Carmen Lebrón – NAVFAC ESC • Dr. David Major, Dr. Erik Petrovskis and Natasha Barros – Geosyntec Consultants • Phil Dennis, Ximena Druar, Elizabeth Ney and Jennifer Wilkinson – SiREM • Dr. Frank Löffler, Dr. Kirsti Ritalahti and Janet Hatt – Georgia Institute of TechnologyDr. Natuschka M. Lee – Technical University of Munich • Dr. Carolyn Acheson – US EPA / ORD • Dr. Elizabeth Edwards, Dr. Melanie Duhamel and Winnie Chan – University of Toronto • Dr. Chris Yeager – SRNL DOE

Background

Overall Project Objective

Technical Approach

Task 1: White Paper

Task 2A: Development of Microbial Surrogates

Task 2B: Development of Calibrated Dehalococcoides Reference Standards

Task 3: Optimization of Sample Processing & Analysis

References Acknowledgements

Evaluate factors affecting quantification and interpretation of nucleic acid biomarkers in groundwater samples, with the goal of developing the foundation for standard procedures for microbial groundwater analysis. The application of quantitative real-time PCR (qPCR) for enumerating Dehalococcoides (Dhc) biomarker genes will serve as a model system.

This work is funded through SERDP project ER-1561.

Special thanks to Greg Davis and Dora Ogles of Microbial Insights

for their participation in this study.

Background Molecular biological tools (MBT) are “tools that that target biomarkers (e.g., specific nucleic acid sequences, peptides, proteins, or lipids) to provide information about organisms and processes relevant to the assessment and/or remediation of contaminants” (SERDP, 2005).

Project ProductA White Paper entitled An Overview of Current Approaches and Methodologies to Improve Accuracy, Data Quality and Standardization of Environmental Microbial Quantitative PCR Methods was produced and approved by SERDP. The document provides an overview of MBT practices in various technlogy fields. Review of these practices led to the implementation of the tasks that follow. For a draft copy of this document please contact: Carmen A. Lebrón at carmen.lebron@navy.mil.

Microbial SurrogatesMicroorganisms which are equivalent to a chemical analogs used for spike and recovery, used to determine efficiency (% recovery) of nucleic acid

extraction/quantification and matrix interferences.

Genetically Modified Surrogate:An E. coli surrogate containing a mutated Dhc 16S rRNA gene has been produced which minimizes the potential for PCR bias related to the surrogate versus

the wild type sequences, while still allowing differentiation of the surrogate and test targets.

Dhc Reference Culture qPCR has become a standard technology for enumeration of Dhc cells, however, the approach has never been calibrated

against a PCR-independent method. To verify the accuracy of the qPCR approach, a pure Dhc culture will be enumerated

using a variety of methods including qPCR, microscopy, flow cytometry, FISH, and quantification of total protein. Once

quantified the Dhc reference culture will be used to determine the efficiency of DNA extraction and determine the

accuracy of qPCR, by sharing amongst analytical laboratories.

Will use Micorbial Surrogates (Task 2A) and Dhc reference culture (Task 2B) combined with selected findings from White Paper (Task 1) to optimize methods from

cell concentration, nucleic acid extraction, qPCR quantification and data analysis/normalization.

SERDP (2005). SERDP and ESTCP Expert Panel Workshop on Research and

Development Needs for the Environmental Remediation Application of

Molecular Biological Tools.

He, J., K.M. Ritalahti, K-L Yang, S.S. Koeningsberg and F.E. Löffler (2003).

Detoxification of vinyl chloride to ethene coupled to growth of an

anaerobic bacterium. Nature 424: 62-65.

Natural Microbial Surrogates: Three naturally occurring aerobic microorganisms with size and morphology not unlike Dhc have been identified as having potential for use as microbial surrogates.

• Micrococcus luteus – small spherical cell (400 - 1,300 nanometers [nm])

• Brevundimonas dimunata – small rod-shaped cell (400 nm x 1,000 - 2,000 nm) used to test filter sterilization equipment/protocols

• Prochlorococcus – small spherical photosynthetic cyanobacterium (600 - 1,000 nm) unlikely to be found in groundwater samples

F QWildtype TaqMan probe

Non-mutated Dhc 16S rRNA gene monitored using green fluorescent probe

F Mutant TaqMan probe

Mutated Dhc 16S rRNA gene in E. coli surrogate monitored using red fluorescent probe

Q

Progress update: Optimization of multiplex (i.e., dual target) qPCR method for co-monitoring mutated and wild type sequences over a wide dynamic range currently underway.

Progress Update: Natural surrogates have been procured from culture collections, development of growth protocols and qPCR methods for quantifying these microorganisms are in progress.

Transmission electron micrograph

of surrogate candidate

Prochlorococcus (A) (Photo

credit: C. Ting) which is

approximately a 600 - 1,000 nm in

diameter compared with up to

800 nm for Dhc BAV1 (B) a small

disk-shaped microorganism (He

et al., 2003).

Progress Update: Growth of pure culture of pure Dhc BAV1 (left) currently in progress.

Progress Update:

Laboratory Round Robin 1(completed): Establish baseline variability of 5 laboratories using Dhc DNA (cloned Dhc 16s rRNA gene)

Laboratory Round Robin 2 (in progress): Groundwater spiked with Dhc cells

Anaerobic Growth apparatus/approach for Dhc BAV1

at Löffler Lab, Georgia Tech. The culture will be

quantified using multiple methods for use as a

calibrated Dhc reference standard.

Results of DNA Round Robin: Three concentrations of Dhc

plasmid DNA (M1, M2, M3) were quantified in 5 laboratories.

Results indicated that inter-lab variability may be statistically

significant, but may not be practically significant from a

remediation perspective. Future round robins will compare

results obtained with Dhc spiked groundwater samples which

may increase inter-lab variability due to the integration of

nucleic acid extraction variability.

A B

1.0E+01

1.0E+03

1.0E+05

1.0E+07

1.0E+09

M1 M2 M3

Gene Copies /

µl

Lab 1

Lab 2

Lab 3

Lab 4

Lab 5

Applied Research Objectives Develop improved methods to characterize data quality for MBT procedures including microbial surrogates and calibrated Dhc reference standard (Task 2).

Optimize and quantify the efficiency of each step in the procedure from sample collection to processing to Dhc quantification (Task 3).

Improve understanding of the relationship between Dhc quantified in groundwater and Dhc in the aquifer matrix (Task 4).

Evaluate the impact of groundwater sampling methods and site conditions on data collected with Dhc-targeted MBT tools (Task 5).

Gen

e C

op

ies/

µL

ER-1561

Inter-laboratory Comparison of Quantitative PCR Test Results for Dehalococcoides