Iranian Journal of Environmental Health, Science and Engineering Iranian Association of Environmental Health (IAEH) ISSN: 1735-1979 Vol. 7, Num. 3, 2010, pp. 199-208

Iranian Journal of Environmental Health, Science and Engineering, Vol. 7, No. 3, 2010, pp. 199-208

Influence Of Bioaugmentation In Biodegradation Of Pahs-Contaminated Soil In Bio-Slurry Phase Reactor

¹S. Nasseri, *²R. Rezaei Kalantary, ³N. Nourieh, ¹K. Naddafi, ¹A. H. Mahvi, ¹N. Baradaran

¹ Department of Enviroment Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
² Department of Enviroment Health Engineering, School of Health, Iran University of Medical Sciences, Tehran, Iran
³ Department of Enviroment Health Engineering , School of Public Health, Lorestan University of Medical Sciences, Khoramabad, Iran

*Corresponding author: E-mail: : rrkalantary@iums.ac.ir Tel: 021 88 77 91 18

Received 13 January 2010; revised 11 May 2010; accepted 20 June 2010

Code Number: se10023

ABSTRACT

Polycyclic Aromatic Hydrocarbons (PAHS) are important pollutants which have toxic, carcinogenic and mutagenic properties and are considered as a serious hazard to human health and environment. Bioremediation of PAHs contaminated soil was studied in the soil slurry phase bioreactor. For enhancement of biodegradation, bioaugmentation (which is the process of adding microorganisms with the potential of pollution biodegradation to the bio-slurry reactor) was applied. Phenanthrene (C₁₄H₁₀), a three-benzene ring PAHs, was added in concentration of 100mg/kg soil. Two isolated species and consortium of bacteria were inoculated to the medium in density of 7×10⁷ CFU/mL. The analysis of variance (ANOVA) was used for finding of optimum levels of type of bacterial culture and presence effect of endogenous factors. The base of the bacteria was petroleum-contaminated soil from around Tehran petroleum Refinery. Control reactor (killed bacteria) showed 5% loss of phenanthrene and biodegradation in the non-augmented reactor (endogenous microorganisms) in a slurry bioreactor was about 17%. In the case of bioaugmentation with Pseudomonas.spp, Pseudomonas aeruginosa and consortium, phenanthrene degradation efficiency were 87.8%, 85.5% and 92.8% ,respectively, presenting the positive effect of biodegradation in consortium augmented compared to the isolated one. Colony forming units (CFUs) variation showed good conformity and agreement with the performance of the reactors with respect to phenanthrene degradation. Hence, the results of this experiment show that bioaugmentation may be considered as an effective method to enhance the bioremediation in removal of PAHs from contaminated soils.

Key words: Polycyclic aromatic hydrocarbons; Phenanthrene; Bioremediation; Bioaugmentation; Native microorganism

INTRODUCTION

Polycyclic Aromatic Hydrocarbons (PAHS) are important pollutants which are introduced into the environment through different ways such as anthropogenic activities, combustion, undesirable discharging of oil tankers, spills around petroleum refineries and gas plant facilities (Liebeg and Cutright, 1999; Leblond et al., 2001; Mahvi and Mardani, 2005; Venkata Mohan et al., 2008; Zhao et al., 2008).

These compounds have toxic, carcinogenic and mutagenic properties (Mrozik et al., 2003 ; Ganjidoust and Naghizadeh, 2005; Samimi et al., 2009 ) and are considered as a serious hazard to human health and environment (Karimi-Lotfabad et al., 1996; Yerushalmi and Guiot, 2001; Delnavaz et al., 2008; Venkata Mohan et al., 2008; Anastasi et al., 2009; Larsen et al., 2009; Muckian et al., 2009; ). PAHs are categorized as priority environmental pollutant by United State Environmental Protection Agency (USEPA) and European commission (EU).

