MXPA00000244A

MXPA00000244A - Inhibition of sulfate-reducing-bacteria-mediated degradation using bacteria which secrete antimicrobials

Info

Publication number: MXPA00000244A
Application number: MXPA/A/2000/000244A
Authority: MX
Inventors: Thomas K Wood; Arul Jayaraman; James C Earthman
Original assignee: Regents Of The University Of California
Priority date: 1998-05-06
Filing date: 2000-01-05
Publication date: 2001-03-05

Abstract

The present invention relates to the field of degradation or corrosion prevention or inhibition through the use of bacteria which secrete antimicrobial chemical compositions. In particular, the invention relates to the use of bacteria which, either naturally or through the use of recombinant technology, secrete chemical compositions which inhibit the growth of sulfate-reducing bacteria on metals, concrete, mortar, and other surfaces subject to corrosion.

Description

INHIBITION OF MEDIATED DEGRADATION BY SULPHATE REDUCING BACTERIA USING BACTERIA THAT SECRETS AN IMMIGRANTS RECIPROCAL REFERENCES TO RELATED REQUESTS This application is a continuation in part of the United States Series No. 09 / 074,037, filed May 6, 1998, the contents of which are incorporated by reference. DECLARATION OF THE RIGHTS OF THE INVENTIONS CARRIED OUT UNDER INVESTIGATION AND DEVELOPMENT SPONSORSHIP FEDERAL ENTE Not applicable. NON-FEDERAL SUPPORT FOR RESEARCH The invention described below was made in the course of, or under a contract, RP8044-02, with the Electric Power Research Institute ("Electric Power Research Institute"). FIELD OF THE INVENTION The present invention is related to the field of the prevention or inhibition of the degradation of surfaces susceptible to degradation through the use of bacteria that secrete antimicrobial chemical compounds.

In particular, the invention relates to the use of bacteria, which, either naturally or through the use of recombination technology, secrete chemical compounds that inhibit the growth of sulfate-reducing bacteria in metals, concrete, cement and other surfaces subject to corrosion or degradation. BACKGROUND OF THE INVENTION Deterioration due to degradation and corrosion implies a huge cost worldwide. In the United States alone, the annual cost of corrosion damage has been calculated as equivalent to 14.2% of the gross national product (Martinez, L. J. Metals, 45:21 (1993)) (hereinafter: Martinez, 1993). These huge costs could be reduced to a large extent through increased USE and better corrosion protection techniques. Microbes contribute significantly to deterioration by degradation and corrosion. When surfaces and particularly metals are exposed to natural environments, they are rapidly colonized by aerobic bacteria present largely in the liquid phase (Geesey, GG, hat is biocorrosion? Presented in the International workshop on bio-contamination and industrial biocorrosion, Stuttgart, Germany, Springer-Verlag, New York (1990)) (from now on, Geesey, 1990). The upper layers of this biological film are aerobic, while in the regions near the surface of the metal they are anoxic due to the reduction of oxygen by the biological film (Blenkinsopp, SA et al., Trends, Biotechnol. -143 (1991), Bryers, JD et al., Biotec, Prog. 3: 57-67 (1987)). Sulfate-reducing bacteria ("SRB") can colonize these anaerobic niches and therefore contribute to corrosion even in aerobic environments (Hamilton, WA Sulfate-reducing bacteria and their role in biocorrosion. International workshop on bio-addiction and industrial biocorrosion, Stuttgart, Germany, Springer-Verlag (1990)) (hereinafter, Hamilton, 1990). SRBs have been implicated in the deterioration of metals in a broad classification of environments (Borenstein, SW Microbiologically Influenced Corrosion Handbook, Woodhead Publishing Limited, Cambridge, England (1994) (hereinafter: "Borenstein, 1994"), Hamilton, WA Ann, Rev. Microbiol., 39: 195-217 (1985) (hereinafter: "Hamilton, 1985"), Hamilton, WA Trends, Biotechnol., 1: 36-40 (1983), Hamilton, 1990). Offshore petroleum pipelines and platforms in the oil and shipping industries (Hamilton, WA Trends, Biotechnol. 1: 36-40 (1983)), cooling water recirculation systems in industrial systems (Borenstein, 1994; Miller, JD Metals, pp. 150-201, In Rose, AH (ed.), Microbial Deterioration, Academic Press, New York (1981)) (from now on: Miller, 1981), sewage treatment plants and pipes (Hamilton, 1985;) Odom, J.M. ASM NEWS 56: 473-476 (1990)), fuel tanks for aircraft in the aviation industry (Miller, 1981) and the power generation industry (Licina, GJ Matera, Perform 28: 55-60 (1989)) (from now on forward: Licina, 1989) all of them have been adversely affected by the growth and colonization of SRB. SRBs can cause corrosion in a wide range of metals, such as low carbon steels (eg Borshchevskii, A.M. et al., Prot. Metals 30: 313-316 (1994); Cheung, C.W.S. and Beech, I.B., Biofouling. 9: 231-249 (1996) (hereinafter: Cheung and Beech, 1996); Dubey, R.S. et al. , Ind. J. Chem. Tech. 2: 327-329 (1995); Gaylarde, C.C. Int. Biodet. Biodeg. 30: 331-338 (1992)) (hereinafter: Gaylarde, 1992); Lee et al. , Biofouling 71: 244-251 (1991); Mollica, A. Int. Biodet. Biodeg. 29: 213-229 (1992); Newman, R.C. et al. , ISIJ International. 3: 201-209 (1991)); Oritz et al. , Int. Biodet. 26: 315-326 (1990)); and copper alloys (Licina, 1989; Wagner, P. and Little, B., Ma ter. Perform. 32: 65-68 (1993)) (hereinafter: Wagner and Little, 1993), all of them of use frequent in processes, shipments and energy industries. ~ The SRBs also contribute substantially to the degradation of non-metallic portions of the world's infrastructure. SRBs produce hydrogen sulfide, which is metabolized by sulphide oxidizing organisms, such as Thiobacillus, converting it into sulfuric acid. Degradation by sulfuric acid due to bacteria has been found to drastically reduce the lifespan of, for example, concrete water conduits. Corrosion damage from SRBs in metals in the United States alone has been about $ 4-6 billion annually (Beloglazov, SM et al., Prot. Met. USSR 27: 810-813 (1991)) (from now on: Beloglazov 1991). Conventional strategies to inhibit corrosion have included the modification of the pH, the redox potential and the soil resistivity in which the equipment is to be installed (Iverson, WP Adv. Appl. Microbiol. 32: 1-36 (1987)) (from now on: Iverson, 1987), inorganic layers, cathodic protection and biocides [Agent used to eradicate living organisms] (Jack, TR et al., Industrial settings control, p.265-292.) In: Barton, LL ( ed.) Sulfate-reducing bacteria, Plenum Press, New York (1995)) (hereinafter: Jack et al., 1995) (Barton's full reference is included here by reference). Inorganic protective coatings such as paints and epoxy resins were used in the past but are not permanent and the maintenance cost and their replacement is substantial (Jayaraman, A., et al., Api. Microbiol. Biotechnol. 42: 62-68 ( 1997) (from now on Jayaraman et al., 1997 a), Martínez, 1993). With cathodic protection, the cathodic reaction is stimulated on the metal surface by coupling to an anode to be sacrificed made of magnesium or zinc, or by supplying a printed current from an external power source through a corrosion-resistant anode . The galvanic or printed current decreases the electrochemical potential over the entire surface of the metal, so it does not form cations and dissolution does not occur. ((Iverson, 1987); Little, B. J. et al., Mater. Perform 32: 16-20 (1993)). However, Wagner and Little (1993) report that the use of cathode potentials of up to -1074 mV, were not able to prevent the formation of the biological film. Biocides have also been used to retard the corrosion reaction in closed systems such as cooling towers and storage tanks (Iverson, 1987) and are probably the most common method to combat biocorrosion (Boivin, J., Mater. 32: 65-68 1995) (hereinafter: Boivin, 1995); Brunt, K. D., Biocides for the oil industry, p. 201-207, in Hill, E.C., Shennan, J.L., Watkynson, R. J. (ed.), Microbial Problems in the Offshore Oil Industry, John Wiley and Sons, Chichester, England (1986); Cheung, C. W. S. et al., Biofouling. 9: 231-249 (1996)) (from now on: Cheung, 1996). Saleh et al. (J. Appl. Bacteriol., 27: 281-293 (1964)) (hereinafter: Saleh et al., 1964) reviewed the use of approximately 200 bactericidal or bacteriostatic compounds against SRB. Oxidizing biocides such as chlorine, chloramines and chlorinating compounds are used in freshwater systems (Boivin, 1995, supra). Chlorine compounds are the most practical biocides; however, its activity depends on the pH of the water and the amount of light and temperature (Keevil, C. W. et al., Int. Biodet. 26: 169-179 (1990)) (hereinafter: Keevil et al., 1990), and are not very effective against biological film bacteria.

(Boivin, 1995, supra). Non-oxidizing biocides such as quaternary salts (Beloglazov, 1991), amine-type compounds, anthraquinones (Cooling III, F. B. et al., Appl. Environ.

Microbiol 62: 2999-3004 (1996)) (hereinafter: Cooling et al., 1996) and the aldehydes (Boivin, 1995) are more stable and can be used in several environments. The use of these biocides is affected by a number of serious disadvantages, including not only the cost of the biocides themselves, but also the environmental cost of adding large amounts of inorganic compounds to the supply water. An additional problem arises from the organization of the biological film on the surface of the material. The glycocalyx (Brown, ML et al., Appl. Environ Microbiol. 61: 187-193 (1995); Hoyle, BD et al., J. Antimicrob. Chemother. 26: 1-6 (1990); Suci, PA et al., Antimicrob Agents Chemother 38: 2125-2133 (1994)), phenotypic changes occurring in the biological film, such as expression for algC of the gene in P. aeruginosa (Costerton, WJ et al., Ann. Rev. Microbiol. 49: 711-745 (1995)) (hereinafter: Costerton, 1995) and the effect of surface chemistry on the metabolic state of the biological film (Keevil et al., 1990) can all be used to increase the resistance of organisms in a biological film to antimicrobial agents beyond that observed in plankton bacteria (Brown, MRW et al., J. Appl. Bacteriol., Suppl 74: 87S-97S (1993)) . The combination of an organic corrosion inhibiting film, a polyacrylate / phosphonate and two biocides have been successfully employed to control corrosion in a water cooling system (Iverson, supra). However, SRBs are inherently resistant to a wide range of antimicrobials (Saleh et al., 1964, supra) and the severe anaerobic environment (created by corrosion products) in which SRBs would thrive also reduces the efficiency of antimicrobials. (Cheung, 1996; Iverson, supra). Once SRBs are firmly established in their niche, it is difficult to remove them from a system without disassembling it (Boivin, 1995, supra). Another strategy to control the corrosion caused by microbes, is the suppression of the growth of the most harmful microorganisms by manipulating the availability of nutrients and therefore, a more benign biological film is created (Jack et al., 1995). Recently, Jansen and Kohnen (J. Ind. Microb., 15: 391-396 (1995)) reported the reduction in adhesion of Staphyl ococcus epidermis KH6 on surfaces by modifying the polymeric surface by ionic bonds of silver ions to the surface and they suggested the development of antimicrobial polymers to prevent bacterial adhesion. Wood, P., et al. (1996) (Appl. Environ. Microbiol. 62: 2598-2602) reported the generation of potassium monopersulfate and hydrogen peroxide on the surface, by the increased activity by catalysis of these biocides in 150 times with respect to the biological film of P. aeruginosa. This method is based on permeabilizing a plastic with the necessary chemical agents and would require substantial diffusions and costly changes in manufacturing techniques to implement it. Finally, the work suggested by others (Pedersen and Hermansson, Biofouling 1: 313-322 (1989) and Biofouling 3: 1-11 (1991)) and our own work have recently confirmed (Jayaraman et al., 1997a and Jayaraman et al. ., J. Ind. Microb. 18: 396-401 (1997) (hereinafter: Jayaraman et al., 1997b), that aerobic bacteria in a biological film can inhibit electrochemical corrosion by two to forty times, possibly Due in part to the fact that bacteria that breathe in a biological film on a metal use part of the oxygen that might otherwise be available to oxidize that metal, however, as mentioned above, this oxygen level reduction also creates the opportunity for the SRB, which are anaerobic, that can colonize the metal. Therefore, in practice, the effectiveness of biological films as a means to inhibit electrochemical corrosion is reduced by consequently improving the rate of corrosion related to SRB. What is required is an effective and less expensive means to prevent or inhibit corrosion caused by SRB or degradation, with a lower addition of toxic agents to the environment. The present invention provides this and other advantages.

COMPENDIUM OF THE INVENTION The present invention relates to the field of prevention of corrosion or inhibition by the use of bacteria that secrete antimicrobial chemical compounds. In particular, the invention relates to the use of bacteria which, either naturally or through the use of recombination technology, secrete chemical compounds that inhibit the growth of sulfate-reducing bacteria in metals, concrete, cement and other surfaces. subject to corrosion or degradation. The invention provides a method for inhibiting the growth of SRBs in a material sensitive to degradation or corrosion. The method consists in applying to the material sensitive to corrosion or degradation, a bacterium that secretes a chemical compound in an amount sufficient to inhibit the growth of the SRB in the material. The material sensitive to corrosion can be a metal, such as iron, aluminum, titanium, copper or its alloys. For example, the metal can be mild steel or one of its various stainless steels. The material sensitive to degradation can be a material such as concrete, reinforced concrete or cement. The bacterium can be an aerobic and can be for example of the genus Pseudomonas, or Bacillus. The chemical compound may be one that is not normally secreted by an uncultivated member of the species of that bacterium and may be an antibiotic, such as gramicidin S, indolicidin, polymyxin or bactenecin; it can be a poly-amino acid, such as polyaspartate or polyglutamate, or it can be a siderophora. The invention further provides a system for inhibiting corrosion, comprising a material sensitive to corrosion or degradation having a biological film on its surface, wherein the biological film includes a bacterium that secretes a chemical compound in an amount sufficient to inhibit the growth of the SRB in the material. The material sensitive to corrosion can be a metal such as those mentioned in the paragraph above; The material sensitive to degradation can be a material such as cement, concrete or reinforced concrete. The bacteria can be aerobic, particularly of the genus Pseudomonas or of the genus Ba cillus. The chemical compound secreted by the bacterium may be one that is not normally secreted by an uncultivated member of the species of that bacterium and may be an antibiotic, such as gramicidin S, indolicidin, polymyxin or bactenecin; it can be a poly-amino acid, such as polyaspartate or polyglutamate, or it can be a siderophora. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Cloning and expression of indolicidin and bactenecin. S = Serine, A = Alanine. Only the relevant restriction sites are shown. Dib. la: Scheme of the expression system used for the cloning and secretion of indolicidin and bactenecin. Dib. Ib: Complementary oligonucleotides used for the cloning of indolicidin. Dib. le: Complementary oligonucleotides used for the cloning of bactenecin. Drawing 2 Cloning and expression of bactenecin with a pro-barnase (pro) protective region. Dib. 2a: Scheme of the expression system used for the cloning and secretion of pro-bactenecin. The one-letter codes for the amino acids represent the pro- and the bactenecin gene. SP denotes the signal of a peptide of an alkaline protease. Dib. 2b: Relevant nucleotides for the cloning of pro-bactenecin. S = Serine, and A = Alanine. Only the relevant restriction sites are shown. Drawing 3 Impedance spectra of stainless steel 304 in Baar 's modified medium with dual cultures (except for control) of B. subtilis BE1500 (with plasmid pBE92 in the absence (empty squares) and presence (filled squares) of SRB, PBE92-Ind (indolicidin) (filled diamonds), pBE92-Bac (Bactenecina) (empty triangles) and pBE92-ProBac (bactenecin with a -pro region) (empty circles) and control bacteria P. fragi K (filled hexagons) and representative SRB D. vulgaris. The data is from a representative experiment. Dib. 3a: Y axis: Logarithm of the impedance. X axis: Frequency in hertz. Dib. 3b: Y axis: phase angle, in degrees. X axis: Frequency in hertz. Note for the interpretation of the impedance spectra shown in Drawings 3-9: Electrochemical impedance spectroscopy is a technique in the science of materials used to investigate corrosion. The upper graph in each of the drawings 3-9 (the "a" drawing) is a graph showing the logarithm of the impedance of the metal referred to, treated as indicated in that drawing, in a range of frequencies. The plateau in the impedance at low frequencies is calls it the polarization resistance and is inversely proportional to the corrosion rate; therefore, if the impedance at low frequencies moves upwards, this reflects that the corrosion rate is moving downwards. While the instrument moves through a range of frequencies shown in the graph, the portion of the graph considered relevant for corrosion studies is the result of the lowest frequency. Because of this, the effect on the corrosion rate of a change in the experiment is determined from the values plotted on the left side of the drawing. Since the y-axis for the "a" graphs represent a number that is a logarithmic function, the difference between each number on the Y axis reflects a difference [multiplied] ten times. In this way, the small differences in the relative position of the data points for the respective lines reflect substantial differences in the corrosion rate. More information about polarization resistance, impedance spectra and other techniques for measuring corrosion can be found in Baboian, R., ed., Corrosion Tests and Standards: Application and Interpretation, American Society for Testing and Materials, Philadelphia (nineteen ninety five) . The lower graph of each Drawing (drawing "b") is a graph that indicates the phase change in the impedance response. These graphs confirm for each experiment, that the impedance plotted in the drawing "a" reflects a single time constant and that the impedance plateau for the low frequencies is the polarization resistance. The X axis for all the graphs in the Drawing 3-9 (for both "a" and "b") is the frequency in Hertz. Figure 4 Upper and lower panels: impedance spectra for 304 stainless steel in modified Baar medium with dual cultures of B. subtilis WB600 (with plasmid pBE92, in the absence (empty squares) and presence (filled squares) of SRB, pBE92-Ind (indolicidin), filled rhombuses, pBE92-Bac (bactenecin), empty triangles, and pBE92-ProBac (bactenecin with a region -pro), empty circles, and P. fragi K (filled hexagons) The term "SRB" remains for the representative SRB D. vulgaris The data is from an experiment Fig. 4a: Y axis: Impedance logarithm X axis: Frequency in hertz Fig. 4b: Y axis: Phase angle, in degrees X axis: Frequency in hertz Figure 5 Upper and lower panels: Impedance spectra for stainless steel 304 in modified medium of Baar with Dual cultures of B. polymyxa (with plasmid pBE92 in the absence (empty triangles and presence (filled circles) of SRB, pBE92-Bac (bactenecin) (empty squares) and the control bacterium P. fragi K (filled hexagons) with representative SRB D. vulgaris The data are from a representative experiment (two exp independent erimentos). Dib. 5a: Y axis: Logarithm of the impedance. X axis: Frequency in hertz.

