Dynamic surface deformation of silicone elastomers for management of marine biofouling: laboratory and field studies using pneumatic actuation

Many strategies have been developed to improve the fouling release (FR) performance of silicone coatings. However, biofilms inevitably build on these surfaces over time. Previous studies have shown that intentional deformation of silicone elastomers can be employed to detach biofouling species. In this study, inspired by the methods used in soft-robotic systems, controlled deformation of silicone elastomers via pneumatic actuation was employed to detach adherent biofilms. Using programmed surface deformation, it was possible to release > 90% of biofilm from surfaces in both laboratory and field environments. A higher substratum strain was required to remove biofilms accumulated in the field environment as compared with laboratory-grown biofilms. Further, the study indicated that substratum modulus influences the strain needed to de-bond biofilms. Surface deformation-based approaches have potential for use in the management of biofouling in a number of technological areas, including in niche applications where pneumatic actuation of surface deformation is feasible.     

Marine Biofilms as Mediators of Colonization by Marine Macroorganisms: Implications for Antifouling and Aquaculture

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.     

Effect of copper on multiple successional stages of a marine fouling assemblage

Copper based paints are used to prevent fouling on the hulls of ships. The widely documented effect of copper on hull assemblages may be primarily due to direct effects on the invertebrates themselves or indirect effects from copper absorbed into the microbial biofilm before settlement has commenced. Artificial units of habitat were exposed to varied regimes of copper to examine (1) the photosynthetic efficiency and pigments of early-colonising biofilms, and (2) subsequent macroinvertebrate assemblage change in response to the different regimes of copper. Macroinvertebrate assemblages were found to be less sensitive to the direct effects of copper than indirect effects as delivered through biofilms that have been historically exposed to copper, with some species more tolerant than others. This raises further concern for the efficacy of copper as a universal antifoulant on the hulls of ships, which may continue to assist the invasion of copper-tolerant invertebrate species.     

Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora.

Survival and growth of Legionella pneumophila in both biofilm and planktonic phases were determined with a two-stage model system. The model used filter-sterilized tap water as the sole source of nutrient to culture a naturally occurring mixed population of microorganisms including virulent L. pneumophila. At 20 degrees C, L. pneumophila accounted for a low proportion of biofilm flora on polybutylene and chlorinated polyvinyl chloride, but was absent from copper surfaces. The pathogen was most abundant on biofilms on plastics at 40 degrees C, where it accounted for up to 50% of the total biofilm flora. Copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila organisms. The pathogen was able to survive in biofilms on the surface of the plastic materials at 50 degrees C, but was absent from the copper surfaces at the same temperature. L. pneumophila could not be detected in the model system at 60 degrees C. In the presence of copper surfaces, biofilms forming on adjacent control glass surfaces were found to incorporate copper ions which subsequently inhibited colonization of their surfaces. This work suggests that the use of copper tubing in water systems may help to limit the colonization of water systems by L. pneumophila.     

Microbe-surface interactions in biofouling and biocorrosion processes.

The presence of microorganisms on material surfaces can have a profound effect on materials performance. Surface-associated microbial growth, i.e. a biofilm, is known to instigate biofouling. The presence of biofilms may promote interfacial physico-chemical reactions that are not favored under abiotic conditions. In the case of metallic materials, undesirable changes in material properties due to a biofilm (or a biofouling layer) are referred to as biocorrosion or microbially influenced corrosion (MIC). Biofouling and biocorrosion occur in aquatic and terrestrial habitats varying in nutrient content, temperature, pressure and pH. Interfacial chemistry in such systems reflects a wide variety of physiological activities carried out by diverse microbial populations thriving within biofilms. Biocorrosion can be viewed as a consequence of coupled biological and abiotic electron-transfer reactions, i.e. redox reactions of metals, enabled by microbial ecology. Microbially produced extracellular polymeric substances (EPS), which comprise different macromolecules, mediate initial cell adhesion to the material surface and constitute a biofilm matrix. Despite their unquestionable importance in biofilm development, the extent to which EPS contribute to biocorrosion is not well-understood. This review offers a current perspective on material/microbe interactions pertinent to biocorrosion and biofouling, with EPS as a focal point, while emphasizing the role atomic force spectroscopy and mass spectrometry techniques can play in elucidating such interactions.     

