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Exploring the role of key bacteria in stream biofilms

What is known about the development of stream biofilms?

All available surfaces in streams are usually well-populated and further colonization is often an “adhere-and-compete” effect rather than fresh formation. Occasionally, sloughing of complete or partial biofilms results in fresh surfaces being made available for biofilm formation. Bacterial colonization of such surfaces will depend on the ability of certain organisms to identify favourable sites, attach, multiply rapidly and dominate within the biofilm. Hence, it is important to know if some of the mature stream biofilm bacterial isolates (obtained from regular sampling of New Zealand streams) possess primary colonization properties and promote the development of functional communities.

As part of the Public Good Science Fund (PGSF) programme, biofilms were being regularly sampled and pure cultures of bacterial isolates obtained. This led us to ask the following question: Can we identify any “key” isolates from the existing bacterial biofilm isolates on the basis of their attachability to a surface?

Origin of bacterial isolates :

As part of the sampling programme, biofilms were collected at regular intervals from New Zealand streams with varying levels of human impact. Biofilm samples were collected, homogenised and a series of biofilm dilutions spread plated onto R2A nutritional media plates to obtain pure cultures which were stored in 20% glycerol at -80oC. Eleven such isolates obtained from the moderately impacted Opanuku Creek, (Auckland) and one isolate from the unimpacted Canterbury site were used in a preliminary screening experiment to investigate their biofilm forming capabilities and how they compared with each other.
Brief description of isolates: - All twelve isolates were motile, Gram negative rods, approximately 1.5-2.5 μm x 0.5 μm, and most isolates showed end-to-end attachment of cells while some showed a palisade cell arrangement. Some cultures showed the presence of intracellular granules.
Preliminary screening experiments to derive the five key attachable isolates: - A surrogate crystal violet biofilm assay was carried out during which 12-well microtitre plates were inoculated with overnight broth cultures of all isolates, and R2A broth was used as the nutrient medium. All plates were incubated statically at 15oC and biofilm formation assessed over 3, 24 and 48 h. Unattached cells were washed off and biofilms were stained with crystal violet. The amount of biofilm biomass was estimated from the amount of crystal violet attached to the wells of the microtitre plates by destaining with alcohol and the absorbances read at 650 nm. Quantitative data was plotted for all twelve isolates and the five key isolates were identified as those with the highest crystal violet absorbances.

Identity of key isolates :

Preliminary screening results showed that isolates 5A, 6A, 8A, #10 and CB007 were the best attachable cultures and were selected as the five key isolates mainly on the basis of the crystal violet assay.
The results obtained were compared with the molecular diversity data already available (Roberts, K.) and the functional presence of the “key” isolates confirmed.


48hrs old biofilms stained with crystal violet and the “key” isolates in red

The biofilm formation capabilities of the key isolates were compared by carrying out crystal violet assays in two nutrient media, 1:10 R2A broth ands stream water. Results indicated that most “key” isolates formed denser biofilms in stream water than in diluted broth. All isolates formed unique but similar biofilm structures in bgoth low-nutrient media. These ranged from simple sheet-like biofilms to “pillar-shaped” microcolonies. Serratia plymuthica formed unique “leaf-shaped” structures in stream water but not in dilute broth.
Now we know that key bacteria exist and play a role in attachment to establish functional presence over time. In natural systems, microbial communities are known to exist as mixed populations. This means that key bacteria must also have the ability to interact with other bacteria they may encounter in the stream flow and contribute to the development of biofilm structure. Hence, an understanding of the mechanisms by which key bacteria obtained from mature NZ streams respond to the presence of other bacteria, key and non-key, is important in determining the structural integrity and functional capability of biofilms.

Ongoing research :

This will mainly involve methods to Gfp-tag key bacteria so that desirable partners of a mix can be differentially labelled (one green and other red for fluorescence microscopy) and their interactions studied by growing the mix in artificially cultivated chambers better suited to provide conditions similar to those encountered in stream biofilms.

 

 

 

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Stream Biofilm Research Group
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The University of Auckland
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© Stream Biofilm Project, The University of Auckland 2008-2010
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