Dib. 5b: Y axis: phase angle, in degrees. X axis: Frequency in hertz. Figure 6 Upper and lower panels: Impedance spectra for SAE 1018 mild steel in modified Baar medium with purified antimicrobial ampicillin added to the P. fragi K cultures before and after the addition of SRB. Control culture of P. fragi K: empty circles; P. fragi and SRB: (D. vulgaris): filled diamonds. P. fragi and SRB, with ampicillin added after the SRB; filled triangles; P. fragi with ampicillin added before [add] SRB: empty squares. The data is from a representative experiment (from a minimum of two independent experiments). Dib. 6a: Y axis: Logarithm of the impedance. X axis: Frequency in hertz. Dib. 6b: Y axis: Phase angle, in degrees. X axis: Frequency in hertz. Figure 7 Upper and lower panels: Impedance spectra for stainless steel 304 see a modified Baar medium with purified antimicrobial ampicillin added before and after the addition of SRB. Control cultivation of P. fragi: empty circles; P. fragi and SRB (D. vulgaris): filled diamonds; P. fragi and SRB, with ampicillin added after adding SRB: filled triangles; P. fragi with ampicillin added before [add] SRB: empty diamonds. The data is from a representative experiment (minimum of two independent experiments). Dib. 7a: Y axis: Logarithm of the impedance. X axis: Frequency in hertz. Dib. 7b: Y axis: Phase angle, in degrees. X axis: Frequency in hertz. Drawing 8 Upper and lower panels: Impedance spectra for 304 stainless steel in Baar modified medium with added antimicrobial purified gramicidin S before the addition of SRB and gramicidin S generated in situ by the recombinant biological film. Filling circles: control bacteria P. fragi and SRB; empty rhombuses: P. fragi and gramicidin S and SRB D. vulgaris (gramicidin S added before SRB); empty squares: gramicidin S with hyperproductive strain B. brevis 18; filled squares: B. brevis and SRB. The data is from a representative experiment (minimum of two independent experiments). Dib. 8a: Y axis: Logarithm of the impedance.

