Environmental and genetic factors influencing biofilm structure

It is increasingly evident that biofilms growing in a diverse range of medical, industrial, and natural environments form a similarly diverse range of complex structures (Stoodley et al., 1999a). These structures often contain water channels which can increase the supply of nutrients to cells in the biofilm (deBeer and Stoodley 1995) and prompted Costerton et al., (1995) to propose that the water channels may serve as a rudimentary circulatory system of benefit to the biofilm as a whole. This concept suggests that biofilm structure may be controlled, to some extent, by the organisms themselves and may be optimized for a certain set of environmental conditions. To date most of the research on biofilm structure has been focused on the influence of external environmental factors such as surface chemistry and roughness, physical forces (i.e. hydrodynamic shear), or nutrient conditions and the chemistry of the aqueous environment. However, there has been a recent increase in the number of researchers using molecular techniques to study the genetic regulation of biofilm formation and development. Davies et al. (1998) demonstrated that the structure of a Pseudomonas aeruginosa biofilm could be controlled through production of the cell signal (or pheromone) N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). In this paper we will examine some of the research that has been conducted in our labs and the labs of others on the relative contribution of hydrodynamics, nutrients and cell signalling to the structure and behaviour of bacterial biofilms

Biodegradation of the chlorophenoxy herbicide (R)-(+)-mecoprop by Alcaligenes denitrificans

An Alcaligenes denitrificans strain capable of utilizing the herbicide (R)-(+)-2(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) as a sole carbon source was isolated from soil and cultured in liquid medium. Crude cell extracts of the bacterium were utilized in spectrophotometric assays to elucidate a biochemical pathway for degradation of mecoprop. Results indicated a reaction sequence analogous to the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). GC-MS analysis provided direct evidence for the biotransformation of mecoprop to the transient metabolite 4-chloro-2-methylphenol (MCP). No NADPH-dependent activity was observed during this reaction. Pyruvate was verified as the second product derived from the aliphatic side chain of mecoprop. MCP was subsequently transformed to a substituted catechol by an NADPH-dependent monooxygenase. When grown on mecoprop, A. denitrificans was adapted to oxidize catechol and its 3- and 3-methylated derivatives indicating the broad substrate specificity of catechol dioxygenase. The microorganism was demonstrated to adopt the ortho mechanism of aromatic cleavage which resulted in the formation of 2-methyl-3-carboxymethylene but-2-en-4-olide, a reaction intermediate of the beta-ketoadipate pathway

Metal removal by sulphate-reducing bacteria from natural and constructed wetlands

The use of wetlands is a promising technology to treat acid mine drainage, yet there is little understanding of the fundamental biological processes involved. They are considered to centre on the complex anaerobic ecology within sediments and involve the removal of metals by sulphate-reducing bacteria (SRB). These bacteria generate hydrogen sulphide and cause precipitation of metals from solution as the insoluble metal sulphide. Sulphate-reducing bacteria have been isolated from natural and constructed wetlands receiving acid mine drainage. Sulphide production by isolates and removal of the metals iron, manganese and zinc were measured, as well as utilization of a range of carbon sources. Marked ecological differences between the wetlands were reflected in population composition of SRB enrichments, and these consortia displayed significant differences in sulphide generation and rates of metal removal from solution. Rates of metal removal did not correlate with sulphide generation in all cultures, suggesting the involvement of other biological mechanisms of metal removal. Differences in substrate utilization have highlighted the need for further investigation of carbon flow and potential carbon sources within constructed wetlands

Influence of electric fields and pH on biofilm structure as related to the bioelectric effect

Mixed species biofilms of Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas aeruginosa were grown in a flow cell fitted with two platinum wire electrodes. The biofilm growing on the wires reached a thickness of approximately 50 microm after 3 days. When a voltage was applied with oscillating polarity, the biofilm attached to the wire expanded and contracted. The biofilm expanded by approximately 4% when the wire was cathodic but was reduced to 74% of the original thickness when the wire was anodic. The phenomenon was reproduced by alternately flushing the flow cell with media adjusted to pH 3 and pH 10 with no electric current. At pH 10 the biofilm was unaltered, but it became compacted to 69% of the original thickness at pH 3. We explained these phenomena in terms of the molecular interactions between charged acidic groups in the biofilm slime and the bacterial cell walls. Contraction of the biofilm under acidic conditions may be caused by (i) the elimination of electrostatic repulsion from neutralization of negatively charged carboxylate groups through protonation and (ii) subsequent hydrogen bonding between the carboxylic acids and oxygen atoms in the sugars. Electrostatic interactions between negatively charged groups in the biofilm and the charged wire may also be expected to cause biofilm expansion when the wire was cathodic and contraction when the wire was anodic. The consequences of the explanation of the increased susceptibility of biofilm cells to antibiotics in an electric field, the "bioelectric effect," are discussed

The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow

Mixed-species biofilms, consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens and Stenotrophomonas maltophilia, were grown in glass flow cells under either laminar or turbulent flow. The biofilms grown in laminar flow consisted of roughly circular-shaped microcolonies separated by water channels. In contrast, biofilm microcolonies grown in turbulent flow were elongated in the downstream direction, forming filamentous 'streamers'. Moreover, biofilms growing in turbulent flow developed extensive patches of ripple-like structures between 9 and 13 days of growth. Using time-lapse microscopic imaging, we discovered that the biofilm ripples migrated downstream. The morphology and the migration velocity of the ripples varied with short-term changes in the bulk liquid flow velocity. The ripples had a maximum migration velocity of 800 micromh(-1) (2.2 x 10(-7) m s(-1)) when the liquid flow velocity was 0.5 ms(-1) (Reynolds number=1,800). This work challenges the commonly held assumption that biofilm structures remain at the same location on a surface until they eventually detach

Genetic link between 2,4-D and Mecoprop degradation

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