Molecular tools can provide lines of evidence for biological remediation

There are 3.5 million suspected contaminated sites within the EU. The EU Soil Framework Directive will soon require member states to compile a national database of these sites, measure the contamination and put in place a national remediation strategy.

It is estimated that over 500,000 of these sites throughout the EU will require remediation. For example, VROM (the Dutch Ministry of Housing, Spatial Planning and the Environment) estimate there are 60,000 sites in the Netherlands requiring urgent clean up. OVAM regulating the Flanders region has identified 76,000 sites of which 24,000 have been investigated and 3,000 have been identified as requiring remediation. This represents an enormous challenge using conventional techniques. Prior to implementation of the EU Landfill Directive (1999/31/EC), the ‘dig and dump’ approach was commonly used to clean up contaminated sites.

However, the Directive has reduced the number of landfill sites that are licensed to take hazardous wastes and made contaminated land liable to the full landfill tax rate, making landfill an increasingly expensive and difficult to source option.

In many cases, the cheapest and the best remediation strategy for sites contaminated with organic pollutants such as petroleum hydrocarbons, BTEX or chlorinated hydrocarbons, is to simply let nature take its course and allow the microorganisms within the site to degrade the pollutants over time. This process known as natural attenuation (NA) has gained acceptance and is increasingly being used within Europe. Where in situ remediation techniques are used, monitoring and clean up of the contaminated groundwater is particularly important as there is a greater risk of the pollution spreading.

The UK Environment Agency defines the process of Natural Attenuation (NA) as: “The effect of naturally occurring physical, chemical and biological processes or any combination of these processes to reduce the load, concentration, flux or toxicity of polluting substances in groundwater. For natural attenuation to be an effective remedial treatment action, the rate at which these processes occur, must be sufficient to prevent polluting substances impacting on identified receptors and to minimise expansion of contaminant plumes into unpolluted groundwater. Dilution within a receptor, such as in a river or borehole is not natural attenuation.”

They further define Monitored Natural Attenuation (MNA) as “Monitoring of groundwater to confirm whether NA processes are acting at a sufficient rate to ensure that the wider environment is unaffected and that remedial objectives will be achieved within a reasonable timescale; this will typically be less than one generation or 30 years.”

The costs of natural attenuation, even allowing for ongoing monitoring can be significantly lower than off site landfill or incineration (see table 1). Natural attenuation is also generally seen as a more benign, environmentally friendly, less invasive approach to cleaning up a site as it is not simply moving the problem to a different location.

Several regulators provide guidance on the use of biological remediation techniques. For example, “Guidance on the Assessment and Monitoring of Natural Attenuation of Contaminants in Groundwater” R&D publication 95 is available online from the UK Environment Agencies web site.

In general MNA will only be considered an acceptable approach where the contaminant plume is shrinking or stable and where there is no significant risk of impact to receptors. Most authorities require lines of evidence to support an MNA approach which include:

  • Historical contaminant data to demonstrate a trend of reduced concentration down-gradient of the source
  • Geochemical and biochemical indicators which demonstrate the natural processes that are resulting in reduction in contaminant concentration
  • Microbiological data to support the occurrence of biodegradation

The microbial processes governing natural attenuation are complex and involve a wide range of microorganisms growing under different conditions. It can be very difficult to measure these processes. Currently, the microbial activities are usually indirectly inferred from analysis of the terminal electron accepters and other geochemical parameters observed in ground water samples collected from around the site. If gradients of dissolved oxygen, nitrate, iron, sulphate, dissolved carbon dioxide and methane etc., coincide with the plume then it is possible to infer biological process are occurring. However, this approach does not provide direct proof that microorganisms are degrading the pollutants.

Abiotic processes or biological degradation of non-pollutant materials can produce similar geochemical profiles. Also, on some sites the ground water analysis can be very difficult to interpret if there are multiple zones of pollution rather than a discrete plume.

