Nanoliter qPCR Platform for Highly Parallel, Quantitative Assessment of Reductive Dehalogenase Genes and Populations of Dehalogenating Microorganisms in Complex Environments Koshlan Mayer-Blackwell, † Mohammad F. Azizian, ‡ Christina Machak, § Elena Vitale, ∥ Giovanna Carpani, ∥ Francesca de Ferra, ∥ Lewis Semprini, ‡ and Alfred M. Spormann* ,†,⊥ † Civil and Environmental Engineering, § Geological and Environmental Sciences, and ⊥ Chemical Engineering, Stanford University, Stanford, California 94305, United States ‡ Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, Oregon 97331, United States ∥ Environmental Technologies - Istituto Eni Donegani - ENI, 20097 San Donato Milanese, Italy *S Supporting Information ABSTRACT: Idiosyncratic combinations of reductive deha- logenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics. 1. INTRODUCTION Bioremediation of groundwater aquifers and sediments con- taminated with chlorinated aliphatic hydrocarbons (CAHs) depends on the activities of reductive dehalogenases that are present in some anaerobic microorganisms. 1,2 Of particular importance are obligate organohalogen-respiring bacteria, such as Dehalococcoides or Dehalogenimonas sp., because reductive dehalogenation is the only known mode of metabolic energy conservation in these microorganisms, and each microorganism can carry up to 36 different nonredundant rdh genes. 3−5 While organohalogen-respiring bacteria have been key for decontaminating polluted sites via biostimulation and bio- augmentation, there are many instances where such treatments have been hindered by the absence of key microorganisms and/ or genes, enzymatic inhibition, 6−9 hydrological complications, 10 or insufficient management of microbial ecology and associated biogeochemistry. 11,12 Remediation of common groundwater contaminants such as tetrachloroethene (PCE), trichloroethene (TCE), 1,1,2-trichloroethane (1,1,2-TCA), and 1,2-dichloro- ethane (1,2-DCA) poses additional challenges since an appropriate assemblage of organohalogen-respiring bacteria plus their supporting microbial communities is required for complete dechlorination of these compounds to a harmless end product. Furthermore, it is unclear whether faithful representa- tives of the well-studied laboratory isolates are dominant organohalogen-respiring bacteria in sediments and groundwater and to what extent their laboratory-studied phenotypes are relevant in the field. Given this uncertainty, managing bioremediation of CAHs requires (i) gauging the structure of the microbial community, in particular the organohalogen- respiring bacteria, and (ii) being able to identify and differentiate between closely related but functionally distinct subpopulations. Such information is crucial for predicting and controlling the ecological responses of the microbial communities to natural or engineered perturbations during bioremediation. Metagenomics, 13 transcriptomics, 14 proteomics, 15 pan-genome- microarrays, 16,17 and functional-gene tiling microarrays 18,19 have been used to study the eco-physiology of organohalogen-respiring bacteria. However, these approaches have not been widely applied Received: February 26, 2014 Revised: June 5, 2014 Accepted: June 27, 2014 Published: July 21, 2014 Article pubs.acs.org/est © 2014 American Chemical Society 9659 dx.doi.org/10.1021/es500918w | Environ. Sci. Technol. 2014, 48, 9659−9667