Biomineralisation of manganese on titanium surfaces exposed to seawater

A 2-year long study was carried out to isolate and characterise various bacterial species present in the biofilm formed on titanium surfaces exposed to seawater and to assess the manganese oxidizing potential of the marine isolates. The amount of manganese present in the biofilm was also measured using atomic absorption spectrometry (AAS). The results showed that titanium was susceptible to biofouling. More than 50% of the culturable marine bacterial isolates were capable of bringing about oxidation of Mn(II). All these manganese oxidizing bacteria were heterotrophic. Autotrophic manganese oxidizing bacteria such as Leptothrix was not isolated in the present study. The AAS results confirmed that the manganese content in the biofilms increased with increasing exposure time. Hence, the study indicates that the titanium surfaces when exposed to seawater were colonised by a large number of heterotrophic bacteria, which have the ability of bringing about biomineralisation of manganese.     

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.     

Settlement of Pomatoceros lamarkii (Serpulidae) larvae on biofilmed surfaces and the effect of aerial drying

The settlement responses of Pomatoceros lamarkii (Polychaeta: Serpulidae) larvae to biofilms of varying age on slate surfaces and to dried biofilms on slate surfaces were investigated in the laboratory. Settlement experiments were performed as multi-treatment, still water assays. Larvae did not settle on clean, non-biofilmed slates but settled on biofilms up to 28 days old. Settlement intensity was closely related to the bacterial density of a biofilm. Drying a biofilmed surface for 1-2 h at 20 degrees C to simulate a single tidal emersion completely negated the former inductive effect of the biofilm. Drying also negated the larval settlement-inducing effect that normally results from the presence of conspecific adults. The settlement inhibition lasted for approximately 5 days following a single drying event. Larvae settled readily on biofilms exposed to formalin and antibiotics. Treating biofilms with formalin or antibiotics before or after drying had no effect upon larval avoidance of dried biofilms. Freeze-drying a biofilm had the same effect as aerial drying. The biofilm drying effect could not be mimicked by exposing biofilms to hyper-saline seawater. The finding that P. lamarkii larvae do not settle on dried biofilms could have significance in explaining the natural distribution of this species in the intertidal.     

Propagule pressure determines recruitment from a commercial shipping pier

Artificial structures associated with shipping and boating activities provide habitats for a diverse suite of non-indigenous marine species. Little is known about the proportion of invader success in nearby waters that is attributable to these structures. Areas close to piles, wharves and piers are likely to be exposed to increasing levels of propagule pressure, enhancing the recruitment of non-indigenous species. Recruitment of non-indigenous and native marine biofouling taxa were evaluated at different distances from a large commercial shipping pier. Since artificial structures also represent a desirable habitat for fish, how predation on marine invertebrates influences the establishment of non-indigenous and native species was also evaluated. The colonisation of several non-indigenous marine species declined rapidly with distance from the structure. Little evidence was found to suggest that predators have much influence on the colonisation success of marine sessile invertebrate species, non-indigenous or otherwise. It is suggested that propagule pressure, not predation, more strongly predicts establishment success in these biofouling assemblages.     

Biological filtration limits carbon availability and affects downstream biofilm formation and community structure

Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (R(2) = 0.92) between the biomass (given by the A(600) in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by Sphingomonadaceae and Caulobacter organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while Betaproteobacteria were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g., Aquabacterium, Caulobacter, and Legionella) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run.     

Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms

The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.     

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.     

Advances in the treatment of problematic industrial biofilms

In nature, microorganisms tend to form biofilms that consist of extracellular polymeric substances with embedded sessile cells. Biofilms, especially mixed-culture synergistic biofilm consortia, are notoriously difficult to treat. They employ various defense mechanisms against attacks from antimicrobial agents. Problematic industrial biofilms cause biofouling as well as biocorrosion, also known as microbiologically influenced corrosion. Biocides are often used to treat biofilms together with scrubbing or pigging. Unfortunately, chemical treatments suppress vulnerable microbial species while allowing resistant species to take over. Repeated treatment cycles are typically needed in biofilm mitigation. This leads to biocide dosage escalation, causing environmental problems, higher costs and sometimes operational problems such as scale formation. New treatment methods are being developed such as enhanced biocide treatment and bacteriophage treatment. Special materials such as antibacterial stainless steels are also being created to combat biofilms. This review discussed some of the advances made in the fight against problematic industrial biofilms.     