X axis: Frequency in hertz. Dib. 8b: Y axis: Phase angle, in degrees. X axis: Frequency in hertz. Drawing 9 Upper and lower panels: Impedance spectra for SAE 1018 mild steel in Baar modified medium with added antimicrobial purified gramicidin S before the addition of SRB and gramicidin S generated in situ by the recombinant biological film. The labels are the same as in Figure 8. The data is from a representative experiment. (minimum of two independent experiments). Dib. 9a: Y axis: Logarithm of the impedance. X axis: Frequency in hertz. Dib. 9b: Y axis: Phase angle, in degrees. X axis: Frequency in hertz. DETAILED DESCRIPTION I. INTRODUCTION This invention provides methods to inhibit the degradation of materials, as well as a system to inhibit degradation. We have recently shown that the presence of aerobic biological films in metals can reduce corrosion by 2 to 40 times. (Jayaraman et al., 1997a and Jayaraman et al., 1997b). This inhibition may be due in part to the reduction of oxygen levels on the metal surface caused by bacterial respiration. However, in natural environments, this reduction in oxygen levels also creates the opportunity for the colonization of sulfate-reducing bacteria, or "SRB," in the metal to occur. While the impact of SRBs was not studied in Jayaraman et al., 1997a or 1997b, SRBs would be expected to substantially increase the corrosion rate over that reported in those studies. Consistently, while the studies by Jayaraman et al., 1997a and 1997b demonstrated that aerobic biological films can serve as a means of inhibiting corrosion, they do not guide on how to reduce the impact of SRB-mediated corrosion. The present invention solves this problem. In this, the usefulness of biological films as a means to inhibit degradation is markedly increased. In short, the invention involves the application of bacteria that secrete antimicrobial substances. Now we have shown that it is possible to create biological films of aerobic bacteria that secrete substances that inhibit the growth of SRB. Substances may be those secreted naturally by uncultivated bacteria that are not normally present in the biological film into which the bacteria are introduced (including the secretion of substances at higher than normal levels due to mutation). The bacterium can also be altered recombinantly to overexpress a substance secreted naturally by the organism, or secrete an antimicrobial not expressed by the uncultivated members of the bacterial species, or both. Corrosion is a problem that affects metals. But other materials are seriously affected by the degradation associated with the colonization of the material by SRB. SRBs produce hydrogen sulfide as a product of their metabolism. Sulfur attacks iron as well as its alloys, including stainless steels; oxidizes copper and its alloys. Hydrogen sulfide is available to be oxidized to sulfate by any amount of sulfur oxidizing organisms, such as Thiobacillus, which produce sulfuric acid. The sulfuric acid formed in this way has been found to be responsible, for example, for the degradation of concrete water channels in Los Angeles and has drastically decreased the expected life of concrete water control systems. While the invention is particularly useful against corrosion or degradation related to SRBs, the method and system of the invention can also be applied to other organisms that increase corrosion or degradation of materials. For example, fungi such as Hormoconis resinae contaminate jet fuel and produce organic acids that increase the corrosion of aluminum alloys in fuel systems. See, for example, H.A. Videla, Manual of Biocorrosion (CRC Lewis Pub., New York) (1996), at 129 (hereinafter: "the Manual;" the complete Manual is incorporated herein by reference). The use of secretory bacteria or designed to secrete antifungal, or both, can reduce the corrosion caused by this source. Similarly, the growth of Pseudomonas in aircraft fuels increases corrosion, whereas Serratia marcescens was found to be protective. Id. At 129-132. The present invention could encompass the engineering of S. marcescens to secrete one or more antimicrobial substances inhibiting the growth of Pseudomonas, as well as SRBs or other microbes that can be considered as causing corrosion. Inhibition of degradation or corrosion mediated by SRB, as well as corrosion caused by other bacteria (such as Pseudomonas) or fungi, is highly desirable by this method. Because bacteria rduce themselves, the population of organisms that secrete the antimicrobial agent refills itself over time. Therefore, a single application can be effective for a long period, in contrast to the application of organic or inorganic chemicals, which must be repeated frequently. Moreover, the secretion of the agent in the biological film itself means that it automatically results in the highest concentration of the agent at its point of action, unlike the exogenously applied chemicals, which are applied on a daily basis in large quantities to ensure an adequate dose capable of reaching SRBs or other target organisms. What is more, the mechanism by which SRBs obtain energy is only slightly favored energetically speaking and the growth of organisms can be inhibited by agents that will not seriously affect other organisms. Therefore, secretions of antimicrobial agents by microbes in the environment can completely inhibit or reduce the resistance of SRBs to other agents, making it possible to inhibit SRB-related corrosion or degradation by the exogenous application of biocides and other toxic agents. at levels much lower than those that would otherwise be required. A further advantage of the invention is that even if the biological film is damaged or is removed in some places due to fluid flow or abrasion, the continuous supply of inhibitor from neighboring regions could preferably favor recolonization by the inhibitor producing bacteria on the exposed metal, or other surface. Since biological films can be rapidly formed on exposed surfaces (Costerton, 1995), judicious selection of the bacterium can result in the exclusion of other bacterial species from the biological film. Finally, the inhibiting effect of corrosion or degradation due to a biological film can be improved by introducing bacteria that secrete corrosion or degradation inhibiting agents, either separately or in combination with antimicrobial agents. Such corrosion or degradation inhibiting agents may include polypeptides such as polyaspartate and polyglutamate, as well as siderophors, such as parabactin and enterobactin. The text below explains some of the many uses of this invention and how to put them into practice. Once the terms are defined, the text talks about the improvement of the anti-corrosive effect of an aerobic biological film by the use of organisms that secrete anti-SRB or anti-fungal agents, including both organisms that naturally secrete said agents, and those that They were genetically engineered to do so. In addition, the use of anti-corrosion or antidegradation agents such as polypeptides and siderophors to further improve the anticorrosive effect is described. The following text describes how to select a suitable organism for the use of the method or system of the invention and how to determine whether the organism produces (before or after being modified) substances that inhibit the growth of SRBs, fungi or other target organisms. The methods for applying the organisms are then discussed either before or after the material to be protected is put into service and describes the uses for both the method and the system. Finally, examples are given. II. DEFINITIONS As used herein, "mild steel" refers to a low grade, cheap steel commonly used for pipes and the like. "Steel 1018 SAE" is a mild steel of particular grade that meets an industrial standard set by the Society of Automotive Engineers. As used here, "304 stainless steel" or "304 stainless steel", refers to a stainless steel of particular grade that complies with an industrial standard for such designation. The term "piece of metal" refers to a small, thin rectangle or circle of metal. Such pieces are used on a daily basis in the medium to compare corrosion characteristics of different metals, agents and inhibitors. As used herein, "corrosion sensitive material" includes all metals subject to corrosion, specifically including iron, aluminum, titanium, copper, nickel, as well as the alloys of each, including mild steel and steels. stainless. "Corrosion" specifically applies to the deterioration of metals, while "degradation" refers to the deterioration of other materials such as concrete, mortar and similar materials. Therefore, as used herein, a "material sensitive to degradation" is a non-metal subject to deterioration from causes related to bacteria. However, for convenience of reference as used herein, the term "corrosion" may also include deterioration to materials other than metals, unless otherwise handled in the context. Dental implants can also be a "material sensitive to degradation." As used herein, "Chemical compound" means a chemical that has a growth inhibitory effect on a microorganism, which can cause corrosion of the metal or degradation of a non-metallic material. The term is in general, but not necessarily used here, as a synonym for the term "antimicrobial agent." The term "application" should be understood as comprising any means mediated or facilitated by human action, whereby the bacteria come in contact with the surface and includes, as appropriate in the context, contacting, spraying, rubbing, hosing or dripping bacteria or a mixture containing bacteria on the material sensitive to corrosion or degradation. It also encompasses bacteria that secrete an antimicrobial compound, such as an anti-SRB compound, into an initial bolus of tap water, for example, a tube, conduit, cooling tower or water system, when the tube, conduit, tower or system is first put into operation. It is intended to include the physical placement of bacteria on a surface, with or without scraping the surface to create a space within an existing biological film. The phrase "in sufficient quantity to inhibit the growth of sulfate-reducing bacteria" means an amount sufficient to reduce the growth of said bacteria in a statistically significant manner, compared to a control population. The range can be as low as the limit of ability to detect a statistically significant difference, until inhibition completely. Preferably, it means that the degree of inhibition is at least 10%, meaning that the growth of said bacteria is approximately 10% lower compared to the growth of the control population. More preferably, it indicates that the degree of inhibition is between 30 and 50%. Even more preferably, it means that the degree of inhibition is between 5O and 90%. The most preferred of all, means that the degree of inhibition is 90% or greater. III. Improvement of the Anticorrosive Effects of the Biological Films A. Improvement of the Anticorrosive Effect in Biological Pelic caused by Secretory Bacteria Antimicrobial 1. General The surfaces exposed to natural environments are rapidly colonized by aerobic bacteria. Metals and other surfaces develop adherent microbial populations, enclosed in a layer of polysaccharides known as glycocalyx (Costerton, 1995). As mentioned in the Background, the inventors' recent work has confirmed that biological films have a protective effect on surfaces when they grow as monocultures (or "axenic" crops). In nature, however, organisms rarely grow in monocultures and the anoxic regions found near the surface of the metal or other material, due to the decrease in oxygen on the part of the aerobic bacteria in the biological film, create the conditions for that the material is colonized by sulfate-reducing bacteria, or "SRB". These bacteria, therefore, are responsible for corrosion even in an aerobic environment. (Hamilton, 1990). The protective effects of the biological film can be improved by the introduction, in an existing biological film, of one or more bacteria that secrete antimicrobial agents that inhibit the growth of SRBs. In a group, the bacteria can be of a class such that, ordinarily, or as a result of a mutation, they naturally produce and secrete an agent that inhibits the growth of SRBs. Alternately, the bacteria can be modified by means of recombination technology, either to secrete antimicrobial agents that are not secreted by unmodified members of their species, or to secrete at higher levels or continuously, an agent that normally they will secretariate at lower levels or only at certain times. 2. Natural Bacterial Secretors. Some bacteria naturally produce agents that are effective in inhibiting the growth of microbes, such as SRBs, which cause corrosion. The biology of bacteria has been studied for decades and a considerable body of knowledge has been developed, including information regarding numerous bacteria that are known to secrete antimicrobial agents. One such bacterium, which overexpresses the antibacterial agent Gramicidin-S as a result of an induced mutation, is described below and was tested for its ability to inhibit SRB-mediated corrosion in the Examples, below (bacteria overexpressing an agent). as a result of a chemical mutation they are considered as natural secretors of the agent for the present purposes). Other bacteria known to secrete antimicrobial agents can be easily tested to determine the effectiveness of their secretions against microorganisms, such as the SRB or the fungus Hormoconis resinae, which cause corrosion according to the tests shown in the Examples, below, or as the medium. 3. Secretors Modified by Recombination a) Chemicals not produced naturally by the bacteria to be used as secretory It will not always be the case that a bacteria can be found that naturally secretes a particular antimicrobial substance, or that the bacteria that secrete the desired antimicrobial substance for a particular application to thrive in the particular environment to which it would be exposed. In this and in other circumstances, a bacterium that does not naturally secrete the antimicrobial agent in question can be modified by recombinant biology techniques, to secrete the desired antimicrobial agent. b) Chemicals produced naturally by the bacteria, but in large quantities or constitutively The techniques can also be used to improve the anti-SRB corrosion properties of bacteria that normally secrete antimicrobial agents by transfection with constructions that include the gene for the agent feasibly linked to a strong constitutive promoter, in order to increase the amount of the secreted agent, or to provide a continuous production of an agent that normally occurs discontinuously or only in response to particular metabolic or environmental conditions. The construction may alternatively take place placing the gene encoding the antimicrobial agent under the control of an inducible promoter, such that the secretion of the agent can be controlled. c) Introduction of DNA constructs in bacterial cells It will be appreciated that numerous techniques are known in the medium for the introduction of DNA into bacterial cells, including heterologous DNA. An example method to accomplish this is shown in the Examples, below. The selection of the particular method for introducing such DNA into bacteria and obtaining their expression is not critical to the practice of this invention. B. Selection of the Antimicrobial Compound 1. Antimicrobial Agents Numerous antimicrobial agents are known in the medium that can be produced by bacteria. For example, Nisin, a 34 amino acid peptide secreted by the bacterium Lactococcus lactis, is used as a food preservative. A polypeptide of 1700 amino acids, secreted by marine bacteria known as D2, has been shown to have a general antimicrobial activity. Any of these antimicrobial agents that are inhibitors of the target organism or organisms, such as SRBs, can be employed in the invention. In a preferred group, the antimicrobial agent is an antibiotic peptide. Antimicrobial peptide agents can be small (typically 10 to 35 amino acids), and small ones can be cloned into bacteria more easily than many conventional antibiotics, for which large activities or several routes may be required to achieve the expression of a single antibiotic. In addition to the above mentioned, several peptide antibiotics are known in the medium, such as Gramicidins S and D (which are discussed in the Examples, below). However, larger antibiotic compounds may be used if desired, for a particular application in question, such that they can be manifested in the bacteria in sufficient quantities. Other small peptides, which are not normally considered antibiotics but have antimicrobial effects, can also be used. For example, indolicidin and bactenecin are cationic antimicrobial peptides from bovine neutrophils known for their activity against a wide range of organisms (detailed information about these compounds, including references in the literature, is described in the Examples, below) . Indolicidin is the smallest antimicrobial linear peptide known. The selection of a particular antimicrobial chemist remains at the practitioner's judicious discretion and will depend on the target organism, the organism selected to secrete the antimicrobial, and the application in which the secretory organism is to be used. The selected antimicrobial must be an inhibitor of the target organism (for example, it must inhibit fungal growth if the target is a fungus; inhibit the growth of Pseudomonas if the target organism is a pseudomonad and so on. Testing tests to determine the inhibitory effect of antimicrobials on the members of a group of organisms are shown in the Examples). In order to allow continuous production of the antimicrobial over time, the selected antimicrobial should typically be more inhibitory to the target organism than the antimicrobial secreting organism (sometimes referred to as the "host organism," if the organism is manifesting an introduced gene). A test test to determine the sensitivity of the host organism to an antimicrobial is set forth in the Examples. However, under some circumstances, continuous production of the antimicrobial may not be necessary; no other organism may be available to secrete a particular antimicrobial or it may be desirable to eliminate the producer species at about the same time that the target species is eliminated or inhibited. In these situations, an antimicrobial may be selected that will inhibit both the host organism and the target organism. Finally, the selection of the antimicrobial will depend in part on the proposed application. Indolicidin and bectenecin, for example, are antimicrobial agents derived from bovine neutrophils. Its release into the environment can, therefore, result in the development of bacterial strains resistant to those natural antimicrobials of at least the bovine immune systems and could result in the development of strains more resistant to similar antimicrobial agents present in the human immune system. For this reason, indolicidin and bactenecin are not preferred antimicrobials for use in open systems (ie, systems in which secretory organisms or secreted antimicrobials are typically circulating in the environment, such as water conduits, drainage pipes and the like). On the other hand, these compounds can be used in closed systems, that is, where organisms and secreted antimicrobials will not typically be circulating in the environment. 2. Anticorrosive agents a) Polypeptides Amino acids, and especially glycine, aspartic and glutamic acids, are known to act as corrosion inhibitors. See, for example, Kalota and Silverman, Corrosion 50 (2): 138-145 (1994) (hereinafter: Kalota and Silverman) and references cited therein. However, many amino acids tend to have more than one acid-base constant, with multiple pK values and different charges, depending on the pH of their environment. Kalota and Silverman found that the ability of low molecular weight amino acids to inhibit corrosion depends on pH and that only at high pH (pH> 10), the corrosion rate was significantly reduced. Based on Kalota and Silverman, it would be desirable to engineer the bacteria to secrete polyaspartate, polyglutamate or polyglycine, or polypeptides consisting of these three amino acids, as corrosion inhibitors only for use in environments where the pH could be close to 10 or higher. While this would be applicable for some industrial uses, the amount of situations involving such high pH values is somewhat limited. Our own studies contradict Kalota and Silverman. We have found that polyaspartate and polyglutamate, for example, protect the metal from corrosion at pH values as low as 7. Hence, according to our data, inhibition of corrosion is possible if the bacteria secrete polypeptides, such as polyaspartate, polyglutamate (or its corresponding acids or salts), polyglycine, or mixtures of these amino acids, if the expected or measured pH value of the metal environment, is close to 7 or higher. In such a way that this would improve the corrosion inhibiting effect of an aerobic biological film if the organisms of the biological film secrete these polypeptides when the pH is close to 7 or higher. b) Siderophors Siderophors such as parabactin (isolated from Parcoccus denitrificans) and enterobactin (isolated from E. coli) are relatively low molecular weight chelating agents, generated and secreted by bacteria that solubilize iron ions to carry it into their cells . (McCafferty and McArdle, J. Electrochem, Soc., 1421447-1453 (1995)). These agents were tested and found to inhibit iron corrosion. Id. To improve the anticorrosive effect of a biological film, the genes of these agents can be placed under the control of a strong constitutive promoter and be manifested at higher than normal levels, or be inserted into the bacteria that normally do not secrete them. . 3. Combinations of antimicrobials, anticorrosives, or both It has been contemplated that bacteria used in the invention may be designed to secrete more than one antimicrobial agent. One of the studies reported in the Examples, to mention one, included the use of a Bacillus that overexpressed Gramicidin S as a result of a mutation, which was also genetically modified to produce another antimicrobial agent. The use of bacteria that secrete two or more antimicrobials is seen as advantageous since it makes it more difficult for the target organism causing corrosion (be it a SRB or a fungus) to develop resistance. Additionally, a bacterium that secretes an antimicrobial agent may have improved its ability to inhibit corrosion by being designed to also produce an anti-corrosive agent, such as polyaspartate, polyglutamate, polypeptides consisting of these two peptides, or parabactin, enterobactin or any other another siderófora. As a practical matter, the limitation in the amount of antimicrobial and anticorrosive agents that the bacteria can be designed to produce them, seems to be a combination of some toxic effects of the antimicrobial agents in the host cell and the metabolic drain in the host cell of the host cell. production of secreted substances. Since different organisms have different metabolic efficiencies and given that the availability of nutrients in the environment seems to play a role, the determination of how many people the selected bacteria will be able to secrete will usually be determined empirically. Such determinations can be easily performed by serial transformation of the bacteria with the desired antimicrobial and anticorrosive agents, in a medium containing the nutrients expected for the site in which it is intended to be used, until a point is reached at which the target cells are completely inhibited, and then select the best combination of (1) the competitiveness of the host cells relative to the natural population of the biological film and (2) the ability of the host cells to secrete a desirable amount of agents. C. Determination of an Appropriate Organism for the Proposed Use 1. Selection of Exogenous Bacteria to the Environment for the Proposed Use In general, the organisms will be selected to secrete antimicrobial agents against SRB, fungi, or other target organisms, according to the intended use. Our findings indicate that aerobic bacteria protect surfaces from corrosion and degradation; in such a way that the organism to choose must be aerobic. In addition, the organism must be able to live in the environment of the intended use. If, for example, the object to be protected is steel and concrete from a bridge anchored and arching in sea water, an organism capable of growing in seawater or saline environment must be selected. Similarly, if the intention is to protect pipes or pipes that carry fresh water containing industrial waste, then the body must be able to grow in fresh water and in the presence of the expected effluents. In addition, the organism must be able to grow under the expected conditions of temperature and pH of the environment. Since bacteria have been studied assiduously for almost one hundred years, the temperature, pH conditions and other environmental needs and tolerances of most species are known and available in the literature. Preferably, the organism must be able to exert a protective effect against corrosion under the anticipated environmental conditions. We have published the results of a study in which we compared the effects of 15 different bacteria representing 7 different genera for metal protection in two different media, one imitating sea water and another fresh water richly linked with nutrients. Jayaraman et al. , Appl. Microbiol. Biotechnol., 48: 11-17 (1997c) (hereinafter: Jayaraman et al., 1997c, this reference is completely included as a reference). The extent of corrosion inhibition varied markedly between the two media by some of the bacteria, while 10 of the organisms were tested to protect the metal significantly in both media. Id., At 397. Following the trials of this study, someone with the skills of the medium can easily determine if any particular bacterial species contemplated for use in the present inventive method or as part of the present inventive system will be able to grow in the medium presented by the supposed environment and if the organism will be protective against corrosion under those conditions. Additionally, it is preferable if the organism is able to grow in a biological film. Often these are organisms that are capable of "losing weight". The genera of the examples are: Bacillus, Pseudomonas, Serra tia and Escherichia (although the Pseudomonas species should not be selected for use in these environments, such as aviation fuel tanks, where it has been found that these organisms cause corrosion.) See, for example, the Manual, supra, at 129. An exemplary method for determining the ability of selected organisms to form biological films is shown in Jayaraman et al. , 1997c, supra. 2. Selection of Endogenous Bacteria to the Environment for the Proposed Use A preferred way to select the appropriate bacteria in connection with the facilities that are already in use, is to let nature do it. Since biological films are penetrating in nature, pipes, ducts, water cooling towers, water tanks of power plants and the like and the facilities will present biological films similar to those already present, which consist of organisms already selected. in a natural way for its ability to grow in that environment. A sample of these organisms can be subtracted from there (for example, by scraping the biological film), cultivated by standard techniques and identified. If the identified species are in some way adequate (which are, for example, suitable for being genetically modified and which is not known to increase corrosion, but rather inhibit it), then they themselves can be modified to secrete the antimicrobial agent wanted. However, if what is desired are pure cultures of the organisms found on the site, they can be purchased or cultivated with extract rather than using the crops that have arisen from the organisms found on the site. Once the organisms were modified to secrete the selected antimicrobial agent, they can be introduced into the conduit, tube, tower or any other facility. The introduction into the installation can be carried out by any convenient means, either scraping the surface at intervals in order to dispose of dispersion in the biological film and with pipette, transferring an aliquot of the culture on the scraping site. In a more chosen method, bacteria are introduced by simply allowing a "plug" of water (ie, a bolus of water) that contains a high concentration of bacteria to pass over the material to be protected. Bacteria will adhere to the biological film through the transverse flow of water and will become an integral part of the biological film, or form one if there is not one present. D. Application Methods 1. Application of the organisms prior to the commissioning of the facility Our research has shown that the reduction of the degradation associated with SRB of a surface is more successful when SRB colonization of said surface is prevented, than when it is intended to remove an established colonization. In such a way that a preferred method for practicing the invention is to treat the material sensitive to corrosion or degradation with bacteria secreting suitable antimicrobial agents, such as those suitable for inhibiting the growth of SRBs, before the equipment, system or installation is put into operation. If the selected bacteria produce spores, the organism can be cultured under conditions that cause the formation of spores; The spores are then applied to the surface of the installation, prior to its use and the surface is wetted to activate the spores just before the installation is put into operation. If the bacterium is not spore-forming, or if it is not convenient for the spore-producing bacteria to form spores at first, because, for example, the time constrains the media containing the bacteria can be applied to the surface by any convenient means, be brushing, spraying with izer, spraying with spray, pipetting, hosing or dripping the crop on the surface. If the surface is irregular or has nooks and crannies, then it will be preferable to spray the surface with an atomizer or aerosol in order to allow a better inoculation in the nooks and crannies. Some facilities, such as outdoor sources, water cooling towers, heating and cooling systems and the like, are designed to recirculate water, oil or other liquids through the system. Such facilities can be conveniently inoculated if a bolus of water is first inoculated that is then used to charge or flow the system. Other systems, such as tubes, which are open or otherwise do not recirculate the liquid placed in the system, can also be inoculated in this manner. 2. Application of the organisms after the installation of an installation The elimination of SRB once they have been established in a biological film is difficult. In some circumstances, it is possible to disassemble a device, equipment or installation, either partially or totally, and to sterilize the surface totally or partially with, for example, concentrated biocides or "live" vapor. In other situations, it is possible that those facilities that can not be disarmed can get wet by flowing a liquid through, with strong biocides or live steam to kill the biological film. The facilities treated in this way can be inoculated by the same means that were described in the preceding section for their treatment before they come into operation. For installations that can not be treated in this way, or in which the biological film can not be removed effectively, the existing biological film can be considered as an advantage by modifying the organisms that are already present, so that they secrete the desired antimicrobials. , as described above. The secretory bacteria of the desired agents can then be reintroduced into the biological film. If desired, organisms can be simply introduced into the liquid or other medium, or brushed or sprayed on the surface. Bacteria can be introduced more successfully by scraping or by interrupting, in some other way, the biological film on the surface. Bacteria can be introduced even more successfully by undermining the biological film before introducing new bacteria to create a space in which they can settle. E. Uses of the Invention 1. Closed Systems There are a large number of closed systems (that is, systems that routinely do not discharge their contents into the environment) that are used in industry, commerce and practical use in frameworks. Examples include steel vessels for storage, which are commonly tested under pressure at the site and are then used to store liquids for long periods of time; water cooling towers, which are used both in power plants and in plants for cooling and heating plants, office buildings and other commercial buildings; heat exchangers (known to exhibit failures due to SRB-related corrosion) and fire protection systems. These systems typically use metal pipes and storage containers. Aerobic bacteria that secrete suitable antimicrobial agents, or anti-corrosion agents or both, can be employed in these frameworks to form a biological film that enhances the ability to inhibit the corrosion associated with SRB. The containers used to store liquids for human consumption, such as milk and beer and which are regularly sterilized, for example, by contact with live steam, will not, however, be specifically protected against corrosion when using this invention. Fuel tanks for aviation and others are also closed systems. As mentioned above, corrosion in these systems can be caused by bacterial (pseudomonad) or fungal contamination. In these frameworks, bacteria such as Serratia, modified to secrete an antimicrobial or antifungal agent that inhibits the target organism (such as a pseudomonad), can be introduced to reduce corrosion by this source. For continuous protection of the system, it is highly desirable that the antimicrobial agent selected for its secretion be little toxic to the organism that will secrete it from what it is for the fungus, pseudomonada or other target organism. 2. Open Systems Systems that routinely or regularly download their contents into the environment (with or without treatment intervention) can be considered as open systems. Such systems include municipal sewage systems, storms, drainage and sewerage systems, which on a daily basis, cover concrete conduits that are subject to repeated or relatively prolonged dives or exposure to water or other liquids. Such conduits are subject to SRB-associated corrosion due to the formation of sulfuric acid by sulfide oxidizing bacteria from the hydrogen sulfide generated by the SRB. Accordingly, the inhibition of SRB in these and other similar concrete conduits, and by this invention, corrosion is reduced in these structures. 3. Structures exposed to the weather A large number of metal and concrete structures, such as bridges, railroad rails, road overpasses and the like, which are exposed to the weather with frequent contact with water, allow the development of films biological on the surface. The corrosion associated with SRB in these structures can be inhibited by the use of this invention. EXAMPLES The invention is illustrated with the following examples. These examples are shown to illustrate, but not to limit, the present invention. Example 1: Architecture of the biological film and its correlation with the inhibition of corrosion The main objectives of this study were to characterize the architecture of the biological film and correlate the constituents of the biological film with the inhibition of corrosion. The biological films were stained for living cells, dead cells and exo-poly-saccharides, visualized using confocal scanning laser microscopy ("CSLM") and quantified to obtain deep profiles. The effect of increasing temperature and growth in saline medium was studied both for the composition of the biological film and for the inhibition of corrosion. Methods and Materials Bacterial strains, growth media and culture conditions. A mutant transponder of a flesh-rotating bacterium resistant to kanamycin, P. fragi ATTC 4973 ("P. fragi K"), (Jayaraman A. et al., 1997a) and a tetracycline-resistant enteric bacterium, E. coli DH5a (pKMY319) (Jayaraman, A. et al., 1997a), were used based on their ability to form biological films (Párolis, LAS et al.Carbohydrate Research 216: 495-504 (1991); Huang, C.-T. et al. Biotechnology and Bioengineering 41: 211-220 (1993)). Both strains were grown without shaking at 23 ° C or 30 ° C in 250 ml Erlenmeyer flasks with multiple metal pieces of SAE 1018 in 35 ml of Luria-Bertani medium (hereinafter: "LB") (Maniatis, T et al., Molecular cloning: A laboratory manual, Cold Spring Harbor, NY (1982)) (hereinafter: Maniatis et al., 1982) and solution of nine salts of Vaatanen ("VNSS," Hernández, G. et al., Corrosion 50: 603-608 (1994)) (hereinafter: Hernández et al., 1994), supplemented with 50 μg ml-1 of kanamycin (Jayaraman, A. et al., 1997a) or 25 μg ml -1 of tetracycline (Yen, K.-M. Journal of Bacteriology 173: 5328-5335 (1991)). All the strains that were scratched were taken from a glycerol extract at -85 ° C on LB agar plates with the appropriate antibiotics. A single colony was taken and used to inoculate 10 ml of the growth medium with the appropriate antibiotics and allowed to grow overnight at 30 ° C and 250 rpm (25 series agitator, New Brunswick Scientific, Edison, NJ). An inoculum (350μl) was used to develop the biological films for the corrosion experiments. The filling of the media was obtained by slow removal of the old medium and soft addition of fresh medium by sliding it through the walls of the Erlenmeyer flask. Preparation of the metallic piece and determination of mass loss. SAE 1018 steel pieces weighing 5.1 grams, with a diameter of 25.5 mm and thickness of 1.2 mm were cut from a common sheet and polished with 240 grit sandpaper (Buehler, Lake Bluff, IL) and prepared as previously reported ( Jayaraman et al., 1997a). the observed specific mass loss (mg / cm2) was determined by dividing the total surface area of the piece (11.18 cm2) and was used as an indicator of the extent of corrosion (Jayaraman et al., 1997a). All the corrosion experiments were carried out with three repetitions. Confocal scanning laser microscopy (CSLM) and determination of the biological film thickness. The metal pieces were removed with biological films attached to their surface from the Erlenmeyer flasks and submerged once in a 0.85% NaCl solution to remove the bulky floating cells. The cells and - polysaccharides were stained for 30 min simultaneously in 4 ml of dye solution using the Baclit protocol for testing the viability of the Live / Dead bacteria (1125 μl mi -1 of each dye compound, Molecular Probes (Eugene, OR)) and calcofluor (300 μg ml-1, Sigma (St. Louis, MO), Stewart et al., 1995). The viability kit for living / dead [cells] distinguishes living and dead cells with red dye committed to the membranes. The dyed pieces were taken to the stage of the confocal scanning laser microscope (MRC 600, Bio-Rad, Hercules, CA) equipped with a krypton / argon laser and at 60X, 1.4 NA oil immersion lenses. To minimize deterioration of the biological film when placed on the inverted microscope stage, a 1.8 cm coverslip (circles No. 1, 1.3 to 1.7 cm thick, Fisher Scientific Co., Pittsburgh, PA) was placed gently on the piece (sustained by capillary action) and the piece (2.55 cm in diameter) was supported by the circular opening of the microscope (2.0 cm in diameter) in the area outside the coverslip. The central area of the biological film was not compressed by the weight of the piece and only that area was visualized. The sample was stimulated at 488 nm and the fluorescent light was imaged using the combination of K1 / K2 filter blocks. The biological films were analyzed using a MRC 1024 confocal microscope (Bio-Rad, Hercules, CA) with a combination of T1 / E2 multipurpose filters. Thin optical sections (horizontal sectioning) from 0.5 to 1.0 μm were collected from the entire thickness of the biological film for a representative position (selected as one of four similar positions in the biological film of a single piece). The thickness of the biological film was found by focusing from the top and bottom of a biological film with a distance traveled corrected by refractive index (Bakke, R. and Olsson, P.Q., Journal of Microbiological Methods 6: 93-98 (1986)) and the thickness was determined as the average of four similar positions analyzed for the same piece. Image analysis The image processing and analysis of all the biological films was done with the COMOS software package, available in the Bio-Rad MRC600. Optical sections were segregated based on pixel intensities to differentiate living dead cells, polysaccharides and empty space. The percentage for each section of area covered by a range of pixel intensities was measured to obtain the relative proportions of the compound in each section; these relative proportions of each compound were plotted as a function of the normalized depth (depth at which the image was obtained divided by the total thickness of the biological film). In such a way that position 0.0 represents the biological-liquid film interface and position 1.0 represents the biological-metal film interface. RESULTS Inhibition of corrosion with P. fragi and E. coli The mass loss in LB medium and P. fragi VNSS with DH5a and E. coli (pKMY319) was examined by 8 days in stationary batch cultures at 23 ° C and 30 ° C in which the growth medium was as either filling in daily or leaving it unchanged for 8 days. The metal pieces immersed in the bacterial suspensions showed a decrease in mass loss after 8 days from 2.3 to 6.9 times, compared to the pieces immersed in sterile media. These results compare well with those of Jayaraman et al. (1997a) and those of Pedersen and Hermansson, 1989), who reported a corrosion reduction eight times for SIS 1146 steel using Pseudomonas S) and Serratia marscens after 19 days of exposure in VNSS medium. Previous work in our laboratory have shown no difference in corrosion for SAE 1018 steel parts in fresh and sterile LB medium and LB medium worn and filtered (Jayaraman et al., 1997a). The eight-day mass loss observed with P. fragi and E. coli DH5a varied with the growth medium and culture temperature. The total mass lost was lower at low temperature for both media; however, corrosion inhibition was comparable (as a percentage of reduction in mass loss of the sterile control) or higher at higher temperatures in both media. The loss of mass with both strains at both temperatures was lower in LB medium compared to the VNSS medium. Daily replacement of the medium did not significantly affect the corrosion inhibition except for P. fragi K in VNSS medium at 30 ° C, where corrosion inhibition was approximately 2.3 times better. Regardless of the medium replacement, E. coli DH5a (pKMY319) resulted in a greater mass loss compared with P. fragi K at 23 ° C and the mass loss in the presence of both strains was similar to 30 ° C. The sterile controls suffered similar corrosion, regardless of whether the medium was replaced daily. Metal parts in most bacterial suspensions suffered corrosion at a rate of approximately 0.03-0.06 mg day_1 / cm2 during the first four days. The corrosion rate decreased as the days went by. The sterile controls suffered corrosion at a slightly faster rate in VNSS medium than in LB medium at both temperatures, and the corrosion rate was relatively uniform for the entire eight-day period. Determining the thickness of the biofilm by CSLM Multiple pieces were removed from the medium after 2, 3, 4 and 8 days, cells were stained and polysaccharides simultaneously and analyzed by CSLM. The biological films observed at a magnification of 100X (without coverslips) and at 600X magnification (with coverslips) showed a similar depth profile of both living and dead cells and polysaccharides. Both the films of P. fragi K and E. coli DH5a (pKMY319) μ day "1 developed a detectable thickness (-10-15 μm) within the first 48 hours of exposure to growth media (data not shown). The biological films of P. fragi K did not vary significantly in thickness for different growth temperatures, media and medium replacement and the biological film was approximately 14 μm thick after four days; the thickness of the biological film was approximately 12 μm after eight days of exposure. E. coli DH5a (pKMY319) exhibited a similar trend with a four-day biological film of 13 μm, being slightly thicker than the eight day biological film (approx. Characterization of biological films Biological films of P. fragi and E. coli DH5a (pKMY319) in LB and VNSS media with and without medium replacement at 23 and 30 ° C were characterized and analyzed using image analysis to create the normalized four-day depth profiles. As a control experiment to verify that Live / Dead staining could be used to quantify populations with live and dead cells, 200 μg ml-1 of kanamycin was added to a 12-hour natural culture of P. fragi and visualized with CSLM after 48 hours. The sample was predominantly red (approximately 75%) with some green and yellow cells. The biological film sample was also scratched with LB agar plates and minimal growth was observed along the scoring route (while cells not exposed to antibiotic grew as a bacterial line along the main streak). Hence, the dye can be used to identify and quantify dead cells. The horizontal sections at each 1.0 μm depth of the films of P. fragi and E. coli DH5a (pKMY319) were obtained with the confocal microscope and the distribution of living cells, dead cells, exo-poly-saccharides (EPS, by its acronym in English) and null spaces. Both the films of P. fragi and E. coli DH5a (pKMY319) consisted of uniform layers of cells and polysaccharides (when present) near the surface of the metal. The proportion of cellular material (living and dead cells) and non-cellular (polysaccharides and water channels) varied with depth, this was for all biological films. The biological films of both strains exhibited a pyramidal architecture, with a dense concentration of cells near the bottom of the biological film (biological film-metal interface) and a dispersed distribution of cells near the biological-liquid film interface. This is in agreement with Lawrence et al., J. Bacteriol. 173: 6558-6557 (1991), who reported a similar pyramidal structure for the biological films of P. fluorescens and P. aeruginosa developed on glass slides in complex and minimal media with continuous cultures. In the work reported there, the upper layers of the biological film consisted predominantly of living cells and the density of living cells decreased near the surface of the metal. Polysaccharides (when present) were usually detected near the bottom of the biological film (typically 3 μm from the metal surface). Thick groups of loose associated cells and dead cells (thickness of 15 to 40 μm) were found in the upper part of the biological film (at the biological film-liquid interface) and were not taken into account for the determination of the thickness of the film. the biological film. The films of E. coli DH5a (pKMY319) also had a thin layer of silt covering the metal pieces after eight days of exposure to the growth medium, which could not be retained on the upper part of the metal during the staining procedure. . The depth profiles of four days were determined for P. fragi in medium LB and VNSS at 30 ° C. What's more, more cell mass was formed for P. fragi at higher temperatures in LB medium, since at 23 ° C, 50% of the biological film had already been formed from living and dead cells, while biological films grown at 30 ° C had 90% of living and dead cells. In the biological films developed in VNSS medium, they comprised 10 to 50% of the biological film, while in LB medium, less than 5% of EPS had been detected. By filling the medium daily, the relative proportions of the constituents of the biological films changed significantly; typically, more living cells were detected in the biological film under all conditions. The architecture of the biological film was also modified with the addition of fresh medium daily with approximately equal proportions of cells observed at all depths of the biological film, instead of a pyramidal architecture. The proportion of cellular material versus non-cellular material remained relatively constant throughout the biological film for most conditions and only polysaccharides were observed in the VNSS medium. The expansion of polysaccharide production (when present) was greater with the replacement of the medium. Less clustering was observed in the upper layers of the biological film and the proportion of living cells also did not decrease significantly towards the bottom of the biological film. Conclusion The CSLM image analysis of the biological films and the quantification of the relative proportions of living cells, dead cells, EPS and empty space, revealed that the maximum density of cells (both living and dead) was obtained after four days of exposure and decreased after four days. For this reason, the biological films of four days in batches, of P. fragi K and E. coli DH5 (pKMY319) grew in metal pieces in LB and VNSS medium from where they were selected for a greater characterization and comparison with biological films that grew with medium that was replaced daily. The composition of the biological films depended on the growth medium, the culture temperature and the medium replacement. The development of the biological films in LB medium beyond four days showed a decrease in the number of cells, suggesting that the absence of a polysaccharide matrix causes a cell detachment. In VNSS medium, the cells were embedded in a polysaccharide matrix and showed a lower tendency to detach from the surface of the metal at exposure greater than eight days (not shown). This matches well with Dewanti and Wong, Int'l. J. Food Microbiol., 26: 147-164 (1995), who observed a similar biological film structure with E. coli 0157: H7, grown in trypticase soybean germ and minimal media. In addition, the physiology and cellular morphology of the biological film of bacteria was different in biological films developed in different media and temperatures. The biological films of P. fragi and E. coli developed in LB media were observed as small and distinct cells; in contrast, the biological films in VNSS medium were elongated and in clusters, probably in response to the tension of the environment. Filling the growth medium daily caused a small decrease in cellular content through the depth of the biological film and less clustering was observed. The continuous availability of nutrients could possibly increase the aggregation of metabolically active cells to the biological film, causing addition of cells up the biological film to replace lost cells and is congruent with the observations of Costerton (1995). The absence of clusters in the biological-liquid film interface could also be explained by the minimal disturbance to the architecture of the biological film caused by the daily addition of fresh growth medium and the staining process. The characteristics of the biological films (distinguished by means of CSLM), when compared to the corrosion results, indicate that increasing the total cells in the biological film increases the inhibition of corrosion. Increasing the temperature from 23 ° C to 30 ° C resulted in a 100% increase in corrosion for the sterile controls and an increase of 1.6 to 4.1 times in the cell mass (calculated as the total average of living and dead cells at along the entire depth profile of the biological film) for six of the eight conditions studied (two bacteria, two media and two temperatures). Corresponding to this increase in cell mass and temperature, there was only a 22% increase in corrosion for six of the eight conditions of the biological films (for E. coli DH5a in VNSS medium with daily replacement, corrosion was increased by 100%; and for P. fragi K in VNSS medium without replacement, corrosion increased by 230%). Therefore, in general, the increase in temperature increases the cell mass and the increase in corrosion for parts protected with biological films was much lower than when they were seen in sterile media (22% versus 100%).