Bioremediation in which microorganisms are used to metabolize petroleum compounds is a cost – effective alternative for cleaning up PAHs-contaminated soils (Akhavan et al., 2008; Wong, et al., 2002). Three important factors affect the effectiveness of bioremediation: environmental, physical and chemical factors. The most important physical factor is availability of contaminant for the microorganisms. Availability is a complicated concept involving affinity of contaminants for the solid and gas phases and presence of the necessary microbial communities. PAHs are nonpolar and tend to partition onto the solid phase, which results in very low liquid phase contaminant concentration. In bioremediation, the most important chemical factors are the molecular structure and biodegradability (Ewies et al., 1998; Rittman and McCarty, 2001). Because of low bioavailability, hydrophobicity, toxicity and complex structures of PAHs derivatives, bioremediation of these contaminants are complex (Venkata Mohan et al., 2008).

For soils in which the microorganisms do not have the ability of biodegradation of these compounds, bioaugmentation is recommended as a process for enhancement of the ratio of bioremediation. Bioaugmentation is the addition of endogenous or genetically engineered microorganisms (GEM) with the potential of pollution biodegradation (Vogel, 1996; Limbergen et al., 1998; Zhang et al., 2000 ; ; Venkata Mohan et al., 2005; Venkata Mohan et al., 2006; Venkata Mohan et al., 2008).

The use of native microflora for bioaugmentation is prefered because these microorganisms have more ability for adaptation to particular pollutant than non-endogenous microorganisms (Silva et al., 2009) and using GEMs may be lead to gene transfer which is not desirable (Urgun-Demirtas et al., 2006). Jacques reported that Kästner by using PAH degrader bacteria could achieve to about six and ten fold increasing in biodegradation of pyrene and anthracene respectively (Jacques et al., 2008).

Phenanteren a three-ring angular PAH was used as a model of PAHs (Samanta et al., 1999; Vossoughi et al., 2002). The bioslurry reactor is a recent area of interest to environmentalists in bioremediation studies because the bacteria and hazardous materials are in a solid-liquid medium under predefined optimized control system (Venkata Mohan et al., 2004). Mixing in slurry-phase results in agitation and surface scrubbing that can result in the release of some of the adsorbed contaminants.

The objective of the present study was to investigate the effect of bioaugmentation in an ex-situ slurry phase by inoculation of isolated and consortium of high potential PAHs biodegrader bacteria.

MATERIALS AND METHODS

Chemical agents

Phenanthrene with purity of 98% and acetonitrile in HPLC grade were purchased from Merck Company. Nutrient agar and R2A agar were purchased form Hi Media. Chemical materials for mineral salt medium (MSM) were purchased from Merck, Sigma and Aldrich Chemical Companies.

Adaptation

Contaminated soil for preparing PAHs microbial culture was taken from surrounding of Tehran refinery because of long time contact and suitable properties(Alimohammadi et al., 2005). Bacteria were grown in an aqueous MSM contained per litre: 0.8 g K₂HPO4, 0.2 g KH₂PO4_, 1 g KNO_3, 0.2 g MgSO₄.7H₂O, 0.1 g CaCl₂.2H₂O, 0.1 g NaCl, 0.01 g FeCl₃.6H₂O and 1 mL trace element solution. The trace element solution contained per liter:23 mg MnCl₂.2H₂O, 30 mg MnCl_4, 32 mg H₃BO_3, 39 mg CoCl₂.2H₂O_, 50 mg ZnCl_2, 30 mg NaMnO₄.2H₂O and 20 mg NiCl₂ (Ressler et al., 1999).

For preparing microbial culture 100 mL MSM was added to 10 g of contaminated soil (10% w:v). The mixture was stirred for 24 hours using a magnetic stirrer. After 20 minutes for settling, one mL of supernatant was added to 50mL sterile MSM plus phenanthrene. The MSM was freshened every week to prevent any deficiency of nutrients and carbon source. After one month, the adapted microorganisms were brought on nutrient agar for supplying later experiments (Rezaei Kalantary and Badkoubi, 2004).

Experimental methods

Two isolated cultures and a consortium of bacteria which had the potential of PAHs biodegradation were used for inoculation in bioagmentation. Soil was passed through 2mm sieve, then was spiked with the phenanthrene at a ratio of 100 mg/kg soil.

The soil was inoculated with bacterial culture which was prepared in MSM. The optical density of the culture at 630 nm was 1 (OD at E_{630 nm}=1) (Ressler et al., 1999). Experiments were conducted in 1L flasks each containing 40g soil and 400mL MSM.

In order to find the optimum levels of type of bacterial culture and presence effect of endogenous factors, full-factorial design was applied. Statistical method was analysis of variance (ANOVA).

The sample had the similar blanks; B₁ was the sterile soil and B₂ was the nonsterile soil (with endogenous microflora). CB₁, CB₂ and CB₃ were the sterile soils, inoculated with the same culture as samples. All of these soils, were spiked before inoculation with the same concentration of phenantherene with their similar samples. MB₁, MB₂ and MB₃ were the sterile soil with the same inoculation and without spike of phenanthrene (Table 1). Samples and blanks were put in the shaker at the velocity of 180 rpm in room temperature for two months.

Extraction and Analysis

Phenanthrene was extracted from the soil according to EPA 3550B, by using ultrasonic (RK31 h, Bandeling Electronic Sonorex). Samples were analyzed by HPLC (KNAUER Company). The column of HPLC was C18 and the solvent was 95% acetonitril and 5% DDW. It was detected at 254nm. Microbial population was measured by using plate count agar or HPC (heterothrophic plate count media)(Arbabi et al., 2004).

RESULTS

After enrichment and isolation, four spices were isolated: Pseudomonas.spp, Bacillus, Pseudomonas aeruginosa and Acinetobacter. The selections of Pseudomonas.spp and Pseudomonas aeruginosa for developing the bacterial inoculation were based on their demonstrated success for PAHs biodegradation (Nourieh et al., 2010). Hence these two cultures and consortium of bacteria were used for bioaugmentation investigation.

An important decrease in the phenanthrene concentration was observed in all systems (augmented and non-augmented), except for abiotic control, during the first 3 weeks (Fig. 1). Bioaugmentation in all cases showed lower values of residual phenanthrene, but this amount in consortium inoculation was more than the others (Figs. 2, 3, 4), however it was not significant (Fig. 1).

The trend of phenanthrene removal in bioaugmentation with consortium is shown in Fig. 2. The abiotic control (B1) shows 5% loss in pollutant and the endogenous microorganisms degraded about 17% of phenanthrene in 2 months (Fig. 2). According to the analysis of variance (ANOVA), the F-ratio for inoculated culture and endogenous microorganisms were about 88.3 and 21.7, respectively (Table 2).

The bacterial population after inoculation to sterile and non-sterile systems was about 7×10⁷ and 5×10⁸ CFU m/L respectively (Figs. 5-6). After initial decreasing about 20-300 fold of magnitude in the first week, it increased in 4-6 fold of magnitude in two months (Fig. 6). The population decrease in the blanks continued till the end of the tests (Figs. 5 and 7).

DISCUSSION

The two cultures with high potential for bioremediation which were used for bioaugmentation investigation were gram negative. Mrozik et al (2003) had reported that most of PAHs degradable bacteria are gram negative (Mrozik et al., 2003).

The phenanthrene concentration on the 60th day in S₁, B₁, B₂ and CB₁ (Table 1) were 7.25, 95, 83and 35 mg/kg soil respectively. The reduction of 5% in B₁ which had not any microorganisms may be related to the bounding between phenanthrene and soil texture and aging after two month (Hwang and Cutright, 2002) or may be lost. Ruberto et al, (2003) in their research, in spite of poisoning the culture, obserred 30% loss in control (Ruberto et al., 2003).

Comparison between the other tests shows that the biodegradation in B₂ (with the endogenous microflora) was less than 20%. Hence the endogenous microflora in a bio-slurry system had a weak efficiency (Jacques et al., 2008) and it was about 75% and 50% less than the system with inoculated of consortium to non-sterile and sterile systems, respectively. The F-ratio would also confirm it.

Hamdi et al, (2007) used bioaugmentation/biostimulation for enhancement of PAHs biodegradation (Hamdi et al., 2007). In our research the bioaugmentation had removal efficiency about 85-93%, while Ruberto et al. (2003) reported only about 75% in the best condition (Ruberto et al., 2003). The phenanthrene removal in co-cultivation of Acinetobacter sp. and rice was 87% (Gao et al., 2006). Comparison between CB₁ and S₁ shows that removal in an augmented system (S₁) was about 25% more than the other one. On the other hand, inoculation to a non-sterile system had more efficiency than inoculation to a sterile system, so augmentation could improve the rate of bioremediation (Venkata Mohan et al., 2008), which does not agree with Silva et al., 2009, who had reported that bioaugmentation had not significantly effect on bioremediation of low molecular weight PAHs (Silva et al., 2009). Yu et al, (2005) did not observe any significant difference between natural attenuation and bioaugmentation in phenanthrene removal, but it was in pyrene elimination. They related it to negative interaction which may have occurred between inoculums and endogenous microbial community (Yu et al., 2005).

The culturable microoganisms which can grow in the presence of phenanthrene can be represented by CFU (Venkata Mohan et al., 2008) which was periodically monitored in the reactors during the experiment time. The bacterial population decreased in all testes in about 7 days; Ramirez et al., 2002 had observed the same decrease in their research (Ramirez et al., 2001). The decreasing in B₂ was prolonged till the end of the test which may be due to nonpotential of native soil microflora in phenanthrene biodegradation (Venkata Mohan et al., 2008).

On the other hand, the bacteria were reproduced on a nutrient agar which is a ready medium for consumption. When these bacteria were inoculated to the soil in which the only carbon source is phenamnthrene, they were transferred from a banquet medium to a famine medium which makes the reduction in population and reproduction (Rittman and McCarty, 2001).

Also the interaction or competition between the autochthonous and the enriched microbes may result in the inhibitory effect in bioaugmentation in the initial days (Yu et al., 2005) which may be the other reason for decreasing in bacterial population. The second decreasing in population may be related to substrate reduction or toxic intermediate production (Ruberto et al., 2003; Gao et al., 2006).

Comparison between the isolated and consortium inoculation (S₁, S₂ and S₃) shows that the phenanthrene biodegradation in augmented consortium was more than the isolated one. It may be due to the ability of certain strains in removal of intermediates produced by other members of consortium which facilites the phenanthrene removal. Leblond et al (2001) showed that a mixture of four different species was more effective than each single one in PAHs biodegradation (Leblond et al., 2001).

Jacques et al (2008) reported that the significant synergistic promotion of PAHs mineralization was occurring by mixture of bacteria (Jacques et al., 2008). Churchill et al. 1999 reported that use of isolated bacteria for biodegradation prolonged more than the time needed for mixulture(Churchill et al., 1999).

Fig. 1 shows that 50% reduction of pollution by isolated Pseudomonas.SPP and Pseudomonas aeruginosa occurred in 18 and 20 days, respectively, but it took 14 days for consortium. The phenanthrene removal by isolated Pseudomonas.SPP, Pseudomonas aeruginosa and consortium were 87.75%, 85.52% and 92.75%, respectively.

The trend of phenanthrene concentration changes shows after a lag phase in pure cases, decreasing was rapid till the 37th day; then it became slow. This is similar to Silva et al, (2009) who reported that the non-available fraction is due to physical interaction with soil matrix (Silva et al., 2009). The rate of biodegradation in S₁, B₂ and CB₁ were 1.55, 0.3 and 1.3 mg/kg /day. The removal in augmented system was about 5 times higher than native microflora.

As a conclusion the results of our experiment show that bioaugmentation may be considered as a valuable method to enhance the bioremediation in removal of PAHs in contaminated soils.

ACKNOWLEDGEMENTS

The authors would like to thank the personel of the laboratories of the Department of Environmental Health Engineering at Tehran University of Medical Sciences for their supportive services.

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