Stable Isotope Probing (SIP)

Stable isotope probing (SIP) uses heavy isotopes to identify and track contaminant fate to evaluate whether biodegradation is occurring at a site. Here, we describe how SIP works in a typical study, outline its advantages and limitations, briefly walk through reporting and background values, introduce a case study use at a petroleum hydrocarbon site, and summarize suggested guidelines for use in other studies.

Related Article(s):

• Molecular Biological Tools - MBTs
• Quantitative Polymerase Chain Reaction (qPCR)
• Metagenomics
• Compound Specific Isotope Analysis (CSIA) For Environmental Investigation and Remediation

CONTRIBUTOR(S): Dora Ogles-Taggart and Dr. Brett Baldwin

Key Resource(s):

• Identification of TBA-utilizing organisms in BioGAC reactors using ¹³C-DNA SIP
• Using DNA‐Stable Isotope Probing to Identify MTBE‐and TBA‐Degrading Microorganisms in Contaminated Groundwater

Introduction

Stable isotope probing (SIP) is used to conclusively determine whether in situ biodegradation of a contaminant is occurring. The “probe” is a synthesized version of the contaminant compound composed of the heavier stable isotope (e.g., ¹³C, ¹⁵N) rather than the more common light isotope of that element (e.g., ¹²C, ¹⁴N). The heavy isotope serves as the “label” to track the environmental fate of the contaminant and determine if biodegradation is occurring.

SIP is used to conclusively determine if biodegradation of some common contaminants like petroleum hydrocarbons (e.g., BTEX, polycyclic aromatic hydrocarbons (PAHs)) and oxygenates (e.g., MTBE, TBA) is occurring. Results are often used to evaluate the feasibility of monitored natural attenuation (MNA) as a site management strategy. Conclusive evidence of contaminant biodegradation increases stakeholder confidence that MNA is more than a “do nothing” alternative. While often performed to assess MNA, SIP can also be used to evaluate the feasibility and performance of engineered bioremediation approaches. In research settings, SIP can also be used with DNA-based analyses to help identify the organisms involved in specific biodegradation processes.

How Does SIP Work

In a typical SIP study, a ¹³C form of the contaminant (e.g., ¹³C-benzene) is adsorbed to a passive microbial sampling device such as Bio-Sep^® beads inside a Bio-Trap^® sampler. Bio-Sep^® beads are an engineered composite of Nomex^® and powdered activated carbon (PAC). PAC adsorbs the ¹³C-labeled compound and also provides a large surface area for microbial colonization and growth. Nomex^® allows the beads to be heat sterilized prior to the study. Such a passive sampler is deployed in a monitoring well (Fig. 1).

Figure 1. SIP in use: Loading, deployment and recovery of Bio-Trap^® passive sampler with ¹³C-labeled benzene.

During the deployment period (30 to 90 days), the ¹³C-labeled contaminant is subject to the same microbial processes as unlabeled contaminant present at the site. Many contaminants, such as petroleum hydrocarbons, are used as a carbon and energy source for microbial growth. Therefore, if biodegradation is occurring, contaminant-degrading bacteria will colonize the Bio-Trap^® and use the ¹³C-labeled contaminant as a carbon and energy source for growth and the ¹³C label will be incorporated into microbial biomass or ¹³CO₂.

After deployment, the Bio-Trap^® is recovered for gas chromatography and isotope ratio mass spectrometry (IRMS) analysis to quantify the ¹³C/¹²C ratio of biomass and dissolved inorganic carbon. The ¹³C label will either be incorporated into microbial biomass or mineralized to ¹³CO₂. Detection of ¹³C-enriched biomolecules (phospholipids, DNA, or protein) and ¹³C-enriched dissolved inorganic carbon (DIC) following deployment unambiguously indicates that in situ biodegradation occurred. Conversely, if biodegradation is not occurring, the ¹³C/¹²C ratio of the microbial biomass and DIC analyzed after in well deployment will be similar to background values. Phospholipid fatty acids (PLFA) are a main component of microbial cell membranes, therefore, ¹³C-enriched PLFA unambiguously demonstrates incorporation of the ¹³C into biomass. Likewise, ¹³C-enriched dissolved inorganic carbon (e.g., CO₂ , HCO₃^-) provides conclusive evidence of contaminant mineralization.

Advantages

• Conclusive: SIP can provide conclusive evidence that in situ biodegradation of the contaminant is occurring.

• Broadly applicable: A SIP study can be conducted for any contaminant that is used as a carbon and energy source, as long as an isotopically-enriched form of the contaminant can be synthesized.

• No prior knowledge needed: No prior knowledge is needed about the type(s) of microorganisms, biodegradation pathway(s), or gene sequences.

• Reasonable cost: For many common contaminants like BTEX, MTBE, TBA, and even naphthalene, the cost to synthesize the ¹³C-labeled compound is reasonable. As an example, the cost for a SIP study with ¹³C benzene including the cost of synthesizing the ¹³C compound, post-deployment analysis, and reporting is ~ $1,000.

Limitations

• Not applicable to all contaminants: SIP is generally not appropriate for compounds that are used as terminal electron acceptors, such as trichloroethylene (TCE) and other chlorinated ethenes, because the ¹³C label is not incorporated into biomass or CO₂ during this microbial process. Tools such as compound-specific isotope analysis (CSIA) performed on the contaminant itself is more applicable for these compounds. For large or more complex compounds, synthesis of the ¹³C-labeled compound can be expensive or simply not available.