Table 1. Comparison of the costs of various treatment options. Taken from Kim et al., (2006)

Technique Cost in Euros per m 3 treated
Off site incineration 885
Off site landfill 231
In situ immobilization 128
Off site biological treatment 167
On site soil washing 116
In situ bioremediation 73
In situ Monitored Natural Attenuation 20

Growing microorganisms from groundwater samples using traditional culturing techniques, can confirm whether or not certain types of organism are present. However, the organisms that will grow well on laboratory media are not necessarily active under the conditions in situ. Many organisms involved in bioremediation will not grow on laboratory media.

With the advent of molecular DNA technology there are now methods of examining microbial activities that do not require traditional culturing techniques. Polymerase Chain Reaction (PCR) can be used to identify the presence of specific DNA sequences or genes in a sample, confirming the presence of bacteria capable of degrading the pollutants.

A development of conventional PCR known as quantitative PCR (qPCR or real time PCR) which addresses the quantification issue through the introduction of fluorescent dyes into the PCR reaction can be used to estimate the numbers of specific types of bacterial. For example, Da Silva and Alvarez (2007) used qPCR to enumerate putative benzene degraders based on 16S rRNA gene sequences. It can also be used to measure catabolic genes involved in the breakdown of pollutants, such as the gene for benzyl succinate synthase (bssA) which catalyses the first activation step in anaerobic toluene degradation. Winderl et al., (2007)

Molecular techniques are particularly useful at detecting Dehaloccoides sp. whose presence is generally accepted to be essential for the biological remediation of sites contaminated with chlorinated solvents such as PCE. Dehaloccoides sp cannot be grown by traditional culturing techniques, but their presence can be detected by qPCR of 16S rRNA genes or catabolic genes such as vinyl chloride reductase. For example, Losi et al., (2004) used PCR to confirm the presence of Dehalococcoides sp. at a site in California contaminated with PCE, TCE, DCE and VC.

The microorganisms degrading pollutants tend to grow in biofilms attached to submerged surfaces within the aquifer, and are not easily sampled. A novel technique that has recently been developed to overcome many of the limitations of groundwater samples is to use in situ sampling devices. These are based on similar principles to passive diffusion samplers, that can be placed directly in a sampling well to collect microbes over time. Probably the best example of this type of microbial sampling device is the Bio-Trap® cartridge developed by the University of Tulsa.

The key to the sampling approach is contained in the unique properties of the beads which are used as the sampling matrix. The beads are 2-3mm in diameter and are engineered from a composite of Nomex and powdered activated carbon (PAC). The adsorption capacity of the PAC allows nutrients and contaminants present within the aquifer to be collected onto the bead matrix and the beads provide a large surface area (~600 m 2 /g) for the microbes to colonise.

The samplers are normally deployed in a ground water monitoring well and incubated for between 30 to 90 days, depending on the conditions within the site, to allow the formation of a mature biofilm. The traps are then removed and DNA and microbial lipids can be extracted from the beads and analysed. These samplers integrate the microbial response over time providing a way to compensate for the inherent variability of groundwater samples.

For example, Bio-Traps® were used to provide evidence of microorganisms capable of degrading BTEX at an Amoco natural gas facility contaminated by condensate water which had leaked from concrete storage tanks. The samplers were lowered into several wells located around the site. Analysis of the genes amplified from traps showed a broad mixture of organisms was present. The levels of Iron reducing / sulphate reducing bacteria, associated with BTEX and MTBE degradation, were significantly elevated within the plume with the highest levels in wells closest to the source of pollution.

All samples, including control samples outside the plume, were positive for the bssA gene indicating that organisms capable of degrading benzene and toluene are common in this area. (Boone, 2004)

Microbial sampling devices incorporating labeled compounds can provide unequivocal proof that the contaminant of concern is degraded by microorganisms in situ. In the guidance document “Monitored Natural Attenuation of Tertiary Butyl alcohol (TBA) in Ground Water at Gasoline Spill Sites” the USEPA recommend “One particularly compelling approach is to amend beads with organic compound that is mass labeled with the stable isotope 13 C. If the compound is biologically degraded some portion of the mass label should find its way into the biomass that develops on the bead.”