Previous work in our laboratory on corrosion inhibition with seven widely varying genus of bacteria (with differing degrees of biological film formation) confirms that a homogeneous biological film is necessary (Jayaraman et al., 1997b): Metals exposed to bacterial suspensions of S treptomyces (which formed a biological film spread with cells distributed in groups), suffered corrosion at a rate comparable to sterile controls. Since the proportion of corrosion in this study after four days with P. fragi with respect to corrosion in similar sterile controls is similar to 23 ° C and 30 ° C with LB medium and VNSS medium; however, corrosion inhibition is provided by the biological film even when the thickness, composition and characteristics of the biological film under four of the conditions are drastically different. Therefore, it seems that only a certain minimum thickness or density of the biological film is required to inhibit corrosion. Similar results occurred with E. coli in VNSS medium at 30 ° C. When the growth medium was replaced daily, it was interesting to note that significant differences in the corrosion inhibition were observed only in the VNSS medium at 30 ° C. Apart from decreasing or increasing the thickness of the biological film, one of the main differences seen in filling the medium was to increase the uniformity of the distribution of the cells through the biological film and an increase in the relative proportion of living cells. This uniform layer of cells could reduce the amount of oxygen available on the metal surface for the corrosion process and thereby inhibit corrosion. The lack of a significant change in the inhibition of corrosion compared to not filling the medium for other conditions also suggests an upper limit for inhibition of corrosion by a particular bacterium, which is quickly achieved in a uniform biological film by a minimum amount of cells that actively breathe. Example 2: Biological films can inhibit the corrosion of copper and aluminum. This example shows that biological films can inhibit the corrosion of copper and aluminum. The toxicity of copper to microorganisms has led to the belief that the corrosion of copper induced by microbes (MIC) is negligible (Iverson, 1987). However, ammonia generated by microorganisms and sulfuric acid generated by Thiobacillus and by sulfate-reducing bacteria (SRB) can cause corrosion of copper alloys (Iverson, 1987); Wagner and Little, 1993). Wagner and Little observed that the presence of a biological film in copper creates differential aeration cells and chloride gradients can cause sting (Wagner and Little, 1993). Corrosion of copper alloys is a problem in the heat exchanger pipes, in the seawater casing of ships and in aircraft fuel tanks (Iverson, 1987, Miller, 1981). Iverson also mentions that corrosion of copper in freshwater and seawater was inhibited by the addition of bacteria and that corrosion increased after the bacteria died (Iverson, 1987). The formation of a passive oxide film in aluminum increases its resistance to corrosion (Iverson, 1987, Wagner and Little, 1993). It has been commonly related to Pseudomonas and Cladosporium with the MIC of aluminum and its alloys (Iverson, 1987). The production of corrosive organic compounds by P. aeruginosa can remove zinc and magnesium from aluminum and its alloys, causing corrosion. Aluminum concavities have been reported from three strains of SRB and a 100-fold increase in weight loss was observed compared to sterile controls (Iverson, 1987). Materials and Methods Bacterial strains and growth media. P. fragi K is a kanamycin-resistant derivative of P. fragi (Jayaraman, A. et al. 1997a) and B. brevis 18 is an overproducing strain of gramicidin S (Azuma, T. et al., Appl. Microbiol. Biotechnol.38: 173-178 (1992)) (hereinafter: Azuma et al., 1992) . The biological films on metal surfaces were developed in continuous reagents with Baar's modified medium, as previously described by Jayaraman et al. (Jayaraman, A. et al. 1997c) since this medium supports the growth of aerobic and SRB. Preparation of samples Plates of common sheets of unalloyed copper and 2024 aluminum alloy were cut (7.5 cm x 7.5 cm and 1.2 cm thick squares), polished with 240 grit sandpaper (Buehler, Lake Bluff, IL) and stored as previously described (Jayaraman, A. et al., 1997c). Continuous corrosion rate using EIS. Impedance data (from a minimum of two experiments) were obtained using an electrochemical measurement unit Solarton-Schlumberger (SI 1280, Schlumberger Technical Instruments Division, San Jose, CA) interfaced with a Macintosh computer (PowerMac 7100/80, Apple Computers, Cupertino, CA) running EISIS electrochemical experimentation software (University of California, Irvine) (equivalent package: THALES Impedance Measurement and Software [Software] of Synthesis / Simulation / Equivalent Circuit Suitability, which are available commercially by Bioanalytical Systems, Inc., West Lafayette, IN) (hereinafter: THALES parcel). The configuration of the reagent and the operating conditions were described previously (Borenstein, 1994, supra). RESULTS AND DISCUSSION The polarization resistance Rp, the capacitance C and the corrosion potential Ecorr, for all experiments with copper and aluminum are summarized in Table I. Corrosion with unalloyed copper in modified Baar medium at 30 ° C was studied using continuous reagents and the impedance spectra were obtained. The sterile reagents (five independent experiments) had a maximum phase angle of about 56 °, after 10 days of exposure. A biological film of P. fragi K that grew in copper (five independent experiments) increased the impedance by 21 times at the lowest measured frequency (1.4 x 10-3 Hz) in the same period of time, indicating a decrease in corrosion. This decrease in corrosion was also corroborated by an increase in the phase angle (c.f., 56 °, against 71 °). Similar impedance spectra (two independent experiments) were also observed when a biological film of B. brevis 18 in copper was developed. The impedance spectra obtained with sterile Baar modified medium with a 2024 aluminum alloy in continuous reagents (two independent experiments), showed a maximum phase angle of 71 ° at low frequencies, after 10 days of exposure. When a biological film of P. fragi K was developed in the aluminum alloy for six days (five independent experiments), the maximum phase angle changed to 78 ° and an 8-fold increase in Rp was also observed. As seen in unalloyed copper, a biological film of B. brevis 18 (three independent experiments) was also able to increase the Rp of aluminum 2024 by 5 times and the angle of phase by 1 °, under similar conditions. The observed increases in Rp and the changes in the impedance spectra are similar to those observed in Jayaraman et al. 1997c, who reported a 40-fold decrease in Rp and a 35 ° increase in phase angle for mild steel SAE 1018 with axenic biological film of P. fragi K compared to sterile controls. Tallo I RP polarization resistance? C capacitance and corrosion potential Ecorr for unalloyed copper and 2024 aluminum alloy in modified medium Baar, at 30 ° C. The data is from a representative experiment (minimum of two independent experiments).

Experiment Rp (Ohm * cm2) C (F / cm2) Ec (mV Sample vs. Ag / AgCl) Sterile note 1 note 1 71 Copper P. fragi K note 2 note 2 18 Copper B. brevis 18 9.66xl05 1.65xl0-3 77 Copper Sterile 3.04xl04 1.78xl0"5 -670 Aluminum 1024 P. fragi K 1.32xl05 4.05xl0"5 -520 Aluminum 2024 B. brevis 2.13xl05 1.69xl0"5 -512 Aluminum 2024 Note 1: It is not possible to estimate parameters based on available equivalent circuit models Note 2: Impedance suggests pitting (C = 8.1xl0-5 F / cm2; Rp = 2.97xl05 Ohm * cm2; Rp? t / F = 3.52xl03 Ohm) Example 3: Antimicrobial Peptide Agents That Inhibit Growth of SRB. This example demonstrates that antimicrobial peptide agents inhibit the growth of SRB. The antimicrobial peptides are small (Marahiel M. et al., Mol.Microbiol., 7: 631-636 (1993); Nakano MM and Zuber, P., Crit. Rev. Biotechnol., 10: 223-240 (1990)). they can be easily cloned in aerobic bacteria forming biological films and can be optimized by protein engineering (Piers KK et al., Gene 134: 7-13 (1993)) (hereinafter: Piers, 1993); thus they are attractive candidates to exclude SRB from biological films. Saleh et al. (1964) and Postgate (Postgate JR The sulphate-reducing bacteria, Cambridge University Press, New York (1984)) (hereinafter: Postgate, 1984) have compiled lists of antimicrobials that are inhibitors of several SRBs, including the peptide Polymyxin B (which inhibits D. Vulgaris at 100 μg / mL). The present study describes the inhibition of the representative SRBs D. vulgaris and D. gigas in suspension cultures by the antimicrobial peptides gramicidin S (a 10-amino acid cyclic peptide from B. brevis (Azuma et al., 1992), gramicidin D ( Linear peptide of 15 amino acids of B. brevis) (van Dóhren H., Peptides, In LC Vining and C. Stuttard (ed.), Genetics and Biochemistry of Antibiotic Production, Butterworth-Heinemann, Boston (1995), amideated indolicidin and no amidated (a linear peptide of 13 amino acids of bovine neutrophils (Falla TJ et al., J. Biol. Chem. 271: 19298-19303 (1996) (hereinafter: Falla et al., 1996); Selsted ME et al. , J. Biol. Chem. 267: 4292-4295 (1992)) (hereinafter: Selsted et al., 1992), bactenecin (a cyclic peptide of 12 amino acids of bovine neutrophils (Romeo D. et al., J. Biol. Chem. 263: 9573-9575 (1988)) (hereinafter: Romeo et al., 1988) and polymyxin B (a branched cyclic decapeptide of 10 a.m. of Bacillus polymyxa (Fuj ita-Ichikawa) Y. and K. Tochikubo, Microbiol. Immunol. 37: 935-941 (1993)). Materials and Methods Bacterial strains and growth medium. D. vulgaris (ATCC 29579) and D. gigas (ATCC 19364) were obtained from the American Type Culture Collection and cultured in 15 mL screw-top tubes, with 10 mL of modified media from Baar (medium ATTC 1249) supplemented with 100 μL each, from harvesters of sodium sulfide sodium at 4% and Oxyrase (Oxyrase Inc., Mansfeld, OH) the initial cultures were worked in glycerol extract at -85 ° C; all subsequent cultures were worked with a 3% inoculum of the initial culture maintained at 30 ° C without agitation. Both SRBs were routinely cultured in tightly closed screw cap tubes exposed to oxygen in laminar flow covers (which does not inhibit cultures, as previously reported by Angeli, P. and White, DC, J. Ind. Microb 15: 329-332 (1995)). The SRB were also periodically cultivated in the presence of 0.1% ferrous ammonium sulfate and the presence of these sulphate reducers was confirmed by detection of black iron sulfide in the culture tubes. The desulfoviridine test was also carried out after each MPN test to confirm the presence of D. vulgaris or D. gigas for its red color under ultraviolet light., due to the release of the chromophore from the pigment desulfoviridine. (Postgate, 1984). Antimicrobial peptides. Indolicidin (in amidated form and in acid-free form) was kindly provided by Prof. Michael E. Selsted of UC Irvine, and the acid free form was synthesized by Genosys Biotechnologies Inc. (The Woodlands, TX) with a purity of 76%. Gramicidin S (96.5% purity) and Gramicidin D (100% purity), and polymyxin B (100% purity) were purchased from Sigma Chemical Co. (St. Louis, MO). Bactenecin was synthesized by Genosys Biotechnologies Inc. with a purity of 32% and shipped in the presence of dithiothreitol ("DTT") (<0.1%). The molecular weights of synthesized indolicidin (acid form, 1907 Da) and bactenecin (1486 Da) were verified using a MALDI-Timem of Flight mass spectrometer (TOF) (Voyager DE 5-2386- 00, Perseptive Biosystems, MA). A Vydac C18 column (Vydac, Hesperia, CA) was used in a reverse phase HPLC chromatograph (Varian Vista 5000 series, Sugar Land, TX) to remove residual DTT from the bactenecine (and to facilitate the formation of a disulfide bond between waste 3 and 11). A mobile phase of acetonitrile / 0.1% trifluoroacetic acid (TFA) in water (20:80) was used to extract bactenecin by means of a solvent. This fraction was considered free of DTT and was used for antimicrobial tests. SRB Inhibitory Tests To determine the viability indices (Romeo, et al., 1988) of the SRB, an exponential late phase culture (O.D6oo 0.16 to 0.19 corresponding to an initial number of cells of 5-9 x 104 cells / mL) was exposed to various concentrations of antimicrobials for 1 hour at 30 ° C. One mL of cells was harvested, washed once in fresh modified Baar's medium to remove cell debris and resuspended in 1 mL of fresh modified Baar's medium supplemented with 10 μL each, of Oxyrase (Oxyrase Inc., Mansfeld, OH) and 4% sodium sulfide. Aliquots of 450 μL were poured into sterile Eppendorf tubes and appropriate amounts of antimicrobials were added and incubated at 30 ° C. The effectiveness of the treatment was determined by the multiple-most-likely-number (MPN) fermentation technique. (Anonymous.) Multi-tube fermentation technique for members of a coliform group, pp. 9-45 to 9-51 In AE Greenberg, LS Clesceri, and AD Eaton (eds.), Standard Methods for the Examination of Water and Wastewater , 18 ed., American Public Health Association, American Water Works Association, and Water Pollution Control Federation, New York (1992)) (hereinafter: Greenberg, 1992).

