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Antibiofilm Potential of Enzymes as Ecofriendly Antifouilng Additives
Abstract
Marine biofilms form is the first step of fouling process and after film layer has formed, macro organisms hold on smoothly over this film and mature the fouling process. Every year billions of dollars have been spent in order to prevent biofouling situation. Whereas heavy metals and biocide have been used more in antifouling coating today studies about improving eco-friendly nontoxic coating have been becoming widespread. For this purpose, in our study, different concentration of varied commercially available hydrolytic enzymes and enzymes mix (Protease, α-Amilase, Lipase, Xylanase, Pectinase, Glucanase, Viscozyme, and Lysing Complex) were experimented as environment friendly antifouling agent over the biofilm bacteria Pseudoalteromonas porphyrae FJ200650 and Pseudoalteromonas elyakovii FJ200649 which were isolated from İzmir Bay. The antibiofilm potential of enzymes was evaluated separately using the fluorescence microplate reader at varying concentrations. The experimental method was based on total biofilm biomass quantification by the fluorescent dye DAPI after 3 and 24 h incubation period. These analyses were performed as prevention and detachment test. As a result of analysis, it is formalized by the adhesion of marine biofilm-forming bacteria was examined in the effectiveness of hydrolases in sea-water throughout at 3 and 24 hours’ incubation processes. Overall, the mixtures of enzymes can be use as eco-friendly antifouling additives in antifouling coatings.
... Lipases from marine sources have shown attractive characteristics such as thermostability [98], high-salt tolerance [99], cold-adaptation [100], extreme pH tolerance [101], organic solvents tolerance [102], and enantioselectivity [103]. These important properties are largely required in industrial and biotechnological applications such as plastic degradation [104] and biodiesel synthesis [105], as well as anti-biofilm and biofouling additives [106]. ...
Epiphytic bacteria from marine macroalgae synthesize enzymes of
industrial and biotechnological interest. In this study, we obtained two
DNA candidate fragments for lipid-degrading enzymes from the total
DNA of Ulva lactuca-associated epiphytic bacteria. First, we evaluated a
method for total bacterial DNA isolation from the surface of U. lactuca
thalli. Then, we designed sets of primers and used them directly for
PCR amplification. The resulting PCR products were sequence-analyzed
and used for expression and functional evaluation with the Escherichia
coli pBAD-TOPO system. We obtained high molecular weight and
good quality total bacterial DNA that served as a template to identify
a fragment corresponding to an Acetyl-CoA C-Acetyltransferase (or
Thiolase), and a candidate fragment for a versatile “true” lipase. We
expressed the possible “true” lipase gene fragment heterologously in
Escherichia coli and obtained proof of hydrolytic activity on Tributyrin,
Tween-20, and Olive-oil media. This study resulted in new knowledge
on U. lactuca-associated epiphytic bacteria as possible brand-new sources
of enzymes such as thiolases and “true” lipases. However, future studies
are required to describe the characteristics and important applications of
these candidate enzymes.
... The assay was performed and modified as described by Leroy et al. [29] and Aykin et al. [30,31]. ...
ARTICLE INFO Abstract In the maritime industry, biofouling is a severe problem and represents a serious matter of economic losses worldwide. The sizable investments are made for the prevention of biofouling by marine related sectors, especially in the paint industries. It is known that antifouling applications contain heavy metal and biocides which are hazardous for the marine environment and, for this reason, new studies are focusing on the development of environmentally and friendly paints. The main target of our investigation was the identification of natural marine compounds from tunicate and sponge species, evaluating their capability to inhibit biofilm progress which is the first step of fouling process, and their plausible usage in industrial products, too. Therefore, in this study, the antibiofilm effects of four marine invertebrates, Styela plicata, Clavelina lepadiformis, Dysidea avara and Spirastrella sp., collected in different seasons along the coasts of Izmir Bay, were evaluated. The extracts from two tunicates (Styela plicata and Clavelina lepadiformis) and a sponge (Dysidea avara) were selected due to their interesting antibiofilm capacities. A bio-active-guided isolation method allowed us to identification of the active fractions which were tested for inhibition of bacterial adhesion at different concentration. The content of the active fractions was identified by NMR spectroscopy, LC-MS and HRMS analysis.
... The assay was performed and modified as described by Leroy et al. [29] and Aykin et al. [30,31]. ...
