Characterization of microbial communities in a pilot-scale constructed wetland using PLFA and PCR-DGGE analyses

Abstract

Phospholipid fatty acid (PLFA) analysis and 16S ribosomal DNA polymerase chain reaction amplification-denaturing gradient gel electrophoresis (PCR-DGGE) were used to determine microbial communities and predominant microbial populations in water samples collected from a pilot-scale constructed wetland system. This pilot-scale constructed wetland system consists of three types: subsurface-flow (SSF), surface-flow (SF) and a floating aquatic plant (FAP) system. Analysis of PLFA profiles indicated primarily eukaryotic organisms, including fungi, protozoa, and diatoms were observed in all three wetland systems. Biomarkers for Gram-negative bacteria were also detected in all samples analyzed while low proportions of biomarkers for Gram-positive bacteria were observed. Biomass content (total PFLA/sample) was highest in water samples collected from both SF and FAP system while highest metabolic activity was observed in FAP system. This is consistent with the observed highest metal removal rate in FAP system. Sequence analysis of the predominant PCR-DGGE DNA fragments showed 0.92 to 0.99 similarity indices to Beta-proteobacteria, Flavobacterium sp. GOBB3-206, Flexibacter-Cytophaga-Bacteroides group, and Gram-positive bacteria. Results suggest diverse microbial communities including microorganisms that may significantly contribute to biogeochemical elemental cycles.

Keywords:

• Microbial community
• characterization
• constructed wetlands
• phospholipid fatty acid (PLFA) analysis
• 16S ribosomal DNA polymerase chain reaction
• amplification-denaturing gradient gel electrophoresis (PCR-DGGE)

Acknowledgments

This study was funded by a grant from Bloomington-Normal, Illinois Water Reclamation District (BNWRD) wastewater treatment facility and an Illinois State University [ISU] research grant. Their support, guidance, and cooperation are appreciated. PLFA and PCR-DGGE analyses were conducted by Microbial Insights, Rockford, TN (http://www.microbe.com, E-mail: [email protected]).

Notes

¹ The cell equivalent value is calculated from experiments with typical bacteria isolated from soil and water. This value is based on 2.0 × 10¹² cells per gram dry weight of cells and 10⁸ picomoles of phospholipid/gram dry weight of cells. The number of cells/gram of dry weight may vary and is dependent on the environmental conditions from which the microorganisms were recovered.

^a Growth rate of the Gram-negative community is assessed by the ratio cy/ω7c fatty acids. Ratios < 0.1 log phase, 0.1 to 5.0 Stationary Phase, > 5.0 Decline Phase.

^b Adaptation of the Gram-negative community to changes in the environment is determined by the ratio of ω 7t/ω 7c fatty acids. Ratios > 0.1 shows adapting to environmentally induced stress.