ENVIRONMENTAL MICROBIOLOGY Spatial Variation in the Bacterial and Denitrifying Bacterial Community in a Biofilter Treating Subsurface Agricultural Drainage J. Malia Andrus & Matthew D. Porter & Luis F. Rodríguez & Timothy Kuehlhorn & Richard A. C. Cooke & Yuanhui Zhang & Angela D. Kent & Julie L. Zilles Received: 15 March 2013 /Accepted: 28 August 2013 /Published online: 29 September 2013 # Springer Science+Business Media New York 2013 Abstract Denitrifying biofilters can remove agricultural ni- trates from subsurface drainage, reducing nitrate pollution that contributes to coastal hypoxic zones. The performance and reliability of natural and engineered systems dependent upon microbially mediated processes, such as the denitrifying biofilters, can be affected by the spatial structure of their mi- crobial communities. Furthermore, our understanding of the relationship between microbial community composition and function is influenced by the spatial distribution of samples. In this study we characterized the spatial structure of bacterial communities in a denitrifying biofilter in central Illinois. Bac- terial communities were assessed using automated ribosomal intergenic spacer analysis for bacteria and terminal restriction fragment length polymorphism of nosZ for denitrifying bacte- ria. Non-metric multidimensional scaling and analysis of similarity (ANOSIM) analyses indicated that bacteria showed statistically significant spatial structure by depth and transect, while denitrifying bacteria did not exhibit significant spatial structure. For determination of spatial patterns, we developed a package of automated functions for the R statistical environ- ment that allows directional analysis of microbial community composition data using either ANOSIM or Mantel statistics. Applying this package to the biofilter data, the flow path correlation range for the bacterial community was 6.4 m at the shallower, periodically inundated depth and 10.7 m at the deeper, continually submerged depth. These spatial structures suggest a strong influence of hydrology on the microbial com- munity composition in these denitrifying biofilters. Under- standing such spatial structure can also guide optimal sample collection strategies for microbial community analyses. Electronic supplementary material The online version of this article (doi:10.1007/s00248-013-0286-0) contains supplementary material, which is available to authorized users. J. M. Andrus : L. F. Rodríguez : T. Kuehlhorn : R. A. C. Cooke : Y. Zhang Department of Agricultural and Biological Engineering, University of Illinois at Urbana–Champaign, Urbana, IL, USA M. D. Porter : J. L. Zilles Department of Civil and Environmental Engineering, University of Illinois at Urbana–Champaign, Urbana, IL, USA A. D. Kent Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana–Champaign, Urbana, IL, USA J. L. Zilles (*) 3230C Newmark Civil Engineering Laboratory, MC250, 205 North Mathews Avenue, Urbana, IL 61801, USA e-mail: jzilles@illinois.edu Present Address: J. M. Andrus Waterborne Environmental, Inc., 2001 S First Street, Suite 109, Champaign, IL 61820, USA Present Address: M. D. Porter Environmental Resources Management, 1701 Golf Road, Suite 1-700, Rolling Meadows, IL 60008, USA Present Address: T. Kuehlhorn Agricultural Informatics, PO Box 17074, Urbana, IL 61803, USA Microb Ecol (2014) 67:265–272 DOI 10.1007/s00248-013-0286-0