Bacterial biofilm growth in aquatic environments can be promoted by natural compounds, such as polysaccharides and proteins. Fucoidan is a group of marine sulfated polysaccharides found in cell wall matrix of brown and red algae. In the present study, efficiencies of fucoidan extracts obtained by two alternate methods from Cystoseira sp. and Padina pavonica were investigated of stimulation of biofilm development of Pseudoalteromonas agarivorans . The fucoidan rich extracts were tested for adhesion of biofilm bacterium in black polystyrene flat-bottom microplates. In the biofilm experiments, the extracts were found to stimulate biofilm development. The maximum mean for the biofilm stimulation efficiencies were 67.52% for SG1 - Cystoseira sp. and 67.50% for SG1 - P. pavonica . The results highlighted that Pseudoalteromonas agarivorans was able to adhere to the plates, and the growth rates were increased with the addition of fucoidan extracts. This research indicated that fucoidan can be effective in biofilm development strategies in aquatic environments.
Due to increased concern about the adverse effects of biocides on the environment, the creation of environmentally friendly coatings is now of considerable interest. This work is yet another attempt to use the enormous potential of marine microorganisms to protect surfaces from fouling. A strain of Pseudomonas aeruginosa 1242 was selected for this study after screening of different microorganisms for ability to inhibit the growth of microorganisms; this strain appeared to be highly active. Using the enzymatic approach, we have chosen the medium and cultivation conditions for the strain 1242, which ensured the maximum production of hydrolytic exoenzymes. The cells and metabolite complex of the strain were then embedded into an epoxy-resin-based commercial anticorrosion coating. It was shown in laboratory experiments that the bacterial cells introduced into the epoxy coating remain viable for a sufficiently long time. The results of trials of the experimental coatings in seawaters of different climatic zones demonstrated that the cells of this strain and the complex of enzymes and secondary metabolites that they produced could prevent adhesion of micro- and macrofoulers.
Current antifouling technologies for ship hulls are based on metals such as cuprous oxide and co-biocides like zinc pyrithione. Due to the persistent adverse environmental effects of these biocides, enzyme-based antifouling paints are proposed as a bio-based, non-accumulating alternative. Here, a hydrogen peroxide-producing system composed of hexose oxidase (HOX, EC 1.1.3.5), glucoamylase (GA, EC 3.2.1.3) and starch is tested for the chemical and physical functionalities necessary for successful incorporation into a marine coating. The activity and stability of the enzymes in seawater was evaluated at different temperatures, and paint compatibility was assessed by measuring the distribution and activity of the enzymes incorporated into prototype coating formulations. We used a biomimetic encapsulation procedure for HOX through polyethylenimine-templated silicaco-precipitation. The co-precipitation and formulation of a powder for mixing into a marine paint was performed in a one-step economical and gentle formulation process, in which silica co-precipitated HOX was combined with GA and starch to form the antifouling system. Silicaco-precipitation significantly improved the stability and performance of the antifouling system in marine-like conditions. For example, encapsulation of HOX resulted in 46% higher activity at pH 8, and its stability in artificial seawater increased from retaining only 3.5% activity after 2 weeks to retaining 55% activity after 12 weeks. A coating comprising the full enzyme system released hydrogen peroxide at rates exceeding a target of 36 nmol cm−2 d−1 for 3 months in a laboratory assay, and had potential for prolonged action through incorporation in a self-polishing coating.
Adhesion of microorganisms to surfaces in marine environments leads to biofouling. The deleterious effects of biofilm growth in the marine environment are numerous and include energy losses due to increased fluid frictional resistance or to increased heat transfer resistance, the risk of corrosion induced by microorganisms, loss of optical properties, and quality control and safety problems. Antifouling agents are generally used to protect surfaces from such a biofilm. These agents are toxic and can be persistent, causing harmful environmental and ecological effects. Moreover, the use of biocides and regular cleaning considerably increase the maintenance costs of marine industries. An improved knowledge of biofilm adhesion mechanisms is needed for the development of an alternative approach to the currently used antifouling agents. The aim of this study is to characterise the chemical composition of the molecules first interacting with stainless steel during the period immediately following immersion in natural seawater and to elucidate the kinetics of the adsorbtion process. Proteins are shown to adhere very rapidly, closely followed by carbohydrates. The distribution on the surface of organic molecules is also examined. The adsorbate on the surface is not a continuous film but a heterogeneous deposit, whose average thickness varies widely. The cleaning procedures used affect the adsorption kinetics. In particular, cleaning with hexane results in slower adsorption of nitrogen-containing species than does cleaning in acetone.
