15 March 2019
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Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.
Often described in a cursory manner as the slime, the extracellular polymeric substances (EPS) are key to the formation, persistence and physicochemical behavior of microbial biofilms across clinical, environmental and industrial settings (Seviour etal., 2012b). Moreover, increased tolerance to antimicrobials is the result of the ability of certain pathogens to produce EPS, which hence constitutes a global threat to the consequences of multidrug resistance (Frieri etal., 2017).
EPS also play significant roles in the successful implementation of water reclamation and purification technologies that have arisen to meet increasing demands for water of different purities, water scarcity (predicted by the United Nations to be the biggest global problem in the coming decade), land shortage and the water-energy nexus. EPS provide structure for anaerobic and aerobic granular sludges, which have emerged over the last thirty years, along with activated sludge and fixed biofilm systems (i.e. trickling filters), as alternatives for biological treatment of industrial and domestic used waters with lower land and energy footprints (Bengtsson etal., 2018). Advances in membrane technologies have made it possible to create drinking water either from sources that were previously considered not available for drinking water production (i.e. brackish water seawater, or wastewater) (Le and Nunes, 2016), or without the addition of chemical disinfectants (Derlon etal., 2012; Madaeni, 1999). However, the hydraulic throughput of these technologies is often limited by membrane fouling, which in many instances is due to biofilm growth.
Biofilms, therefore, feature prominently in many of the challenges facing water technology implementations. As the number of antimicrobial-resistant strains increases, and the range of water reclamation and purification technologies grows, so too does the need to control or predict EPS production. Yet, despite decades of research, we know very little about the molecular composition and function assigned to individual EPS components, and we are not in a position to control the formation and composition with any meaningful predictable outcome. This limits our ability to manage biofilms effectively. We need to enhance our efforts to deliver improved analytical methods and unravel biochemical production pathways, and most importantly, discontinue the use of methods that misrepresent the roles and significance of EPS. The current practice of dismissing EPS, or relegating them to merely a perfunctory study as a footnote in process optimization, should be abandoned. It is essential to identify and reveal how EPS composition determines the microscopic and macroscopic behavior of biofilm systems.
We propose that identifying functional biofilm EPS is the critical path to address key questions in biofilm control. This will not be possible if we persist with the current practice of applying general, superficial and correlative characterizations alone. However, prior to suggesting a roadmap for achieving an in depth understanding of EPS, it is first necessary to explain why so little progress has been made in identifying and characterizing extracellular polymers present in biofilms.
The extracellular matrix
The EPS of biofilms are a complex mixture of interlaced biological polymers. They provide mechanical stability and scaffolds that allow biofilm cells to establish synergistic microconsortia, enhance water retention and nutrient sorption, provide protection against viruses, predation, antimicrobials and disinfectants, and ultimately act as nutrient recycling yards (Flemming and Wingender, 2010). These functions can be provided by a large variety of biopolymers, particularly polysaccharides,
A solution for the insoluble?
The range of techniques required to extract and solubilize known biopolymers, such as the polysaccharides cellulose, chitin and alginate (examples of neutral, cationic and anionic polysaccharides respectively), highlights the need for even harsh extraction methods (i.e. non-aqueous, extreme pH or temperature) (Zhang etal., 2017). Combinations of mechanical pre-treatments (grinding, ultrasonication, homogenizers), acidification (demineralization), enzymatic hydrolyses, alkalinization (for
Do the same extracellular polymers provide the same functions across systems?
