Oily wastewaters treatment using Pseudomonas sp. isolated from the compost fertilizer
© Azhdarpoor et al.; licensee BioMed Central Ltd. 2014
Received: 19 November 2012
Accepted: 16 April 2014
Published: 28 April 2014
Discharging the oily wastewater in the environment causes serious problems, because of the oil compounds and organic materials presence. Applying biological methods using the lipase enzyme producer microorganisms can be an appropriate choice for treatment of these wastewaters. The aim of this study is to treat those oil wastewaters having high concentration of oil by applying lipase enzyme producer bacteria.
Materials and methods
Oil concentration measurement was conducted using the standard method of gravimetric and the wastewater under study was synthetically made and contained olive, canola and sunflower oil. The strain used in this study was Pseudomonas strain isolated from compost fertilizer. The oil under study had concentration of 1.5 to 22 g/l.
The oil removal amount in concentrations lower than 8.4 g/l was over 95 ± 1.5%. Increase of the oil's concentration to 22 g/l decreases the amount of removal in retention time of 44 hours to 85 ± 2.5%. The best yield of removing this strain in retention time of 44 hours and temperature of 30°C was achieved using Ammonium Nitrate as the nitrogen resource which yield was about 95 percent.
The findings of the research showed that Pseudomonas bacteria isolated from the compost fertilizer can degrade high concentration oils.
Oily wastewaters produced in oil factories provide various environmental problems, because of having different pollutant compounds [1, 2]. Reduction of the surface water's oxygen and its effect on the aquatic organism strains are among the problems . Various physical, chemical and biological methods have been used to remove such compounds [4, 5]. Cheryan and Rajagopalan show that the conventional treatments such as gravity separation, air flotation, coagulation are not efficient to solve this problem, especially when the oil droplets diameters are less than 20 μ. Fischer et al. worked on a combination of gravity separation and a downstream microfiltration. Sarakulski et al. proposed to treat oily wastewater by a combination of ultrafiltration and reverse osmosis processes. However, for real wastewaters is observed a membrane fouling or a high regeneration frequency .
In biological methods, the microorganisms with high enzyme activities can degrade oil. Lipase is an enzyme which can degrade fats into glyceride and fatty acids . Many microorganisms including fungi, yeast and bacteria are able to produce lipase enzyme. They have been studied in many researches. Penicillium, Yarrowia, Geotrichum, Bacillus, Acinetobacter and Serratia are samples of these microorganisms, among which bacteria are more applied in oily wastewaters treatment [8–10]. Dongzhi et al., worked on construction of a whole-cell catalyst displaying a fungal lipase for effective treatment of oily wastewaters. They declared that 96% of oil (5 mg/l oil) and 97% of COD were removed . Lan et al. investigated biodegradation of oil wastewater by free and immobilized Yarrowia lipolytica. Their results showed that immobilized Y. lipolytica might be applicable to a wastewater treatment system for the removal of oil . Bacteria which are able to produce lipase can be found in various places, including dairy industries and oil wastes, hot springs and soils contaminated with oils [13, 14]. Hasanuzzaman et al. separated a novel, oil-degrading bacterium from a hot spring in Japan. The 16S rRNA gene sequence analysis revealed it as a new strain of Pseudomonas. Selva et al., examined isolation of lipase-producing Bacillus strains from the soil sample of coconut oil industry. Results indicated that the lipase activity was maximum (2.2U/ml) for Bacillus sp. in 1.5% concentration . Adding lipase-producer bacteria into the biological treatment units can increase the yield of oily wastewater treatment systems . In fact, these bacteria speed up the treatment process through fat degradation. In this study, strains were isolated from compost fertilizer obtained from a solid waste disposal plant. Various researches have shown that compost fertilizer is suitable for isolating the resistant bacteria with a high ability to decompose organic compounds. Anna et al. (1998) showed that methanotrophic bacteria isolated from compost successfully converts CFCs into simpler products . In other experiments, Ghazifard et al. (2001) reported the isolation of heat resistant microorganisms from a composting mass . Since the compost fertilizer is suitable for the growth of various microorganisms which are able to degrade resistant compositions and since the related bacteria have been in contact with various oil combinations (edible and industrial) and on the other hand, there has not been any similar research on degrading the oil combinations by the lipase-producer bacteria isolated from compost fertilizer, we decided to conduct such an experiment. In this research a Pseudomonas strain was applied to treat oily wastewater whose removing oil capability was much more than that in other researches. Particularly interesting is the residual oil that oily wastewater contains in variable quantities, thus making this waste a potentially suitable growth culture for lipolytic bacteria. The interest of this study is to show that isolated Pseudomonas sp can be a strong and appropriate strain for bioaugmentation of aerobic treatment of oily wastewaters with high oil levels. In addition, optimal conditions of the degradation process were identified and proposed.