The goal of the research was to isolate and identify biofilm bacteria on static panels, and to determine the minimum inhibitory concentrations (MICs) of the antifouling agents preventing the growth of biofilm bacteria. The effectiveness of four types of antifouling paints containing additives based on zinc oxide, copper oxide, tributyltin, fluorine and triazine diamine as well as one type of rustproof paint against biofilm formation on static panels was determined. Bacterial strains were isolated from biofilms formed on test panels embedded in the two marinas located at Izmir region. Bacterial growth was found on all test panels and counts of them on the surfaces of the panels rapidly increased in the period of 7-15 days. Twenty-six strains were cultured, and a total of 13 unique strains were identified. Phylogenetic analysis using 16S rDNA sequences in-dicated that all of the strains belonged to the ã-Proteo-bacteria and Firmicutes subclasses (Pseudoalteromonas agarovorans, P. haloplanktis, P. marina (3 strains), P. elyakovii (2 strains), P. porphyrae (2 strains), Alteromonas genoviensis, A. alvinella, Vibrio lentus and Exiguobacterium homiense). The paint, which contains triazine diamine and copper oxide, showed strong antibacterial activity against each individual strain. None of the isolates grew in all concentrations (1/2 to 1/1024) of this paint. Physico-chemical parameters of seawater and viable bacteria counts displayed relations during the study period. However, it was not found any strong correlation between viable bacteria density and nutrient concentrations.
The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.
The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most effective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some enzymatic preparations tested such as Amano protease were not only ineffective but also increased the number of adhered bacterial cells. Enumeration using epifluorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and DAPI stained adhered D41 cells confirmed these observations. Overall, these results demonstrated that hydrolases could either prevent adhesion and remove adhered bacterial cells effectively, or conversely increase bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.
Leading experts from all over the world present an overview of the use of enzymes in industry for: The production of bulk products, such as glucose, or fructose. Food processing and food analysis. Laundry and automatic dishwashing detergents. The textile, pulp and paper and animal feed industries. Clinical diagnosis and therapy. Genetic engineering. The book also covers identification methods of new enzymes and the optimization of known ones, as well as the regulatory aspects for their use in industrial applications. Up to date and wide in scope, this is a chance for non-specialists to acquaint themselves with this rapidly growing field. '...The quality...is so great that there is no hesitation in recommending it as ideal reading for any student requiring an introduction to enzymes. ...Enzymes in Industry - should command a place in any library, industrial or academic, where it will be frequently used.' The Genetic Engineer and Biotechnologist. 'Enzymes in Industry' is an excellent introduction into the field of applied enzymology for the reader who is not familiar with the subject. ... offers a broad overview of the use of enzymes in industrial applications. It is up-to-date and remarkable easy to read, despite the fact that almost 50 different authors contributed. The scientist involved in enzyme work should have this book in his or her library. But it will also be of great value to the marketing expert interested in the present use of enzymes and their future in food and nonfood applications.' Angewandte Chemie. 'This book should be available to all of those working with, or aspiring to work with, enzymes. In particular academics should use this volume as a source book to ensure that their 'new' projects will not 'reinvent the wheel'.' Journal of Chemical Technology and Biotechnology.
The aim of this work is to study the antifouling performance and water uptake behaviour of coatings formulated with papain (an environmentally friendly pigment). Antifouling coatings have been formulated using rosin (natural resin) as matrix and papain adsorbed in activated carbon as pigment. Electrochemical impedance spectroscopy (EIS) measurements were used to evaluate the behaviour of the formulated coatings in the marine environment and to calculate the apparent water coefficient of diffusion (D). FTIR and XPS analyses confirm the presence of papain adsorbed inside the activated carbon pores and the release of papain in water. Immersion tests in the Mediterranean Sea were carried out for 7 months to verify the degree of biofouling of the tested coatings. These field assays clearly indicate the excellent behaviour of papain-based antifouling coatings; the results being similar to those achieved using a commercial coating. Additionally, the EIS technique is shown to be a great tool to predict the coating diffusivity of antifouling coatings before immersion tests. Furthermore, the use of biodegradable papain as a nature-friendly antifouling agent can eliminate the negative environmental impact caused by metals and chemical biocides typically used in current commercial formulations.
