DE19646324A1 - Use of secondary metabolites of freshwater and marine organisms - Google Patents

Abstract

Use of secondary metabolites of freshwater and marine organisms, as natural antifouling agents, is new.

Description

The invention called "Use of biogenic agents from the Cyanobacterium Scytonema hofmanni and the common starfish Asterias rubens as natural antifouling agents "relates to the use of the above-mentioned agents as antifouling agents. Active ingredients to protect surfaces against fouling by fouling organisms.

Underwater structures such as B. ship hulls, sea mark, port and off-shore facilities for economic and ecological reasons against fouling, which results from a complex layer of fouling organisms such as bacteria, microalgae, macroalgae, Barnacles and mussels are protected. Especially in the case of ships due to the overgrowth of the ship's hull the frictional resistance in the water, resulting in a greater fuel consumption, increased susceptibility to corrosion and increased CO₂ emissions leads. The docking intervals are shortened; increase the maintenance and maintenance costs themselves.

The most effective and effective method in this field, which is predominantly used worldwide therefore in a coating of the corresponding surfaces with highly toxic Antifouling paints. The antifouling paint usually consists of a binder, one or more highly toxic biocides and pigments.

As a binder z. B. rosin, fatty acid derivatives, poly (meth) acrylates, polyesters, Polyurethanes, epoxy compounds, chlorinated rubber, resins or other film-forming systems used. Within the biocides, due to their biodiversity, are considered vegetation organisms in question animal, plant and microorganism groups broadband poisons (mainly heavy metals and, more recently, copper (I) salts such as Copper (I) oxide or organic tin compounds such as. B. tributyltin oxide) is used. As Pigments are either poorly water-soluble pigments such as B. zinc oxide or water insoluble pigments such as B. titanium dioxide or iron oxide. The water-insoluble Due to their properties, pigments delay the rapid dissolution of the Color system.

The development of antifouling paints led to the following different ones Types (Holzapfel & Rother 1988):

• 1. Antifoulings with soluble matrix (binder: rosin, fatty acid derivatives):
  Simultaneous removal of biocide and binder, growth protection through continuous release of biocide up to the limit of the life of the layer, relatively short life of the layer, soft antifouling layer, increase in roughness.
• 2. Antifoulings with insoluble matrix (binder: chlorinated rubber, vinyl resins):
  Biocide diffuses out of the binder matrix, no uniform biocide release due to different concentration gradients, used antifouling layer must be removed, roughness increase.
• 3. Self-polishing antifoulings (binder: tin-containing acrylate copolymers):
  Biocide is released continuously by hydrolysis of the binder, the remaining polymer matrix is water-soluble and is "polished off".

The use of biocides in the first two types is in high concentration required in self-polishing antifoulings in a significantly lower concentration expedient.

The above Broadband poisons, however, in addition to preventing the settlement of Fouling organisms also cause serious damage to "non-target organisms" (e.g. immune weak and reduced reproductive capacity in fish, sterility in snails and Mussels) and pollute our waters considerably. Thus, the necessary alternative methods to combat fouling.

The extraordinarily diverse attempts to find alternative strategies for using copper and To develop tin-free antifoulings have become a variety of different ones physical / mechanical, physical / chemical and biological / biochemical approaches prevention of fouling, which are not yet fully usable in practice (Von Oertzen et al. 1989, Abarzua & Jakubowski 1995).

Within the biological / biochemical methods to combat fouling, the Substitution of the toxic biocides used for antifouling paints by biogenic, not Toxic active ingredients the most promising and environmentally friendly method of Prevention of fouling (Abarzua & Jakubowski 1995).

The invention is based, biogenic active ingredients from freshwater and the task Isolate marine organisms and embed them in certain surface coatings, to prevent colonization by vegetation organisms without toxic effects to evoke. This object is achieved in that the biogenic active substances benthic cyanobacteria (blue-green algae) and marine invertebrates (invertebrates) isolated which are known to produce a number of secondary metabolites which can protect them against vegetation pressure. As a result of the screening of 30 benthic Cyanobacteria and marine invertebrate species crystallized the benthic  Cyanobacterium Scytonema hofmanni and the marine common starfish Asterias rubens as effective producers of specific active ingredients to protect against vegetation pressure. The diatom Nitzschia pusilla is used as a model organism in the laboratory.

After concentration and purification of the extracts by chromatography Secondary metabolites are isolated for the structures described so far in the literature to be proposed:

According to the invention, organic solvents such as ether, hexane, benzene, ethanol, Chloroform, xylene or naphtha suitable solvents for cyanobacterin; Butyl acetate and propyl acetate for saponins.

The isolation (extraction) of the specific secondary metabolites from the benthic Cyanobacterium Scytonema hofmanni and the common starfish Asterias rubens and their biological testing for the diatom Nitzschia pusilla (agar diffusion test, adhesion test, Toxicity test) will be explained using the following examples.

example 1 Cultivation of Scytonema hofmanni

Scytonema hofmanni Ag. UTEX B 1581 is obtained from the Austin Collection of the University of Texas (USA). S. hofmanni is in a cyanobacterial medium according to Jüttner et al. (1983) (pH = 7.7) at 25 ° C and steady light (15 µE m ^-2 · s ^-1 ) cultivated in batch culture. The ventilation is carried out by an air / CO₂ mixture (0.3 vol .-%). The cyanobacteria producing the biogenic active ingredients are harvested after 21 days of cultivation.

Example 2 Isolation of Rohcyanobacterin (Extraction from S. hofmanni)

The cells of S. hofmanni are filtered, the resulting pellet in small parts cut up. The fresh mass is resuspended in Tris-HCl buffer (pH = 7.5) Homogenized ultrasonic bath, centrifuged and filtered again, then lyophilized and ground. 1 g of lyophilized and ground dry matter from S. hofmanni is mixed with 50 ml TBE, chloroform, benzene or hexane extracted for 30 min in an ultrasonic bath and 2 × Room temperature re-extracted. After centrifugation, the crude extracts are combined and filtered and the remaining raw cyanobacterin solution was evaporated to dryness.

Example 3 Collecting Asterias rubens

Several kilograms of the common starfish Asterias rubens (size 3-15 cm) are in the Baltic Sea (Danish Baltic Sea coast, Little Belt, Fredericia) collected at a depth of about 20 m.

Example 4 Isolation of raw saponin (extraction of A. rubens)

The fresh starfish are cleaned and ethanol (final concentration 80%) is poured over them and left overnight. After filtration through paper filters, the crude extract is reduced to 1/3 the total volume was concentrated in vacuo and then shaken with chloroform. The chloroform phase is separated off and the crude saponins are precipitated out with acetone the concentrated aqueous-ethanolic extract solution to dryness as a compact white Precipitation won. The raw saponins are then cleaned by precipitation interfering substances (macromolecules, salts) from the aqueous raw saponin solution Ethanol. The interfering substances are filtered off as a crystalline precipitate and the  remaining aqueous purified crude saponin solution is dried in vacuo constricted.

Example 5 Cultivation of Nitzschia pusilla

Nitzschia pusilla is a typical type of diatom found in microfouling growth. N pusilla is axenically cultivated in batch culture at 15 ° C. and continuous light (20 μE · m ^-2 · s ^-1 ) (light cooling chamber) in an ASW medium (artificial seawater medium) (Van Baalen 1962) (pH = 7.8) . Since N. pusilla is obtained by sampling from mud flats of the Jade Bay, sub-area Dangast and Vareler outer harbor (North Sea) and then isolated, besides sterile cultivation, continuous treatment with an antibiotic solution (streptomycin: penicillin: amphotericin = 40: 20: 0 , 25; necessary when using 4 mg · ml ^-1 streptomycin) (Round et al. 1990). It is ventilated with an air mixture (0.03 vol .-% CO₂).