The MPN test to enumerate the SRBs was carried out in three 12 L tubes with an inoculum of 1000 μL SRB, three 12 mL tubes with 500 μL inoculum and three 12 mL tubes with 100 μL inoculum. The nine tubes contained a final volume of 10 mL of Baar's modified medium supplemented with 100 μL each of 4% sodium sulfide and Oxyrase (Oxyrase Inc., Mansfeld, OH). The tubes were followed for 72 hours to determine the number of tubes that were positive for growth. Growth was determined by the increase in crop turbidity and the MPN / mL index was calculated using the Thomas formula (Greenberg 1992). RESULTS AND DISCUSSION D. vulgaris and D. gigas were incubated in the presence of several antimicrobial peptides and their variability was determined after one hour of exposure. Ampicillin was used as a positive control for D. vulgaris, since ampicillin and chloramphenicol were found to inhibit this strain at 20 μg / mL, which is in accordance with previous reports (Odom and Singleton, The sulphate-reducing bacteria: contemporary perspectives, Springer Verlag, New York (1993) (from now on: Odom and Singleton, 1993), however, neither ampicillin nor chloramphenicol were effective in inhibiting D. gigas at 100 μg / mL. SRB to several additional antibiotics (kanamycin, tetracycline, thiostrepton, penicillin G and nalidixic acid), inorganics (ammonium molybdate, sodium molybdate and anthraquinone) and peptides (nisin and polymyxin B) were also evaluated using stationary phase SRB cultures. D. gigas was inhibited by anthraquinone at 100 μg / mL (Cooling et al., 1996) and both SRB were inhibited by sodium molybdate at 100 μg / mL This is similar to the observations of Saleh, et al., 1964, q who did a follow-up study of approximately 200 compounds for the inhibitory activity of their SRB and noted that SRBs show a higher degree of resistance to inhibitory compounds (Id.). The MPN test was used to determine the viability index of D. gigas and D. vulgaris for the antimicrobial peptides. For D. gigas, both the gramicidins S and the amidated form of indolicidin, Ind-NH2 (which is the natural form in bovine neutrophils (Falla et al., 1996), Selsted et al., 1992), were able to reduce the viability of a late exponential phase culture by 92 to 96% after one hour exposure at 25 μg / mL. For D. vulgaris, Ind-NH2 at 25 μg / mL was slightly more effective in inhibiting growth (reduced viability by 99.3%), whereas gramicidin S was less effective and reduced viability by 93% at 100 μg / mL. The acid form of indolicidin (Ind-OH) was 10 times less effective than the amidated form of indolicidin against D. gigas and 174 times less effective against D. vulgaris at 25 μg / mL. This is not surprising since it is believed that post-translational amidation increases the potency of indolicidin (Falla, et al., 1996). The antimicrobial peptides gramicidin D, polymyxin B and bactenecin (Postgate, 1984) were also able to decrease the viability of D. vulgaris and D. gigas by approximately 90% at 100 μg / mL. The results of these MPN tests were also corroborated by similar results obtained when d. vulgaris was exposed to gramicidin S, gramicidin D, indolicidin and bactenecin for one hour, on Desulfovibrio agar plates (medium 42 of ATTC) and incubated in anaerobic GasPak chambers (Fisher Scientific Co., Pittsburgh, PA). These results indicate that antimicrobial peptides such as gramicidin S, indolicidin, polymyxin B and bactenecin have the potential to be used in the inhibition of SRB growth and decrease microbially influenced corrosion in steel. Indolicidin is able to inhibit Escherichia coli and Staphylococcus aureus by 99.9% at 5-25 μg / mL (Romeo et al., 1988); Selsted et al., 1992), but, in this study, D. gigas and D. vulgaris exhibited great resistance to indolicidin. Bactenecin inhibits E. coli by 95% at 100 μg / mL (Romeo et al., 1988) and demonstrated similar inhibition for D. gigas and D. vulgaris (90%) in this study. Gramicidin S is also known to completely inhibit the growth of Gram-negative bacteria at 3-12.5 μg / mL (Kondejewski L. et al., Int. J. Peptide Protein Res. 47: 460-466 (1996)) and demonstrated an inhibitory effect against both Gram-negative SRBs in this study at 50-100 μg / mL. Based on their activity against SRB in suspension cultures, all the antimicrobial peptides tested in this study were more potent at similar concentrations, than commercially available antibiotics such as kanamycin, nalidixic acid, and tetracycline and that inorganic compounds such as molybdate sodium and anthraquinone. Example 4: Exclusion of SRB from biological films that use cloned antimicrobial agents of secretory bacteria This example shows the cloning and expression of a chemical antimicrobial agent in bacteria and its use to exclude SRB from a biological film in stainless steel. Antimicrobial peptides have been isolated and identified from several bacteria (Hancock, REW et al., Adv. Microb. Physiol. 37: 135-175 (1995), plants (Hancock, RW et al., Cationic peptides: a class of antibiotics able to access the self-promoted uptake pathway across the Pseudomonas aeruginosa outer membrane, pp. 441-450, In T. Nakazawa (ed.), Molecular Biology of the Pseudomonads, ASM press, Washington DC (1996)) (from now on further: Hancock et al., 1996), insects (Boman, HG et al., Eur. J. Biochem., 20: 23-31 (1991)) and mammals (Frank, RW et al., Annu., Rev. Immunol. : 105-128 (1993); Zasloff, M. Proc. Nati Acad. Sci. 84: 5449-5453 (1987)) (hereinafter: Zasloff, 1987). These peptides can be broadly classified into magainins, (Zasloff, 1987), defensins (Cullor, JS et al., Arch. Ophtalmol. 108: 861-864 (1990)) (hereinafter: Cullor et al., 1990), cecropins (Calloway, JW et al., Antimicrob Agents Chemother, 37: 1614-1619 (1993)) (hereinafter: Calloway et al., 1993); melitins (Piers, KL et al., Mol.Microbiol.12 (6): 951-958 (1994)) (hereinafter: Piers, et al., 1994) and have been shown to exhibit antimicrobial activity against Gram bacteria. -Negative and Gram-positive, as well as against yeast and fungi (Hancock et al., 1996). Most cationic peptides have multiple residues of lysine and arginine and hydrophobic and hydrophobic faces (Hancock et al., 1996) and kill microorganisms by increasing the membrane permeability of bacterial cells or by inhibiting DNA synthesis (Hancock et al., 1996; Romeo et al., 1988). Indolicidin (Cullor et al., 1990; Del Sal, G. et al., Biochem Biophys. Res. Conm. 187 (1): 467-472 (1992)) and bactenecin (Frank, RW et al., J. Biol. Chem. 265 (31): 18871-18874 (1990); Romeo et al., 1988)) are cationic antimicrobial peptides isolated from bovine neutrophils.

(Lehrer, R.I. et al., Annu., Rev. Immunol., 11: 105-128 (1993)).

Indolicidin is a tridecapeptide belonging to the family of defensins (Selsted et al., 1992) and consists of only six different amino acids with the highest proportion of tryptophan in any known protein (39%) (Falla et al., 1996). Indolicidin is also the smallest linear antimicrobial peptide known and its carboxyl end is amidated in its natural form of appearance (Falla et al., 1996; Selsted et al., 1992). Bactenecin is a cyclic dodecapeptide rich in arginine and contains a disulfide bond that maintains the cyclic structure (Romeo et al., 1988). Some attempts have been made to produce antimicrobial peptides in prokaryotic and eukaryotic expression systems for commercial applications Piers et al. (Piers et al., 1993 and Piers et al., 1994) have described methods for synthesizing and purifying the human neutrophil peptide 1 (HNP-1) and a hybrid cecropin / melittin peptide in bacteria using an expression system Staphylococcus aureus. These peptides were synthesized as Protein A fusions, secreted into the culture medium and purified using affinity chromatography (Piers et al., 1993). Calloway (Calloway et al., 1993) tried to manifest cecropin A in E. coli and concluded that post-translational modification of the carboxyl end was required for greater antimicrobial activity. Hara and Yamakawa (Hará, S. and M. Yamakawa, Biochem. Biophys. Res. Comm. 224 (3): 877-878 (1996)) have produced the peptide moricin in E. coli as a fusion to the maltose binding protein. and found similar activity in the native protein. Haugh et al. . { Biotechnol, Bíoeng. ) 57: 55-61 (1998)) have reported the production of the antisense recombinant P2 antimicrobial peptide in E. coli as inclusion of bodies using a bovine prochymosin fusion; high levels of the protein were expressed (approximately 16% of the total protein in the cell). Pang et al. (Gene, 116: 165-172 (1992)) (hereinafter: Pang et al.) Have attempted to manifest and secrete the scorpion insectotoxin I5A in bacteria, yeast and tobacco plants (no measurable activity was detected). All these approaches were aimed at the production of purified, large-scale, non-expensive antimicrobial peptides rather than in vivo applications. In the previous example, we show that the purified antimicrobial peptides indolicidin, non-amidated indolicidin and bectenecin inhibit anaerobic SRBs in suspension cultures. This example shows that the production of antimicrobial peptides in aerobic bacteria forming biological films can exclude SRBs from biological films and inhibit the metal corrosion associated with SRB. In particular, this example demonstrates the expression of the cationic antimicrobial peptides indolicidin and bactenecin in Bacillus Gram-positive and their use to exclude SRBs from biological films in 304 stainless steel. The indolylidin and bactenecin have been cloned as fusions to the signal of alkaline protease sequence (apr) and has been constitutively expressed using an apr promoter. The barnase pro-region (an extracellular Rnasa from B. amyloliquefaciens) has also been used to produce bactenecin as a pre-propeptide in Bacillus. The capacity of these strains to inhibit the growth of SRB in 1018 SAE mild steel and 304 stainless steel in continuous reagents has been characterized. Materials and Methods Bacterial strains, plasmids and growth media. E. coli XLI (Blue) < RecAl endAlgyrA96 thi-1 hsdR17 supE44 relAl lac [F'proAB laclq ZDM15 TnlO (Tet ')] í > it was purchased from Stratagene (La Jolla, CA). B. subtilis BE1500 < TrpC2, metBIO, lys-3,? AprE66,? Npr-82,? SacB:: ermCr * and plasmid pBE92 containing the alkaline protease promoter apr, signal sequence and the alkaline phosphatase reporter gene were obtained from E.I. du Pont de Nemours Inc. (Wilmington, DE). The protease deficient B. subtilis WB600 (Wu, X.-C. et al., J. Bacteriol 173 (16): 4952-4958 (1991)) (hereinafter: Wu et al., 1991) <; itrpC2,? nprE,? aprA,? epr,? bpf,? nprBr was provided by Dr. Sui-Lam Wong (University of Calgary, Alberta, Canada). B. polymyxa was obtained from the American Type Culture Collection, (ATCC 10401). D. vulgaris (ATCC strain 29579) was used as the reference SRB in this study. All corrosion experiments with B. subtilis BE1500 and P. fragi K (Jayaraman, A. et al., 1997a) were carried out in Baar modified medium (medium ATCC 1249) for sulfate-reducing bacteria. Corrosion experiments with B. polymyxa were carried out in Baar's modified medium supplemented with 1/10 ° [tenth] volume of TY lOx medium (10 g tryptone, 5 g yeast extract in 100 mL H20). Enzymes and Chemicals All restriction enzymes, T4 DNA ligase and Taq polymerase were obtained from Promega (Madison, WI). BCIP (5-bromo-4-chloro-3-indolyl phosphate) was purchased from Sigma Chemical Co. (St. Louis, MO). Indolicidin (acid free form, purity 76%) was synthesized by Genosys Biotechnologies Inc. (The Woodlands, TX). Plasmid constructions The methods for recombining DNA were carried out, as described by Maniatis (Maniatis, T. et al., 1982) and Rodríguez and Tait (Rodríguez, RL et al., Recombinan t DNA techniques. Benjamin / Cummings Publishing Company Inc., Menlo Park, CA (1983)). The DNA plasmid was isolated from Bacillus according to the procedure of Bramucci and Nagarajan (Bramucci, MG and V. Naragajan, Appl. Environ. Microbiol .62 (11): 3948-3953 (1996)) (hereinafter: Bramucci, 1996). The amino acid sequences were used for the non-amidated indolicidin [NH2-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-OH] (Selsted et al., 1992) and bactenecin NH2-Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg-OH] (Romeo et al., 1988) to design oligonucleotides that encode the genes for these peptides. Plasmid pBE92-Ind was designated to express non-amidated indolicidin since a 12 amino acid peptide was fused to the apr signal sequence; pBE92-Bac was designated to express bactenecin, since a peptide of 13 amino acids was fused to the signal sequence of apr and pBE92-ProBac was designated to express the bactenecina fused to the pro-region of the barnase RNase extracellular of B. amyloliquefaciens (Paddon, CJ et al., J. Bacteriol 171: (2): 1185-1187 (1989)) (hereinafter: Paddon et al., 1989) and the signal sequence apr. Synthetic oligos (Drawing 1) were synthesized by Gibco-BRL Life technologies (Long Island, NY) on a 200 nmole scale, with purification by polyacrylamide gel electrophoresis (PAGE, for its acronym in English). The oligos were synthesized with restriction sites Hind III and Nhe I in the flanks with six additional bases in any of the endings for an effective restrictive digestion. Two complementary oligos of each structure were suspended again in TE buffer (50ng / μL), mixed in equimolar proportions and incubated in boiling water for 3 minutes. The oligos created rings in the water bath as they cooled to room temperature (approximately two hours). The ringed oligos were digested with Hind II and Nhe I overnight, precipitated with ethanol at -85 ° C for one hour and resuspended in distilled and deionized H20. The plasmid vector pBE92 was isolated from the extracts of E. coli XLI cells (Blue) using an idi plasmid kit (Qiagen Inc., Chatsworth, CA) and the DNA was digested with Hind III, Nhe I and Sal I simultaneously by the same protein. hours at 37 ° C. the triple digested vector and the grafted antimicrobial gene were ligated at 16 ° C for 17 hours at an injurious molar ratio: vector of 28: 1. The ligation mixture was extracted with phenol / chloroform / isoamyl alcohol (25: 24: 1), the ethanol precipitated and was resuspended in 30μL of H20 dd. The pro-bactenecine was synthesized as two oligo filaments with a complementary pair region of 21 bases with restriction sites Hind III and Nhe I at the end of the two filaments (Drawing 2). A Not I site was also created downstream of the stop codon, which was used to enter a single site within pBE92. The two filaments were ringed as described above and the complementary regions were completed using Taq polymerase (one cycle, 30 secs at 94 ° C, followed by 30 secs at 55 ° C and two hours at 72 ° C) with a Perkin-Elmer thermal cycler N801-0150 (Perkin-Elmer, Norwalk, CT). The final product was extracted with phenol / chloroform / isoamyl alcohol, the ethanol precipitated at -85 ° C in the presence of lmM MgCl2 for one hour and resuspended in 50μL of H20 dd. The transformants were identified by restrictive digestion with BglI (indolicidin), BssHII (bactenecin) and Not I (Pro-bactenecin) and was confirmed using a modification of the colony survey of Boehringer-Mannhein. Two hundred nanograms of DNA plasmid (from mini-preparations of E. coli putative transformants with antimicrobial genes) were found in positively charged nylon membranes (Product No. 1209272, Boehringer Mannheim, Indianapolis, IN) and tested according to the specifications of the manufactured using a synthetic antimicrobial gene of oligo DNA (Drawing 1) labeled using the primitive random DNA labeling protocol of Boehringer Mannheim. Transformation of __ *. coli and Bacillus.