BACKGROUND: Chemical methods used to control biofilm formation in filtration systems are not always effective. Two commercially available proteases and an amylase were immobilized onto polymer nanofibers that are used in the production of filtration membranes.
RESULTS: The enzymes were immobilized onto poly acrylonitrile nanofibers that were chemically activated by imidoesterification to allow the covalent immobilization of enzymes. The immobilized enzymes retained above 80% of the specific activity of the free enzymes. For each of the immobilized enzymes, just below 30% of initial activity was retained after 10 repeated cycles of use. No biofilm formation was observed on nanofibers that were coated with the enzymes, where biofilms formed on non‐coated nanofibers.
CONCLUSION: When considering the combined advantages of this effective immobilization process, the robustness of the enzymes used in this study, and the results of this study indicating activity against biofilms, a valuable addition has been made to filtration membrane technology. © 2012 Society of Chemical Industry
The enzyme complement of a selection of commercial food-grade peptidase and lipase preparations was investigated. Each preparation was assayed for protein content, proteinase activity at pH 5.5 and 7.0 at 37°C using azocasein and semi-quantitatively assayed for lipase, peptidase, proteinase, phosphatase and glycosidase activity by the API-ZYM system. Each peptidase preparation was also assayed for various endo-, carboxy-, amino- and di-peptidase activities at pH 5.5 and 7.0 at 37°C, using chromogenic or fluorogenic substrates, while each lipase preparation was assayed for esterase and lipase activity at pH 7.0 at 37°C using p-nitrophenol substrates. All enzyme preparations were found to contain enzyme activities in addition to their stated main activity. According to the API-ZYM system the peptidase preparations contained varying levels of lipase, proteinase, peptidase, phosphatase and glycosidase activity, with the lipase preparations containing lipase, phosphatase and glycosidase activity. Only two peptidase and two lipase preparations contained significant amounts of proteinase activity as measured by azocasein. The peptidase and lipase activities of the preparations appeared to be dependent upon source. Most peptidase preparations had significantly more activity at pH 7.0 than at 5.5.
The attachment of motile spores of the green alga Enteromorpha to the substratum is an active process involving an irreversible commitment to adhesion and the secretion of an adhesive. This paper provides an overview of the spore adhesion processes and outlines the results of an experimental approach towards the molecular characterisation of the adhesive, based on the use of monoclonal antibody (mAb) technology. Hybridomas were produced to settled spores displaying secreted adhesive. Candidates producing mAbs to putative adhesive were selected using a range of criteria based on cellular localisation, time of secretion and functional inhibition of adhesion. MAb Ent 6 immunolabelled fibrillar material which was secreted during the early stages of adhesion and low (nM) concentrations of this mAb, or its F(ab)(2) fragments, strongly inhibited the attachment of zoospores. A related antibody (Ent 1) also labelled the spore adhesive apparatus, but the antigen appeared to be secreted later during the adhesion process and was predominantly associated with the developing cell wall. Ent 1 also inhibited settlement in spore adhesion assays but the effect was most pronounced at later time points which suggests that this antigen does not have a role in the earliest stages of adhesion. Immunolocalisation showed that both antigens were absent from the cytoplasm or organelles of vegetative tissue Tout labelled the vegetative cell wall, suggesting a relationship between cell wall components and materials involved in primary adhesion. Both mAbs labelled the Golgi region of settled spores, suggesting continued synthesis of both antigens after adhesion. Both mAbs recognised a 110kDa N-linked polydisperse and heterogeneous glycoprotein in extracts of swimming spores under denaturing conditions. In native form the antigens behaved as high molecular weight aggregates (M-r > 1.3 x 10(6)). The antigens became progressively insoluble after zoospore attachment. Taken together, the data suggest that the two antibodies recognise closely related, polydisperse, self-aggregating cell wall glycoproteins in, which there is some structural variation to suit alternative roles in primary adhesion and cell wall formation. The two mAbs Ent 1 and Ent 6 partially discriminate between these structural and functional variants. A model for zoospore adhesion is discussed in which adhesion is viewed as an extension of cell wall synthesis, with cross-links between glycoproteins and other cell wall matrix components providing a strong physical continuum between the cell and the adhesive at the substratum interface.
