The main objective of this study was to isolate and identify PCB-degrading bacteria from PCB-contaminated soil and test them for their ability to degrade PCBs in natural habitat conditions. Whereas Pasteurella pneumotropica could remove 24% of the PCBs, Burkholderia cepacia 21% of the PCBs and the mixed culture removed 23%.
General Background
The production and illegal use of PCBs was regulated by the United States Environmental Protection Agency (USEPA) in 1978 (Kafilzadeh et al., 2011; Gray, 2004). In general, PCBs can enter the environment during their production, use and disposal (Brody et al., 2008).
Problem Statement
One of the best methods to clean up contaminated soil is to use certain bacteria that degrade PCBs through bioremediation processes (Broding et al., 2008; Fatima et al., 2005; ATSDR 2001). Bioremediation processes are simple to perform, applicable to large areas, cost-effective and usually lead to the complete destruction of the contaminant in question (Hatamian-Zarmi et al., 2009; Begonja et al., 2007; Fatima et al., 2005).
Motivation
Hypothesis
Research Objectives
General Objectives
Specific Objectives
Introduction
However, the appropriate degradation technique depends on many factors, such as treatment time, cost and type of environmental medium, site sensitivity, climatic factors, PCB molecular weight, and PCB concentration (Field and Sierra-Alvarez, 2008; Leigh et al. , 2006). Jong-Su Seo et al., (2009) identified a high risk to humans and the Chinese white dolphin (Sousa chinenses) from the consumption of PCB-contaminated marine species (Jong-Su Seo et al., 2009).
Origin of Polychlorinated Biphenyl (PCBs)
The impact of PCBs on the Environment
The impact of PCBs on Air
Field et al., (2008) and Al-khalid et al., (2012) indicated that the atmosphere is the main transport pathway for PCBs worldwide, especially those congeners with 1 to 4 chlorine atoms. Animals that inhale a certain amount of airborne PCBs can develop liver damage, anemia and thyroid damage (Al-khalid et al., 2012; Field et al., 2008; Woods et al., 1999).
The impact of PCBs on Soil
Human intake of PCBs occurs through consumption of termite soil, fish, and fruits and vegetables (Al-khalid et al., 2012). Adeyemi et al., (2009) reported that leakage of PCBs contaminates soil and consequently causes health hazards in water sources.
The impact of PCBs on human health
Polychlorinated biphenyls do not degrade due to their high solubility in water, they bioaccumulate in the entire food chain (Duffy et al., 2002). Previous studies have shown that PCBs alter estrogen levels in the body and contribute to reproductive problems (Adeyemi et al., 2009; Atagana 2009).
Polychlorinated Biphenyl (PCBs) in South Africa
Polychlorinated biphenyls have affected pregnant women exposed to relatively high concentrations of PCBs (Aluyor and Ori-Jesu, 2009). The removal of PCB-containing transformers began in 2007 and they destroyed 10.0 tons of PCB-containing equipment, in 2008 they destroyed 17.0 tons and in 2009 they destroyed 505.6 tons and dumped them in a registered landfill (Eskom, 2009 ).
Methods for PCBs remediation on the contaminated Soils
- Physico-chemical methods of remediation of PCBs contaminated soil
- Microbiology and biochemistry of PCB biodegradation
- Role of bacteria in PCBs degradation
- Biological factors
- Environmental factors
Joutey et al., (2013) determine absorption as an analogous process where a contaminant penetrates the bulk mass of the soil matrix. It affects the rate and process of biodegradation to take place (Hank et al., 2010).
Methods of studying microbial biodiversity of soil
- Biochemical-based techniques to study microbial population
- Sole carbon Source utilization (SCSU) patterns
- Plate Counts
- Fatty Acid Methy Ester (FAME) Analysis
- Molecular-Based Techniques
- Guanine plus Cytosine (G + C) content
- Nucleic acid reassociation and hybridization
- PCR – Based techniques
- Denaturing Gradient Gel Electrophoresis (DGGE)
- Restriction Fragment Length Polymorphism (RFLP)
- Terminal Restriction Fragment Length Polymorphism (T-RFLP)
- Ribosomal Intergenic Spacer Analysis (RISA)/ Automated Ribosomal Intergenic
- Highly Repeated Sequence Characterization or Microsatellite Region
Which includes PCR-based methods such as Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), Ribosomal Intergenic Spacer Analysis (RISA) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) techniques (Kirk et al., 2004). They compare the common species present in soil samples using nucleic acid reassociation and hybridization techniques (Bing-Ru et al., 2006; Kirk et al., 2004). It can also be used to measure the spatial and temporal change in bacterial communities (Bing-Ru et al., 2006; Kirk et al., 2004).
