GB2316684A - Shewanella putrefaciens strains - Google Patents

Abstract

Strains of S.putrefaciens are used, preferably with a solid support (especially cellulose foam beads) in an immobilised cell bioreactor, for the adsorption of (anthra)quinones, or in the degradation of azo bonds or azo groups (especially azobenzenes or azo dyes) or of reactive dyes. A specific bacterium, designated J18 143 (ECACC 96081914), is disclosed.

Description

DE GRADATIVE BACTERIA The present invention concerns bacteria, particularly used in degrading azo bonds, for example those of reactive dyes, and used in adsorbing quinones such as anthraquinones, together with methods for same.

Reactive dyes are extremely commercially important for the dyeing and printing of cellulose fibres, the dyes being characterised by their brilliant shades, excellent wet-fastness and good stability to photochemical and chemical attack.

However, thousands of tonnes of these organic dyes are wasted every year (Anliker, R., 1977, Rev. Prog. Col., S : 61-62). Losses of dye from reactive dye-baths can be as high as 50%, resulting in highly coloured alkaline washwater containing unreacted dye(s), hydrolysed dye(s) and salt(s). There is an increasing need for such washwater to be treated and the reactive dye to be removed prior to the discharge of the washwater.

However, there is presently no commercially available process for the complete removal of reactive dyes from a coloured textile effluent - approximately 90% ofthe reactive dyes in textile effluents passes through sewage treatment works (Pierce, J., 1994 (April), J.S.D.C., 110:131-133).

The present inventors have isolated and purified a bacterium which (see 'Experimental' below) is capable of degrading reactive dyes.

According to the present invention there is provided bacterium 518 143 (also referred to as 143; Deposited at the ECACC on 19 August 1996 under the provisions of the Budapest Treaty; accession number 96081914) or a derivative thereof.

Derivatives may include progeny of the micro-organism in which it has for example given up DNA to, or accepted DNA from, another micro-organism, the derivative exhibiting the same, or substantially the same, degradative characteristics as the parent microorganism.

The bacterium may degrade azo bonds in molecules.

Preliminary characterisation of the bacterium has identified it as being a variety ofthe species Schewanellaputrefaciens and so according to the present invention there is also provided Schewanellaputrefaciens or a derivative thereof used in degrading azo bonds in molecules.

The bacterium (i.e. J18 143 or Schewanellaputrefaciens) may degrade azo groups. It may for example degrade azobenzene or derivatives thereof, for example azo dyes such as reactive dyes.

The bacterium may for example degrade Reactive Red 120, Reactive Orange, Reactive Blue 222, Reactive Blue 217, Reactive Red 1 or Reactive Black 5.

Experiments (below) have also shown that J 18 143 may be used to adsorb quinones from a mixture, and so the bacterium (i.e. J18 143 orSchewanellaputrefaciens) may also adsorb quinones (for example anthraquinone).

The degradation may create no, or substantially no, toxins.

Experiments (below) have shown the single culture J18 143 to be as effective in decolorising dye effluent as the mixed culture J18. The use ofjust J18 143 means that only a single pure culture needs maintaining, rather than a complex mixed culture, for example J18 (see 'Experimental' below) in which individual bacteria may come to dominate, possibly reducing the efficacy of the culture.

The bacterium may for example be used with a solid support, for example cellulose foam beads, although any other material to which the bacterium may be attached may be used.

The bacterium may be used in an immobilised cell bioreactor.

Also provided according to the present invention is a method for degrading azo bond-containing molecules comprising the use of J18 143 or Schewanella putrefaciens. For example, the azo bond-containing molecule may be contacted with the becterium in an immobilised cell bioreactor.

Also provided according to the present invention is a method of adsorbing quinones comprising the use of 518 143 or Schewanella putrefaciens. For example, the quinone may be contacted with the bacterium in a mixture or with immobilised bacteria.

Bacteria in a mixture may then be filtered from the mixture, thereby removing the quinone from the mixture.

The invention will be further apparent from the following description and accompanying figure which, by way of example only, illustrates forms of the invention.

Figure 1 shows an immobilised cell bioreactor.