An overview of the current technologies developed for use as antifouling (AF) coatings and the progress made in Fouling Release Coatings (FRC) from the end of the 1990s is presented. Olsen et al. proposed four requirements for enzyme-based AF systems. These include enzymes must retain activity when mixed with coating components, enzymes must not deteriorate coating performance, enzymes must have a broad-spectrum AF effect, and enzyme activity must have long-term stability in the dry coating and after submersion of a coated surface in the sea. Schultz et al. proposed a model to correlate the measurements of the adhesion strength of barnacles and FR performance on a ship. DeSimone and co-workers pursued their studies on photocurable PFPE with the synthesis of amphiphilic PFPE/PEG networks as FRCs.
As early as 1975, significant and repeated disturbances were observed to occur in the Crassostrea gigas oyster farms of the Arcachon Bay along the French Atlantic Coastline. TBT contamination of the local breeding waters was found to be responsible for failure to reproduce and for anomalies occurring in the shell calcification of adult oysters leading to stunted growth. On 17 January 1982, the French Ministry of the Environment issued a 3 month ban on the sale of marine anti-fouling paints with an organotin compound content exceeding 3%.The Ministerial ban was subsequently renewed and reinforced and ultimately converted into an enforcement decree transcribing an European Union directive into French law, in October 1992. The ban was the first official action in response to a growing awareness to the TBT-related pollution of some coastal areas and to its unacceptable impact on shellfish farming.These measures were adopted as soon as the first scientific evidence of a causal relationship was established between tributyltin and oyster shell anomalies. Since then numerous research studies have been conducted on the geochemical behavior of tributyltin, its ecotoxicity and on TBT-related risks presented by antifouling paints. The knowledge gained on this topic since 1982 reveals in particular that:•TBT is extremely toxic to aquatic organisms in general and to molluscs in particular for which the NOELs are below 1 ng l-1(imposex, calcification anomalies),•Contamination levels can reach very high values in port areas, and even exceed no effect levels beyond which mollusc reproduction and growth are affected, in estuaries or semi-enclosed areas,•Contamination of breeding waters has a significant economic impact, jeopardizing the sustainable development of some highly exposed marine activities,•The ban on TBT use in antifouling paints for crafts under 25 m represents an efficient way of reducing TBT inputs in coastal areas and restoring proper water quality,•The regulations adopted by many countries to ban or restrict TBT use on ships sailing along the coastline, have prompted manufacturers to develop less toxic paints.Fifteen years after TBT was first implicated as a major cause of coastal contamination, the issue no longer lies in finding out under which conditions TBT could still be used in antifouling paints, but rather in developing environmentally-harmless formulations. While the ban on TBT-based paints may be justified due to their harmful impact on coastal ecosystems, substitutes based on the release of biocides (copper oxide, pesticides …) also give rise to major concerns. Prevention of marine biofouling is not motivated merely by esthetics: it responds to economic needs and to navigation safety requirements. Protection of the ships while preserving the coastal ecosystems remains a major challenge for future research.
The imminent ban of environmentally harmful tributyltin (TBT)-based paint products has been the cause of a major change in the antifouling paint industry. In the past decade, several tin-free products have reached the commercial market, and claimed their effectiveness as regards the prevention of marine biofouling on ships in an environmentally friendly manner. The main objective of this review is to describe these products in as much detail as possible based on the knowledge available in the open literature. This knowledge has been supplemented by means of performance data provided, upon request, by some of the paint-producing companies. An exhaustive review of the historical development of antifouling systems and a detailed characterisation of sea water are also included. The need for studies on the behaviour of chemically active paints under different sea water conditions is emphasised. In addition, the most common booster biocides used to replace TBT-containing compounds are listed and described. It must be stressed that there is still a lack of knowledge of their potential environmental side effects.
In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.