• Dilute plumes: Data obtained from typical SIP studies where concentrations of ¹³C-labeled compounds are relatively high may not necessarily extrapolate to biodegradation of the contaminant present in a dilute plume where contaminant concentrations are approaching closure levels.

Reporting SIP Results

SIP results are reported as ¹³C/¹²C ratios in delta notation (δ¹³C) with units of parts per thousand (“per mil” ‰), where δ¹³C is defined as follows:

Where:

R_sample is the ¹³C/¹²C ratio of the sample and

R_standard is the ¹³C/¹²C ratio of the Vienna Pee Dee Belemnite (VPDB) standard.

Background δ¹³C Values

The ¹³C/¹²C ratio of the VPDB standard (1.1112328% ¹³C) is greater than most other natural carbon-based substances. Because R_standard is typically larger than R_sample , calculated δ¹³C values of most naturally-occurring materials will be negative values (0‰ to -110‰).

The isotopic composition of subsurface microorganisms and their metabolic products (e.g., CO₂ ) will directly reflect the isotopic composition of the compounds used as carbon and energy sources. Thus under natural conditions, background δ¹³C values for microbial biomass and DIC would be expected to be negative and are often between -20‰ and -30‰ . In a SIP study, δ¹³C values for microbial biomass and DIC will be greater than these background levels if biodegradation is occurring because the contaminant degraders are utilizing ¹³C from the synthesized contaminant as a carbon and energy source.

Use for Evaluating MNA at Petroleum Hydrocarbon Sites

At complex petroleum hydrocarbon sites, observing decreasing trends in contaminant concentrations and plume stability may be insufficient to conclusively evaluate the feasibility and performance of MNA. SIP can provide a critical third line of evidence to conclusively determine if biodegradation of a specific contaminant is occurring under the existing subsurface conditions and improve stakeholder confidence in MNA as a site management strategy.

Figure 2. Comparison of δ¹³C values obtained from (A) DIC and (B) PLFA biomarkers from each Bio-Trap® unit to typical background δ¹³C values observed in samples not exposed to ¹³C-enriched compounds.

In an ongoing study, SIP was conducted at a former manufactured gas plant (MGP) to determine whether naphthalene biodegradation was occurring under existing site conditions and whether MNA would be effective. Bio-Traps^® amended with ¹³C-naphthalene were suspended in two monitoring wells (UMW-7C and UMW-22) located within the dissolved plume for approximately 60 days. After the deployment period, the Bio-Traps^® were recovered for analysis.

• Detection of ¹³C-enriched DIC in Bio-Traps^® recovered from UMW-7C and UMW-22 demonstrated mineralization of ¹³C naphthalene under existing site conditions (Fig. 2A).
• Detection of ¹³C-enriched PLFA demonstrated ¹³C incorporation into biomass (Fig. 2B).

Overall, the SIP study conclusively demonstrated biodegradation of naphthalene under the existing site conditions. This was a critical line of evidence in support of an MNA remedy.

SIP Study Guidelines

As with any site assessment tool, study design and proper implementation are needed to ensure the relevance and accuracy of the results. Here are some suggestions:

• Study locations: SIP studies with a ¹³C labeled contaminant should only be performed in impacted areas within the dissolved plume. Comparisons of results to literature δ¹³C values for DIC and PLFA provide meaningful conclusions. If desired, site specific δ¹³C values for DIC and PLFA can be determined from water samples or standard Bio-Traps^® (not amended with a ¹³C compound).

• Deployment period: Typical deployment periods are between 30 and 90 days although longer deployment periods have been used to investigate biodegradation of more recalcitrant compounds.

• Handling: Clean latex gloves (or similar) should be worn at all times when handling Bio-Traps^®. Bio-Trap^® samplers should be kept in sealed bags and refrigerated, but not frozen until deployment. After deployment, Bio-Traps^® should be bagged and immediately placed on ice. Samplers should be shipped on ice using an overnight carrier.

Summary

For contaminants that microbes can use as a carbon and energy source (Table 1), stable isotope probing (SIP) is a useful tool for conclusively determining whether in situ biodegradation is occurring. While SIP can aid in evaluating MNA at any site, SIP studies are particularly valuable at complex sites where stakeholders may be reluctant to accept MNA as a site management strategy. Definitive evidence of contaminant biodegradation increases stakeholder confidence that MNA is more than a “do nothing” alternative. At the remedy selection phase, SIP studies can also be performed as part of a pilot study to assess active bioremediation approaches. Greater ¹³C incorporation into DIC and biomass in Bio-Traps^® deployed in wells influenced by site activities (pilot study, injection area, system operation) relative to wells outside of the radius of influence strongly suggests that the remediation technology enhanced biodegradation. In research settings, SIP is used with DNA-based analyses to help identify the organisms involved in specific biodegradation processes.

Table 1. Examples of appropriate contaminants for SIP studies.

See Also

• Stable Isotope Probing – Fact Sheet
  
• Stable Isotope Probing (SIP)
  
• Development of Biomarkers for Assessing In Situ RDX Biodegradation Potential ER-1606
  
• New Approaches to Evaluate the Biological Degradation of RDX in Groundwater ER-1607
  
• Characterization, Assessment & Monitoring
  
• Online Lecture Course - Stable Isotope Probing