Example 6 Agar diffusion test with N. pusilla

The agar diffusion test with the diatom N. pusilla is used to screen various extracts from S. hofmanni and A. rubens for growth inhibition effects. The agar plates are prepared as follows: 10 ml of nutrient medium consisting of ASW medium (Van Baalen 1962) with the addition of 1.5% agar are poured sterile into petri dishes (⌀ 50 mm). After the agar plates have cooled, 0.2 ml of a suspension of N. pusilla from the logarithmic phase (4-5 · 10⁶ cells · ml ^-1 ) (see Example 5) is spatulously spread out on the agar plates. After the algae suspension has dried, a hole (⌀ = 7 mm) is punched out of the agar layer using a flamed cork borer in the center of the agar plate. The raw cyanobacterin from S. hofmanni (see Example 2) obtained by various extraction agents is dissolved in tert-butyl methyl ether (TBE), the raw saponin from A. . rubens in aqua dest. (see Example 4) dissolved in a concentration of 100 mg · ml ^-1 . Appropriate dilutions are made from these solutions to determine the minimum inhibitory concentration. The amount of the applied extract solution per petri dish is 50 µl in all cases. The inhibition of growth is measured from the edge of the hole to the edge of the inhibition zone (in mm) after 8 days of incubation (3 parallels) in the light cooling chamber for diatoms (15 ° C, 20 µE · m ^-2 · s ^-1 ). The inhibition of growth of the corresponding solvent control is subtracted.

Table 1 shows the influence of different concentrations by different Extracted raw cyanobacterins from S. hofmanni on the growth of N. pusilla.  

Table 1

It can be clearly seen from Table 1 that the crude cyanobacterin from S. hofmanni, regardless of the extracting agent used, has a strong growth inhibition on N. pusilla. The growth inhibition increases with increasing concentration, with 0.002 mg · ml ^-1 (= 0.1 µg per agar plate) making up the minimum inhibitory concentration in all cases.

The following table (Table 2) shows the effect of different concentrations of Raw saponins from A. rubens on the growth of N pusilla.

Table 2

The minimum inhibitory concentration for this application is 2 mg · ml ^-1 (= 100 µg per agar plate).

Example 7 Adhesion test with N. pusilla

To test the antifouling activity on a laboratory scale, the adhesion test is carried out with a 1-algae culture of the diatom N. pusilla. After 5-6 days of cultivation, N. pusilla is harvested in a batch process (see Example 5), taken up in 6 l of sterile ASW medium (Van Baalen 1962) and adjusted to a cell number of ∼ 2 · 10⁶ cells · ml ^-1 . This algae suspension is then distributed to sterile Erlenmeyer flasks (200 ml) with a wide neck (5 cm). Preparation of the PVC plates: PVC plates (20 × 40 mm), which are provided with 2 small holes on the sides, are mixed with 2 layers of a mixture of binder + extract (raw cyanobacterin or raw saponin) or binder + solvent 40: 1 (1 g binder + 25 mg extract / 0.25 ml solvent) or pure binder coated, solid yarn pulled through the sides and dried hanging for 24 hours. Unless otherwise stated, the extract is applied in a concentration of 100 mg · ml ^-1 , ie for a ratio of binder: solvent = 40: 1, the extract is applied in 2.5% by weight. The PVC platelets are immersed in the above-mentioned algae suspension of N. pusilla (platelets must be completely covered and upright). Then the Erlenmeyer flasks provided with the algae suspension of N. pusilla and the PVC plates are incubated for 5-90 days at 15 ° C and steady light (20 µE · m ^-2 · s ^-1 ) (light cooling chamber). The N. pusilla suspensions are shaken with the appropriate PVC plates in a rotary shaker at 180 rpm ^-1 .

After incubation of the PVC test platelets for 5-90 days, the evaluation of the Adhesion test in the form of a visual assessment (growth in%; inhibition of Growth in%) and by exact determination of the number of cells in the growth. For this the Gently scraped off vegetation with rubber wipers, in 5 ml sterile aqua dest. added and fixed with 0.5 ml Lugol's solution. The cell number is determined using the Counting chamber according to THOMA under the light microscope (Olympus CH-2).