The technique is used to identify bacterial genomic fingerprints of chromosome structures (Ranjard et al., 2000).
Summary of Literature Review
Highly repeated sequence characterization or microsatellite region technique contains the repetitive short DNA sequence that is 1-10 base pair long. The similarity can be compared using the indices to examine the difference between the interspecific and intraspecific level (Kirk et al., 2004). The highly repeated sequence characterization or microsatellite region technique faces certain limitations depending on the complexity of the bacterial community.
The technique is good for developing probes for detecting any change in the microbial community caused by environmental change (Bing-Ru et al., 2006; Kirk et al., 2004).
The Study Area
Soil samples collection
Assessment and Quality Control of PCB in the Laboratory
Infrared Spectroscopy (IR) Analysis of PCBs
Gas Chromatography-Flame Ionization Detector (GC-FID) Analysis of PCBs
Transformer oil was extracted from contaminated soil samples by taking 10 g of contaminated soil and soaking in 100 ml of heptane and mixing with a spatula. The conditions were optimized and summarized as follows: high purity nitrogen gas was used as the carrier gas at a flow rate of 1 ml/min. The composition gas of the FID detector was ultra-high purity hydrogen gas at a flow rate of 25 ml/min.
A sample volume of 0.5 µL was manually collected with a syringe and injected into the injector without splitting.
Isolation and testing of PCB degrading Bacteria
- Culture Medium
- Chemicals and reagents
- Isolation of PCB degrading bacteria from contaminated soils
- Identification of the isolated Bacteria Strains
- Total plate count
In the laboratory, soil samples were air-dried and passed through a 2 mm sieve (Sigma-Aldrich Chemie CH-9471 Buchs; Switzerland). A total of 10g of PCBs contaminated and uncontaminated soil samples were separately homogenized in 90 ml of sterile quarter strength Ringer's solution in 250 ml Erlenmeyer flasks and allowed to shake in a shaker incubator at 200 rpm, and temperature was set at 37oC for 60 min maintained. . Soil samples were prepared as follows; a total of 40 g of soil samples were collected within the University of Venda campus that contained no PCBs.
On day 1, after 24 hours of incubation, soil samples were transferred to a shaking incubator to mix at 200 rpm, and the temperature was maintained at 37°C for 60 minutes.
Statistical Analysis
Total plate counts were performed as follows: the experiment was performed in triplicate from day 0, day 1, and day 2.
Assessment and Quality Control of PCBs in the Laboratory
Infrared Spectroscopy (IR) analysis of PCBs
The typical IR Stretching Bond Absorptions of PCB peaks were found in the range from 2951.73 to 1376.43 wavelength cm-1 resolution in transmission. It can be concluded that due to the large and strong band of PCB solution, the area of 2951.73 cm-1 corresponds to carbon-hydrogen (C-H) stretching vibrations of PCB.
Gas Chromatography-Flame Ionization Detector (GC-FID) Analysis of PCBs
In general, PCBs are illustrated in GC in increasing order of the number of chlorine atoms in the biphenyl molecule. To obtain the linearity, the areas of the chromatograms of the mg/l solutions were used. In such a process, the number of calibration concentrations was representative of the matrix to be analyzed.
Isolation and testing of PCB degrading bacteria
Burkholderia cepacia showed the highest growth with 0.43 x 108 cfu/ml on the contaminated soil, on the unpolluted soil the growth showed a growth of 0.36 x 108 cfu/ml. In contrast, Enterococcus feacalis showed the lowest growth of 0.0051 x 108 cfu/ml on the uncontaminated soil, on the contaminated soil the growth of 0.00153 x 108 cfu/ml. In contrast to Pasteurella pneumotropica and Enterococcus faecalis, Burkholderia cepacia was the bacterium that showed the highest growth rate on the contaminated and uncontaminated soil samples.
The Biodegradation ability of the isolated bacteria on PCBs in soil
Degradation ability of Burkholderia cepacia
According to Goris et al., (2004), Burkholderia cepacia has characteristic pathways for PCB degradation, both at the genetic and molecular level. A review by Mailin and Firdausi, (2006) on PCB degradation by Burkholderia cepacia has shown the model system for bacterial degradation of PCBs (Mailin and Firdausi, 2006; Goris et al., 2004). Burkholderia cepacia showed the ability to break down PCB congeners, the chlorine content when it rises, it affects PCB metabolizing processes by the bacteria (Ines et al., 2007).
The trend decreases with increasing levels of chlorine as a by-product of decomposition and causes death of the strain (Field and Sierra-Alvarez, 2008; Ines et al., 2007).
Degradation ability of Pasteurella pneumotropica
Burkholderia cepacia and Pasteurella pneumotropica showed the same degradation average for PCBs as all PCB-degrading bacteria require the respective set of powerful enzymes to biodegrade and metabolize biphenyls (Ashrafosadat et al., 2009; Mailin and Firdausi, 2006 ). The species is able to use PCBs as the only carbon source (Mailin and Firdausi, 2006). Chlorobenzoic acid can be further co-metabolized and result in the production of carbon dioxide, water, chlorine and biomass (Hank et al., 2010; Ashrafosadat et al., 2009; Mailin and Firdausi, 2006).
Degradation ability of Enterococcus faecalis
The availability of sufficient amounts of nutrients and oxygen is also necessary in the co-metabolizing process of the PCBs (Hank et al., 2010). Joutey et al., (2013) and Hank et al., (2010) reported that biological enzymes involved in the degradation pathway have an optimum temperature. The trend shows that the ability of mixed bacterial culture growing in the different PCB concentrations decreases with the increase in the chlorine level (Figure 4.10) (Joutey et al., 2013; Hank et al., 2010; Field and Sierra-Alvarez, 2008).
While many bacteria are capable of degrading high concentrations of PCBs, a single bacterium does not possess the enzymatic capacity to degrade all or even most of the PCB congeners in a contaminated soil (Joutey et al., 2013).
Comparison of the effect of bacteria on different PCB concentrations
The increase in chlorine content as a by-product of PCB degradation inhibits bacterial growth and gradually the bacteria will no longer be able to metabolize the PCB congeners (Ines et al., 2007). The trend shows that the ability of Burkholderia cepacia to degrade PCBs decreases with an increase in chlorine content (Field and Sierra-Alvarez, 2008; Ines et al., 2007). Mixed bacterial cultures showed lower growth corresponding to PCB degradation by 23% in the high concentration of 100 mg/l PCBs respectively (Figure 4.12) (Field and Sierra-Alvarez, 2008; Ines et al., 2007).
According to Joutey et al. (2013) and Prasanne et al. (2008), mixed cultures are more effective in degrading PCBs than pure strains.
Correlation between PCB degradation and growth of bacteria isolates
Statistical analysis showed that means with the same letter or letters do not differ significantly at the 5% significance level (Snedecor & Cochran, 1980). Statistical analysis indicated that means with the same letter or letters do not differ significantly at a 5% significance level (Snedecor & Cochran, 1980). A significant positive correlation was found between bacterial growth and treatment with correlation coefficient (r) =0.1459 and p value <0.001.
The correlation between PCB degradation and treatment was not significant with correlation coefficient (r and p-value <0.056).
Conclusion
Recommendation
Al-khalid T and El-Naas M.H (2012) Aerobic biodegradation of phenols: a comprehensive review; Journal of Environmental Science and Technology. Isolation of biphenyls and polychlorinated biphenyl-degrading bacteria and their degradation pathways; Journal of Applied Biochemistry Biotechnology. Effects of polychlorinated biphenyls (PCBs) on expression of neurotrophic factors in C6 Glial cells in culture; Journal of Neurotoxicology.
Determination of petroleum-degrading bacteria isolated from crude oil-contaminated soil in Turkey; African Journal of Biotechnology. Degradation of Polychlorinated Biphenyls (PCBs) by Staphylococcus xylosus in liquid media and meat mixture; Journal of Food and Chemical Toxicology. High-performance phenol-degrading microorganisms isolated from wastewater and oil-contaminated soil; Journal of Microbiology, 2: (2).
