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Final Report FINAL REPORT Application of Mi croarrays and qPCR to Identify Phylogenetic and Functional Biomarkers Diagnostic of Microbial Communities that Biodegrade Chlorinated Solvents to Ethene SERDP Project ER-1587 January 2012 Lisa Alvarez-Cohen University of California at Berkeley This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the Department of Defense. 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Z39.18 TALBE OF CONTENTS TALBE OF CONTENTS ................................................................................................................. i LIST OF FIGURES ........................................................................................................................ ii LIST OF TABLES ......................................................................................................................... vi LIST OF ACRONYMS ................................................................................................................ vii ACKNOWLEDGEMENT ........................................................................................................... viii ABSTRACT .................................................................................................................................... 1 PROJECT BACKGROUND .......................................................................................................... 3 OBJECTIVES ................................................................................................................................. 3 PROJECT ACCOMPLISHMENTS ............................................................................................... 4 Objective 1: Identification of supportive microorganisms present and active in Dehalococcoides-containing communities. ............................................................ 4 1.1 Characterization of four TCE-dechlorinating microbial enrichments grown with different cobalamin stress and methanogenic conditions .................................................. 5 1.2 Phylogenetic microarray analysis of a microbial community performing reductive dechlorination at a TCE-contaminated site ..................................................................... 12 1.3 Construction and operation of the continuous-flow chemostat ...................................... 24 Objective 2: Construction of defined consortia containing Dehalococcoides and identification of rRNA-based phylogenetic biomarkers. (Part of this study was published in Men et al., ISME J, 2012) ..................................................................................... 25 Objective 3: Identification of mRNA-based functional biomarkers by application of genus- wide microarray on different TCE-dechlorinating microbial communities. ........ 36 3.1 Design, validation and application of a genus-wide microarray..................................... 36 3.2 The development of fluorescence-activated cell sorting (FACS), whole genome amplification (WGA) and microarray method ................................................................ 41 3.3 Metagenomic analysis of a TCE degrading microbial community (ANAS) .................. 46 Objective 4: Evaluating correlations between quantitative biomarker detection and solvent degrading activity.................................................................................................. 49 4.1 Quantification and correlation of RDase gene copies with 16S rRNA gene of Dehalococcoides at a TCE-contaminated site at Ft. Lewis East Gate Disposal Yard (EGDY) (Tacoma, Washington) ..................................................................................... 49 4.2 Investigation of the correlation between growth phases and identified biomarkers (this manuscript is currently under preparation for submission) ............................................. 52 4.3 Investigation of the correlation between dechlorination and cobalamin riboswitches ... 54 4.4 Identification of diverse corrinoid forms in Dehalococcoides-containing microbial communities .................................................................................................................... 58 CONCLUSIONS AND IMPLICATION FOR FUTURE RESEARCH ....................................... 65 LITERATURE CITED ................................................................................................................. 68 APPENDIX ................................................................................................................................... 72 i LIST OF FIGURES Figure 1. Dechlorination and growth curves of Dhc (A: MethB12; B: Meth; C: NoMethB12; D: NoMeth, indicates the re-amendment of lactate and TCE, indicates the re-amendment of lactate according to the feeding regimes) and H2 production in the enrichment cultures (E: H2 in MethB12 and Meth; F: H2 in NoMethB12 and NoMeth; Note: the H2 concentration scales are different) ................................................................................... 8 Figure 2 16S rRNA gene copy numbers of dominant OTUs in the four enrichments at the end of a feeding cycle. (Note: P.D.S represents a group of Pelosinus_GW, Dendrosporobacter_GW and Sporotalea_GW) ............................................................ 10 Figure 3. Principal component analysis (PCA) plot based on a time-course 16S rRNA gene copy numbers of 6 OTUs in the 4 enrichments (numbers next to each dot represents incubation days) ............................................................................................................. 11 Figure 4. A schematic of the treatment plots at Ft. Lewis EGDY (Tacoma, WA). MW = monitoring well; IW = injection well; EW = extraction well. ....................................... 15 Figure 5. Molar concentrations of chlorinated ethenes and ethene in groundwater samples over the 1-year monitoring period for treatment plots 1 and 2. Concentrations are reported as averages for eight sampling locations within each treatment plot, and error bars represent analytical error. .............................................................................................. 16 Figure 6. Phylogenetic tree of the expressed 16S rRNA sequences. Neighbor-joining algorithm with the filter lanemaskPH was used for phylogenetic analysis in order to exclude highly variable positions of the alignment. A total of 102 phylotypes were found (number in brackets) based on 97% sequence homology. Scale bar indicates 10% estimated sequence divergence. ..................................................................................... 18 Figure 7. Principle component analysis (PCA) of the PhyloChipanalyzed samples. The dotted circle highlights the samples that were collected within the treatment plot at different time points...................................................................................................................... 19 Figure 8. Changes in hybridization intensity relative to the background sample of the 393 subfamilies immediately after biostimulation with whey (July 2005) in the treatment plot. Phyla are color-coded and ordered alphabetically from left to right starting with the archaeal domain followed by the bacterial domain. Each bar represents a subfamily with positive bars indicating subfamilies that increased in abundance relative to the background after receiving whey and negative bars represent subfamilies that decreased in abundance. A 1000-unit change in hybridization intensity is
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