QuantArray®-NSZD: Bioremediation Testing for Natural Source Zone Depletion

Comprehensive Microbial Analysis for LNAPL Biodegradation

Natural source zone depletion (NSZD) is the process where light non-aqueous phase liquid (LNAPL) petroleum hydrocarbons are lost from the subsurface due to naturally occurring processes, including dissolution, volatilization, and biodegradation. NSZD takes place at most petroleum sites where LNAPL is present. However, its contributions to remediation have historically been underestimated. A better understanding of NSZD at a site can improve the site conceptual model as well as projections of the mobility, distribution, and longevity of LNAPL.

QuantArray^®-NSZD provides a direct line of microbial evidence to assess the potential for LNAPL biodegradation in the source zone. QuantArray^®-NSZD quantifies members of the diverse microbial community involved in biodegradation, including methanogens, methanotrophs, sulfate reducers, iron reducers, denitrifiers, fermenters, acetogens, biosurfactant producers, and slime formers.

QUANTARRAY^®-NSZD ADVANTAGES:

ACCURATE

Direct analysis of sample DNA removes the need to grow the bacteria, thus eliminating biases associated with traditional approaches (e.g., plate counts and MPNs).

QUANTITATIVE

Absolute quantification of the concentrations of specific microorganisms and functional genes encoding enzymes responsible for contaminant biodegradation gives site managers a direct line of evidence to evaluate remediation options and monitor remedy performance. Results reported as cells/mL, cells/g, etc.

COST EFFECTIVE

In a single analysis, QuantArray^®-NSZD quantifies a broad spectrum of microorganisms, processes, and genes involved in biodegrading LNAPL. QuantArray^® provides the same accuracy as CENSUS^® but is more comprehensive and cost-effective.

INFORMATIVE

Is that a low, medium or high concentration of contaminant degraders? With the MI Database, clients can retrieve percentile rankings of their QuantArray^® results to answer that question based on the tens of thousands of samples MI has received from sites around the world.

SENSITIVE

The Method Detection Limit (MDL) is 10 cells/sample and the Practical Quantification Limit (PQL) is 250 cells/sample.

SPECIFIC

Target specific microbial groups (e.g., sulfate reducers, iron reducers, methanogens) and functional genes (e.g., methane monooxygenases) responsible for contaminant biodegradation in the source zone.

FLEXIBLE

Analysis can be performed on almost any type of sample (water, soil, sediments, Bio-Traps^®, and others).

HOW TO USE QUANTARRAY^®-NSZD:

In NSZD, biodegradation is an important, although complex, mechanism for contaminant destruction. Temporal and spatial changes in the diverse microbial community contributing to NSZD are expected as electron acceptors are consumed and redox conditions move from aerobic to nitrate-reducing, iron-reducing, sulfate-reducing, and ultimately methanogenic conditions. In addition to collecting chemical and geochemical data, shifts in concentrations of associated microbial groups (e.g., denitrifying bacteria, iron reducing bacteria, and sulfate reducing bacteria) can provide insight into current conditions in the subsurface.

The presence of organisms that produce biosurfactants can greatly enhance the remedial process in the source zone. These biosurfactants act as emulsifying agents to enhance the solubilization of hydrocarbons and increase their bioavailability.

NSZD Diagram

Fermenters and acetogens have been directly implicated in NSZD processes. Hydrocarbon-degrading fermenters produce acetate and hydrogen from the anaerobic degradation of hydrocarbons while acetogens can utilize the hydrogen and carbon dioxide produced during the anaerobic degradation of hydrocarbons to produce acetate.

Methanogenesis is another important process to NSZD. Methanogens utilize acetate and hydrogen produced from the anaerobic degradation of hydrocarbon to produce methane. Methanogenesis is an exothermic process, so vertical temperature profiles of the vadose and saturated zones are sometimes collected to detect this biogenic heat.

Methanotrophs in the vadose zone can oxidize the methane and produce carbon dioxide. Soil gas measurements showing an increase in methane and carbon dioxide and a decrease in oxygen can provide another line of evidence for NSZD.

