Decolorization of olive mill wastewaters by co-culture of Geotrichum candidum and Lactobacillus plantarum

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1 Proceedings of International Symposium on Environmental Pollution Control and Waste Management 7-0 January 00, Tunis (EPCOWM 00), p Decolorization of olive mill wastewaters by co-culture of Geotrichum candidum and Lactobacillus plantarum Lamia AYED, BEN OTHMAN and Nada HAMDI MOKTAR,.Ecole Supérieure des Industries Alimentaires de Tunis (ESIAT) 58 Avenue Alain Savary 00 Tunis- Tunisie..Institut National des Sciences Appliquées et de Technologie (INSAT) Boulevard de la terre. BP Tunis- Tunisie. . insat.rnu.tn. ABSTRACT Biodegradation system shows some advantages that make these suitable for phenolic compounds removal. The microbial degradation of OMW which contains many different chemical compounds was most extensive in mixed culture of Geotrichum candidum and Lactobacillus plantarum. Geotrichum candidum used for the treatment of OMW induces the COD and OD removals which are respectively 0 % and % after five days of culture. Lactobacillus plantarum has the capacity to reduce the red-ox potential of OMW and to avoid auto-oxidation of phenolic compounds. Lb growth removes the COD of 6% and the OD of 6 %. The use of co-culture of the two micro-organisms for OMW treatment has given a best result faster than with pure cultures. The yield of the biodegradation has doubled compared to these of pure cultures. The decolorization becomes extensive (6% of color removal and 5 % of COD removal) after five days when the culture is not flushed with oxygen in spite of the high charge of initial COD (70g/). This decolorization of OMW resulted to depolymerization of high molecular mass aromatics combined with biodegradation of a wide range of monoaromatic compounds. KEYWORDS Geotrichum candidum, Lactobacillus plantarum, co-culture, OMW, decolorization. Introduction The manufacturing process of olive oil yield a liquid waste called olive mill wastewaters (OMW). OMW induce a serious problem, especially in Mediterranean countries. The OMW cause an important environmental problem that needs to be solved. The ecological problem is mainly due to the phenolic compounds, which are responsible for the dark color. In addition to its high polluting power, OMW posses high anti-microbial activity exerted mainly by various phenolic compounds (Borja et al., 995). Several biological processes have been evaluated for the use of OMW. Among these some aerobic biological methods seem to be especially suitable since the treatment leaves a residue lower toxicity and diminishes phenol content (Hamdi, 99, Sayadi et al., 99). The use of Geotrichum candidum for the practical treatment of OMW was investigated because the fungus can reduce the color of the effluent and hydrolyses phenolic compounds with high molecular weight and degrades many simple 6

2 Decolorization of olive mill wastewater phenolic compounds (Assas et al., 000). This fungus secretes a peroxidase enzyme which has the ability to decolorize a wide range of dyes (Kim et al., 999). Unfortunaly aerobic biodegradation of OMW was limited by auto-oxidation of phenolic compounds into polyphenolic compounds with high molecular weight (Hamdi, 99). The use of Lactobacillus plantarum with reductive potential which stops auto-oxidation phenomenon of polyphenols and induces their hydrolysis and removals (Ayed et Hamdi,00). It has shown that a mixed of micro-organisms may be more effective than any strain actives alone. The efficiency of co-culture of Lactobacillus plantarum and Geotrichum candidum on the OMW decolorization was evaluated. Material and methods Microorganisms and culture procedures. Lactobacillus plantarum was isolated from fermented olives and maintained on MRS. Geotrichum candidum was isolated from the aerated pilot scale bubble column fed continuously with OMW (Assas et al., 000). Biodegradation tests of OMW by the co-culture of the two micro-organisms were conduct in 0- litres Plexiglas bubble column with 0 cm in diameter and 50 cm in height. The bubble column was filled with 8 litres of OMW supplemented with g/l of yeast extracts and g/l of ammonium sulphate (initial COD = 70 g/l; initial ph= 6). Analytical methods. Total suspended solid. TSS were obtained by centrifugation at 000g during 5 mn, the settled solids were then dried overnight at 05 C. The chemical oxygen demand. COD was determined by the method of Knechtel (978). The soluble COD was measured on the centrifuged OMW at 000 g during 0 min. Decolorization assay. Decolorization assay was determinate by the measurement of the absorbance at 90 nm (Spectrophotometer CECIL-CE 00). Molecular mass distribution of polyphenolics. Gel filtration on sephadex G-50 was used to analyse the polyphenolic compounds present in different samples of OMW. Three ml of sample were filtered and placed on a sephadex coarse G-50 column (,5 x 50 cm ) previously equilibrated with distilled water. This column was washed with 00 ml of distilled water at a flow rate of 0,6 ml / mn. The effluent was collected on the basis of ml / tube. The optic density of these fractions were measured spectrometrically at 80 nm. Results and discussion Lb. plantarum growth on OMW Lb. plantarum growth on the OMW reached quickly the maximum after two days of culture and then remained constant. Lactobacillus growth decreased the ph because of lactic acid production. Biodegradation of OMW by Lactobacillus growth induces a 6% of COD and 6% of OD removals after five days of culture (figure ). 6