ExDerimental A mixed bacterial culture (J18) was isolated from a textile effluent sample and maintained on agar-soy broth slants at 3 "C. Agar No. 2 and Tryptone Soy Broth were obtained from Lab M, Topley House, Wash Lane, Bury, England. All other chemicals (AnalaR grade) were supplied by Aldrich Chemicals Co. (Gillingham, UK), BDH Ltd.

(Poole, Dorset, UK), Vickers Laboratories Ltd. (Burley-in-Wharfedale, Nr. Leeds, UK) and Fluke Chemicals Ltd. (Gillingham, Dorset).

Biodegradation of Reactive Dyes (i) Selection of a bacterial culture for reactive dye degradation Six different bacterial populations were each tested for their ability to remove colour from reactive dye-soy broth solutions. The bacterial populations tested were the mixed bacterial culture (J18), three single bacterial populations (J18 143, J18 149 and J18 150) separated from culture J 18, and samples of Bacillus benzoevorans and Pseudomonas sp., supplied by the National Collection of Industrial and Marine Bacteria (NCIMB) Ltd.

Two tests were performed. The first test comprised an investigation of the ability of the six bacterial cultures to remove colour from Reactive Red l-soy broth solutions. In the second test, soy broth solutions containing Reactive Red 1 or Remazol Black B were employed. The bacterial culture was in excess when compared to the reactive dye substrate. The first test was also repeated using Remazol Black B-soy broth solutions.

Soy broth solutions (30 g dim.3) containing 40-50 mg dm of Remazol Black B were inoculated with a loopful of one of the bacterial populations. The solutions were incubated for 24 hours at 30 "C under aerobic conditions, without shaking. The D value (below) of each of the reactive dye-soy broth solutions was recorded after 24 hours.

During incubation, the visual appearance of the solutions was monitored. A spread plate of each of the solutions was prepared after 24 hours of incubation to ensure that the bacterial cultures remained pure. A control was used which was not inoculated with bacteria.

The D value is the percentage change in the absorbance or optical density (OD) of a reactive dye solution following decoloration. The greater the D value, the greater the decoloration of the solution. The absorbance is measured at the Xmax of the reactive dye solution after filtration of the solution through a 0.45 micron nitrocellulose membrane filter. The D value is calculated as follows: D= [OD initial - ODfinal] X 100 % OD initial - CDfinaI] X1OO% OD initial OD initial = optical density of reactive dye solution before decoloration test OD final = optical density of reactive dye solution after decoloration test Culture J 18 143 removed the colour from the Remazol Black B-soy broth solution more rapidly than did the other bacterial cultures. In the solutions containing the mixed bacterial culture and the single bacterial population, J18 143, there was a visible colour change from blue-black to mustard yellow by the end of incubation. No significant colour change was seen in the other solutions. Culture J18 143 generated less than 50 % ofthe biomass created in the decolorised solution containing the mixed bacterial culture.

In the first test, the D values recorded for the decolorised solutions containing Reactive Red 1 or Remazol Black B and the single bacterial culture, J18 143, were greater than 80 after 8 hours of incubation. The D values after 24 hours of incubation are given in Table 1.

Table 1: D values of decolorised soy broth solutions containing Reactive Red 1 or Remazol Black B

Culture/ J18 143 J18 149 J18 150 Bacillus Pseudo. J18 J18 143 Control Dye benzoev sp. J18 149 J18 150 Reactive 96 10 11 1 3 97 98 0 Red 1 Remazol 98 68 68 50 49 97 97 3 Black B A characteristic "end point" in the decoloration was observed in the decolorised reactive dye-soy broth solutions containing cultures J 18 143, J 18 and a combination of the three single populations separated from J18. The decolorised Remazol Black B-soy broth solutions were mustard yellow in colour and the decolorised Reactive Red l-soy broth solutions were bright yellow in colour.

(ii) Analysis of culture JIS 143 Genus level identification The pure culture, J18 143, has been provisionally identified as Schewanella putrefaciens.

The preliminary identification of the culture was based upon tests performed by the NCIMB Ltd. The results of the test are given in Table 2. The test methods are as described by Cowan and Steel (1993, Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed).