GENE TARGETS INCLUDED IN A SINGLE QUANTARRAY^®-NSZD qPCR ANALYSIS

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Total Eubacteria	EBAC	Index of total bacterial biomass
Total Archaea	ARC	Index of total archaeal biomass
Sulfate Reducing Bacteria	APS	Quantification of sulfate reducing bacteria provides an additional line of evidence when evaluating redox conditions and terminal electron accepting processes.
Iron and Manganese Reducing Bacteria - Other	IRB	Iron reducing bacteria (IRB) are capable of reducing insoluble iron oxides to soluble ferrous iron. Some IRB can also reduce insoluble manganese oxides to soluble manganese.
Iron and Manganese Reducing Geobacter	IRG	Many Geobacter spp. are capable of reducing insoluble iron and manganese oxides to soluble forms.
Iron and Manganese Reducing Shewanella	IRS	Many Shewanella spp. are capable of reducing insoluble iron and manganese oxides to soluble forms.
Denitrifying Bacteria	DNF	Denitrifying bacteria are involved in the reduction of nitrate to nitrogen gas (denitrification).
Acetogens	AGN	Acetogens can utilize hydrogen and carbon dioxide produced during the anaerobic degradation of hydrocarbons to produce acetate.
Fermenters	FER	Some fermenters in anaerobic LNAPL zones are capable of biodegrading hydrocarbons to form dissolved hydrogen and/or acetate which can be utilized by methanogens to form methane.
Methanogens	MGN	Methanogens utilize the hydrogen, acetate, and other substrates that are produced by fermenters and other hydrocarbon degraders as byproducts during the anaerobic biodegradation of hydrocarbons.
Acetoclastic methanogens	AMGN	Acetoclastic methanogens can utilize the acetate produced by fermenters and other hydrocarbon degraders for the production of methane.
Particulate methane monooxygenase	PMMO	Some methanotrophs utilize the particulate methane monooxygenase (PMMO) genes to convert the methane into carbon dioxide which is then released to the surroundings along with heat. The PMMO genes are also capable of degrading C1-C5 linear alkanes and alkenes.
Soluble methane monooxygenase	SMMO	Some methanotrophs utilize the soluble methane monooxygenase (SMMO) genes to convert the methane into carbon dioxide which is then released to the surroundings along with heat. The SMMO genes are also capable of degrading linear C1-C8 alkanes and alkenes as well as cyclic and aromatic hydrocarbons.
Glycolipid biosurfactants	SurG	Glycolipid surfactants are composed of lipids with a carbohydrate attached by a glycosidic bond. The SurG assay quantifies two genes from Pseudomonas spp. responsible for the production of rhamnolipids which are involved in the uptake of low polarity hydrocarbons.
Liposaccharide biosurfactants	SurL	Liposaccharide biosurfactants are high molecular weight, water soluble compounds composed of a hydrophobic lipid section, a hydrophilic core polysaccharide chain, and a repeating hydrophilic oligosaccharide side chain. The SurL assay quantifies the weeA and alnB genes involved in the production of emulsan and alasan in hydrocarbon degrading Acinetobacter spp.
Lipopeptide biosurfactants	SurP	Lipopeptide biosurfactants are composed of a lipid connected to a peptide molecule. The SurP assay quantifies the SrfAC, licC, aprE genes involved in the production of Surfactin, lichenysin, and Subtilisin in Bacillus spp. as well as the visC gene involved in the production of viscosin in Pseudomonas spp.
Trehalose biosurfactants	SurT	This is a specific group of glycolipid biosurfactants that are produced by Rhodococcus and Mycobacterium spp. and are involved in the uptake of low polarity hydrocarbons. The SurT assays quantifies two genes responsible for the production of these biosurfactants.
Burkholderia cepacian exopolysaccharide	BCE	Targets a functional gene involved in exopolysaccharide (EPS) production by slime-forming Burhkholderia cepacia.
Deinococcus spp.	DCS	Deinococcus spp., most notably Deinococcus geothermalis, are considered eﬀicient primary biofilm formers.
Meiothermus spp.	MTS	Along with Deinococcus, Meiothermus are considered primary biofilm formers functioning as an adhesion platform for secondary biofilm bacteria.

FREQUENTLY ASKED QUESTIONS

How soon can I expect to receive the QuantArray®-NSZD results?

The turnaround time to receive a QuantArray^®-NSZD report is 14-21 calendar days.

What type of sample should I submit for QuantArray®-NSZD testing?

QuantArray^®-NSZD can be performed on a variety of sample types, including groundwater from the saturated zone, soil from the vadose zone, and even liquid samples containing LNAPL. More information on sample collection can be found here.

What analysis is recommended to assess the biodegradation potential for specific classes of hydrocarbons like BTEX or PAHs?

QuantArray^®-Petro analysis provides simultaneous quantification of 20+ functional genes and microbial groups responsible for aerobic and anaerobic biodegradation of BTEX, PAHs, fuel oxygenates, and a variety of short and long chain alkanes.

How is QuantArray® different from CENSUS® qPCR and multiplex qPCR?

In many respects, QuantArray^® is the same as conventional qPCR, so you can expect the same level of accuracy and precision. Other methods like multiplex qPCR have been described that achieve some level of parallel quantification. However, there is a fundamental difference between the QuantArray^® and multiplex qPCR. For multiplex qPCR, multiple primer sets are added to a reaction mixture to quantify multiple gene targets whereas QuantArray^® employs discrete through-holes for individual qPCR reactions ensuring that reaction kinetics are not compromised. Download our “CENSUS^® versus QuantArray^®” white paper for further information.