Phenolic compounds and COD removal from olive mill wastewater by chemical and biological procedures.

In this study biological and chemical treatments were applied to olive mill wastewater (OMW) to reduce its phenolic compounds and chemical oxygen demand (COD) contents in order to reduce its toxicity. The biological treatment was carried out by using two fungal isolates (Aspergillus wentii and Aspergillus niger) isolated from the OMW and compared with Pleurotus ostreatus. A. wentii was more efficient than A. niger and Pleurotus ostreatus in removing of COD and phenolic compounds from OMW. The optimum OMW dilution was 10%, in which the maximum COD removal (62.2%) and maximum phenolic compounds reduction (80.9%) were obtained by A. wentii. This was followed by 59.3% removal of COD and 79.6% reduction of phenolic compounds obtained by P. ostreatus at the same OMW dilution (10%). Lower biodegradation percentages of COD (28% at 40% OMW dilution) and phenolic compounds reduction (72.9% at 30% OMW dilution) were obtained by A. niger. Different chemical treatments were applied primary on tannic acid solution to select the best 2 2 of them for OMW application. These treatments were UV alone and combined with H O , Diefferent Fenton systyems and photocatalysis by titanium dioxide. The maximum degradation of tannic acid 2 2 obtained in the first treatment was 47% at H O concentration 0.11M at pH 9 under UV radiation. The maximum tannic acid degradation obtained by Fenton system was 70%. It was obtained at both Fe 2 2 concentrations (0.0024M and 0.0048M) and 0.11M H O in dark and UV radiation at pH 9. While 2 the maximum degradation obtained by photocatalysis by titanium dioxide was 35% at TiO 50mg/l and pH 5. The maximum degradation of phenolic compounds (74.4%) was obtained when OMW 2 2 treated with 0.55M of H O in dark after 40 min . 2 Key word: Olive mill wastewater; Fenton; Photo-Fenton; UV plus hydrogen peroxide and TiO INTRODUCTION Olive mill wastewater (OMW) composed of many complex substances that are not easily degradable. The acid pH and the high amounts of organic matter and phenols make it very difficult to be purified. Deterioration of natural water bodies due to olive mil wastes is a serious problem as indicated by coloring, appearance of an oily shine, and increased oxygen demand. It affects the soil quality, toxic to plants and soil micro flora when disposed into the soil. Therefore, direct discharge of olive mill wastewater into receiving media is not permissible and certain measures must be taken before disposal of the OMW into the environment (Azbar et al., 2004 and Ergu et al., 2008). Several studies carried out to reduce the phenolic compounds and COD content of this waste by using different treatment methods. Biological treatments by different microorganisms may be fungi such as Aspergillus niger (Hamdi et al., 1991) and Borja et al. (1995); Phanerochaete chrysosporium (Ahmadi et al., 2006), Pleurotus ostreatus (Fountoulakis et al., 2002 and Aggelisa et al., 2003); Lentinula edodes (Annibale et al., 2004); yeasts such as Yarrowia lipolytica and Candida tropicalis (Ettayebi et al., 2003), bacteria such 2 as Azotobacter vinelandii (Piperidou, et al., 2000). Chemical treatments such as photocatal ysis using TiO (Oppenländer, 2003; Konstantinou and Albanis, 2004 and Parsons, 2004); by Fenton system (Benitez et al., 2001; Gernjak et al., 2004 and Dincer et al., 2008). Other treatments were also used such as ultrafiltration (Akdemir and Adem, 2008); electrochemical oxidation (Longhi et al., 2001 and Gotisia et al., 2005).The present study was aimed to reduce the phenolic compounds and COD contents of olive mill wastewater (OMW) by using biological and chemical treatment methods. Aust. J. Basic & Appl. Sci., 3(2): 1087-1095, 2009 1088 MATERIALS AND METHODS Materials: Olive mill wastewater (OMW) was obtained from Horticultura department, Agricultural Research Center, Giza, Egypt. Pleurotus ostreatus (NRRL–2366) and Phanerocheate chrysosporium (NRRL-6361) were provided from Northern Regional Research laboratory, Illinois, Peoria, USA and used as standard strains in the present study. The microorganisms were subcultures every 30 days. Rose-Bengal chloramphenicol agar medium (The Oxoid, 1979) used for isolation of fungi from OMW. Potato Dextrose Agar medium (PDA) (Martin; 1950) used for maintenance of the isolated fungi. Some of fungal isolates isolated from OMW on the same medium. Methods: Isolation and Identification of Fungal Isolates from OMW: According to the method described by Gilman (1982) the specialists in plant pathology department (A.R.C), Giza, Egypt identified the fungal isolates that were under study. Ten milliliter of OMW was serial diluted up to 10 . One ml of each dilution was plated (in triplicate) on both Rose-Bengal chloramphenicol and -7 Martin’s media. The plates were incubated at 28°c for 24 hr. Determination of Chemical Constituents of OMW: Total lipids were determined according to the method described by Halmiton and Shiela (1992). Total nitrogen was determined by Kjeldahl method (Cottenie et al., 1982).Total sugars were determined by the method of (Dubois et al., 1956). Ash content was determined according to (AOAC, 1990).Tannins were determined according to the procedure described by Hagir et al. (1997). Phenolic compounds were extracted according to the method of (Elena De Marco et al., 2006). 10.0 ml of OMW was mixed with 15 ml of hexane; the mixture was vigorously shaken and centrifuged for 5 min at 3000 rpm. The phases were separated and the washing was repeated two times successively. Extraction of phenolic compounds was then carried out with 10 ml of ethyl acetate after acidification by HCl to pH 2. The phases were separated and the extraction was repeated four times successively. The ethyl acetate was evaporated under vacuum and the dry residue was dissolved in 3 ml of methanol and this solution was used for determination of phenolic compounds. Total phenols were determined spectrophotometrecally as described by Swain et al. (1959). Minerals (K, Ca, Na, Zn, Mn, Cu, Fe, Mg, and P) were determined according to the method described by Cottenie et al. (1982) 5 using Atomic Absorption Spectrophotometer (Perkin-Elmery model: 372). Biochemical oxygen demand (BOD ) was determined as the method described by Lenore et al. (1992).Chemical oxygen demand (COD) was determined according to the method described by Arnold et al. (1992). Biological Treatments: According to Warcup (1957) the ability of isolated fungi to degrade the phenolic compounds and reducing the COD contents in OMW was examined as the following: Seven fungal species that were previously identified namely Aspergillus clavatus, Mucor strictus hagem, Penicillium nigricans, Penicillium cilreaviride, Aspergillus wentii, Penicillium chermesinum and Aspergillus niger were tested. Each fungus was inoculated into 250 ml conical flasks containing 50 ml of sterilized OMW (20%). The flasks were shaken (150 rpm) for 1 week at 25°C. Phenolic compounds and COD content were determined as descried above. Chemical Treatments: Several chemical oxidation treatments were carried out primarily on tannic acid solution and one of these treatments was chosen for OMW treatment: It was treated as the method described by Fotiadis et al. (2007) and Benitez et al. (2001). Chemical oxidation of tannic acid solution was conducted for 2.5 hours at 25°C by using several oxidizing agents: aUV radiation at 254 nm. bCombination of UV radiation with two different concentrations of hydrogen peroxide (0.05M and 0.11M) at three pH values 5, 7 and 9. cSingle Fenton's reagent: A solution consists of hydrogen peroxide and ferrous sulfate. Two different 2 2 concentrations of ferrous sulfate 4.6 X 10 and 2.3 X 10 M were used with 0.05 M and 0.11 M H O -3 -3 (four treatments) at three pH values 5, 7 and 9. Aust. J. Basic & Appl. Sci., 3(2): 1087-1095, 2009 1089 dCombination of Fenton reagents with UV radiation at three pH values 5, 7 and 9. 2 eTitanium dioxide treatments: different amounts of TiO (25, 50, 75, 100 and 125 mg) were added to one liter of tannic acid solution and exposed to UV radiation. In all of the previous experiments the tannic acid concentration was determined in a sample every 30 min along the treatment period (150 min). Chemical Treatment of OMW: 2 2 The OMW is oxidized by three concentrations of H O (0.11, 0.55 and 2.0 M) at pH 9 in the presence and absence of UV radiation. A sample was taken every 30 min. along the treatment period (150 min) for determination of total phenols. RESULTS AND DISCUSSION Chemical Composition of OMW: Olive mill wastewater was analyzed for its content. Tables (1) and (2) show the chemical composition of OMW samples as g/100 ml OMW. While the mineral analysis showed that the major element in this waste was potassium (475 mg/100ml), followed by calcium and sodium (250 and 175 mg/100ml, respectively). These results were in agreement with the results obtained by Paredes et al. (1999) who analysed the OMW in deferent localities from deferent verities and recorded a range for each measurement parameter. He found that total phenols ranged were from 1.32-3.99 mg %, while these results were recorded 3.46 mg % for total phenoles. Biological Treatment of OMW: As shown in Table (3), the results indicate the maximum reduction of COD was 68.4% by A. wentii while the minimum COD reduction (7%) was obtained with P. chrysosporium. Where as the maximum degradation of the phenolic compounds (70%) was obtained by A. niger. It was similar to that obtained by P. ostreatus and P. chrysosporium their values 70.6%, 75.0% and 70.2% respectively. On the other hand, P. nigricans recorded the minimum reduction of phenols (54%). These results were in agreement with the results obtained by Hamdi et al. (1991). They reported that Aspergillus niger removes 61% of COD and 58% of total phenolic compounds of OMW. Also Chwei et al. (2003) found that A. niger removes 35% of COD of OMW in batch culture. Sayadi and Radhouane (1993) reported that p. chrysospo