Fouling on marine underwater surfaces causes critical and economic problems such as important material biodamages and industrial performances reduction. We chose to test antifouling potential of enzymatic commercial preparations like hydrolases (proteases, glycosidases and lipases) in order to inhibit the first fouling adhesion step: bacterial biofilm formation. An evaluation test of antifouling properties onto marine bacterial adhesion was designed using a mono-incubation of Pseudoalteromonas sp. D41 in microtiter plate and in sterile natural sea water. This test was adapted to screen agents for bacterial adhesion removal or inhibition activities and allowed to test enzymatic preparations toxicity on non adhered bacteria. Inhibition rates according to logarithm of enzymatic preparation concentration exhibits a sigmoid shape like dose-response curves. Among hydrolases, proteases like subtilisin are the most efficient enzymes. The efficiency of amylase, lipase and protease activity mixture was evaluated and showed a high synergistic inhibition on Pseudoalteromonas sp. D41 adhesion in microtiter plate. Studies on polymeric extracellular substances from Pseudoalteromonas sp. D41 in fermentation and in biofilm will be helpful in the understanding of the organic molecules nature involved in the adhesion inhibition. L'adhésion de salissures marines sur les structures immergées en eau de mer cause de sévères dégâts et problèmes économiques par une accélération de la dégradation des matériaux et une diminution des performances industrielles. Nous avons choisi de tester le potentiel antisalissure de préparations enzymatiques commerciales de type hydrolases (protéases, glycosidases et lipases) sur les premières étapes d'adhésion des salissures marines : le biofilm bactérien. Un test d'évaluation de propriétés antisalissures concernant l'adhésion d'une bactérie marine du genre Pseudoalteromonas sp. D41 en microplaque et en eau de mer naturelle stérile a été mis au point. Ce test est adapté au criblage d'activités de prévention ou de nettoyage d'un biofilm marin et permet de tester la toxicité des préparations enzymatiques sur les cellules non adhérées. Les taux d'inhibition exprimés en fonction du logarithme de la concentration en enzyme consiste en une courbe sigmoïde de type dose-réponse. Des hydrolases testées, les protéases dont la subtilisine sont les plus efficaces. Un mélange d'activités enzymatiques amylases, lipases et protéases a montré une forte synergie d'activité pour inhiber l'adhésion de Pseudoalteromonas sp. D41 en microplaque. L'étude de la composition de substances polymériques produites par Pseudoalteromonas sp. D41 en fermenteur et au sein d'un biofilm a permis de mieux comprendre la nature des molécules organiques cibles impliquées dans l'inhibition de l'adhésion.
The biofilm matrix is a dynamic environment in which the component microbial cells appear to reach homeostasis and are optimally organized to make use of all available nutrients. The major matrix components are microbial cells, polysaccharides and water, together with excreted cellular products. The matrix therefore shows great microheterogeneity, within which numerous microenvironments can exist. Although exopolysaccharides provide the matrix framework, a wide range of enzyme activities can be found within the biofilm, some of which will greatly affect structural integrity and stability.
Microbes often construct and live within surface-associated multicellular communities known as biofilms. The precise structure, chemistry and physiology of the biofilm all vary with the nature of its resident microbes and local environment. However, an important commonality among biofilms is that their structural integrity critically depends upon an extracellular matrix produced by their constituent cells. Extracellular matrices might be as diverse as biofilms, and they contribute significantly to the organization of the community. This review discusses recent advances in our understanding of the extracellular matrix and its role in biofilm biology.
A systematic overview is presented of the literature that reports the antifouling (AF) protection of underwater structures via the action of enzymes. The overall aim of this review is to assess the state of the art of enzymatic AF technology, and to highlight the obstacles that have to be overcome for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers to the use of enzymes to release an active biocide with AF activity. For direct AF, several patents have been granted, and a commercial product has been launched. However, the achievement of an efficient broad-spectrum AF coating based on a single or a few enzymes has not yet been achieved. An indirect AF coating is not yet available commercially. The technology is mainly limited by the instability of substrate supply, whether the substrates are found in the surrounding seawater or in the coating itself. Legislative issues regarding which part(s) of an enzyme system should be regarded as biocidal for product registration purposes are also considered. The above question currently remains unanswered for technologies utilising indirect enzymatic AF.
Antifouling coatings are used extensively on marine vessels and constructions, but unfortunately they are found to pose a threat to the marine environment, notably due to content of metal-based biocides. Enzymes have repeatedly been proposed as an alternative to traditional antifouling compounds. In this review, the enzymes claimed to hold antifouling activity are classified according to catalytic functions. The enzyme functions are juxtaposed with the current knowledge about the chemistry of settlement and adhesion of fouling organisms. Specific focus will be on bacteria, microalgae, invertebrate larvae and macroalgae zoospores. Two main concepts in enzyme-based antifouling are identified: breakdown of adhesive components and catalytic production of repellent compounds in-situ. The validity of the various modes of action is evaluated and the groups of enzymes with the highest potential are highlighted.
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