Electrocompetent E. coli XLI (Blue) cells were made according to the method of Smith and Iglewski (Smith, A.W. et al., Nuc.Acids, Res. 17: 10509 (1989)). Ten μL of the ligation mixture was used to electroporate the bacterium (1.2 kV7cm, 200 Ohms, 25μF) using a pulser / pulse controller gene (Bio-Rad Laboratories, Hercules, CA), and the clones containing the correct graft (pBE92 -Indolicidin, pBE92-Bactenecin and pBE92-ProBactenecin) were selected on LB agar plates containing 100 μg / mL of ampicillin and 40 μg / mL of BCIP using a blue / white selection technique (transformants with the appropriate graft produced white colonies while that the colonies that returned to enclose the vector turned blue). B. subtilis BE1500 was made competent and transformed according to the two step method of Cutting and Vander Horn (Genetic analysis, pp. 27-74, In C.R. Harwood and S. Cutting, M. (ed.), Molecular biological methods for Bacillus k, John Wiley & Sons, New York (1990)). The competent cells of late exponential phase were incubated with DNA plasmid (approximately lμg isolated from E. coli XLI (Blue)) for 30 minutes. The cultures were diluted with 1-mL of 10% yeast extract and incubated on a rotary shaker (New Brunswick Scientific, Edison, NJ, agitator series 25) at 37 ° C before being placed on LB agar plates containing 25μg / mL kanamycin. The competent cells of B. polymyxa were prepared according to the procedure of Rosado et al. (J. Microbiol. Meth. 19: 1-11 (1994)). Approximately 1 μg of DNA (pBE92-base structures) isolated from B. subtilis BE1500 (Nagarajan, V. et al., Gene 114: 12-126 (1992)) using the procedure of Bramucci and Nagarajan (Bramucci, 1996) was used pair electroporar B. polymyxa (6.25 kV / cm, 200 Ohms, 25 μF). The cells were then incubated at 37 ° C for 3 hours with shaking and selected on LB agar plates containing 150 μg / mL kanamycin. SDS-PAGE B. subtilis BE1500 containing plasmid pBE92 with base-constructs, expressing the antimicrobial peptide genes were cultured in 25 mL of LB medium at the late exponential phase (O.D.600 = 0.70-1.0) at 37 ° C. the cells were harvested by centrifugation at 10,000 x g for 10 minutes at 4 ° C and the supernatant was concentrated 25 times using a SpeedVac concentrator (Model 200H, Savant Instruments Inc., Holbrook, NY). The concentrated supernatant was mixed with a 2X sample buffer (0.125 M Tris-base, 0.4% SDS, 20% glycerol and 0.1 mL of lgm / mL of blue bromophenol with 5μL 2-mercapto ethanol added for each 100 μL of 2X buffer) , boiled for 5 minutes and subjected to electrophoresis in a 16.5% Tris-Tricine gel (Bio-Rad, Hercules, CA). Continuous corrosion experiments.

Corrosion experiments with batch cultures using SAE 1018 mild steel pieces (2.5 cm in diameter, 1.2 mm thick) were carried out in triplicate in 250 mL Erlenmeyer flasks at 30 ° C without shaking, as described previously (Jayaraman et al., 1997a). A continuous reagent (Jayaraman et al., 1997c) was also used to develop biological films in stainless steel 304 and corrosion monitoring was performed using electrochemical impedance spectroscopy (EIS) in a minimum of two independent reagents using an electrochemical measurement unit of Solarton-Schlumberger (SI 1280, Schlumberger Technical Instruments Division, San Jose, CA) in interface with a Macintosh computer (PowerMac 7100/80, Apple Computers, Cupertino, CA) running EISIS electrochemical experimentation software (University of California , Irvine) (the similar commercial parcel, THALES, can also be used). The open circuit potential (OCP) was measured as the potential between the metal specimen and a reference electrode Ag / AgCl and the polarization resistance (Rp) was determined as the value of the impedance at low frequency (where the imaginary part of the impedance was zero or negligible). The continuous corrosion rates of the crops were estimated as the inverse of the polarization resistance (Macdonald, DD and MCH McCubre, Applications of impedance spectroscopy, pp. 262-267) In JR Macdonald (ed.), Impedance Spectroscopy: Emphasizing solid ma terials and systems John Wiley &; Sons, New York (1987); Stern, M., Journal of Electrochemical Society, 105 (11): 638-647 (1958)). Antimicrobial tests To determine the susceptibility of B. subtilis BE1500 and B. Polymyxa hosts to the expressed antimicrobials, these strains were cultured from a single colony in 25mL of LB medium with shaking at 37 ° C to an O.D.6oo of 0.40-0.45. Aliquots of one mL were collected, washed in fresh LB medium and resuspended in lOOμL of fresh LB medium with shaking, at 37 ° C in sterile Eppendorf tubes. The antimicrobials indolicidin and bactenecin (50-100μg / mL) were added and the tubes were incubated at 30 ° C for one hour without agitation. The appropriate dilutions were poured onto LB agar plates and incubated overnight at 37 ° C to determine the vastness of survival. The results were confirmed by performing two independent experiments. The expression of indolicidin and bactenecin in Bacillus was determined in duplicates by exposure of E. coli BK6 in suspension to the supernatants of concentrated cultures. E. coli BK6 was cultured in an O.Dgoo of 0.20-0.25, took a spherical shape at room temperature and resuspended in different volumes (50 or 100 μL) of the concentrated supernatant. The cell suspension was incubated at 30 ° C for 1 hour without aeration and appropriate dilutions were placed on LB agar plates to determine the antimicrobial activity of the supernatant. The ability of the supernatant of B. subtilis constructs to inhibit SRB in suspension was determined by resuspending 500μL of late exponential phase D. vulgaris culture (O.D6oo = 0.15-0.20) in an equal volume of 25 times concentrated culture of Supernatant of B. subtilis BE1500 with the antimicrobial constructs under anaerobic conditions, as described in the previous example. The cells were incubated at 30 ° C for one hour and the surviving SRBs were enumerated using the most-probable-number (MPN) test for three tubes (Greenberg, 1992). To determine the number of viable SRBs in the biological film by the MPN method for three tubes, the biological film was rinsed once in sterile water to remove weakly associated cells, scraped from the 304 stainless steel pieces (2.5 cm in diameter) , 1.2 mm thick), resuspended and serially diluted in fresh Baar modified medium under anaerobic conditions as described by Jayaraman et al. The number of aerobic bacteria in the biological film was determined by placing appropriate dilutions on LB agar plates. RESULTS Susceptibility of host expression to antimicrobial peptides. B. subtilis BE1500 and B. subtilis WB600 showed susceptibility in the order of several thousand times more to the purified antimicrobial peptide indolicidin (non-amidated form, 50 and 100 μg / mL) and susceptibility in the order of several hundred times more to the purified antimicrobial peptide bactenecin (50 μg / mL) after exposure for one hour at 30 ° C, of which B. polymyxa (see Table II). It follows that B. polymyxa is a better host than the other species tested for the expression of the antimicrobial peptides tested, given their resistance to both non-amidated indolidycin and bactenecin. Table II. Susceptibility of host strains to purified antimicrobials after one hour exposure at 30 ° C Reduction in how many times in number of cells Indolicidin Indolicidin Bactenecin Bacteria 50 μg / mL lOOμg / mL 50μg / mL B. subtilis BE1500 6000 10,000 100 B. polymyxal0401 4 2 B. subtilis 20,000 40,000 400 Cloning of antimicrobial peptides using an E. coli impulse vector Bacterial expression systems were constructed using the E. coli-Bacillus pBE92 impulse vector to generate pBE92-Ind, pBE92-Bac and pBE92-ProBac, which utilize the apr promoter. and the signal sequence to constitutively express and secrete the antimicrobial peptides in Bacillus K. The alkaline phosphatase gene in pBE92 was replaced by the Nhe I-HindIII graft containing the last three amino acids (Ser-Ala-Ser) of the signal sequence of apr and the complete antimicrobial gene. Detection of antimicrobial peptides secreted by Bacillus. Purified indolidicin (non-amidated form) was detectable with Coomassie stain when it was loaded at 230ng / cavity, but was not detected when it was loaded at 23 ng / cavity. The purified indolicidin and bactenecin were also not detected using silver dye at a load of 250ng / cavity. Western blots with polyclonal antibodies generated by rabbits to indolicidin (dilution 1: 250) using culture supernatants of B. subtilis BE1500 (pBE92-Ind) (25-fold concentrates) did not reveal a band corresponding to indolicidin; however, the antibody was not specific to indolicidin and bound to many cellular proteins. The primary amino acid sequence of bactenecin indicated significant difficulty in the generation of polyclonal antibodies (Dr. Shing-Erh Yen, Zymed Laboratories Inc., personal communication); therefore, polyclonal antibodies against this peptide were not synthesized. Antimicrobial activity of indolicidin and bactenecin in supernatants of Basillus cultures against E. coli and D. vulgaris in suspension cultures and biological films. The capacity of the concentrated supernatant of the culture of B. subtilis BE1500 with the antimicrobial plasmids to kill E. coli BK6 and D. vulgaris was determined. No reduction in the viability of E. coli BK6 and D. vulgaris was observed for the negative control experiments, in which the supernatants of B. subtilis BE1500 (pBE92) and B were used. subtilis BE1500 (pBE92-Ind) (Table III); however, about 93% of E. coli BK6 annihilators and 83% of D. vulgaris annihilators were observed with supernatants of B. subtilis BE1500 (pBE92-Bac) and B. subtilis BE1500 (pBE92-Pro-Bac). This result indicates that the bactenecine was expressed, secreted in the culture supernatant and that the disulfide bond was adequately processed in the extracellular environment to form active cyclic bactenecin. (E. coli was used in these studies as a positive control to show that the peptide was expressed and active, as well as to show that the inhibition of SRB was due to the peptide and not to exposure to oxygen or other exogenous causes). The amount of viable SRB after five days in a biological film in 304 stainless steel with B. subtilis BE1500 expressing the cloned antimicrobials was counted with the three tube MPN test (Table IV). Approximately 60 times less SRB was found in the biological film formed by B. subtilis BE1500 (pBE92-Bac) than in the biological films formed by B. subtilis BE1500 (pBE92) and B. subtilis BE1500 (pBE92-Ind), while ten times less SRB were found with B. subtilis BE1500 (pBE92-Probac). Table III Susceptibility of E. coli K and D. vulgaris to concentrated supernatants of B cultures. subtilis BE1500 expressing antimicrobials. The data are the average of two independent experiments.

E. coli BK6 D. vulgaris CFU / mL Inhibition MPN / mL Inhibition (%) (%) fresh medium 9 x 107 29 x 10 'buffer-f-kanamycin 100 μg / mL 4 x 103 99,996 B. subtilis BE1500 (pBE92) 8.7 xlO7 29 x 10- B. subtilis BE1500 (pBE92-Ind) 7.9 xlO '12 1.43 x 10- B. subtilis BE1500 (pBE92-Bac) 6 xlOe 93 1.43 x 10-87 B. subtilis BE1500 (pBE92-ProBac) 7 xlO6 92 1.43 x 10- Table IV Inhibition of SRB in aerobic biological film (determined by the MPN test) in biological film of B. BE1500 expressing the antimicrobial plasmids in stainless steel 304 after five days. The data is from a biological film, in two independent experiments.

Plasmido SRB viable Inhibition B. subtilis MPN / mL viable BE1500, CFU / mL pBE92 5.13 x 10- 2.3 x 10 'pBE92- Indolicidin 3.59 x 10- 30 2.3 x 10'? BE92- Bactenecin .64 x 10; 98 1.9 x 10 'pBE92-Pro bactenecina 5.13 x 104 90 6.2 x 10' Corrosion studies in continuous and batch cultures with Bacillus strains that produce antimicrobial cloned peptides. The ability of antimicrobial producing constructs to inhibit the growth of SRB in mild steel SAE 1018 in shake flasks at rest was studied. When an addition of SRB (O.DTOO = 0.16-0.20) was made to a non-antimicrobial producing P. fragi K culture, a strong smell of hydrogen sulphide was detected in less than 18 hours. This was also accompanied by the formation of a precipitate of iron sulfide that indicated the growth and colonization of SRB in the aerobic biological film developed on the surface of the metal. B. subtilis BE1500 was able to retard the emergence of SRB corrosion in 36-48 hours, as compared to P. fragi K (as evidenced by the retardation of occurrence of an iron sulphide precipitate and the smell of hydrogen sulfide) .

The three antimicrobial production constructions in B. subtilis BE1500 were able to retard the emergence of SRB corrosion in 96-120 hours compared to P. fragi K and B. subtilis BE1500. Replacement of the growth medium after seven days, however, resulted in the appearance of a black precipitate within 36 hours with all strains. The addition of SRB to a 304 stainless steel continuous reagent with P. fragi K decreased the impedance value at the lowest measured frequency (1.4 x 10 ~ 3 Hz) by 5 times within 36 hours [after] the addition of SRB. This decrease was also accompanied by the smell of hydrogen sulphide from the reagent landfill, and the reagent became gray due to the formation of iron sulphide. The low frequency phase angle also decreased (c.f., 82 ° to 68 °). A similar change in the impedance spectra was also observed with the negative controls of B. subtilis BE1500 (data not shown) and B. subtilis BE1500 (pBE92) (Figure 3), although the change had a delay of 24 hours. In contrast, the three antimicrobial-producing constructs were able to decrease the vastness of the impedance spectra change (Figure 3). The indolicidin construct was the least effective at inhibiting SRBs and the low frequency phase angle changed from 80 ° to 69 °; however, it was still less than that observed with the pBE92 control (80 ° to 61 °). The bactenecin constructs (with and without the pro region) were more effective than the indolicidin construct and the low frequency phase angle decreased only to 76 °. These results indicate that the growth of SRB in 304 stainless steel has been significantly inhibited by the bactenecine constructs. Similar results were also obtained with B. subtilis WB600 (a strain deficient in six extracellular proteases) (Wu et al., 1991) expressing the cloned antimicrobials (Drawing 4). The addition of SRB to the biological films of B. s? Btilis WB600 (pBE92) and B. Subtilis WBN600 (pBE92-Ind) in stainless steel 304 decreased the phase angle at low frequency by 35 ° and 17 ° respectively; Correspondingly, the impedance at low frequency also decreased by 7 and 5.5 times respectively. However, it was observed that there was no such decrease for both biological films expressing bactenecine although the construction of bactenecina seemed to be slightly more effective than the construction probactenecina (Drawing 4). This suggests that the process of the pro-region to put mature bactenecine into circulation was inefficient in this protease deficient strain. Studies of continuous and batch corrosion with Bacillus strain producing a cloned antimicrobial in addition to the naturally occurring antimicrobial. The capacity of B. polymyxa ATCC 10401 (which produces the antimicrobial peptide polymyxin) to inhibit the colonization of SRB in mild steel, was studied in continuous and batch cultures. In batch cultures, B. polymyxa was able to retard the emergence of SRB corrosion by 60 * hours, compared with non-antimicrobial P. fragi K. Replacement of the growth medium did not result in immediate metal colonization by the SRBs (as seen with P. fragi K and B. subtilis BE1500) and no black precipitate was detected for 72 hours. Hence, B. polymyxa producing polymyxin was able to retard the growth of SRB in batch cultures. In continuous reagents with 304 stainless steel, the addition of SRB did not modify the impedance spectra for approximately 250 hours (as opposed to 36 hours for the impedance spectra at change with P. fragi K, Figure 5). No sulfur odor was detected in the reagent landfill and the reagent did not increase turbidity, as was observed for P. fragi K and B. subtilis BE1500 and B. subtilis WB600. Hence, it follows that B. polymyxa was able to inhibit the growth of SRB on 304 stainless steel in continuous reagents. Similar inhibition of corrosion was also observed with B. polymyxa that presented the antimicrobial construction (Figure 5) and extension of the inhibition was distinguishable for that uncultivated type of strain. DISCUSSION The cationic antimicrobial peptides indolicidin and bactenecin were constitutively expressed in B. subtilis BE1500 as fusions to the peptide signal of the alkaline extracellular protease (apr) by an approximation similar to Piers et al. , 1993 and Pang et al. (1992). Synthetic oligos for indolicidin and bactenecin were designed as precise fusions to the signal sequence, so no additional amino acids were added to the N-terminus of the peptide. This ensured that the expressed peptide should be active as much as possible and avoid the inappropriate process observed by Pang et al. (1992), whose expression system added 7 amino acids to the N-terminus of the Scorpion insectotoxin I5A. Bactenecin was also produced as a pre-pro-peptide by grafting DNA sequence from the pro-portion of B's barnase. amyloliquefaciens (Paddon et al., 1989) between the peptide signal and the bactenecin gene. A similar fusion of pre-pro defensin resulted in complete prevention of proteolytic degradation of the peptide secreted in S. aureus (Piers et al., 1993) and has been attributed to the formation of a secondary structure between the anionic pro-region and the cationic peptide. Indolicidin was expressed in Bacillus as the acid form, whereas in its natural form of appearance in bovine neutrophils, it is amidated in the C-terminus. The viability of B. subtilis was decreased by four orders of magnitude by indolicidin, while that B. polymyxa did not exhibit the same degree of sensitivity to indolicidin. This suggests that B. subtilis would not be an ideal expression host for expressing indolicidin in biological films, especially as in a biological film, indolicidin would not diffuse as much as it would in a suspension culture and thus could attack host cells. The sensitivity of E. coli BK6 to the supernatants of the concentrated cultures of B. subtilis BE1500 was used as an indicator of the antimicrobial activity of the supernatant since this bacterium is commonly used to evaluate the antimicrobial activity of the cationic peptides (Romeo et al. ., 1988); Selsted et al. , 1992). Our results indicate that the supernatant of B. subtilis BE1500 (pBE92-Ind) was not an inhibitor for E. coli while the supernatant of B. subtilis BE1500 (pBE92-Bac) and B. subtilis BE1500 (pBE92-Probac) were active in reducing viability for E. coli BK6. In our continuous reagent experiments, we observed that there was no difference in the growth of B. subtilis BE1500 (pBE92) and B. subtilis BE1500 (pBE92-Ind), which suggest a poor expression of indolicidin. This was also corroborated by the lack of inhibition of the SRBs demonstrated with this construction in continuous reagents, as was inferred from the changes in the impedance spectra (Figure 3). B. subtilis 1500 was more resistant to bactenecin than it was to indolicidin by a factor of 60, which could explain the ability of bactenecine constructs to inhibit SRBs in stainless steel. Continuous reagent experiments with 304 stainless steel clearly showed that the growth of SRB was inhibited (based on both qualitative indicators, such as hydrogen sulfide odor and iron sulphide precipitate and the quantitative decrease in polarization resistance Rp ). The bactenecin constructs were more effective than the indolicidin construct in the inhibition of SRB growth, which suggests that bactenecin was expressed and processed appropriately to form a disulfide bond since the defensins are usually inactive with inadequate disulfide bond processing ( Piers et al., 1993). However it was apparent that the SRB were not completely excluded from the biological film since all the reagents had increased turbidity when added SRB and a slight sulfur odor was still detected from a reagent with B. subtilis BE1500 (pBE92-Bac). The inhibition of SRB with bactenecine-producing constructs compared to the pBE92 control was also corroborated by the 36-fold decrease in viable SRB present in a biological film from a seven-day batch culture in 304 stainless steel (Table IV). Nevertheless, about 1 x 104 SRB / mL were detected even in the presence of bactenecin, confirming that the SRB were not completely eliminated by the cloned antimicrobial peptides. The B. Polymyxa producer of polymyxin (uncultivated type) was also able to delay the growth of SRB (and the emergence of SRB-induced corrosion) in batch cultures in mild steel for 60 hours. The addition of antimicrobial-producing plasmids to 73. polymyxa did not significantly improve its ability to kill SRB in mild steel. But the B. polymyxa developed in 304 stainless steel in continuous reagents was able to inhibit the growth of SRB completely (up to 275 hours). Our observations that D. vulgaris is unable to grow as a monoculture in stainless steel (while it can do so in mild steel) could also explain the effectiveness of these antimicrobials in the inhibition of SRB only in stainless steel. The data set forth in this example demonstrates that the growth of SRB in stainless steel 304 can be controlled by the generation of antimicrobial peptides from within the biological film and illustrate their potential for use in the prevention of corrosion influenced microbiologically in steel. The effectiveness of B. polymyxa in inhibiting the growth of SRB provides the basis to optimize a dual eliminator system to combat SRB-induced corrosion, where low levels of two antimicrobials (naturally produced and cloned antimicrobials) could act simultaneously to inhibit SRB. Example 5: inhibition of SRB colonization and corrosion in mild stainless steel by bacteria secreting antibacterial agents. This example demonstrates the inhibition of SRB colonization and anaerobic corrosion in biological films in mild steel and in stainless steel by the use of bacteria secreting antimicrobial agents. The commonly used antibiotic, ampicillin, was used as a reference antimicrobial in this study to show that the addition of an antimicrobial agent prior to the colonization of SRB may be a viable approach to reduce SRB-induced corrosion. As shown in the previous example, the 10-amino acid cyclic peptide, gramicidin S, inhibits SRB and was also added externally as an antibiotic peptide model to demonstrate the feasibility of producing antimicrobial peptides in biological films to inhibit corrosion of the mild steel and stainless steel. What is more, a strain of Bacill us brevis overproducing gramicidin S (Azuma, et al., 1992) was used to establish the biological film that secretes gramicidin S and inhibit SRB in stainless steel. Materials and methods Bacterial strains, medium and creeping conditions All aerobic bacteria were cultured from a single colony in lOmL of modified Baar medium (medium 1249 ATCC) at 30 ° C and 250 rpm (25 series agitator, New Brunswick Scientific, Edison , NJ) and was used as the inoculum for the development of the biological film. D. vulgaris was cultured in 15mL screw cap tubes containing 10mL of modified Baar medium supplemented with 100μL each of the oxygen collector in 4% sodium sulfide and Oxyrase (Oxyrase Inc., Mansfeld, OH). Initial cultures were developed from glycerol extract at -85 ° C; all subsequent cultures were developed with a 3% inoculum of the initial culture at 30 ° C without shaking. D. vulgaris was routinely cultivated in tightly closed screw cap tubes and exposed to oxygen from the air without any difficulty in its cultivation, as reported by Angelí and White (1995, supra). The cultures of D. vulgaris were also developed periodically in the presence of ferrous ammonium sulphate at 0.1% and the presence of sulfate-reducing agents was confirmed by the detection of black iron sulfide in the culture tubes. The desulfoviridine test (Postgate, 1984) was also carried out routinely with the detection of a pink color under ultraviolet light confirming the presence of D. vulgaris Gramicidin S was obtained from Sigma Chemical Company (St. Louis, MO), chloramphenicol from Fisher Scientific (Pittsburgh, PA) and ammonium molybdate, from Aldrich Chemical Company (St. Louis, MO). Preparation of the piece of metal. SAE 1018 mild steel parts for the batch culture experiments (25.5 mm diameter and 1.2 mm thickness) and SAE 1018 mild steel plates and stainless steel were cut from common leaf for continuous culture experiments and prepared as previously reported (Jayaraman, et al., 1997a). Corrosion experiments for batch crops. Corrosion experiments for batch cultures were carried out in Erlenmeyer flasks of 250 mL at 30 ° C without shaking as previously described (Jayaraman, et al., 1997a). The pieces of mild steel (triplicate) exposed to D. vulgaris were cleaned by sweeping the surface with 0.01% chromic acid followed by repeated washing of warm water; all other parts were cleaned as previously described (Jayaraman et al., 1997a). The loss of specific mass (in mg / cm2 for the total surface area of the piece, 11.18 cm2) was used as an indicator of the extent of corrosion, which was assumed to be uniform. The growth medium was filled every 7 days and replaced (with appropriate antibiotics) with mild addition along the walls of the flasks. A 3% inoculum (vol / vol) of SRB was added to the flasks after three days of aerobic biological film development. Corrosion experiments with continuous cultures using EIS. A continuous reagent was used to develop the biological film on metal surfaces as previously described (Jayaraman, et al., 1997c). Electrochemical impedance spectroscopy (EIS) was used to obtain the impedance data in at least one duplicate of the experiments using an electrochemical measurement unit Solarton-Schlumberger (SI 1280, Schlumberger Technical Instruments Division, San Jose, CA) in interface with a Macintosh computer (PowerMac 7100/80, Apple Computers, Cupertino, CA) running EISIS electrochemical experimentation software (University of California, Irvine) (THALES, a similar commercial package can also be used). The open circuit potential (OCP) was measured as the potential between the metal specimen and the reference electrode (Ag / AgCl) and the polarization resistance was determined as the limit of the impedance using the package software [software] ANALEIS developed by Mansfeld et al. (ASTM Special Technical Protocol 1154: 186 (1992)). The continuous crop corrosion rates were estimated from the experimental polarization resistance Rp based on the Ster-Gray equation Rp = B / Icorr, where B is a parameter that depends on the gradual attenuations of Tafel and iCorr is the current density of corrosion which can be converted to corrosion rate using Faraday's law (Mansfeld, F., The polarization resistance technique for measuring corrosion currents, In Fontana, MG, Staehele, RW (ed.), Advances in Corrosion Science and Technology, Plenum Press, New York (1976)). An inoculum of SRB at 3% (vol / vol) culture of 24-48 hours of age was added to the reagent after 3 to 5 days of aerobic biological film development. Based on the minimum inhibition concentrations available in the literature (Saleh et al., 1964 and also in the data generated in this laboratory about the susceptibility of SRB cultures in suspension to several inorganic and antimicrobial, ampicillin (200μg / mL) , chloramphenicol (200μg / mL), ampicillin (200μg / mL), and chloramphenicol (100μg / mL), and ampicillin (200μg / mL) and ammonium molybdate (200μg / mL), were added to the reagents (before or after that SRBs had colonized the metal) in an attempt to inhibit SRB All antimicrobials were added simultaneously to the nutrient feeder and to the reagent at appropriate concentrations Enumeration of viable SRBs in the biological films Aerobic biological films were developed on stainless steel pieces (25.5 mm in diameter, 1.2 mm thick) in 250 mL Erlenmeyer flasks for two days in Baar modified medium at 30 ° C. 1.0% (vol / vol) ass of D. vulgaris (O.D60o = 0.16-0.18) was added and allowed to colonize the biological film for four additional days. The metal pieces were carefully removed from the flasks and rinsed twice by immersion in distilled water to remove the weakly added cells. The biological film was then scraped with a sterile spatula and resuspended in 500μL of modified Baar medium. Aerobic bacteria were determined by plaque counting and viable SRBs were enumerated by the MPN test for three tubes (Greenberg, supra). RESULTS Continuous and batch corrosion with P. fragi K and D. trulc aris not producing antimicrobials in mild steel SAE 1018. The mass loss for SAE 1018 mild steel pieces was examined for 28 days in Baar modified medium in the presence of of P. fragi K and D. vulgaris in stationary cultures in batches at 30 ° C. In the cases in which D. fragi was present in the biological film, the pieces were covered with a thick black deposit and it was difficult to clean it. A dual culture of P. fragi K and D. vulgaris produced a 1.8-fold increase in the rate of corrosion after 21 days of exposure, compared with a monoculture of P. fragi K; however, the corrosion rate observed in both cases was always lower than that observed in a modified sterile Baar medium (Table V). The corrosion rate observed for a monoculture of D. vulgaris in steel SAE 1018 was much higher than in a sterile medium after 14 days (1.4 times) and 21 days (2.5 times, extrapolated from Table V). When ampicillin (100 μg / mL) was added to the flasks before allowing colonization of the metal piece by D. vulgaris, the mass loss observed was 40% (1 week) to 14% (3 weeks) less that observed when ampicillin was added after the SRB (Table V). The macroscopic examination of the metal pieces exposed to D. vulgaris revealed the presence of numerous gaps for all these experiments. The anaerobic D. vulgaris grew in continuous reagents as a monoculture with an air flow rate of 200 mL / min to the head space as indicated by the development of a black precipitate of iron sulfide and hydrogen sulfide odor of the reagent landfill. The growth of D. vulgaris in continuous reagents increased the Rp by 90 times after 72 hours, compared with the sterile controls. The addition of 200μg / mL of ampicillin after 240 hours of SRB growth did not change the Rp and the reagent remained black with the distinctive sulfur odor from the exhaust (Table VI). The combination of 200 μg / mL of ampicillin and 200 μg / mL of ammonium molybdate after 320 hours clarified the supernatant of the reagent; however, the sulfur odor was still detected, indicating that the corrosion rate did not decrease and SRB growth was not inhibited. The addition of D. vulgaris to a continuous reagent of P. fragi K decreased the Rp of the mild steel by 3 times after 36 hours and modified the frequency dependence of the phase angle; the reagent turned black and the sulfur odor was detected from the reagent spillway (Table VI and Figure 6). Before the addition of D. vulgaris, the impedance tended to a stable asymptotic value at low frequency (4.52 x 104 Ohms »cm2); however, within 24 hours of the addition of SRB, the reagent turned black, sulfur odor was detected and the impedance did not return to an asymptotic value at the lowest frequency (1.4 x 10 ~ 3 Hz). The addition of 200μg / mL of ampicillin (Table VI) and the combination of 100μg / mL of ampicillin and 25μg / mL of chloramphenicol after 120 and 150 hours of SRB growth (data not shown) also did not change Rp to its previous value before the addition of SRB, indicating that there was no inhibition of SRB.

Table V Corrosion Loss for SAE 1018 steel in batch cultures with dual cultures of aerobic bacteria and representative SRB * Corrosion loss, mg / cm Antimicrobial 3 days 14 days 21 days 7 days 10 days 28 days 32 days Bacterial strain (s) Produced sterile medium 1.03 ± 0.04 2.05 ± 0.11 0.54 ± 0.08 0.77 ± 0.11 P. fragi K None 0.33 ± 0.05 0.04 ± 0.01 0.43 0.04 ± 0.01 - 0.19 ± 0.05 P. fragi K + SRB None 0.52 ± 0.08 0.04 ± 0.08 0.86 ± 0.17 0.04 ± 0.01 - 0.35 ± 0.01 10 P. fragi K + SRB + Amp 100 None 0.42 ± 0.04 0.04 ± 0.08 0.65 ± 0.07 0.04 ± 0.01 - 0.35 ± 0.01 P. fragi K + Amp 100 + SRB None 0.33 ± 0.04 0.35 ± 0.07 0.04 ± 0.01 - 0.25 ± 0.04 D. vulgaris ATCC 29579 None 0.095 0.191 1.225 3.83 B. subtilis ATCC 6633 Subtilin 0.13 ± 0.01 0-45 ± 0.08 0.57 ± 0.08 B. subtilis ATCC 6633 + SRB Subtilin 15 0.13 ± 0.01 0.52 ± 0.03 0.81 ± 0.04 B. brevis ATCC 35690 Edeins 0.07 ± 0.01 0.16 ± 0.03 0.19 ± 0.01 B. brevis ATCC 35690 + SRB Edeinas 0.28 ± 0.03 0.07 ± 0.01 0.23 ± 0.04 B. brevis 18 Gramicidin S 0.28 ± 0.06 0.40 ± 0.06 0.09 ± 0.01 0.16 ± 0.02 B. brevis 18 + SRB Gramicidin S 0.30 ± 0.07 0.44 ± 0.06 0.19 ± 0.02 twenty 13 * The order in which the entries are listed in the first column indicate the order in which they were added to the crops. For example, "P. fragi K + SRB + Amp 100" indicates that P. fragi K bacteria were added to the culture and allowed to establish as a biological film; SRB were added and allowed to settle into the biological film and ampicillin was then added to the culture medium.

Table VI. Behavior of corrosion of SAE 1018 steel in continuous reagents with dual aerobic and SRB cultures after several methods to kill SRB. * Time elapsed Antimicrobial or later aggregate to kill addition Rp (Ohm * Rp (Ohm * Rp (0hm * SRB experiment μg / mL of anti-Characteristic cm2) cm2) cm2) spectro microbial ace of after then after EIS, hr Reagent D. vulgaris Ampicillin 200 The Reagent 3.58 x 3.43 No (1 of 2 (200 μg) became 103 105: i) show experiments! added black and (0) (10) after landfill 240 hours of had sulfur growth odor. No SRB changes were observed after the addition of the antimicrobian P. fragi K + Ampicillin 200 The Reagent 4.52 x 1.35 x 3.76 x Drawing SRB + (200 μg) became 104 10? 5-10 'ampicillin added black and the (2) (7! (14) (1 of 2 after landfill experiments) 120 hours of had odor of p (g ni ni ni ni ni i sul sul. No No No No No No No No No No No No No No No No No No SR No No No No No No No No No No SR No No No No No No No No No No No No No.

P. fragi K + Ampicillin 100 The reagent 4.52 x 2.60 x 2.00 x Drawing Ampicillin + (100 μg) did not become 104 104 104 SRB added black and the (2) (9) (10) (1 of 3 before landfill no experiments) addition of smelt to SRB sulfide P. fragi K + Gramicidin S Reagent 4.52 x 3.89 x Note 1 Drawing Gramicidin S (100 μg) did not become 104 104 + SRB added black and (2) (6) 16 (1 of 1 before landfill experiment) addition of had SRB sulfur smell B. brevis 18 Gramicidin S 48 Reagent 3.43 x 5.78 x 2.34 x Drawing + SRB (produced in did not become 104 104 104 (1 of 2 situ before black and the (3) (8) (14) experiments) of addition of Weir SRB had sulfur odor B. brevis Edeinas 250 Reagent 3.75 x No No No 35690 [produced in did not become 104 calculated compud shown (1 of 2 situ before black and the (3) o or experiments) addition of landfill SRB had sulfur odor * The order in which the entries are listed in the first column indicate the order in which they were added to the crops. For example, "P. fragi K + SRB + Amp 100" indicates that P. fragi K bacteria were added to the culture and allowed to establish as a biological film; SRB were added and allowed to be established in the biological film the ampicillin was then added to the culture medium. Note 1. It is not possible to calculate Rp of the impedance spectra based on available equivalent circuit models.

Rates of continuous corrosion with P. fragi K not producing antimicrobials and D. vulgaris in stainless steel S.S. 304. No differences were observed between the impedance spectra for the sterile Baar medium with P. fragi K in 304 stainless steel after 900 hours of exposure. D. vulgaris did not grow as a monoculture in 304 stainless steel and the addition of D. vulgaris to a P. fragi K reagent changed the frequency dependence of the impedance at low frequencies within 48 hours (Figure 7). The phase angle showed a minimum value with the addition of SRB indicating the emergence of a new time constant at very low frequencies (Figure 7) and the maximum value for the phase angle decreased from 81 ° to 69 °. Changes in the impedance spectra were accompanied by the detection of sulfur odor from the reagent landfill and the reagent also turned gray. The addition of 200μg / mL of ampicillin (Figure 7), both of ampicillin 200μg / mL and of chloramphenicol 100μg / mL (Table VII, second column), or 200μg / mL of ampicillin and 200μg / mL of ammonium molybdate (data not shown) to a reagent with a dual culture did not modify the. impedance spectra for the behavior of the simple constant of a time observed before the addition of D. vulgaris (Figure 7) or stopped the production of hydrogen sulphide and iron sulfide, indicating that the SRB had not been annihilated.

