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A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy
Published on: January 25, 2017
Microscopic observation of multispecies biofilm of various structures on whey concentration membranes
A N Hassan1, S Anand, M Avadhanula
1Midwest Dairy Foods Research Center, Dairy Science Department, South Dakota State University, Brookings 57007, USA. Ashraf.Hassan@sdstate.edu
Abstract:
The objective of this study was to evaluate biofilm formation on polyamide reverse osmosis (RO) whey concentration membranes. Biofilms were observed with scanning electron and fluorescence microscopy. For scanning electron microscopy, pieces of 6-, 12-, and 14-mo-old membranes were allowed to air dry at room temperature (22 degrees C) for 24h followed by sputter coating with a 5-nm layer of gold and microscopic observations. Scanning electron microscopy images revealed that the hydrophilic layer, used to prevent membrane plugging, was not evenly distributed on the surface. Although this hydrophilic layer seemed to prevent the attachment of proteins, it supported biofilm formation. Three different structures of multispecies biofilm were observed on the retentate side of the membrane: 1) a mono layer, 2) a 3-dimensional structure of a dense matrix of extracellular polymeric substances where different types of bacterial cells were embedded, and 3) cell aggregates. In some of the biofilms, a smooth layer (shell) covered cell aggregates. In the 6-mo-old membranes, part of the shell layer was broken off. Biofilms as observed on the RO membrane were described as having a hill-and-valley type of structure, with hills showing a mushroom-like appearance and valleys comprising dense matrices of extracellular polymers with embedded bacterial cells. Fluorescence microscopy showed live cells on the surface of the biofilm. It is concluded that both cells in the deep layers of biofilm and surface cells may resist cleaning and sanitation. The extent of biofilm formation and the presence of live cells on RO membranes after regular clean in place cycles indicate the need for a more effective cleaning regimen customized for dairy separation systems.
Insights
Biofilm formation on polyamide reverse osmosis (RO) membranes used in whey concentration was evaluated. Findings indicate that despite a hydrophilic layer, biofilms develop, necessitating improved cleaning protocols for dairy systems.
Area of Science:
- Membrane science
- Microbiology
- Food science
Background:
- Polyamide reverse osmosis (RO) membranes are crucial for whey concentration in the dairy industry.
- Biofilm formation on membranes can impede efficiency and compromise product safety.
- Understanding biofilm structure and resilience is key to effective sanitation.
Purpose of the Study:
- To investigate and characterize biofilm development on polyamide RO whey concentration membranes.
- To assess the impact of membrane surface properties on biofilm formation.
- To evaluate the viability of biofilm cells and their resistance to cleaning.
Main Methods:
- Scanning electron microscopy (SEM) was used to visualize biofilm structures on membranes of varying ages (6, 12, 14 months).
- Fluorescence microscopy confirmed the presence of live microbial cells within the biofilms.
- Membrane surface characteristics, including hydrophilic layer distribution, were examined.
Main Results:
- The hydrophilic layer, intended to prevent fouling, was unevenly distributed and supported biofilm formation.
- Three distinct biofilm structures were observed: monolayer, 3D matrix with embedded cells, and cell aggregates, some with a protective shell layer.
- Biofilms exhibited a hill-and-valley morphology with mushroom-like hills and dense extracellular polymeric substance valleys containing bacterial cells.
- Live cells were detected on the biofilm surface and within deeper layers.
Conclusions:
- Biofilm formation occurs on polyamide RO whey concentration membranes, despite surface treatments.
- Both surface and deep-layer biofilm cells demonstrate resistance to standard cleaning procedures.
- Current cleaning-in-place (CIP) regimens may be insufficient, highlighting the need for customized, more effective sanitation strategies for dairy processing systems.
