity at the genus level was observed.
Ecological indexes calculated based on the presence and abundance of identified diatoms revealed that diatom communities that developed during summer exposure had greater richness (S, Margalef) and diversity (H'-loge) than winter-exposed biofilms during the first eight weeks.Changes in diatom richness and diversity in the following weeks resulted in similar values for both types of biofilms (Table 1).Particularly, the twelve-week winter biofilms differed from the previous assemblage due to the settlement of new genus, such as Achnanthes sp., Entomoneis sp., Grammatophora sp., Melosira sp., Proschkinia sp., and Lyrella sp.Finally, in both seasons, a considerable diversity of diatoms was seen within the biofilm harvested at the endpoint of exposure (week 15).
The identification of different diatoms that have settled on the surface of the SS plates was accomplished using their key morphological features (Figure 4).In total, 24 different diatom genera were detected, with 21 present for the summer and 18 for the winter.Their distribution over time at each season is presented in Table 1.Among the diatoms identified on biofilms SS surfaces during summer and winter are the genera Navicula sp., Melosira sp., Amphora sp., Nitzschia sp., Trachyneis sp., and Achnanthes sp. that were most dominant, irrespective of the season.Cocconeis sp., Plagiotropis sp., Actinocyclus sp., Cylindrotheca sp., Triceratum sp., and Tabularia sp. were only observed during summer, while Pseudotaurosira sp., Surirella sp., and Fallacia sp. were only observed during winter.