Example 8 Adhesion test with N pusilla using various binders / solvents combinations

The selection of a suitable carrier for the active ingredient d. H. of a binder, plays one very important role for its success as an antifouling system. Furthermore, the Use of a suitable solvent to dissolve the active ingredient and to incorporate it of great importance in the paint. The following must be compiled Criteria for the use of the appropriate binders and solvents in the adhesion test get noticed:

With regard to these criteria, numerous test series with different Combinations of binders and solvents carried out and evaluated.

The following are used as binders in these experiments:

• - the color samples B and E from BÜFA-BAEUERLE, vinyl resin red, White vinyl resin, red acrylic resin and white acrylic resin from BROHL CHEMIE.

For comparison, the special antifouling paint Clean-Snip 200, 522-4039 is used as representative commercial antifouling paint carried.

The following are used as solvents:

• - ethanol, naphtha, glycol ether, xylene.

The following tables (Tables 3 and 4) show an extract from the test series. As the best Combination of binder / solvent with regard to the above. Criteria crystallize the mixture of white vinyl resin and naphtha (see Tables 3 and 4), since:

• 1. good solubility and mixing of vinyl resin white and naphtha as well as good Film formation are guaranteed (Table 4)
• 2. strong vegetation is induced, which is clearly visible on a white background and which is still stable after 90 days (Table 4)
• 3. Vinyl resin white as a binding agent is mechanically stable (belongs to the antifoulings insoluble matrix) (Table 4)  
• 4. no toxic effects are found (Table 4)
• - no attachment inhibition
• - no inhibition of growth (see example 10a))
• 5. almost all extracts dissolve well in naphtha (exception: aqueous extract of A. rubens, here solvent butyl acetate)

With this mixture of white vinyl resin and naphtha, the adhesion test is then carried out with N. pusilla with embedding of the raw cyanobacterins from S. hofmanni listed in Example 9 and the raw saponin from A. rubens.

Example 9 Adhesion test with N. pusilla embedding Rohcyanobacterin from S. hofmanni and Raw saponin from A. rubens

Rohcyanobacterin from S. hofmanni obtained by extraction with TBE is dissolved in naphtha in a concentration of 10, 50 and 100 mg · ml ^-1 , and crude saponin obtained from A. rubens by extraction with ethanol in a concentration of 100 mg · ml ^-1 in Butyl acetate. Since the raw saponin from A. rubens is best in aqua dest. dissolves, must be used as a solubilizer to bind in the paint butyl acetate. The dissolved extracts are then embedded in the binder vinyl resin white in a ratio of 40: 1 (1 g binder + 25 mg extract / 0.25 ml solvent) (see Example 7) and the growth is evaluated (see Example 7). Table 5 shows the effect of Rohcyanobacterin (100 mg · ml ^-1 ) and Rohsaponin (100 mg · ml ^-1 ) on the growth by N. pusilla.

When 100 mg · ml ^-1 crude cyanobacterin from S. hofmanni is used, an approximately 98% inhibition of the growth is achieved (see Table 5). The low inhibitory effect of crude saponin from A. rubens (100 mg · ml ^-1 ) (approx. 55% inhibition of growth) can be attributed to the fact that only 20% of the crude saponins are incorporated into the paint using this method (see Table 5) .

When using raw cyanobacterin of different concentrations from S. hofmanni, it can be seen that embedding a concentration of 10 mg · ml ^-1 (0.25% by weight) is sufficient to achieve a significant inhibition of the growth (see Table 6).

Example 10 Growth and vitality tests with N. pusilla

To clarify whether the test coat in the adhesion test only the adhesion of the diatom N pusilla prevents or whether released substances due to the leaching effect The vitality of the algae in the suspension will also impair growth and growth Vitality (percentage of living cells) of N. pusilla before, during and after incubation of the appropriate PVC plate examined in the adhesion test. Following specific tests are carried out:

a) Measuring the absorption of the algae suspension

To determine the growth of the N. pusilla suspensions surrounding the PVC platelets the absorption on the spectrophotometer at 750 (turbidity), 680 (chlorophyll a content measured in vivo) and 490 nm (carotene content in vivo).

