WO2007059793A1 - Controlling cyanobacteria - Google Patents

Abstract

The invention relates to use of at least one bacterium of the species Brevibacillus parabrevis for controlling or reducing the concentration or proliferation of cyanobacteria.

Description

 CONTROL OF CYANOBACTERIA

The invention relates to the use of at least one bacterium of the species Brevibacillus parabrevis for controlling or reducing the concentration or proliferation of cyanobacteria.

Certain cyanobacteria, such as Microcystis aerigunosa or Spirulina subsalsa, secrete substances which can be highly toxic to a wide variety of organisms (see eg Repavich, WM et al., (1990) Water Research, 24, 225-231 or Lightner, DV (1978) Journal of Invertebrate Pathology 32, 139-150). Furthermore, secreted metabolites, such as 2-methylisoborneol, may result in inedible drinking water (Terauchi, N. et al. (1995) Water Sci., Technol. 31, 229-235), or the taste of organisms grown in aquaculture Boyd, CE (2003) ACS Symposium Series, 848 or Lovell, RT and Broce, D. (1985) Aquaculture, 50, 169- It is therefore of economic interest to control or minimize the concentration and / or proliferation of cyanobacteria, especially Microcystis aeruginosa.

It is thus an object of the invention to keep the concentration and / or proliferation of cyanobacteria, especially Microcystis aeruginosa, as low as possible.

According to a first aspect, this object is achieved by the subject matter of claim 1.

Thus, it has surprisingly been found that by using at least one bacterium of the species Brevibacillus parabrevis a particularly efficient control or reduction of the concentration or proliferation of cyanobacteria, in particular in aquacultures, can be achieved. Furthermore, it is advantageous that Brevibacillus parabrevis not only represents a bacterium harmless to aquatic organisms, but also provides a positive health-promoting effect in aquatic organisms and a control of important pathogens of aquatic organisms such as Vibrio harveyi.

The use according to the invention of at least one bacterium of the species Brevibacillus parabrevis can take place in the case of already present cyanobacteria (in aquacultures) or prophylactically or as a precaution. What is meant by cyanobacteria (also called "blue-green algae"), in particular Microcystis aerigunosa, is known to the person skilled in the art and need not be explained in more detail.

The application according to the invention is preferably carried out in aquacultures, in particular for the cultivation or storage of aquatic organisms. In practice, commercial important aquacultures, especially in basins, containers, tubs, cages, aquariums, tanks, ponds or the like in the foreground. Of course, the method according to the invention is also useful in private housing or breeding of aquatic organisms.

Preferred aquatic organisms for which the use according to the invention may be carried out include, but are not limited to, crustaceans, e.g. Penaeus spec. (Shrimp), fish, aquatic plants, mollusks.

According to the invention, living cells of Brevibacillus parabrevis are preferably used. Preferably, they are still capable of replication. However, according to one aspect, dead or destroyed cells, eg cells destroyed by heat sterilization, lysis or fragmentation, can also be used for Brevibacillus parabrevis cells which are no longer replicable. The amount or concentration of the cells (cultures) of Brevibacillus parabrevis used according to the invention can vary within wide limits, but will generally preferably be at least 10 ¹ cells, preferably at least 10 ² cells per ml of the aquaculture medium to be treated, in particular water ,

According to a preferred embodiment of the invention, the at least one bacterium of the species Brevibacillus parabrevis is used in the form of a composition containing at least one bacterium of the species Brevibacillus parabrevis. Preferably, 1 g of the composition according to the invention contains at least 10 ³ cells, in particular 10 ⁴ cells, more preferably at least 10 ⁵ cells (or spores), more preferably at least 10 ⁶ cells (or spores), more preferably at least 10 ⁷ cells (or spores), more preferably at least 10 ⁸ cells (or spores). According to a preferred embodiment of the invention, Brevibacillus parabrevis can be used together with other probiotic microorganisms, such as, for example, species of the genus Bacillus or Lactobacillus, Vibrio, Cellulomonas, Pseudomonas or Bifidobacterium spp. For example, probiotic bacteria may be selected from strains of: Bacillus subtilis, Bacillus laterosporus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus azotoformans, Bacillus circulans, Bacillus pumilus, Bacillus firmus, Paenibacillus polymyxa, Paenibacillus macarans or Alteramonas spp., Lactobacillus acidophilus , Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus casei subsp. tolerans, Lactobacillus jensenii, Bifidobacterium bifidum, Vibrio alginolyticus, Pseudomonas stutzeri.

Furthermore, according to a further embodiment of the invention, any other additional components may be included in the composition of the invention, e.g. Feed ingredients, veterinary components such as antibiotics, fungicides or the like, vitamins, nutrients, coloring agents or the like. Such additives are well known to those skilled in the art and can be readily selected depending on the particular body of interest, the aquaculture species, or the desired dosage unit of the composition of the invention, and also provide positive effects in addition to Brevibacillus parabrevis.

In general, the composition used according to the invention may be a liquid or solid composition. The shape and (dosage) size of the composition can be chosen arbitrarily depending on the desired application. In the context of the method according to the invention, for example, an addition into the water or culture medium can take place. Same is a treatment of the individual components of aquaculture or the containers used, pipes, pumps, tubs, basins, cages, aquariums, tanks, ponds or the like possible. Again, the composition can be used in liquid or solid form or sprayed, for example. According to a preferred embodiment of the invention, the treatment with the composition according to the invention is carried out at aquaculture sites where the cyano bacteria are predominantly found.

According to a preferred embodiment, the composition is sprayed, applied or applied in a similar manner to the aquaculture or parts or surfaces of the basins or containers.

According to another possible embodiment, introduction into solid form in or on the water, the basin or container surfaces, or existing sediment or the like takes place. The application or incorporation of the composition can also be done directly on the cyanobacteria.

It is possible in principle for the composition to be introduced before, during or after the cultivation of the aquatic organisms. As far as cyanobacteria often occur in new establishments or water changes in aquacultures, the addition is preferably carried out when re-establishing or water change in aquaculture.

The at least one bacterium of the species Brevibacillus parabrevis can generally be used in any desired form (also on a solid support material), preferably in the form of a bacterial culture, a bacterial concentrate, a seed culture, or in the form of spores or lyophilisates of Brevibacillus parabrevis. Preferably, viable or replicable cells or spoilage cells are present in the composition. of Brevibacillus parabrevis, also in lyophilised form. As far as spores are used, induction of germination, especially by heat treatment of the composition, may be expedient prior to introduction into aquaculture. It is also possible to use parts, for example cell fragments or cell extracts of Brevibacillus parabrevis.

As such, Brevibacillus parabrevis is well known and commercially available to those skilled in the art. A particularly suitable isolate (isolate 2) of Brevibacillus parabrevis was on 16.12.2004 under the accession number DSM 16986 in the name of Süd-Chemie AG ₇ Munich, DE, according to the Budapest Treaty in the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH , Mascheroder Weg Ib, D-38124 Braunschweig, deposited.

According to a preferred aspect, the present invention relates to a method comprising the following method steps:

a) providing a composition containing at least one bacterium of the species Brevibacillus parabrevis,

b) contacting the composition of (a) with cyanobacteria, especially in an aquaculture.

According to a particularly preferred embodiment, the composition is added directly to the water or the aquaculture medium, optionally via an existing water exchange circuit or a pump system, or by mixing the composition with the water or aquaculture medium.

In principle, the composition of the invention before, during or after the cultivation of the. introduced into aquatic organisms. This is usually the case with aquacultures after the harvest of aquatic organisms carried out a purification of the plants. In many cases, it may be useful, therefore, to carry out the inventive method before the onset of a new generation of aquatic organisms in the aquaculture.

The invention will now be explained in more detail with reference to the following non-limiting examples:

EXAMPLES

Detection of inhibition of Microcystis aerigunosa

Microcystis aerigunosa. was grown in a preculture to a cell density of 10 ^δ cells per milliliter. For this purpose, 200 ml HBPD water (composition see below) were inoculated with the microorganism and at 25 ⁰ C in 12 h

Light / dark rhythm incubated. For the detection of the inhibition 200 ml of HBPD water with a Microcystis aerigunosa concentration of 10 ³ cells / ml in 1000 ml shake flasks without baffles were submitted. The cultivation was carried out in a 12h light / dark rhythm at 25 ⁰ C with shaking.

Composition of HBPD water:

Tap water with:

pH-Wert: 7,8 Härte: °dH ca. 7,5 Konduktivität : 54,8 mS/m

pH value: 7.8 Hardness: ° dH approx. 7.5 Conductivity: 54.8 mS / m

The blank sample was not supplemented with Brevibacillus paparrevis. In the test series "Brevibacillus", 4 μl of a preculture of Brevibacillus parabrevis were added, which had been grown to a cell density of 4.0 × 10 ⁸ cells / ml The starting concentration of Brevibacillus parabrevis in these experiments was thus 8 × 10 ³ cells / ml . the cultivation of the seed culture of Brevibacillus brevis para carried out in SWMl- medium at 30 ⁰ C and 150 rpm.

All Brevibacillus parabrevis used were freshly streaked on SWM 1 agar before use in the experiments and precultivated in SMW 1 liquid medium.

* Seawater: 19 g of sea salt in 500 ml deionised water

The cell number of Microcystis aeruginosa was determined as follows: 1 ml of the culture was taken. 20 μl of it was added undiluted to a Thoma counting chamber under the coverslip so that a film was formed between the counting chamber and coverslip. At a magnification of 1: 400, the cells were then counted according to the following scheme: 16 small squares out of a total of 256 small squares were randomly selected and the number of cells in the selected small squares was determined by counting. The cell numbers were averaged and the microorganism concentration determined according to the following formula (area of a small square: 0.0025 mm ² , chamber height 0.1 mm):

Number of microorganisms per small square * 10 ³

Microorganism concentration [Z / ml] = area of a small square [mm ² ] * chamber height [mm]

test results

The results are summarized in Tables 1 (blank) and 2 (comparative sample with addition of Brevibacillus pa. Rabrevis), each for two parallel samples (A and B). Table 1: Microcystis aerigunosa growth without addition of Brevibacillus parabrevis (blank sample)

"Z" = cells, "E + XX" = '1 (T ^*

Table 2: Microcystis aerigunosa growth with addition of Brevibacillus parabrevis (comparative sample)

The results clearly show the reduction of the growth of Microcystis aerigunosa with the addition of Brevibacillus parabrevis. Other Bacillus spec. showed significantly worse results.

Claims

PATENT CLAIMS 1. Use of at least one bacterium of the species Brevibacillus parabrevis for controlling or reducing the concentration or proliferation of cyanobacteria. 2. Use according to claim 1, characterized in that living, preferably reproducible cells of at least one bacterium of the species Brevibacillus parabrevis be used. Use according to any one of the preceding claims, in an aquaculture for the breeding or storage of aquatic organisms. 4. Use according to one of the preceding claims, characterized in that the aquatic organisms are plant or animal organisms, in particular crustaceans, such as 2.B. Penaeus spec. (Shrimp), fish, aquatic plants or mollusks. 5. Use according to one of the preceding claims, characterized in that the aquaculture takes place in a container, a tub, a basin, a cage, an aquarium, a tank, a pond or the like. 6. Use according to one of the preceding claims, characterized in that at least one bacterium of the species Brevibacillus parabrevis is used in the form of a liquid or solid composition. 7. Use according to one of the preceding claims, characterized in that in the composition at least one of the species Brevibacillus parabrevis is immobilized on a solid support. 8. Use according to one of the preceding claims, characterized in that the composition contains: a bacterial culture or a bacterial concentrate, a seed culture, spores, a lyophilisate, cell fragments or cell extracts of Brevibacillus parabrevis, and mixtures thereof. 9. Use according to one of the preceding claims, characterized in that the composition contains further constituents, in particular other probiotics such as probiotic microorganisms, feed components, veterinary components such as antibiotics or the like, vitamins, nutrients, binders, enzymes, biopolymers such as alginates, colorants , Fungicides or the like. 10. Use according to one of the preceding claims, characterized in that it is the at least one bacterium of the species Brevibacillus parabrevis to the isolate 2 according to the deposits DSM 16410 and DSMZ 16986. 11. A method for controlling or reducing the concentration or proliferation of cyanobacteria, characterized by the following method steps: (a) providing a composition containing at least one Brevibacillus parabrevis bacterium, (b) contacting the composition of (a) with cyanobacteria, especially in an aquaculture. 12. The method according to the preceding claim, characterized in that the composition is added directly to the water or aquaculture medium, optionally via an existing water exchange cycle, or while mixing the composition with the water or aquaculture medium. 13. The method according to any one of claims 11 or 12, characterized in that the composition is sprayed on the aquaculture or parts or surfaces of the basins or containers, applied or applied in a similar manner. 14. The method according to any one of claims 11 to 13, characterized in that an introduction in solid form in or on the water, the basin or container surfaces, or existing sediment or the like takes place. 15. The method according to any one of claims 11 to 45, characterized in that the composition is introduced before, during or after the cultivation of the aquatic organisms. 16. The method according to any one of claims 11 to 15, characterized in that the application or introduction of the composition takes place directly on the cyanobacteria. 17. The method according to any one of claims 11 to 16, characterized in that the concentration of Brevibacillus rababrevis, after contacting with the cyanobacteria or the addition of the composition to the aquaculture medium at more than 10 ² / ml, preferably more than 10 ³ / ml.