WASTE WATER TREATMENT METHOD USING MICROORGANISMS

Abstract: A method of treating waste water, based on the use of a strain of the white-rot basidiomycete fungus <i>Phanerochaete chrysosporium</i>, is described.

Description: WASTE WATER TREATMENT METHOD USING MICROORGANISMS FIELD OF INVENTION The invention relates to a micro-organism, the white-rot basidiomycete fungus Phanerochaete chrysosporium Burdsall, called Ml, isolated from waste water originating from oil mills in Morocco, and the enzymes produced by it, for use in bioprocesses for the treatment of pollutant and toxic waste from the agrifood industry. This treatment reduces the toxicity and concentration parameters of the pollutant molecules that characterise some waste waters generated by the agrifood industry and waste water originating from the processes used to manufacture lignocellulose pulp from traditional sources and/or alternative materials (such as straw) with a high lignin and cellulose content. BACKGROUND TO THE INVENTION The waste water generated by various industrial processes generally contains very high concentrations of soluble organic substances. The factors that cause the toxicity of such waste water include phenols, present in the waste water generated by various industries, such as oil refineries, textile mills, paper mills, oil mills, etc. Practically all phenols and their derivatives are toxic and considered as high-risk pollutants; the removal of phenols from industrial waste water is therefore a major environmental problem. Traditionally, waste water has been treated to remove phenols and their derivatives by means of chemical and physical processes including extraction with solvents, ultrafϊltration, adsorption on activated carbon, and chemical oxidation. These processes involve numerous disadvantages, including high cost, low purification efficiency, and applicability to waste with limited phenol concentrations only. More recently, attention has focused on biological treatment methods, which constitute an attractive alternative to conventional systems. In industrial paper manufacturing processes, delignification is performed by means of expensive, polluting chemical methods. The possibility of applying biological delignification processes or using lignin in the manufacture of high-quality chemicals or biomasses is therefore viewed with great interest. In the agrifood industry, oil mill wastewater (OMW) has a high environmental impact because of the concentration of its pollutant content and the quantity of waste water produced. The pollutant content of such waste in terms of COD (chemical oxygen demand) may be hundreds of times greater than that of urban waste water. Moreover, the production of OMW amounts to approx. 30 million cubic metres per annum in the Mediterranean countries. Although this waste is of natural origin, its disposal presents various problems due to its high content of phytotoxic compounds, the seasonal nature of production, and the fact that oil mills are scattered all over the country. The problem of disposing of such waste has not yet been solved, because a cost- effective treatment system has not yet been found. Among the various biological methods proposed, anaerobic bio-treatments could present some advantages, although the fact that OMW contains phenol compounds and other antimicrobial substances necessitates pre-treatment before those substances can be removed. This means that the method is not cost-effective. In the ambit of biological treatments, the possibility of using white-rot basidiomycete fungi to detoxify waste water with a high content of aromatic compounds has been extensively investigated. Various fungi have been selected on the basis of their ability to decolourise and detoxify the waste water originating from the production of lignocellulose pulp and the bleaching process. They include Phanerochaete chrysosporium, Coriolus versicolor and Schizophyllum commune, which produce a considerable reduction in colour content and COD. Encouraging results have been obtained with some strains of Coriolus versicolor in the removal of colour and phenol content from molasses stillage. As regards the treatment of OMW with white-rot basidiomycete fungi, the results obtained with Pleurotus ostreatus and Phanerochaete chrysosporium are reported. In these cases, the growth of the fungus and reduction in the parameters relating to colour, phenol content and COD content is only obtained after pre-treatment of the OMW (by ultrafiltration, centrifuging, etc.) and/or by adding nutrients or ligninolytic enzyme inducers to the culture medium. DESCRIPTION OF THE INVENTION A new strain of the white-rot basidiomycete fungus Phanerochaete chrysosporium Burdsall has now been isolated which efficiently reduces the toxicity parameters and concentration of pollutant molecules in OMW and waste water originating from industrial paper manufacturing processes. The micro-organism has proved particularly effective in reducing the colour, phenol content and COD of waste water. A sample of the strain Phanerochaete chrysosporium Ml, which forms the subject of this invention, has been deposited at the DSMZ collection centre under no. 13583. This strain was isolated from OMW originating from Moroccan oil mills on dishes of agar containing potato dextrose (24 g/1), yeast extract (5 g/1) and OMW in various dilutions. Selection was performed on the basis of ability to grow and decolourise the dilute OMW, ie. to form colonies surrounded by pale decolourisation halos. The selection process used was based on classic microbiology techniques. The fungus was subsequently identified as Burdsall. Colonies of the fungus grow very quickly on dishes of malt extract-agar (16 mm per day at 25°C), and exhibit a positive reaction to laccase if tested with guaiacol. As regards morphology, the diameter of the hyphae is 4-5 mm, while no clamp connections are formed. The isolate may be used to treat water containing toxic and pollutant agents, in particular in the treatment of OMW or waste water generated by paper manufacture, possibly in combination with other fungi belonging to the basidiomycete family or with bacteria. The micro-organism can be added in the form of a suspension or immobilised or inactivated cells, with or without the addition of nutrients and/or oxygen to the water to be treated. Another aspect of the invention relates to extracts and/or isolates with enzymatic activity obtained from the strain of Phanerochaete chrysosporium described here, for the above-mentioned uses. The examples set out below illustrate the invention in greater detail. Example 1 Treatment of OMW with a strain of Phanerochaete chrysosporium Burdsall isolated from OMW Treatment: the fungus is pre-inoculated into 300 ml PDY (24 g/1 potato dextrose and 5 g/1 yeast extract) and incubated for 5 days at 28°C under constant agitation (120 rpm). The pre-inoculum is extensively homogenised; 25 ml of homogenate is transferred to a 1-litre Erlenmeyer flask containing 250 ml of OMW and diluted 5 and 10 times in distilled water in the absence or presence of nutrients (PDYm = 2.4 g/1 potato dextrose, 0.5 g/1 yeast extract and 1% maltose). The cultures are incubated at 28°C under constant agitation on a stirrer plate (120 rpm) for 12 days. Composition of crude OMW used for treatment with P. chrysosporium * COT = Total organic carbon ** BOD = Biological oxygen demand Determination of absorbance of OMW at 395 nm during treatment with P. chrysosporium Determination of phenol content of OMW during treatment with P. chrysosporium The phenol concentration of OMW is determined with a colorimetric test which uses 4-aminoantipyrine. The reaction mixture contains 2.4 mM aminoantipyrine, 0.25 mM H 2 0 2 , 0.0025 U/ml horseradish peroxidase, 40 mM sodium phosphate buffer pH 7.4 and the sample to be analysed, in a final volume of 1 ml. The reaction between the oxidised phenol compounds and the reagent under the test conditions used leads to the appearance of a deep pink colour, which is evaluated by spectrophotometry at 510 nm. The test involves the definition of a calibration line obtained with standard quantities of phenol in a concentration range of 10-50μM. Determination of COD of OMW during treatment with P. chrysosporium Determination of BOD of OMW during treatment with P. chrysosporium Determination of toxicity of OMW after 12 days' treatment The micro-organism used in the toxicity tests is Bacillus cereus, strain E/2. Growth is effected in a liquid culture containing 1.5% tryptone under agitation at 37°C to a value of approx. 0.5 OD measured at 600 nm. The toxicity test is conducted by spectrophotometrically monitoring the growth of the Bacillus in the presence of OMW treated with P. chrysosporium and untreated OMW. The final volume of the medium is 5 ml, containing 500 μl of TY 10X broth, 100 μl of bacterial suspension, and the OMW to be tested. Toxicity is determined by evaluating the highest concentration of treated and untreated OMW able to inhibit bacterial growth in 24 hours. Example 2 Treatment of OMW diluted 5 times using the isolate of Phanerochaete chrysosporium Burdsall adapted to OMW The fungus isolated from OMW, Phanerochaete chrysosporium Burdsall, is transferred to and cultured at 28°C on agar containing 2.4 g/1 potato dextrose, 0.5 g/1 yeast extract and 1% maltose; OMW at different final concentrations with 5% intervals is added. Treatment: the fungus is grown in a liquid medium with the addition of 20% OMW under the same conditions as described in the procedure for treatment 1. The cultures are incubated at 28°C, and the flasks are kept under constant agitation on an stirrer plate (120 rpm) for 15 days. Composition of crude OMW used for treatment with adapted P. chrysosporium Determination of absorbance of OMW at 395 nm during treatment with adapted P. chrysosporium Determination of phenol content of OMW during treatment with adapted P. chrysosporium Determination of COD of OMW during treatment with adapted P. chrysosporium Determination of BOD of OMW during treatment with adapted P. chrysosporium Determination of toxicitv of OMW after 15 days' treatment