b) Plate test

By using the plate test, the recultivation ("regrowth") of the respective N pusilla suspensions, in which the corresponding PVC plate with binder and extract or solvent are hung, followed on agar plates. The agar plates are prepared as described in Example 6.

c) Vital stains with trypan blue

The percentage of living cells is determined on the basis of vital staining with trypan blue (Lindl & Bauer 1994). 50 µl algae suspension of N. pusilla are mixed with 50 µl 0.4% Trypan blue solution added, incubated for 5 min at room temperature and then the cell number non-stained and stained cells using the THOMA counting chamber under the Light microscope (Olympus CH-2) determines: Living cells are not stained. Dead cells are colored blue throughout. The percentage of living cells is calculated following scheme:

Tables 7 and 8 show the results of the growth tests used to determine the toxicity in addition to the investigations to prevent fouling by applying Rohcyanobacterin from S. hofmanni and Rohsaponin from A. rubens.

The embedding of Rohcyanobacterin from S. hofmanni leads to impairment of vitality when using a concentration of 100 mg · ml ^-1 , which increases over the course of the investigation period, which suggests that the Rohcyanobacterin is gradually released from the binder vinyl resin white (leaching effect) ( see table 7).

When 10 mg · ml ^{-1 was applied} , the toxicity was very low (see Table 8), although the inhibition of fouling is just as strong (Table 6). Vital staining (staining with trypan blue) shows that there is no toxicity when using 10 mg · ml ^-1 crude cyanobacterin (97% of the cells are viable). In the case of embedding the raw saponin from A. rubens (100 mg · ml ^-1 ), only a low toxicity (25%) is detected (see Table 7).

literature

• - Abarzua, S .; Jakubowski, S. (1995): Biotechnological investigation for the prevention of biofouling. I. Biological and biochemical principles for the prevention of biofouling. Mar. Ecol. Prog. Ser. 123: 301-312
• - Holzapfel, H .; Rother, J. (1988): Antifouling paint systems for shipbuilding. Color and Room 4: 120-123
• - Jüttner, F .; Leonhardt, J .; Möhren, S. (1983): Environmental factors affecting the formation of metisyl oxide, demethylalylic alcohol and other volatile compounds excreted by Anabaena cylindrica. J. Gen. Microbiol. 129: 407-412
• - Lindl, T .; Bauer, J. (1994): Cell and Tissue Culture, Introduction to the Foundations and selected methods and applications. Gustav Fischer Verlag, ISBN 3-437-30736-3, pp. 189-190
• - Round, F.E .; Crawford, R.M .; Mann, D.G. (1990): The diatoms. Biology and Morphology of the genera. Cambridge University Press, ISBN 0-521-36318-7, pp. 13
• - Van Baalen, C. (1962): Studies on marine blue-green algae. Bot. Mar. 4: 129-139

Claims (8)

1. Use of secondary metabolites from freshwater and marine organisms as natural antifouling agents. 2. Use of secondary metabolites from freshwater and marine organisms according to Claim 1, wherein the secondary metabolites by extraction of cyanobacteria (blue-green algae) be won. 3. Use of secondary metabolites from freshwater and marine organisms according to Claim 1, wherein the secondary metabolites by extraction of marine invertebrates (Invertebrates). 4. Use of secondary metabolites from freshwater and marine organisms according to Claims 1 and 2, wherein the freshwater organisms are the Cyanobacterium Scytonema hofmanni acts. 5. Use of secondary metabolites from freshwater and marine organisms according to Claims 1 and 3, wherein the marine organisms are the common starfish Asterias rubens acts. 6. Use a mixture of the binder white vinyl resin and the solvents Naphtha and butyl acetate for embedding the antifouling active ingredients according to claim 1. 7. Preparation of a mixture of Rohcyanobacterin from Scytonema hofmanni and Naphtha in the form of a solution, mixed with the binder vinyl resin white at 0.25% Weight percentages according to claims 1, 2 and 4. 8. Preparation of a mixture of raw saponin from Asterias rubens and butyl acetate in the form of a solution, mixed with the binder vinyl resin white to 2.5% Weight percentages according to claims 1, 3 and 5.