Despite the complexity and diversity of EPS in multi-species biofilms, we assume that particular roles performed by EPS are conserved across biofilms, e.g. gel formation and adhesion (Lin etal., 2013). The more information we acquire on the mechanical, biophysical and structural aspects of the extracellular polymers contributing to these functions, the easier it will be to identify and monitor their expression. This could involve information derived from metaproteomic analysis, specific
Agreeing on model biofilms for EPS characterization
Full-scale biological systems in the water sector are often represented by highly diverse microbial communities (Saunders etal., 2016). We would expect the EPS to be similarly complex at a molecular level. Hence, full-scale systems may not be the ideal starting point for isolating and characterizing reference polymers. We should therefore improve the resolution of characterization of EPS from biofilms comprising organisms known to contribute to key water and wastewater biofilm functions, such
Sequencing approaches for EPS characterization
The application of next generation DNA-sequencing methods in conjunction with bioinformatic analyses may allow for the identification of signature extracellular polymers across a vast number of environmental biofilms, and to elucidate their regulation. Metagenome assembled genomes (MAGs) representing individual community species can be described relatively inexpensively (Albertsen etal., 2013), and when coupled with long-read sequencing technologies, such as PacBio and Nanopore sequencing,
In situ approaches have an important role to play
New and combined imaging techniques offer the opportunity to link the production of specific EPS components with specific bacterial groups in situ, as well as validate whether the isolated polymers are indeed extracellular. Imaging provides a link between genomic information and how the EPS are distributed throughout the biofilm (i.e. with regards to location), whereby changes in microbial cells and matrix composition can be monitored over time and together with changes in environmental
Can EPS recovery help us to move towards a circular economy?
A better understanding of the EPS matrix will lead to improved strategies for both resource recovery and biofilm management in water and wastewater treatment systems. The growing interest in renewable resources highlights a focus on the production of EPS from waste biomass, and their conversion into bioproducts and biomaterials, as an appealing route for contributing to a reduced economic dependence on fossil fuels (More etal., 2016) and enhanced sustainability and economics of wastewater
Improved bioprocess control through EPS management
The optimum strategy for biofilm control depends largely on whether EPS production is beneficial (e.g. granular sludges) or detrimental (e.g. membrane bioreactors, infections or biofouling). For both outcomes, altering the mechanical properties of biofilms may improve the process management. Changing either the EPS constituents that are present or how they interact with each other, will modify biofilm cohesive strength, viscosity or elasticity. This can allow for easier removal of biofilms from
A better understanding of the EPS will increase the breadth of strategies available for controlling biofilms in water, wastewater and medical systems alike, which are currently unreliable, empirical and binary (at best). A variety of complementary approaches is required, to overcome extraction and analysis biases, as well as knowledge constraints regarding, for example, exopolymer references in databases. Required developments include:
Extraction methods targeting full solubilization of key
The collaboration was supported by Singapore National Research Foundation and Ministry of Education under the Research Centre of Excellence Programme, by a program grant from the National Research Foundation (NRF), project number 1301-IRIS-59 (TS); by the SIAM Gravitation 024.002.002, the Netherlands Organisation for Scientific Research and KNAW 530-6CDP15, Koninklijke Nederlandse Akademie van Wetenschappen (YL); by the European Union’s Horizon 2020 research and innovation programme under the
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Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms
The application of membrane technology for water disinfection
The chemical and mechanical differences between alginate-like exopolysaccharides isolated from aerobic flocculent sludge and aerobic granular sludge
Sustainable polysaccharide-based biomaterial recovered from waste aerobic granular sludge as a surface coating material
Characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant
Materials and membrane technologies for water and energy sustainability
Colorimetric measurement of carbohydrates in biological wastewater treatment systems: a critical evaluation
Extraction and characterization of chitin and chitosan from (Labeo rohit) fish scales
Recent applications of hyperspectral imaging in microbiology
J.Infec. Pub. Health
Biofilm structures (EPS and bacterial communities) in drinking water distribution systems are conditioned by hydraulics and influence discolouration
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Linking composition of extracellular polymeric substances (EPS) to the physical structure and hydraulic resistance of membrane biofilms
Predation influences the structure of biofilm developed on ultrafiltration membranes
The challenge and promise of glycomics
Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ
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Mapping glycoconjugate-mediated interactions of marine Bacteroidetes with diatoms
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Treatment of municipal wastewater with aerobic granular sludge
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