Discussion
Sustained interest in understanding the importance of environmental variations in biocorrosion and biofouling development prompted the investigation on the seasonality of diatom assemblages and its implications for corrosion and fouling of SS of submersed infrastructure in and around coastal environments.
The initial settling of diatoms presented a patchy distribution irrespective of the seasons, similar to what was described by Acuña et al. (2006).This initial recruitment of diatoms was mainly associated with the areas on the SS surface with a deposited material layer (Figure S1).The origin of this material has not been determined, as it was not the aim of this investigation.It likely consists of natural organic matter (NOM), cells, and metabolic products (e.g.extracellular polymeric substances EPS; composed of polysaccharides and proteins) of primary colonizers such as bacteria and microalgae, as well as steel corrosion products (multiple minerals possible) (Chamberlain 1992;Callow and Callow 2006).These deposits, often observed in depressions/imperfections on the SS surface, were also directly associated with attached diatoms (Figure S1).It has been reported that colonization of SS starts at grain boundaries when field-exposed in seawater (Geesey and Costerton 1979) and under laboratory conditions (Geesey et al. 1996;Beech et al. 2000;Sreekumari et al. 2001;Javed et al. 2013;Jogdeo et al. 2017).Grain boundaries can act as surface defects, thus promoting microbial colonization.Grain boundaries can also likely serve as an accessible source of electron donation or acceptance for colonizing cells (Hamilton 2003).The observations herein were possible with the experimental design, which utilized time series analysis to enable the observation of the settlement of diatoms with a resolution of 24 h, with the subsequent analysis of their spatial distribution during the process.These findings underscore the importance of conducting interdisciplinary studies to better understand the relationship between diatom settlement and multivariate problems such as marine corrosion.Among the effects potentially resulting from surface modification by primary colonizers, we found that during winter, diatom colonization was delayed and, when it began, resulted in an increase in corrosion current but not potential values (Figure 2).This may be due to surface passivation until the initial settlement of diatoms is more complete after three to five weeks.However, during summer exposure, the brief ennoblement observed appears to correlate with the colonization of diatoms.The displacement of E corr toward more positive values after one week of exposure (Figure 2) indicates ennoblement, as previously described for SS in natural seawater (Scotto et al. 1985;Little et al. 1990;Scotto and Lai 1998;Martin et al. 2007;Little et al. 2008;Little et al. 2012;Abi Nassif et al. 2020).This effect can be associated with active marine primary biofilms mainly composed of bacteria, which can cause the rupture of the passive SS layer and lead to increased pitting corrosion (Pendyala et al. 1996;Beech et al. 2000;Trigodet et al. 2019).It is not yet clear whether the noted ennoblement directly affects the attachment of diatoms.Nevertheless, data indicated that the attachment of diatoms modified the values of E corr and increased the SS corrosion current.The increase in oxygen availability resulting from the photosynthetic metabolism of diatoms would drive the cathodic process, i.e. influence oxygen reduction rates.It has been stated that photosynthetic microorganisms activate the surface and adversely influence corrosion (Liu et al. 2018).Although diatom colonization and associated corrosion are addressed in this study, the findings do not provide evidence for causal relationships between these two processes.
In addition to the mechanism described above, the observed decrease in E corr during summer after the first week of exposure could have been indirectly influenced by the increase in diatom density and interactions among the microorganisms that favour the transport of electrons from the bulk metal to oxygen, i.e. to the ultimate electron acceptor (Hamilton 2003).Laboratory conditions can influence E corr behaviour itself (Messano et al. 2013).However, given the time frame in this case, the conditions reached in the first week of exposure in summer suggest that the rapid colonization of diatoms was facilitated by the presence of a previous biofilm on the surface and/or by the presence of organic matter in the surface (Figure S1).Alternatively, the development of this biofilm may have been facilitated by its community composition and the greater availability of resources that support diatom metabolism during summer, such as light and nutrients.Additionally, the increase in oxygen availability in the biofilms due to photosynthetic activity of the diatoms could promote bacterial activity of facultative aerobes that influence the electrochemical behavior of the surface.For example, nitrate-reducing bacteria like Marinobacter sp. that are known to interact with diatoms (Sterling et al. 2023) but are also able to produce MIC (Yuk et al. 2020).Some of the known diatom-bacteria associations are specific (Shibl et al. 2020), so in future work it will be interesting to analyze whether the bacterial community associated with the dominant diatoms from early times has a role in the electrochemical modifications observed in this study (Figure 2).Regarding the availability of nutrients, the Chilean coast is characterized as an area with upwelling events during the summer season (Aguirre et al. 2012), which may have led to the observed variability in the concentration of nutrients such as silicate and nitrate (Table S1), crucial for diatom development (Stief et al. 2022).This geochemical variability may have contributed to the abundance of diatoms in the water column and the relative rapidity of their colonization during summer (Figure 2).These results highlight the importance of conducting local studies to distinguish whether an effect is due to seasonal environmental changes or direct surface alterations by primary colonizers.Morphological diatom community analyses showed that although exposure during winter had delayed initial recruitment (Figure 2), the resulting diatom community had 60% similarity with the one developed during summer (Figure 3).In addition, the richness of diatoms in winter reached higher values than those of summer in only four weeks (between weeks 8 and 12 of exposure) (Table 1).Finally, after fifteen weeks of exposure, the winter assemblage reached 60% similarity to that of the eight-week summer community (Figure 3).Ecological indexes revealed that after this period, diatom diversity between the seasons was comparable (Table 1), thus indicating that both communities reached a mature stage in succession, as reported for epilithic biofilm development (Jackson et al. 2001).This study may also reflect seasonal diatom deposition/reservoirs on SS coupons that represent localized and/or regional oceanic diatom blooms over time.
The observed diatom succession is likely to result in undesirable settlement and accumulation of biofouling on submerged surfaces (Lau et al. 2005), which could facilitate the development of failures in marine equipment and infrastructure (Li and Ning 2019).Some of the identified genera (Table 1) were widespread and previously described in other biofouling systems.
To put them into context, Amphora sp., Nitzschia sp., Navicula sp., Cylindrotheca sp., and Pleurosigma sp. were found after exposure to metallic surfaces in seawater from the South Atlantic (Messano et al. 2009;Messano et al. 2014).Cocconeis sp., Amphora sp., Nitzschia sp., and Melosira sp. were found in river waters in France (Marconnet et al. 2008;Richard et al. 2017).Cocconeis sp., Amphora sp., Nitzschia sp., Cylindrotheca sp., and Achnanthes sp., were associated with metals exposure to estuarine waters in India (Mitbavkar and Anil 2000;Mitbavkar and Anil 2008).Navicula sp. was found in the latter but also in metals exposed to seawater in the Indian Ocean (Eashwar et al. 2009).
Notably, some of these genera can successfully colonize surfaces protected with antifouling coatings in marine environments (Teran- Arce et al. 2004;Zargiel et al. 2011;Muthukrishnan et al. 2014;Zargiel and Swain 2014) and even be transported due to the release of ship hull coatings (Sweat et al. 2017).To the extent of knowledge, this investigation is the first study focused on increasing the list of possible candidate diatoms that generally contribute to the fouling of metallic surfaces and ought to be considered when planning infrastructure deployment in the Eastern South Pacific Ocean.Although these diatoms contribute to biofouling during both seasons, whether and to what extent they can influence the corrosion of SS and other metallic materials remains to be determined.Some diatom genera in the assembly maintain temporary patterns of colonization, irrespective of the season.Navicula sp. and Thalassiosira sp. are the first diatoms to settle.Subsequently, Trachyneis sp., Amphora sp., and Nitzschia sp.joined the biofilm, and after its settlement, Thalassiosira sp.disappeared.Then, Melosira sp. and Achnantes sp.joined the assembly.This pattern indicates that the above genera followed a sequence of recruitment even when they were delayed during winter.The summer-exclusive genus Cocconeis sp. was part of the assembly since its early stages, and its presence has been observed in other systems, exclusively during spring-summer seasons (Molino et al. 2009).The rest of the season-exclusive genera appeared in the biofilm after 15 weeks of exposure, suggesting that they do not have the capabilities to colonize the metal directly and/or need particular interactions with other species for their settlement.Diatom colonization under those conditions and its long-term importance for the recruitment of macro-organisms and their cumulative impact on MIC remains to be evaluated in further research.
The data reported herein represent an advance toward a comprehensive understanding of marine diatom seasonal dynamics associated with SS in coastal environments.These data demonstrate that the succession and composition of diatom assemblages differ under winter and summer during a shortterm exposure of 15 weeks.The seasonal effect reveals the dominance of different, season-dependent diatoms' genera, despite equivalent values of diversity (Table 1).These results can lead to early detection of these genera as a warning sign of biocorrosion/biofouling and act as bioindicators in the development of new mitigation strategies for MIC.

Figure 1 .
Figure 1.Schematic representation of the exposure system and materials.Aquarium diagram: Design of the acrylic experimental unit utilized to expose the plates to a continuous flow of seawater.The direction of the flow of water in it is indicated in blue.Plates holder: An acrylic holder was used to group the plates and place them in the aquarium.At each sampling time, one holder was collected from each aquarium.Plate dimensions: The size and perforations of the plate are indicated.

Figure 2 .
Figure 2. Characterization of the succession of diatoms on AISI 316 grade stainless steel surfaces and the effect on its electrochemical behavior.Upper panel: Representative ESEM images of biofilms on surfaces of AISI 316 grade stainless steel coupons colonized during summer and winter exposures.The two different panels per season correspond to biological replicates.The quantified cover percentage is shown over time (n ¼ 2-9).Lower panel: corrosion potential (E corr ) and corrosion current (I corr ) measurements during summer (orange) and winter (blue) exposures (n ¼ 2-4).

Figure 3 .
Figure 3. NMDS plot depicting sequential settlement of different genera of diatoms during summer and winter.Similarity among the composition of the diatoms is indicated with gray dashed lines, and dark gray corresponds to 60% and light gray to 40% similarities.The seasonal trajectory of the diatom community composition is marked with colored dashed lines.

Table 1 .
Ecological indexes and succession of diatoms over stainless steel during summer and winter exposure to natural seawater.