Samplers loaded with both 13 C- labeled and unlabeled benzene and toluene were used at a former hydrogenation plant in Zeitz, Germany to evaluate organisms responsible for benzene and toluene degradation. The site had been heavily contaminated with benzene (up to 850 mg L -1 ) and toluene (up to 50 mg L -1 ). Analysis of the isotopic ratio of microbial lipid fatty acids demonstrated significant enrichment of 13C confirming the indigenous microbial community were involved in the degradation of both benzene and toluene at this site.

In another example, samplers loaded with 13 C- labeled benzene were incubated in a site contaminated with BTEX in Maine. 13 C from the labeled benzene was taken up into the microbial biomass and lipids. Approximately 78% (well DP-13) and 43% (well DB-04) of the 13 C- labeled benzene was degraded in 30 days. First order degradation rates were 0.050 day -1 for DP-13 source area well and 0.020 day -1 for DB-04 mid plume well.

Microbial sampling devices can also be used to predict the likely impact of amendments on the microbial community. The central space of the cartridge can be loaded with compounds that are being considered for injection into the site. The compound will slowly leach out through the outer layer of beads mimicking the injection process.

The traps preferentially collect microbes that are stimulated by the additive. For example, traps loaded with various concentrations of either Hydrogen Releasing Compound (HRC) or Low Sulphate Hydrogen Releasing Compound (HRCS) were used to predict whether injection of these compounds would stimulate the degradation of chlorinated solvents in a PCE contaminated aquifer. Standard samplers (without amendments) showed that Dehalococcoidies spp. were not present at a level that would support bioremediation.

Traps baited with 100 ppm HRC stimulated the growth of Dehalococcoidies spp. Traps baited with HRCS gave a stronger stimulation of at lower concentrations.

In conclusion, the combination of in situ microbial samplers and molecular tools such as qPCR, make it possible to:

  • sample microorganisms actively growing within the aquifer
  • confirm the presence organisms capable of degrading pollutants
  • provide direct evidence that the pollutants are biologically degraded under the conditions in the aquifer
  • assess the rate of pollutant breakdown
  • predict the likely stimulatory impact of amendments to the site
  • quantify changes in the microbial community over time

These techniques provide powerful tools to develop and optimise a remediation strategy based on natural attenuation or bioremediation, and provide unambiguous lines of evidence to the regulators to support a remediation strategy that involves microbiological processes.


Cary, M.A., Finnamore J.R., Morrey, M.J. and Marsland (2000) Guidance on the Assesment and Monitoring of Natural Attenuation of Contaminants in Groundwater. R&D Publication 95, Environment Agency

Da Silva, M.L.B. and Alvarez, P.J.J. (2007). Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR. Environmental Microbiology 9: 72-80

Kim, S.S., Han, S.J., Kang, M.S. and Kim, K.N. (2006). Application of In Situ Hybrid Electrokinetic Remediation Technologies on a Lead-Contaminated ‘Clay’ Shooting Range. pp.302-309 in 5th ICEG Environmental Geotechnics. Vol 1, Ed. H.R. Thomas

Losi, ME., J.S. Oslick, A. Eloskof, A. D. Peacock, D. C. White, E. Dienzo, T. Macchiarella (2004) Monitoring of Subsurface Bioprocesses Using Quantitative Biomarker Analyses In Proceedings of the Fourth International Conference on Land Remediation Remediation of Chlorinated and Recalcitrant Compounds. May 24-27, 2004. Monterey CA.

Wilson, J.T. and Adair, C. (2007) Monitored Natural Attenuation of Tertiary Butyl Alcohol (TBA) in Ground Water at Gaoline Spill Sites EPA/600/R-07/100, Cincinnati, OH: U.S. Environmental Protection Agency

Winderl, C, Schaefer, S. and Lueders, T. (2007). Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker Environmental Microbiology 9 : 1035-46

Published: 10th Mar 2008 in AWE International