Claims: CLAIMS 1. Strain of Phanerochaete chrysosporium deposited with DSMZ under no. 13583. 2. Extracts or isolates of cultures of the micro-organism referred to in claim 1 with enzymatic activity. 3. Use of the strain of Phanerochaete chrysosporium or its extracts/isolates as claimed in claims 1 and 2 to reduce the concentration of toxic and pollutant agents present in waste water. 4. Use as claimed in claim 3, wherein the said waste water originates from the agrifood or paper manufacturing industry. 5. Use as claimed in claim 4, for the treatment of olive mill water. 6. Use as claimed in claims 3-5 of the strain of Phanerochaete chrysosporium or its extracts/isolates in combination with other fungi belonging to the basidiomycete family or with bacteria. 7. Use as claimed in claims 3-6, wherein the cells of the micro-organism are added to the water to be treated in the form of a suspension or immobilised or inactivated cells, with or without the addition of nutrients and/or oxygen.

Inventors: ASSOBHEI OMAR, GIARDINA PAOLA, KISSI MERYEM, MELE ANTONIO, MOUNTADAR MOHAMMED, PALMIERI GIANNA, SANNIA GIOVANNI, ASSOBHEI, OMAR, GIARDINA, PAOLA, KISSI, MERYEM, MELE, ANTONIO, MOUNTADAR, MOHAMMED, PALMIERI, GIANNA, SANNIA, GIOVANNI
Applicants: ASSOBHEI OMAR, BIOREN S R L, GIARDINA PAOLA, KISSI MERYEM, MELE ANTONIO, MOUNTADAR MOHAMMED, PALMIERI GIANNA, SANNIA GIOVANNI, ASSOBHEI, OMAR, BIOREN S.R.L., GIARDINA, PAOLA, KISSI, MERYEM, MELE, ANTONIO, MOUNTADAR, MOHAMMED, PALMIERI, GIANNA, SANNIA, GIOVANNI
Application year: 2001
Main classification: C02F3
IPC codes: C02F3, C12N1