Table VII Corrosion behavior for stainless steel 304 in continuous reagents with dual aerobic cultures and after several methods to kill SRB * μ__ Experiment Antimicrobial Time Characteristics EIS spectra aggregated for elapsed Reactive kill SRB, μg / mL after addition of the antimicrobial, hr P. fragi K + SRB Ampicillin (200 490 Reagent was Drawing 7 + Ampicillin (1 μg) after turning gray and the 3 170 hrs and weir had experiments) chloramphenicol sulfur odor (lOOμg) after the 15 additions addition of SRB. after 400 No hours of changes were observed after growth of the addition of the antimicrobial SRB P. fragi K + Ampicillin (100 120 Reagent never Drawing 7 Ampicillin + SRB μg) added turned gray and (1 of 3 before adding the landfill no experiments) of SRB had sulfur odor after the addition of SRB 10 P. fragi K + Gramicidin S 130 Reagent never Drawing Gramicidin + SRB (100 μg) turned gray and (1 of 1 added before landfill no experiments) addition of SRB had 15 sulfur odor after addition of SRB B. brevis + D. Gramicidin S 150 The reagent never drawn vulgaris (1 of 3 (produced in turned gray and experiments) in situ before the spill addition of SRB) had a slight sulfur odor after the addition of SRB B. brevis 18 + Gramicidin S 190 Reagent not shown (Nagano) + D. (produced in turned gray and the vulgaris in situ before landfill had (1 of 2 addition of SRB) sulfur odor experiments) after the addition of SRB * The order in which the entries are listed in the first column indicate the order in which they were added to the crops. For example, "P. fragi K + SRB + Amp" indicates that P. fragi K bacteria were added to the culture and allowed to establish as a biological film; SRB were added and allowed to settle into the biological film and ampicillin was then added to the culture medium. fifteen Rates of continuous corrosion with the biological film exposed to purified antimicrobials, SRB inhibitors, before the addition of D. vulgaris. To determine whether antimicrobials were effective in inhibiting SRB when they were added before SRB colonization, biological films of P. fragi K non-antimicrobial producers on mild steel SAE 1018 and stainless steel 304 were exposed to 100 μg / mL ampicillin or gramicidin S for 24 hours before D. vulgaris was added. P. fragi K grew to saturation in overnight suspension cultures, exposed to lOOμg / mL of both antimicrobials; from there it is seen that they were not affected by the addition of these antimicrobials. When D. vulgaris was added to mild steel and stainless steel reagents after the addition of ampicillin, the impedance and Rp spectra did not change for up to 100 hours (Drawings 6 and 7, Tables VI and VII). No sulfur odor was detected in the reagent landfill; then, D. vulgaris was completely inhibited in the reagents by this antimicrobial. The external addition of the cyclic decapeptide antimicrobial gramicidin S at 100 μg / mL was also completely effective in inhibiting the growth of D. vulgaris in the 304 stainless steel experiments, as evidenced by the capacitive nature of the impedance spectra (Drawing 7 and Table VI); however, with the mild steel, the reagent turned gray although there was no increase in Rp after 80 hours of exposure to the SRBs (Drawing 8 and Table VII). Hence, once the emergence of corrosion induced by D. vulgaris in mild steel was retarded compared to P. fragi K and D. vulgaris without gramicidin S present. Rates of continuous and batch corrosion with Bacilli producer of antimicrobials and D. vulgaris on mild steel SAE 1018 and stainless steel 304. Batch corrosion studies on steel pieces SAE 1018 with D. vulgaris and biological films of Bacillus producers of antimicrobials (based on the recorded production of antimicrobial peptides, Table V), showed that all Bacilli were able to restrict the colonization of D. vulgaris for up to 1 week (evidenced by small increases of 1.2 to 1.4 times in corrosion rates, compared to the large 1.8-fold increases for P. fragi K in Baar's modified medium, Table V, as well as the lack of appearance of a black color and sulfur odor). When the medium was filled after 7 days, however, in all the flasks except those containing B. brevis 18 they turned black, with hydrogen sulphide being detected within 24 hours. The increase in the rate of corrosion with D. vulgaris in the presence of Bacilli (1.2 to 1.5 times increase) was similar to that observed with P. fragi K and D. vulgaris (1.6-fold increase). The mass loss observed with B. brevis 18 and SRB was comparable to that of P. fragi alone and about two times better than with P. fragi K and SRB (Table V). No sulfur odor was detected during the whole experiment. The effectiveness of gramicidin S in inhibiting the growth of SRB in batch cultures was also corroborated by the decrease in third-order magnitude in the viable SRBs detected (by the MPN test for three tubes) in a biological film of B. brevis 18 on stainless steel 304 after four days of growth, compared to the biological film of P. fragi not producing antimicrobials (c.f., 5.47 x 102 / mL against 8.47 x 105 / mL). Corrosion rates in continuous crops with B. brevis 18, a hyperproductive strain of gramicidin S (Azuma, et al., 1992) were obtained in the presence of D. vulgaris on mild steel SAE 1018 (Drawing 9 and Table VI); the increase in Rp when observed after the addition of D. vulgaris to P. fragi K on mild steel was delayed by 24 hours. Sometimes, it appears that the SRBs colonized the biological film as evidenced by the smell of hydrogen sulphide from the reagent's landfill; however, the Rp remained constant at 5.78 x 104 ohms "cm2 as opposed to 3.43 x 104 ohms" cpi before the addition of SRB.

Figure 8 and Table VII show that the addition of D. vulgaris to a biological film of B. brevis 18 in stainless steel type 304 did not decrease to Rp after 120 hours, even though the sulfur odor was detected in the reagent landfill 48 hours after the addition of D. vulgaris. Therefore, the gramicidin S that produces B. brevis 18 was able to inhibit the colonization of SRB in 304 stainless steel, while it was only able to slow the growth of SRB in mild steel SAE 1018. DISCUSSION D. vulgaris was selected as the representative sulfate reducing bacteria to study the effectiveness of antimicrobials produced in situ to inhibit anaerobic corrosion, since it has been reported to accelerate corrosion (Gaylarde, 1992) and strains of this species have the ability to withstand oxygen tension (Hardy, JA, Hamilton, WA, Curr. Microbiol .6: 259-262 (1981)). D. vulgaris showed a remarkable resilience in growing as a monoculture in stationary batch crops and continuous reagents with an oxygen saturated head space and corroded metal pieces, as evidenced by the black discoloration of the medium (Gaylarde, 1992, supra). D. vulgaris was also able to decrease within an aerobic biological film in shake flasks under oxygen saturation conditions at the upper end above the liquid as well as in the continuous reagents with an air flow rate of 200 mL / min at the upper end. Growth conditions for D. vulgaris in this study were very similar to those of Gaylarde (1992, supra) as well as those of Hamilton and Lee (1995) (Biocorrosion, pp. 243-264, in Barton, LL (ed.) , Sulphate-reducing Bacteria, Plenum Press, New York) and has been defined as more aggressive the smaller the amount of oxygen that is present in an SRB culture, which leads to maximum corrosion rates. The mild steel pieces exposed to batch crops with P. fragi K and D. vulgaris showed an increase in corrosion rates compared to exposure to P. fragi K monocultures, which is similar to that reported by Jack et al. (Corr. Sci. 33: 1843-1853 (1992)) and Gaylarde (1992, supra). The addition of various combinations of antibiotics to the batch cultures to inhibit the growth of D. vulgaris did not prove successful in inhibiting corrosion (Table V). However, batch cultures of Bacilli producers of antimicrobials were able to retard the emergence of SRB-induced corrosion compared to a control monoculture of up to 7 days. This inhibitory effect of SRB decreased considerably with most Bacillus after the growth medium was filled. Since most antimicrobials are secondary metabolites (Bailey, JE, Ollis, DF, Biochemical Engineering Fundamentals Second ed. McGraw-Hill Publishing Company, New York (1986)) and occur during the stationary phase of growth (Doi, RH , McGlougin, M. 1992. Biology of Bacilli, Application to Industry, Butterworth-Heinemann, Boston, MA (1992)), the filling of the growth medium after 7 days may have removed most of the antimicrobials present in the film. biological, allowing D. vulgaris to colonize the surface of the metal before antimicrobial inhibition levels were produced again. However, B. brevis 18 completely inhibited the growth of SRB for up to 28 days due to the overproduction of gramicidin S as a result of the mutagenesis used to make this strain (Azuma, et al., 1992). This result points out the potential of gramicidin S to annihilate SRB and demonstrated not only that antimicrobials could be introduced successfully before the colonization of SRB via other bacteria in the biological film, but also that antimicrobial compounds introduced in this way would inhibit successful way the growth of SRB. The impedance spectra of mild steel and stainless steel were used to characterize the corrosion behavior observed in continuous cultures with these metals. The addition of D. vulgaris to P. fragi K in SAE 1018 mild steel reagents increased Rp, which indicates that the corrosion rate was decreased. This seemingly contradictory observation could be explained due to the formation of an oxide layer on the surface of the metal; since Baar's modified medium has a pH of 7.5 (neutral), the formed rust layer does not dissolve as would be expected in a more acidic environment. This rise in rust could cause an increase in Rp and an apparent decrease in the corrosion rate. The validity of the conclusions from EIS with respect to the inhibition of SRB can be verified by the correct correlation between the values of Rp calculated for mass loss experiments of batch crops and for those obtained with EIS for mild steel (Table VII). A single time constant (OTC, for the acronym in English for "one-time constant") was observed with P. fragi K and P. fragi K + ampicillin + SRB (Figure 6) in mild steel, which is characterized by uniform corrosion in neutral media (Mansfeld, F., Lorenz, WJ, Electrochemical impedance spectroscopy (EIS): Application in corrosion science and technology, in Varma, R., Selman, JR (ed.), Techniques for characterization of electrodes and electrochemical processes, John Wiley &Sons, New York (1991)) (hereinafter: Mansfeld and Lorenz, 1991), and the values for Rp and capacitance (C) obtained from these two experiments were similar (Table VII) . For P. fragi K + SRB in mild steel, the dependence of the frequency of the phase angle f to the lower frequencies, suggests the appearance of a new time constant, which could be due to pitting, while the symmetric dependence of the frequency of f for P. fragi K + SRB + ampicillin and the step of the whole curve of the impedance compared to that of P. fragi K could be due to a higher Rp (Dibujod). Similar capacitance values were indicated in the spectra for P. fragi K + SRB and for P. fragi K + SRB + ampicillin in mild steel; however, it was not possible to accommodate these spectra to a simple equivalent circuit and obtain quantitative values of Rp and C. The impedance spectra for B. brevis 18, B. brevis 18 + SRB and for P. fragi K + gramicidin S + SRB in mild steel exhibited the frequency dependence commonly observed for uniform corrosion and Rp could be determined as the limit (f = 0 °) of the absolute value of the impedance | Z | (Drawing 9). The stainless steel samples exposed to the reagents with sterile medium also did not reach stable values for the impedance at low frequencies. The lack of difference in the impedance spectra between the sterile controls and the reagents in stainless steel 304 with P. fragi K or B. brevis indicate that very little corrosion occurred during the period. This result is similar to the observations of Hernández et al. , 1994, who did not see a stable value for impedance at low frequencies with mild steel in solution of nine salts for 20 days, attributing this to lack of corrosion. The spectra for stainless steel 304 for P. fragí K and for P. fragi K + ampicillin + SRB were capacitive with high values of Rp, close to 2 x 10 'Ohm * cm2 and capacitance values between 100 and 200 μF / cm2; this indicates characteristic uniform corrosion of stainless steel in neutral media (Mansfeld and Lorenz, 1991). A deviation of the pure capacitive behavior similar to that of mild steel was observed with P. fragi K + SRB and for P. fragi K + SRB + ampicillin in stainless steel 304 (Drawing 7) and it was not possible to accommodate this impedance data to a simple EC. A new time constant was observed for P. fragi K + SRB, as indicated by the minimum of f near 0.01 Hz (Figure 7) the impedance spectra for B. brevis 18, B. brevis + SRB and for P. fragi K + gramicidin S + SRB were all capacitive and the values for Rp and C observed with B. brevis 18 and B. brevis 18 + SRB were similar to those observed with P. fragi K (Figure 8). While the extent of changes in corrosion rates for all exposure conditions can not be determined carefully without accommodating the experimental data to adequate equivalent circuits, one can conclude that the increased rates of corrosion due to the production of hydrogen sulfide when adding SRB. Similarly, changes in the phase angles of stainless steel 304 after the addition of SRB (Figure 7b) indicate the appearance of additional electrochemical processes and suggest localized corrosion (Mansfeld and Lorenz, 1991). Hence, the absence of changes in the impedance spectra when purified antimicrobials were present before the addition of SRB or when the gramicidin S was generated by the biological film demonstrates the inhibition of SRB in stainless steel (Drawing 8). Ampicillin and chloramphenicol are known to inhibit D. vulgaris suspension cultures at 1 μg / mL and 3 μg / mL, respectively (Odom and Singleton, 1993). Since biological films are known to be 10 to 1000 times more resistant to biocides (Cheung and Beech, 1996), up to 200 μg / mL of both antibiotics were used in this study. However, when they were added after the SRBs had colonized the surface of the metal, these additives did not stop the subsequent production of sulfur or decreased corrosion rates for each type of steel. This is consistent with the observations of Franklin et al. (1991) (Corrosion 47: 128-134), who observed that SRBs may be able to survive exposure to halogen biocides in a span of at least 26 hours and with those of Franklin et al. (1989). (An analogous MIC system with specific bacterial consortia, to test the effectiveness of the selection of materials and countermeasures, Presented at Corrosion 89, New Orleans, LA, National Association of Corrosion Engineers, Houston, TX), who reported decreases of the 3rd and 4th order of magnitude in a population of a biological film with the addition of biocides, but noted that the population reached its pre-treatment density within 24 hours of stopping the dose of biocide. A similar observation was made with mild steel reagents in this study; when the medium containing ampicillin was discontinued, the outbreak of SRB-induced corrosion was evident within a period of 36 hours. Example 6: Use of the method for inoculating a water cooling tower against SRB-related corrosion This example demonstrates the use of the invention in "inoculating" a water cooling tower against SRB-related corrosion. A new water cooling tower is installed in a power plant. When the tower is ready to go into operation, a culture of acillus polymyxa modified by recombination to secrete bactenecina at a concentration of between 1-10 μg / mL is added to the water supply at approximate concentrations of between 103 to 106 cells / mL, preferably in dilution, of a substance not expensive complex nutrients, such as Luria Bertani, which is circulated through the water tower. Before the water used to "inoculate" the tower is dry, the normal service of the tower is connected and the normal operation of the tower begins.

Example 7: Using the method to protect an existing structure This example demonstrates the use of the method to treat a pipeline that is already in operation. A scraping is performed on a wet portion of the pipe surface and the bacteria in the biological film of the pipe are cultivated. One or more of the Gram-positive cultured bacterial species (preferred because said bacteria have only a single cell membrane, through which they secrete antimicrobial or anticorrosive agents, or both) are selected, based on criteria such as ease and reliability of genetic transformation and cultivation. The selected Gram-positive bacteria are then transformed by conjugation with a chromosomal insertion vector, such as a pCNB5, which encodes the gene for bactenecin. The transformed bacteria are cultured and then tested to confirm the secretion of the anti-SRB agent. The cultures that give positive results for the secretion of the anti-SRB agent are then cultured in quantity and aliquots of the resulting culture are introduced into the pipe at intervals, with resulting concentrations of between 103 to 106 cells / ml. All publications and patent applications cited in this specification are hereby incorporated by reference in their entirety, as if each separate publication or patent application was specifically and individually indicated to be incorporated by reference. Although the disclosed invention has been described in some details as an illustration and example for the purposes of clarity of understanding, it will appear without difficulty for someone with the usual skills in the medium and in light of the teachings that this invention, although with Certain changes and modifications that may occur, this does not depart from the essence and scope of the claims that are attached.

Claims

CLAIMS 1. A method to inhibit the growth of sulfate-reducing bacteria in a material selected from a group of materials sensitive to corrosion and from materials sensitive to degradation; said method includes the application to the material of a bacterium that secretes a chemical compound in an amount sufficient to inhibit the growth of sulfate-reducing bacteria in the material.
2. The method of claim 1, wherein the corrosion sensitive material is a metal.
3. The method of claim 2, wherein the metal is steel.
4. The method of claim 3, wherein the metal is mild steel.
The method of claim 3, wherein the metal is a stainless steel.
6. The method of claim 2, wherein the metal is aluminum or an aluminum alloy.
The method of claim 2, wherein the metal is copper or a copper alloy.
The method of claim 2, wherein the metal is selected from a group consisting of titanium, nickel, a titanium alloy and a nickel alloy.
The method of claim 1, wherein the material sensitive to degradation is concrete.
The method of claim 1, wherein the material sensitive to degradation is reinforced concrete.
The method of claim 1, wherein the material sensitive to degradation is cement.
12. The method of claim 1, wherein the bacterium is an aerobic.
The method of claim 1, wherein the bacterium is of the genus Pseudomonas.
The method of claim 1, wherein the bacterium is of the genus Bacillus.
The method of claim 1, wherein the chemical compound is one not secreted by the uncultivated members of the bacterial species applied to the material.
16. The method of claim 1, wherein the bacterium has been altered recombinantly to secrete the chemical at higher levels than the uncultivated members of its species do.
17. The method of claim 1, wherein the chemical compound is an antibiotic.
18. The method of claim 16, wherein the antibiotic is selected from the group of gramicidin S, indolicidin, polymyxin and bactenecin.
19. The method of claim 1, wherein the chemical compound is polyaspartate.
20. The method of claim 1, wherein the chemical compound is polyglutamate.
21. The method of claim 1, wherein the chemical compound is polyglycine.
22. The method of claim 1, wherein the chemical compound is siderophore? 23.
A system for inhibiting corrosion of a material selected from a group of corrosion sensitive material and a material sensitive to degradation, including a material with a biological film on its surface, said biological film including bacteria that secrete a compound chemical in an amount sufficient to inhibit the growth of sulfate-reducing bacteria in the material.
The system of claim 23, wherein the corrosion sensitive material is a metal.
25. The system of claim 24, wherein the metal is steel.
26. The system of claim 25, wherein the steel is mild steel.
27. The system of claim 25, wherein the metal is a stainless steel.
28. The system of claim 24, wherein the metal is aluminum or an aluminum alloy.
29. The system of claim 24, wherein the metal is copper or a copper alloy.
30. The system of claim 24, wherein the metal is selected from a group consisting of titanium, nickel, a titanium alloy and a nickel alloy.
31. The system of claim 23, wherein the material sensitive to degradation is concrete.
32. The system of claim 23, wherein the material sensitive to degradation is reinforced concrete.
33. The system of claim 23, in which the material sensitive to degradation is cement.
34. The system of claim 23, wherein the bacterium is of the genus Bacillus.
35. The system of claim 23, wherein the bacterium is of the genus Pseudomonas.
36. The system of claim 23, wherein the chemical compound is one not secreted by the uncultivated members of the bacterial species applied to the material.
37. The method of claim 23, wherein the bacterium has been altered recombinantly to secrete the chemical at higher levels than the uncultivated members of its species do.
38. The system of claim 23, wherein the chemical compound is an antibiotic.
39. The system of claim 38, wherein the antibiotic is selected from the group of gramicidin S, indolicidin, polymyxin and bactenecin.
40. The system of claim 23, wherein the chemical compound is selected from the group polyaspartate, polyglutamate, polyglycine and siderophors.