Biofilm formation on reverse osmosis membranes is initiated and dominated by Sphingomonas spp

Abstract

The initial formation and spatiotemporal development of microbial biofilm layers on surfaces of new and clean reverse osmosis (RO) membranes and feed-side spacers were monitored in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The feed water of the RO system had been treated by the sequential application of coagulation, flocculation, sand filtration, ultrafiltration, and cartridge filtration processes. The design of the flow cells permitted the production of permeate under cross-flow conditions similar to those in spiral-wound RO membrane elements of the full-scale system. Membrane autopsies were done after 4, 8, 16, and 32 days of flow-cell operation. A combination of molecular (fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis [DGGE], and cloning) and microscopic (field emission scanning electron, epifluorescence, and confocal laser scanning microscopy) techniques was applied to analyze the abundance, composition, architecture, and three-dimensional structure of biofilm communities. The results of the study point out the unique role of Sphingomonas spp. in the initial formation and subsequent maturation of biofilms on the RO membrane and feed-side spacer surfaces.

FIG. 1.

Schematic outline of the reverse osmosis (RO) system of a full-scale water purification plant. The fresh surface water (F) was extensively treated by the sequential application of coagulation, flocculation, and sand filtration (CSF), the ultrafiltration (UF), and the cartridge filtration (CF) processes and used as the feed to the 2-stage RO system and to the connected flow cells. The plant produced process quality water (P).

FIG. 2.

Photographs of fouled reverse osmosis membranes (A) and their feed-side spacers (B). The membranes and spacers were removed from the flow cells after 4 (column 4d), 8 (column 8d), 16 (column 16d), and 32 (column 32d) days of operation. The direction of the feed water flow along the length of each flow cell was from left to right.

FIG. 3.

Scanning electron micrographs of the surface of the RO membrane after 4 and 16 days. (A) Rod-shaped gels embedded in an extracellular fibrillar material structure (square 1). Compact aggregates are visible on top of this biofilm (square 2). The RO membrane surface is visible at the bottom (under the biofilm layer) as a rough-appearing texture. Bar, 1 μm. (B) Typical microcolony formed on the surface of the RO membrane after 16 days. Bar, 5 μm.

FIG. 4.

Epifluorescence micrographs depicting mode of initial formation and spatiotemporal development of biofilm structures by pioneer colonizers of RO membrane surfaces. Horizontal panels (A to D) represent images of microcolonies (red and pink fluorescence) as follows: “carpets” of Sphingomonas spp. (A) and “patches” of members of the Betaproteobacteria (B), Gammaproteobacteria (C), and CFB (D). The ages of the biofilms are represented in the vertical columns, with columns 1 to 4 showing images from 4, 8, 16, and 32 days, respectively. Red fluorescence in the images was acquired from the Cy3-labeled probes (SPH120, BET42a, GAM42a, and CF319a), while blue is from the DAPI-stained cells or from Calcofluor white-stained β-1,4-linked polymers of the biofilm EPS matrix, and green is from the positive interaction of FITC-ConA with α-d-glucose and α-d-mannose. Bars, 5 μm (C1) and 10 μm (the other images).

FIG. 5.

Representative CLSM images of RO membrane biofilms depicting complex architecture of mature microbial communities after 32 days of operation. Series of horizontal (x-y) (A) and sagittal (x-z) (B) optical sections were taken at 1-μm intervals across the z axis of the biofilm. The sections show shapes and spatial arrangements of bacterial cells and EPS matrix within mixed-species biofilm communities. The main distribution of cells and polysaccharides was at the top of the RO membrane surface. Cells of Sphingomonas spp. were stained with Cy3-labeled SPH120 probe (red fluorescence), and cells of remaining community members with DAPI (blue fluorescence). α-Polysaccharides of biofilm EPS matrix were stained with FITC-ConA (green fluorescence). Z-scan positions in μm from the top of the RO membrane surface are indicated in each image. Bars, 10 μm.

FIG. 6.

A schematic representation of the observed biofilm structure in a mature RO membrane biofouling layer. Single planktonic cells of Sphingomonas spp. and clumps of Beta- and Gammaproteobacteria present in the feed water colonize surfaces.