Schewanella putrefaciens belongs to the family Vibrionaceae and was originally classified under the genus Pseudomonas. It is the only Gram-negative, non-fermentor that produces H2S in the Triple Sugar Iron Agar (TSI) test (Topley and Wilson, 1990, Principles of Bacteriology, Virology and Immunity, 8th Ed., Volume 2 - Systematic Bacteriology). The non-diffusible, salmon-pink pigment, produced during the growth of culture J18 143, is also characteristic of this species.

Schewanella putrefaciens is a strict aerobe and is found widely distributed in nature and is a well recognised fish-spoilage organism.

Table 2: Preliminary examination of Culture J18 143: NCIMB Test Results

Test I Result Incubation Temperature ("C) ' 30 Cell Morphology x 630 magnification Rods with straight, parallel sides (phase contrast) and rounded ends

Colony Morphology Round, regular, entire, buff, semi translucent smooth, glossy, low convex, 1-1.5 mm diameter Gram Stain I Gram-negative Catalase Test [ Catalase positive Oxidase Test Oxidase positive Spores Non-sporing Cell Motility/Movement Motile Oxidation or Fermentation (OF) of Glucose Alkaline (negative result) Growth Temperature ("C) Growth at 37; No growth at 41 Triple Sugar Iron Agar Test Positive result (H2S produced) Aerobic and anaerobic growth Agar-soy broth streak plates of culture J18 143 were prepared and incubated at 30 "C for 48 hours, under aerobic and anaerobic conditions. No growth occurred on the streak plates incubated under anaerobic conditions. However, bacterial growth was observed within 24 hours of introducing the anaerobic plates to aerobic conditions. The bacterial growth was accompanied by the production of a salmon-pink pigment characteristic of culture J 18 143. The same result was observed when the anaerobic plates were refrigerated at 4 "C for 48 hours. However, bacterial growth was slower than at 30 "C.

These results suggest that whilst anaerobic conditions prevented growth of the single bacterial population, the organism was not killed by incubation in an oxygen-deficient atmosphere, within the incubation period stated.

Reactive dye adsorption Adsorption of Reactive Red 1 onto the dead biomass of the single bacterial culture has been shown to contribute to less than 10 % ofthe colour removal from the dye-soy broth solution. Solutions of dead biomass comprising the cultures of Table 1 were incubated at 30 "C for 24 hours under aerobic conditions without shaking. A control was used in the test. The control was an unsterilised Reactive Red 1-soy broth solution inoculated with a loopful of culture J 18 143. The visible colour of this solution changed from red to yellow during incubation. The D value of the control decolorised solution after 24 hours of incubation was 95. In comparison, the test solutions, which contained dead biomass, did not change colour during incubation and gave a D value of 9 after incubation for 24 hours. Streak plates of the test solutions were prepared to ensure that the biomass was non-viable.

A similar result to that observed during the assessment of Reactive Red 1 was obtained when adsorption of Remazol Black B onto the dead biomass of the single culture was tested. The test solution did not change colour throughout the incubation period and the D value of the solution, after incubation for 24 hours, was 2. In contrast, the control containing live biomass exhibited a visible colour change from blue-black to mustard yellow and gave a D value of 97, in 24 hours.

The low D values recorded for the control solutions containing Reactive Red 1 or Remazol Black B and dead biomass suggest that colour removal via adsorption was negligible. Furthermore, the visible colour change observed in the decolorised reactive dye-soy broth solutions containing live biomass may have been due to the formation of less conjugated structures as a result of dye degradation. The changes in the UV visible spectra of reactive dye-saline solutions during decoloration were previously shown to support this concept.

Extracellular enzyme activity The possibility ofthe presence of extracellular reactive dye-degrading enzymes in culture J18 143 was investigated. 30 g dm3 soy broth solutions containing 40-50 mg dm-3 of Reactive Red 1 or Remazol Black B were inoculated with a loopful of the single bacterial population and then incubated for 24 hours at 30 "C, under aerobic conditions, without shaking. The initial absorbance of each of the solutions was measured at the max of the dye solution before incubation. After incubation for 4 hours, the reactive dye-soy broth solutions had changed colour and became turbid. The Reactive Red 1-soy broth solution was red-orange in colour and the Remazol Black B-soy broth solution was purple in colour.