3 Ayed et al Residuals COD (%) and OD (%) Figure. Time course of TSS (o);residual COD ( ) and residual OD (x) after incubation of Lb. plantarum on OMW. The sephadex G-50 chromatograms of OMW treated with Lb. plantarum (figure ) showed the disappearance of some phenolic compounds with high molecular weight and the appearance of others with an intermediate weight which could be explained by depolymerization and adsorption phenomenon. Geotrichum candidum growth on OMW The determination of biomass concentration (TSS) versus incubation time showed that the mycelium grew especially during the first two days (figure ). The Geotrichum growth decreased the ph favourable for higher color removal because the production of ligninase enzyme. The COD removal and OD removal were respectively 0 % and % (figure ). 5,5,5,5, Residuals COD (%) and OD (%) Figure. Time course of TSS (o);residual COD ( ) and residual OD (x) after incubation of Geotrichum candidum on OMW. Geotrichum candidum hydrolysed phenolic compounds with high molecular weight and removed many simple phenolic compounds contained in OMW. In fact, gel filtration on sephadex analysis of phenolic compounds after five 6

4 Decolorization of olive mill wastewater days of culture showed that some of them had disappeared, where as others had accumulated due to polyphenols hydrolysis ( figure ). The co-culture of Geotrichum candidum and Lb. plantarum growth on OMW Previous results have shown the efficacity of the two microorganisms for OMW decolorization. Where the interest to mixed the two micro-organisms to improve the yield of the biodegradation and decolorization. The mixed culture was able to decolorize fresh OMW more and faster than the pure cultures (figure ). In fact, the color removal was 6 % whereas the colour removal by pure culture did not exceed 5 % in spite of the high charge of COD. However, the COD and OD removals exceed 50% with pure culture of Lb. plantarum or Geotrichum candidum when the initial COD is less than 50 g/l after eight days of culture. The OD removal has doubled compared to these of pure cultures because of the complementary biodegradation of phenolic compounds by the two micro-organisms in addition, to the decrease of ph induced by the Lactobacillus growth which was favourable to the lignin peroxidase activity. Kirk et al. (978) had shown that a ph of.5 was optimal for the expression of lignolytic activity of Phanerochaete chrysosporium Residuals COD (%) and OD (%) Figure. Time course of TSS (o); residual COD ( ) and residual OD (x) after incubation of co-culture on fresh OMW. After 5 days of incubation the sephadex G-50 chromatogams of OMW treated with the co-culture showed that all phenolic compounds with high molecular weight disappeared and that many simple phenolic compounds were biodegraded (figure ). CONCLUSION The co-culture of Geotrichum candidum and Lb. plantarum seems to be an interesting way to improve the decolorization of OMW without dilution. The decolorization is not influenced only by the micro-organism but also by the culture duration and the storage and dilution of OMW. The OD removal increases when the OMW are more diluted. 65

5 Ayed et al.,5 OD at 80 nm,5, Fractions Figure. Changes in molecular mass distribution of untreated OMW ( ), treated OMW by Lb. plantarum (x), by Geotrichum candidum ( ) and by The co-culture (O) after five days of culture. REFERENCES Assas N, Marouani L, Hamdi M (000) Scale down and optimization of olive mill wastewaters decolorization by Geotrichum candidum. Bioprocess Eng : Ayed L, Hamdi M (00) Reductive decolorization of phenolic compounds contained in olive mill wastewaters by Lactobacillus. Plantarum. (submitted) Borja R, Martin A, Alonso V, Garcia I, Banks CJ (995) Influence of different aerobic pretreatments on the kinetics of anaerobic digestion of olive mill wastewaters. Water Res 9 (): Hamdi M, Kadir A, Garcia JL (99) The use of Aspergillus niger for bioconversion of olive mill wastewaters. Applied Microbiol Biotechnol : Hamdi M. ( 99 ) Futur prospects and contraints of olive oil mill wastewater use and treatment: A Review. Bioprocess Eng. 8: 08-. Kim SJ, Shoda M (999) Purification and caractérisation of a novel peroxidase from Geotrichum candidum Dec involved in decolorization of dyes. Applied Environ. Microbiol. 65: Kirk TK, Schultz E, Connors WJ, Lorenz JF, Zeikus JG (978) Influence of culture papameters on lignin metabolism by Phanerochaete chrysosporium.arch. Microbiol. 7: Knechtel R J (978) Amore economical method for the determination of chemical oxygen demand. Water Control May-June, 5-9. Sayadi S, Ellouz R (99) Decolorization of olive mill waste-waters by the white -rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system. Applied Microbiology and biotehnolog 7: