Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 37 PSEUDOMONAS SP. AND AEROMONAS SP. SELECTION FOR TREATMENT OF GOOSE DOWN WASHING WASTEWATER Aleksander SLAVOV1, Zapryana DENKOVA2, Mima HADJIKINOVA1, Nikolai MIHALKOV3 1Department “Environmental engineering” University of Food Technologies, Plovdiv, Bulgaria, aleksander_slavov@yahoo.com , mimi_uht@abv.bg 2Department “Microbiology” University of Food Technologies, Plovdiv, Bulgaria, zdenkova@abv.bg 3”Water projects” Ltd, Plovdiv, Bulgaria vodniproekti@abv.bg *Corresponding author Received 28 August 2012, accepted 4 September 2012 Abstract: 10 bacterial strains, isolated from municipal wastewater and active sludge that belong to Aeromonas sp. (3) and Pseudomonas sp. (7), were investigated. Strains were analyzed for their ability to treat pre-centrifuged and diluted in different ratios goose down washing wastewater. It is shown that Aeromonas sp. exhibit higher purification degree of goose down washing wastewater compared to the strains of Pseudomonas sp. only at a dilution of 1:1, and the highest purification degree is determined for strains Aeromonas 2 and Aeromonas 2AS with 64,94% each. At a dilution of goose down washing wastewater 1:5 the highest purification degree is at a strain Pseudomonas 1 – 86,49%, and at a dilution of goose down washing wastewater 1:10 Pseudomonas 3AS – 87,27 %. Keywords: Aeromonas sp., Pseudomonas sp., goose down washing wastewater, purification degree 1. Introduction From poultry processing as by-product large quantities feathers are produced - 7% of live weight [1]. Utilization of feathers include using of various technologies – composting, in food industry after hydrolysis of keratin [2, 3, 4, 5, 6], as adsorbent of heavy metals [7], filling material for clothes and bags [8], in composite wood MDF [9]. Obtaining quality feather include its preliminary treatment with NaCl and HCl solutions, followed by washing it with detergents and, if necessary - discoloration. As a result large quantities of wastewater with high pollution levels are produced. Biological methods have application in wastewater treatment from food industry, and many of them are based on the specific action of particularly selected bacterial strains. The aim of this work is the selection of Aeromonas sp. and Pseudomonas sp. strains for treatment of goose down washing wastewater. 2.Materials and methods 2.1.Microorganisms In this work are used 10 strains of bacteria. Bacterial strains Aeromonas sp. Aeromonas 2 are isolated from municipal wastewater, while Aeromonas 1AS, Aeromonas 2AS – from activated sludge. Bacterial strains Pseudomonas sp. Pseudomonas 1, Pseudomonas 3, Pseudomonas 4 were isolated from municipal wastewater, while Pseudomonas 1AS, Pseudomonas 2AS, Pseudomonas 3AS, Pseudomonas 4AS – from activated sludge. mailto:aleksander_slavov:@yahoo.com mailto:mimi_uht:@abv.bg mailto:zdenkova:@abv.bg mailto:vodniproekti:@abv.bg Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 38 2.2.Nutrient mediums 2.2.1.Luria – Bertany glucose agar medium (LBG) with composition (g/dm3): triptone (Difco) – 10 g, yeast extract – 5 g, NaCl – 10 g, glucose (Scharlau) – 10 g; agar – 20 g. pH=7,5. The medium is sterilized for 25 minutes at 121º C. 2.2.2.Glutamate-starch-phenol red agar medium (GSP) with composition (g/dm3): sodium glutamate - 10 g, starch (soluble) - 20 g, KH2PO4 - 2 g, MgSO4, Phenol - red - 0,36 g, agar - 12 g. pH 7,2 ± 0,2. All ingredients are dissolved and medium is sterilized for 15 min at 121° C. Cool to 45- 50° C and aseptically add to it 100 IU/ml penicillin G, and if necessary, 10 g/ml pymaricyn. The resulting composition is mixed thoroughly and dispensed in sterile petri dishes. 2.3.Wastewater: In the experiments goose down washing wastewater to slaughterhouse is used. Wastewater is pre- centrifuged at 3000 min-1 for 10 min to remove insoluble substances and fat and diluted with non-sterile distilled water in ratios 1:1, 1:5 and 1:10. 2.4.Cultivation and storage of microorganisms. Isolated strains are grown in LBG agar medium at 30º C in thermostat for 48 h and stored in a refrigerator at 4º ± 2° C for 2 weeks. 2.5.Analytical methods. 2.5.1.Isolation of Aeromonas sp. Pseudomonas sp. strains. Isolation of strains of Aeromonas sp. and Pseudomonas sp. was carried out by seeding samples of municipal wastewater, and active sludge on GSP agar medium and their subsequent development at 30° ± 2° C in a thermostat for 24 h. Colored in yellow colonies belong to Aeromonas sp., while red relate to Pseudomonas sp. 2.5.2.Development of isolated strains of bacteria in the diluted wastewater. With biomass from developed on LBG agar medium at 30° ± 2° C for 48 h bacterial strains 5 cm3 of diluted wastewater is inoculated. Wastewater samples with investigated cultures are cultivated for 72 h at 30° ± 2° C. 2.5.3.Purification degree wastewater determination using the permanganate oxidizability method. Developed for 72 h at 30° ± 2° C in wastewater cultures are centrifuged at 3000 min-1 for 10 min. The resulting supernatant is analyzed for permanganate oxidizability in accordance with standard BS 17.1.4.16- 79. 100 cm3 sample or lower volume brought to 100 cm3 with distilled water is placed in a 250-300 cm3 flask, which has added pumice. 5 cm3 H2SO4 (1+2) and 20 cm3 0,01 N KMnO4 solution are added. The composition is heated so as to boil for no more than 5 min and boiling for 10 min. To the hot solution 20 cm3 0,01 N solution of HOOC-COOH are added. Hot discoloured solution is titrated with 0,01 N KMnO4 solution. The temperature of the solution during titration should not be below 80° C. If the boiling solution is discolored or brown precipitates are formed, the determination is repeated with a smaller sample size. During titration should not spend more than 12 cm3, and not less than 4 cm3 of KMnO4 solution for diluted sample. Similarly, a blank is done with wastewater. Oxidizability (X) in mg/dm3 oxygen is given by the formula: V Nba X 8000.).( , where – VKMnO4, spent for titration of the sample, cm3; b – VKMnO4, spent for titration of blank, cm3; N – exact normality of KMnO4 solution; Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 39 V – volume of sample taken for analysis, cm3. Similarly oxidizability of a control sample of analyzed wastewater at the corresponding dilution of 1:1, 1:5 and 1:10 is determined. Purification degree PD, in %, is determined using the formula: ,%100 s sc PD , where Xc – oxidizability of control sample, mg/dm3 oxygen; Xs – oxidizability of sample, mg/dm3 oxygen. 3. Results and discussion. In a set of experiments the ability of isolated Aeromonas sp. and Pseudomonas sp. strains to break down pollutants in washing goose down wastewater, diluted with distilled water in different ratios, is investigated. Summarized results are presented in Fig. 1, Fig. 2 and Fig. 3. Experimental data show an average level of treatment for all strains of Aeromonas sp. and Pseudomonas sp. at wastewater dilution 1:1, 1:5 and 1:10, respectively. 54,11 35,53 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P ur ifi ca ti on d eg re e, % Aeromonas sp. 1:1 Pseudomonas sp. 1:1 Figure 1. Average purification degree of goose down washing wastewater, diluted in ratio 1:1 with Aeromonas sp. and Pseudomonas sp. It is noteworthy that with increasing dilution increases the degree of purification as in Aeromonas sp., and in Pseudomonas sp. Exceptions are strains Aeromonas sp. with the degree of dilution 1:5 (Fig. 2). Strains Aeromonas sp. purify wastewater better than the strains Pseudomonas sp. when diluted 1:1 with 18,58% (Fig. 1), while at 1:5 and 1:10 dilutions of the strains Pseudomonas sp. show a higher level of treatment - with 34,86% (Fig. 2) and 29,73% (Fig. 3). 