The D value of each of the solutions was recorded at this stage. A 10 cm3 sample was removed, using an aseptic technique and then filtered through a 0.2 micron sterile syringe filter into a sterile conical flask. The D value of the filtered solutions was recorded and then the stoppered flasks were incubated alongside the unfiltered solutions for a further 20 hours.

The unfiltered solutions continued to change colour during the incubation period. After 24 hours, the Reactive Red l-soy broth solution was bright yellow in colour and the Remazol Black B-soy broth solution was mustard yellow in colour. The average D values of the decolorised, unfiltered solutions were 96 and 92 respectively. In contrast, no visible colour change or turbidity was observed in the filtered solutions. The average D values for the filtered Reactive Red 1-soy broth and Remazol Black B-soy broth solutions, after incubation for 20 hours, were 60 and 54 respectively. The average change in the D values between the initial filtration time and the end of incubation was less than 3. These results suggest that the contribution by extracellular enzymes to the removal of colour from the soy broth solutions containing Reactive Red 1 or Remazol Black B was negligible. Decoloration by culture J18 143 may therefore be an intracellular process.

Decoloration of different reactive dyes in soy broth solutions Soy broth solutions (30 g dm-3) were inoculated with a loopful of culture J 18 143 and incubated at 30 "C under aerobic conditions, without shaking. After incubation for 16 hours, a sample of a reactive dye was added to each solution, using an aseptic technique, to give a dye concentration of 40-50 g dm-3. The dyes tested are listed in Table 3. The dyes were generally unpurified commercial samples (ICI, Blakeley, UK; L.J.

Specialities, Wigan, UK; Sandoz, Leeds, UK), the exception being Reactive Red 1 which was synthesised in the laboratory and purified before use.

Table 3: Reactive Dyes Tested and their D Values

Reactive Dyes Colour Index Chemical Type D Generic Name (%) Procion Red HE-3B Reactive Red 120 azo 92 Procion Blue MX-R Reactive Blue 4 anthraquinone 31 Procion orange MX-2R Reactive Orange azo 95 Sumifix Supra Navy BF Reactive Blue 222 azo 88

Kyacelon React Dark Blue Reactive Blue 217 azo 88 C-NR Drimarene Golden Yellow Reactive Yellow azo 15 K-2R 125 Reactive Red 1 Reactive Red 1 azo 96 Remazol Black B Reactive Black 5 azo 98 The absorbance of each of the reactive dye-soy broth solutions was measured after filtration through a 0.45 micron nitrocellulose membrane filter. The dye-soy broth solutions were then incubated at 30 "C for 24 hours under aerobic conditions without shaking. Following incubation, the D value of each solution was recorded (Table 3).

A visible colour change was generally observed in the dye-soy broth solutions, the exception being those solutions containing Procion Blue MX-R and Drimarene Golden Yellow K-2R. The colour of the incubated solutions at the "end-point" of decoloration was yellow and the D value for 75 % of the decolorised solutions was greater than 88.

The D value for the Procion Blue MX-R-soy broth solution was 31. However, there was no visible colour change in the solution during incubation. The filter membranes from the filtration ofthe solution before and after the test were retained and dried. The second filter membrane was observed to be darker blue in colour than the first membrane. This was not observed with any ofthe filter papers from the other decolorised solutions. This suggests that the decoloration of this dye could be via adsorption onto the biomass of culture J 18 143. The visible colour change in the other decolorised solution could be the result of dye degradation. This may suggest that the single bacterial culture J18 143 removes the colour from reactive azo dye solutions via dye degradation and that when anthraquinone-based reactive dyes were used the removal of colour was achieved via dye adsorption.

The D value obtained for Drimarene Golden Yellow K-2R may be misleading. The Alma, of the dye solution was at 340 nm, in the region where the soy broth solution strongly absorbs. Hence, after decoloration, the absorbance of the dye-soy broth solution at 340 nm would remain high. The absorbance of the decolorised dye soy broth solution in the 400-700 nm region of the spectrum was greatly reduced. This suggests that dye degradation may have occurred and that less conjugated molecules with lower absorbances in the spectrum were produced.

Interaction of culture J18 143 with dextran Dextran was added to a soy broth solution containing culture J18 143 to give a dextran concentration of 1 % . The soy broth solution was previously inoculated with the single bacterial culture and incubated at 30 "C, under aerobic conditions for 24 hours.