The correlation between biofilm biopolymer composition and membrane fouling in submerged membrane bioreactors

Biofouling, the combined effect of microorganism and biopolymer accumulation, significantly reduces the process efficiency of membrane bioreactors (MBRs). Here, four biofilm components, alpha-polysaccharides, beta-polysaccharides, proteins and microorganisms, were quantified in MBRs. The biomass of each component was positively correlated with the transmembrane pressure increase in MBRs. Proteins were the most abundant biopolymer in biofilms and showed the fastest rate of increase. The spatial distribution and co-localization analysis of the biofouling components indicated at least 60% of the extracellular polysaccharide (EPS) components were associated with the microbial cells when the transmembrane pressure (TMP) entered the jump phase, suggesting that the EPS components were either secreted by the biofilm cells or that the deposition of these components facilitated biofilm formation. It is suggested that biofilm formation and the accumulation of EPS are intrinsically coupled, resulting in biofouling and loss of system performance. Therefore, strategies that control biofilm formation on membranes may result in a significant improvement of MBR performance.     

The application of nitric oxide to control biofouling of membrane bioreactors

A novel strategy to control membrane bioreactor (MBR) biofouling using the nitric oxide (NO) donor compound PROLI NONOate was examined. When the biofilm was pre‐established on membranes at transmembrane pressure (TMP) of 88–90 kPa, backwashing of the membrane module with 80 μM PROLI NONOate for 45 min once daily for 37 days reduced the fouling resistance (R_f) by 56%. Similarly, a daily, 1 h exposure of the membrane to 80 μM PROLI NONOate from the commencement of MBR operation for 85 days resulted in reduction of the TMP and R_f by 32.3% and 28.2%. The microbial community in the control MBR was observed to change from days 71 to 85, which correlates with the rapid TMP increase. Interestingly, NO‐treated biofilms at 85 days had a higher similarity with the control biofilms at 71 days relative to the control biofilms at 85 days, indicating that the NO treatment delayed the development of biofilm bacterial community. Despite this difference, sequence analysis indicated that NO treatment did not result in a significant shift in the dominant fouling species. Confocal microscopy revealed that the biomass of biopolymers and microorganisms in biofilms were all reduced on the PROLI NONOate‐treated membranes, where there were reductions of 37.7% for proteins and 66.7% for microbial cells, which correlates with the reduction in TMP. These results suggest that NO treatment could be a promising strategy to control biofouling in MBRs.     

A synergistic d-tyrosine and tetrakis hydroxymethyl phosphonium sulfate biocide combination for the mitigation of an SRB biofilm

Microbiologically influenced corrosion (MIC) is a major problem in various industries such as oil and gas, and water utilities. Billions of dollars are lost to microbiologically influenced corrosion (MIC) each year in the US. The key to MIC control is biofilm mitigation. Sulfate-reducing bacteria (SRB) are often the culprits. They are also involved in souring and biofouling. SRB biofilms are notoriously difficult to eradicate. Due to environmental concerns and increasing costs, better biocide treatment strategies are desired. Recent studies suggested that D: -tyrosine and some other D: -amino acids may signal biofilm dispersal. Experimental results in this work indicated that D: -tyrosine is an effective biocide enhancer for tetrakis hydroxymethyl phosphonium sulfate (THPS) that is a green biocide. Desulfovibrio vulgaris (ATCC 7757) was used in biofilm prevention and biofilm removal tests. It was found that 100?ppm D: -tyrosine alone and 50?ppm THPS alone were both ineffective against the SRB biofilm. However, when 1?ppm D: -tyrosine was combined with 50?ppm THPS, the synergy between the two chemicals successfully prevented the establishment of the SRB biofilm on C1018 mild steel coupon surfaces in batch treatment tests. It also eradicated established SRB biofilms from coupon surfaces in both 1 and 3-h shock treatment tests.     

Uniaxial compression measurement device for investigation of the mechanical stability of biofilms.

The mechanical stability of biofilms is important for biotechnology, as sloughing of the biomass due to mechanical failure of the biofilm matrix can lead to severe interferences with biofilm processes. In cases of biofouling, biofilms have to be removed, in which case their mechanical stability must be overcome. The apparent modulus of elasticity and the yield strength as obtained from uniaxial compression experiments can be taken as parameters indicative for the mechanical stability of a biofilm. A film rheometer is presented which allows for the determination of these quantities, using model biofilms of Pseudomonas aeruginosa grown on membrane filters. The compressive stress-strain behaviour up to the point of failure is recorded at a compression speed of 1 microm s(-1). In accordance with the stress-strain curve, the investigated biofilm can be described as viscoelastic material, which demonstrates plastic flow properties. The extracellular polymeric substances (EPS), which keep biofilms together, form a temporary network of fluctuating junction points. Above the yield point, the gel structure fails and the system behaves as a highly viscous fluid. The apparent modulus of elasticity and the yield point are considered to be useful parameters for characterizing the mechanical properties of biofilms.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.