Materials and methods
Isolation and Inoculums preparation
Oil degrading bacteria were isolated from different locations included (1) hot spring, (2) oil wastewater treatment system, (3) refinery and (4) compost fertilizer. A 10-mL sample of the oily wastewater (or supernatant derived from 5 gr compost fertilizer) was added to an Erlenmeyer flask containing 100 mL of olive oil 2% and KH2PO4 0.2% and ammonium chloride 0.4% (OPY), at 35°C and shaked (100 rpm) for 48 h. Samples were serially diluted, plated onto tween 80 agar and incubated at 35°C for 72 h. Its lipase activity was distinguished in Tween 80 culture , which composed of peptone (1%), Tween 80 (1%), sodium chloride (0.5%), calcium chloride (0.1%), sodium chloride (0.5%), calcium chloride (0.1%) and agar (1.5%). The bacteria had a white halo around the colony. The strain was distinguished using biochemical tests and the bacteria's morphological characteristics . The bacteria were kept on the YPA culture (peptone, 0.5%, yeast extract, 0.3% and agar 1.5%) at 4°C temperature.
The bacteria's lipase activity was measured using polyvinyl alcohol and olive oil emulsion as substrate at 35°C. Cells were separated from the cultivation medium by centrifugation at 6000 rpm for 30 min and the supernatant was used as the source of extracellular lipase. Oil emulsion prepared by mixing 25 ml of olive oil and 75 ml of poly vinyl alcohol 2% solution in homogenizer for 3 min at 5000 rpm. The reaction mixture containing 5 ml of olive oil emulsion, 4 ml of 0.1 mol Tris buffer (pH = 8), 1 ml of 0.1 mol CaCl2 and 1 ml of the culture supernatant was incubated at 37°C for 20 min through orbital shaking. The emulsion was immediately disrupted after incubation by the addition of 15 ml of acetone–ethanol mixture (1:1 v/v), and the liberated free fatty acids were titrated with 0.05N NaOH. One unit of lipase activity was defined as the amount of enzyme required to release one micromole of fatty acid per minute under the test conditions .
The cell mass was calculated through measuring the amount of culture's absorbance in wavelength of 600 nm (OD600) by the spectrophotometer. Then it was calculated based on g/l by the cell mass and the absorbance amount . Cell concentration was equal 0.48 OD600. Three factors indicated the survival the Pseudomonas in synthetic and real wastewater: 1) Survival of Pseudomonas was investigated using re-culture (subculture) of samples after 44 hours. 2) Also, no bacteria growth was observed in the control sample (without Pseudomonas). 3) The medium color changes from clear to milky color indicated the viability of the Pseudomonas.
Synthetic and real wastewater
Characteristics of oily wastewater supported from Behshahr factory
26000 ± 500
20 ± 5
300 ± 10
5500 ± 100
Oil and grease
Oil concentration measurement
Oil concentration measurement was conducted using the standard method of gravimetric, during which the oil was extracted three times using the Hexane and transferred to a distilling flask with a specified weight. Then the Hexane was evaporated at 85°C temperature, and then the flask was reweighted. The oil concentration (g/l) was calculated through dividing the first and the second weight difference by the sample volume .
Results and discussion
The ability of 3 strains, which presented the highest lipolytic activity or predominated to growth and degrade an oily wastewater medium, was studied. The selected strains were identified as belonging to the Flavobacterium (oily wastewater), Pseudomonas (compost), and Acinetobacter (refinery) genera. The bacteria originating from compost showed highest removal of oil. Oil removal efficiencies were 30, 47 and 95% by Flavobacterium, Acinetobacter and Pseudomonas, respectively. The strain which belonged to a special kind of Pseudomonas sp. was applied for the next stages. Lipase activity of the strain was obtained 1.7-2.2 ± 0.1 U/ml in medium supplemented with 2% olive oil and in 21–68 hours after inoculation. All the Erlenmeyer flasks were incubated at 30°C temperature and oil removal rate was monitored in 24 to140 hours. The effect of different nitrogen resources is among the parameters which must be studied for degrading oil biological combinations. This has been usually ignored in similar studies. Seven materials (each sample was 4 g/l) were used as the nitrogen resource in the synthetic wastewater.
The findings of the research revealed that Pseudomonas sp. which was isolated from the compost fertilizer can degrade the high concentration of oil. The Pseudomonas sp. as a lipase producer is able to remove such oils at mesophilic temperature range in a short retention time; and the increase of the oil concentration and the oil type do not affect the yield. Therefore, this strain may develop the biological treatment in processes of the oily wastewater treatment.
This work was supported by the environmental and safety health department, medical college, Tarbiat Modares University.
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