Following the addition of dextran, 40-50 mg dm-3 of Remazol Black B were added and the solution was reincubated for 24 hours. The colour was removed from the dextran-dye-soy broth solution at the same rate as the colour was removed from a similar solution that did not contain dextran. Furthermore, the D values for both solutions, after incubation for 24 hours, were greater than 80.

These results suggest that dextran does not affect the decoloration of the Remazol Black B-soy broth solution by culture J18 143. Hence dextran may be used in the immobilisation of culture J18 143.

Preparation of the Cellulosic Support (i) Regenerated cellosefibres The use of a ram extruder for the preparation of regenerated cellulose fibres was investigated. It was found that the fibre structure was not stable in aqueous systems. Over a period of 24 hours, small fibrils were released from the ends of the fibres into the solution. The instability of the fibres made them unsuitable for use in the immobilisation ofcultureJl8 143.

(ii) Regenerated cellulose beads A solution of wood pulp cellulose in N-methylmorpholine-N-oxide/H,O was pipetted dropwise into cold distilled water, whilst stirring, to form irregular beads of regenerated cellulose. The beads were left to regenerate in the water overnight and then washed several times with boiling distilled water. The method was repeated with 4 % of potassium carbonate incorporated into the cellulose/amine oxide/water solution.

Regeneration was achieved in 1 M aqueous HCl solution. The result was the formation of irregular, cellulose foam beads with a porous structure and greater rigidity than that possessed by the beads with had contained no potassium carbonate. The foam beads were stable, in aqueous solution, for at least 96 hours. The average diameter of the wet beads was 4 mm.

Immobilisation of Culture J18 143 Immobilisation of culture J18 143 by adsorption onto cellulose foam beads The single bacterial culture was adsorbed onto and within the cellulose foam beads by incubating the beads in a saline solution containing the culture. After 24 hours, the beads were removed and suspended in sterile saline. Remazol Black B (50 g din.3) was also added and the solution incubated for a further 24 hours at 30 "C. Decoloration was not observed during incubation. However, the addition of a sterile soy broth solution, to give a concentration of 0.3 %, resulted in decoloration of the dye-saline solution in 8 hours.

The solution became turbid and yellow in colour.

Design of an Immobilised Cell Bioreactor A small scale column bioreactor (Figure 1), for the decoloration of reactive dye solutions, was assembled and tested. A glass column (10) was attached to a peristaltic pump (20) via a recirculation cell (30) and a stirred reservoir (40). The reservoir was filled with the reactive dye solution (41) and the column loosely packed with the immobilised support. Loose packing of the foam beads helps maximise the contact between the culture and the reactive dye substrate. Culture J18 143 was immobilised onto the cellulose foam beads either via aggregation with dextran or via natural adsorption.

Claims (15)

1. Bacterium J1 8 143 or a derivative thereof.

2. Bacterium J18 143 or a derivative thereof according to claim 1, the bacterium degrading azo bonds in molecules.

3. Schewanella putrefaciens or a derivative thereof used in degrading azo bonds in molecules or adsorbing quinones.

4. A bacterium according to any one of the preceding claims, the bacterium degrading azo groups.

5. A bacterium according to claim 4, the bacterium degrading azobenzene or derivatives thereof.

6. A bacterium according to claim 5, the bacterium degrading azo dyes.

7. A bacterium according to any one of the preceding claims, the bacterium adsorbing quinones.

8. A bacterium according to claim 7, the bacterium adsorbing anthraquinones.

9. A bacterium according to any one of the preceding claims, the bacterium degrading reactive dyes.

10. A bacterium according to any one of the preceding claims, the degradation creating no, or substantially no, toxins.

11. Bacterium according to any one of the preceding claims used with a solid support.

12. Bacterium according to claim 11 used with cellulose foam beads.

13. Bacterium according to either one of claims 11 or 12 used in an immobilised cell bioreactor.

14. A method for degrading azo bond-containing molecules comprising the use of J 18 143 or Schewanella putrefaciens.

15. A method of adsorbing quinones comprising the use of J 18 143 or Schewanella putrefaciens.