Influence of bioaugmentation on biodegradation of phenanthrene-contaminated soil by earthworm in lab scale
© Asgharnia et al.; licensee BioMed Central. 2014
Received: 26 July 2014
Accepted: 14 December 2014
Published: 24 December 2014
Use of earthworm to eliminate the phenanthrene from the soil (bioaccumulation) is developed as an economical method. Bioaugmentation of microorganism was used for promotion of bioaccumulation by earthworm. The aim of this study was to determine the bioaccumulation or biodegradation of phenanthrene by Eisenia fetida and bacterial consortium in polluted soil.
The amount of 0.4 kg of the polluted soil in the ratio of 10 and 30 mg phenanthrene per kg of dry soil was transferred into each pot. Afterwards, bacteria and earthworms were added to each pot in separate and combination. The samples were kept under field conditions, and the retention concentrations of phenanthrene were analyzed after 8 weeks.
Results showed that the Eisenia fetida was able to significantly remove phenanthrene from the polluted soil samples. Bioaccumulation and bioaugmentation alone have the removal efficiency of 60.24% and 50.3%, respectively. In the combined mode, phenanthrene removal efficiency was 63.81%.
The current study indicated that the use of earthworms, could improve both phenanthrene bioavailability and microbial activity, which led to enhancing removal of carbon-based pollutants.
Polycyclic Aromatic Hydrocarbons (PAHs) are a group of more than 100 various chemicals substances formed during the incomplete burning of coal, oil, gas, rubbish and tobacco ,. PAHs are known as the mutagen and carcinogen substance. They are toxic and persistent in the environment ,. Methods based on the techniques used to purify the contaminated soils were divided into three categories: physical, chemical and biological ,. Bioremediation based on the physical and chemical engineering technologies has high energy consumption and therefore their use is expensive, especially when a large volume of surface of soil is contaminated. The physical and chemical interactions of the soil structure are possibly damaged that is unusable for agriculture. A physical remediation method is led to eliminate the pollutants from soil - water composition, which can be used for further refining ,. Advantage of chemical oxidation includes the use of on-site contamination. Due to the relatively low cost for degradation of pollution, the contamination is eliminated. Disadvantages of chemical oxidation are the ability of creating dangerous byproducts and added chemicals (such as hydrogen peroxide, manganese dioxide and iron oxide) which can have a negative impact on soil organisms or chemical composition. The advantages of bioremediation are the possibility of cleaning the site and the lack of relocation and transportation of toxic waste. Ensure that the permanent removal of polluting and combining of this technology to other methods of purification, the survey of producing is mentioned but the disadvantages of bioremediation can be mentioned as following:
The inability of microorganisms to break down all pollutants
Biodegradation process is slower in compared to other methods
Microbiological degradation, bioaccumulation and transformation are the main methods for the remediation of the phenanthrene from the environment ,. Several groups of earthworms, bacteria and fungi are able to partly decompose, co-metabolically oxidize or mineralize the phenanthrene to harmless products ,. Earthworms are generally resistant to many chemicals, including heavy metals and organic pollutants in soil . They are usually able to accumulate the phenanthrene in the body wall by intestinal uptake during the passage of soil via the gut . Several earthworm species have been found to remove heavy metals, pesticides and lipophilic organic micropollutants like the phenanthrene from the soil such as Eisenia fetida, Eisenia Andrei, Eiseniella tetraedra, Pontoscolx corethrurus, Lumbricus terrestris, Lumbricus rubellus, Dendrobaena rubida, Dendrobaena veneta, Aporrectodea tuberculata and Allobophora chlorotica,. Earthworms stimulate and increase microbial activity by creating the favorable conditions for bacteria and improving soil aeration. This can be used to remedy the contaminated sites and can occur on- or off-site, which is facilitation by mixed microbial consortia and/or pure microbial strains and plants . These organisms have an important influence on the distribution and activities of the soil micro flora, also they modify the structures, physical and chemical features of soil and act in the organic matter (O.M) degradation, nutrient cycling, chemical exchange and humidity holding through ingesting, burrowing, and casting activities . During the Vermiremediation process of the soil, the population of earthworms increases for benefiting significantly the soil remediation in several ways included promoting the fragmentation, soil aeration and soil turning and scattering brings. Earthworms accumulate many Polycyclic Aromatic Hydrocarbons (PAHs) from the soil, not only throughout inactive absorption of the dissolved fraction through the body wall, but also by intestinal uptake during the passage of soil through the gut . Castellanos et al. (2013) showed that the earthworm (Pontoscolex corethrurus) can be used in bioremediation of hydrocarbon contaminated soil . Monard et al. (2008) found that earthworms (Lumbericus terrestris) could improve atrazine degradation by changing the microbial community structure of indigenous microorganisms and species bioaugmented . Moreover, Butenschoen et al. (2009) showed that earthworms improved the microbial activity and thus mineralization of phenolic compounds .