39,91 74,77 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P ur ifi ca tio n de gr ee , % Aeromonas sp. 1:5 Pseudomonas sp. 1:5 Figure 2. Average purification degree of goose down washing wastewater, diluted in ratio 1:5 with Aeromonas sp. and Pseudomonas sp. 55,21 84,95 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P u ri fic at io n de gr ee , % 1Aeromonas sp. 1:10 Pseudomonas sp. 1:10 Figure 3. Average purification degree of goose down washing wastewater, diluted in ratio 1:10 with Aeromonas sp. and Pseudomonas sp. A comparative characteristic between strains of Aeromonas sp. and Pseudomonas sp. for the purification degree of goose down wastewater at different dilution ratios is done. Results from these studies are presented in Figure 4, Figure 5 and Figure 6. The experimental data represented in Figure 4 show that with purification degree over 50% are strains Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 40 Aeromonas 2, Aeromonas 2AS, Pseudomonas 3, Pseudomonas 2AS and the highest is the degree at Aeromonas 2 and Aeromonas 2AS – 64,94%. Strain Pseudomonas 3 exceeds the other three Pseudomonas sp. with the purification degree 59, 09 %. 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P ur if ic ai o n d eg re e, % Aeromonas 2 (1:1) Aeromonas 1 S (1:1) Aeromonas 2 S (1:1) Pseudomonas 1 (1:1) Pseudomonas 3 (1:1) Pseudomonas 4 (1:1) Pseudomonas 1 S (1:1) Pseudomonas 2 S (1:1) Pseudomonas 3 S (1:1) Pseudomonas 4 S (1:1) Figure 4. Purification degree of goose down washing wastewater, diluted in ratio 1:1 with Aeromonas sp. and Pseudomonas sp. 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P ur ifi ca tio n d eg re e, % Aeromonas 2 (1:5) Aeromonas 1 S (1:5) Aeromonas 2 S (1:5) Pseudomonas 1 (1:5) Pseudomonas 3 (1:5) Pseudomonas 4 (1:5) Pseudomonas 1 S (1:5) Pseudomonas 2 S (1:5) Pseudomonas 3 S (1:5) Pseudomonas 4 S (1:5) Figure 5. Purification degree of goose down washing wastewater, diluted in ratio 1:5 with Aeromonas sp. and Pseudomonas sp. At a dilution ratio 1:5 (Figure 5) strain Pseudomonas 1 shows the highest degree of purification – 86,49%, followed by strain Pseudomonas 3 – 79,48 %. Third place is shared by strains Pseudomonas 4 and Pseudomonas 3AS - with average value 78,70 %. From Aeromonas sp. with the highest purification degree is strain Aeromonas 2 with 54,23%. At 1:10 dilution ratio (Figure 6) the highest purification degree has strain Pseudomonas 3AS – 87,27%, followed by Pseudomonas 3 and Pseudomonas 1 with 87,19% and 87,01%, respectively. Purification degree of Aeromonas sp. strains reached only 57,95% of Aeromonas 2AS, followed by Aeromonas 2 and Aeromonas 1AS with 56,92% and 50,77%, respectively. Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 41 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 P u ri fic at io n d eg re e, % Aeromonas 2 (1:10) Aeromonas 1 S (1:10) Aeromonas 2 S (1:10) Pseudomonas 1 (1:10) Pseudomonas 3 (1:10) Pseudomonas 4 (1:10) Pseudomonas 1 S (1:10) Pseudomonas 2 S (1:10) Pseudomonas 3 S (1:10) Pseudomonas 4 S (1:10) Figure 6. Purification degree of goose down washing wastewater, diluted in ratio 1:10 with Aeromonas sp. and Pseudomonas sp. 4. Conclusion. As a result of investigated analys s on goose down washing wastewater treatment the following more important conclusions can be made: 1.The more dilution of goose down washing wastewater is done, the greater is its purification degree. Exception is made for strains Aeromonas sp. with dilution factor of the effluent 1:5; 2.Strains Aeromonas sp. exhibit higher purification degree of goose down washing wastewater compared to Pseudomonas sp. strains only at a dilution factor 1:1. 3.At a dilution factor of goose down washing wastewater 1:1 the highest purification degree is determined at strains Aeromonas 2 and Aeromonas 2AS with 64,94%, at dilution ratio 1:5 – at strain Pseudomonas 1 – 86,49% and at dilution factor 1:10 strain Pseudomonas 3AS – 87,27%. 5. References. [1] LORTSCHER, L. L., G. F. SACHSEL, D. WILKELMY JR. AND R. B. FILBERT JR., Processing poultry by products in poultry slaughter plants. 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