There are few bacterial strains with the capacity of the degradation of phenanthrene -. Bacteria are capable of degrading hydrocarbons, but when the earthworms are located in the soil, they will improve the ventilation, motivate the microbial action and therefore biodegradation may be enhanced ,. Previous study indicated that, the bioaugmentation was as an effective method to enhance the bioremediation in removal of phenanthrene from contaminated soils ,, but Yu et al. (2005) stated that there was no significant difference between natural attenuation and bioaugmentation in phenanthrene degradation due to the negative interaction between the inoculums and the indigenous microflora .
The objectives of this study were: 1) To determine the phenanthrene removal from contaminated soil by co-existing of earthworm and bacteria; 2) to explore the effect of OM content on the phenanthrene removal efficiency by earthworms and bacteria.
Material and methods
Phenanthrene and acetone were obtained from Aldrich chemical company (USA), with a >98% purity and J. T. Baker (USA) with purity 99.7%, respectively. Solvents used for extractions include the acetone and methanol (Merck, Germany) which were of high performance liquid chromatography (HPLC) grade, and other chemicals were of analytical grade.
Experimental set up
The chemical and texture characteristics of the soil
Treatment of experimental design
State if condition
Bacteria (staphylococcus epidermidis + bacillus subtellis)
Earthworm (Eisenia fetida)
For extraction of phenanthrene from the soil, method of 3550B EPA was used with some modification that was previously defined by Sheng-wang et al. (2008) . Soil samples were carefully collected and homogenized. 2 grams of samples were placed in an Erlenmeyer flask and mixed with 10 ml acetone. Subsequently, 2-minute ultrasonic (cleaning, UK) of these samples and magnetic mixture (Hidolf, Germany) were done during1 hour at a velocity of 200 rpm. The sample was purified with the micro filters with a pore size of 0.22 μm. The chemical analysis was performed using the High Performance Liquid Chromatography (HPLC, CECIL 4100, USA) tool having a C18 column with a length of 25 cm, an internal diameter of 4.2 mm, a UV/VIS detector and a mobile phase of methanol, water v/v 80:20), operating at a flow rate of 1 ml/min at a wavelength of 220 nm.
Bacterial counts were done by using the serial dilution method. Initially, 1gr of the soil sample was suspended with 10 ml and sterilized in normal saline to access a dilution of 10−1. Then, 1 ml of this suspension was injected with 9 ml of normal saline. The serial dilution was done up to 10−12. 1 ml of all dilutions was cultivated on nutrient agar (Merck, Germany) by use of pour plate method. The plates were incubated at 35°C for 24 hours and then the colony count was performed and reported as the number of colony-forming units per g dry soil (CFU/g) .
For extraction of phenanthrene from the earthworm, the recommendation set by the Organization for Economic Cooperation and Development (OECD, 1984) was used with some modification that was previously defined by Kelsey et al. . To assess the ability of the phenanthrene removal from the soil, the Eisenia fetida were used through bioaccumulation after separation of the other earthworms from the soil. Initially, the earthworms were washed by using the tap water, then, weighted by using the digital scaling next, placed in the moist filter paper for 24 hours due to the exertion of the gut. After rewashing, killing of earthworms was done by cooling in the refrigerator and then their corpses transferred into the oven at 65°C for 10 minutes. Then 10 ml acetone was added and mixed for 1 hour in the 200 rpm. After that, the purification and chemical analysis of these samples was performed similar to the phenanthrene analysis in soils. The accumulation of phenanthrene was reported by means of the nanogram per gram of body weight of the earthworm.
After collecting data by the use of Excel 2013 and SPSS (version 14.0), the statistical analysis was performed using the design expert (version 7.0) and One-way Anova test.
Results and discussion
Post Hoc Test for phenanthrene removal efficiency
Mean ± SD (removal percentage)
48.93 ± 1.78
55.47 ± 9.11
Bacteria + earthworm
58.73 ± 8.72
16.37 ± 3.69
55.47 ± 9.11
Bacteria + earthworm
58.73 ± 8.72
16.37 ± 3.69
48.93 ± 1.78
Bacteria + earthworm
58.73 ± 8.72
Bacteria + earthworm
16.37 ± 3.69
48.93 ± 1.78
55.47 ± 9.11
Anova for phenanthrene removal efficiency
Sum of squares
The results of the current study indicated that the bacteria and earthworms could effectively cooperate to remove phenanthrene from soils in two approaches: earthworm uptake and enhanced biodegradation. More experiments about the mechanisms of the actuated biodegradation showed that earthworms led to enhance the microbial activity and an increased bioavailability of phenanthrene, that in turn worked synergistically of facilitate the microbial degradation of phenanthrene in soils. The current study supports the use of earthworms, together with microbial degradation technologies, to produce the comprehensive, innovative remediation approach, according to ecological roles in the removal of carbon-based pollutants.
Authors appreciated Iran University of Medical Sciences for financial supports. All authors like to thank to Dr. Masoud Hashemi and Dr. Abazar Pournajaf because of the microbial analysis.
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