3D Structure

Paul Stoodley P.Stoodley at exeter.ac.uk
Fri Feb 14 09:39:17 EST 1997

I would like to respond to some of the comments made by Julian and Rick
in response to my original message on biofilm structure. There are a
number of points I would like to address:

1) I believe the structure I described is a variation of the one that
Doug Caldwell and John Lawrence’s group and Bill Keevil were describing
before we (the Bozeman we) started using confocal microscopy to get
detailed images of fully hydrated biofilms. The main feature of these
biofilms is that they are heterogenous, with or without a basal layer of

Some references to such heterogenous biofilms: 

Lawrence J.R., Korber D.R., Hoyle B.D., Costerton J.W., and Caldwell
D.E. (1991) Optical sectioning of microbial biofilms. J. Bacteriol.,
173, 6558-6567.

Eighmy T.T., Maratea D., and Bishop P.L. (1983) Electron microscope
examination of wastewater biofilm formation and structural components.
Appl. Environ. Microbiol., 45(6), 1921-1931.

Massol-Deya A.A., Whallon J., Hickey R.F., and Tiedje J.M. (1995)
Channel structures in aerobic biofilms of fixed-film reactors treating
contaminated groundwater. Appl. Environ. Microbiol. 61(2), 769-777.

Bryers, J. and Characklis, W.G. 1981. Early fouling biofilm formation in
a turbulent flow system: overall kinetics. Water Research 15:483-491.

McCoy, W.F., Bryers, J.D., Robbins, J., and Costerton, J.W. 1981.
"Observations of fouling biofilm formation." Canadian Journal of
Microbiology. 27:910-917.

2) How relevant is this heterogenous biofilm structure to the real world
? i.e. is it worthy of study ? It is suggested that the types of
biofilms that we (myself, deBeer, Roe, and Lewandowski) have grown were
limited to flat plate reactors,  with Pseudomonas, growing on glucose in
Bozeman. This is not true, I have also been growing them in Exeter !
Actually we used 3 species of bacteria as referenced in:

Stoodley P., De Beer D., and Lewandowski Z. (1994) Liquid flow in
biofilm systems. Appl. Environ. Microbiol., 60(8), 2711-2716.

The biofilms were also grown in a porous media flow cell (same
reference). Similar biofilms were grown on stainless steel.

Ref: Pendyala, J., Avci, R., Geesey, G.G., Stoodley, P., Hamilton, M.,
and Harkin, G. 1996. "Chemical effects of biofilm colonization on 304
stainless steel."  Journal of Vacuum Science and Technology.

I have used 400 ppm and 40 ppm glucose, and non sterile tap water (no
additional nutrients) and seen similar biofilms. However, the time taken
for the biofilm to accumulate and the final biomass amount (volume -
because I am observing the biofilm, not weighing it) may vary
considerably. A variation that I see is the formation of moving
streamers from the clusters at high Reynolds number.

Ref: Lewandowski, Z. and Stoodley, P. 1995. "Flow induced vibrations,
drag force, and pressure drop in conduits covered with biofilm". Wat.
Sci. Tech. 32(8):19-26.

I have also seen similar biofilms growing on water pipes taken from
industrial systems (nuclear and chemical production facilities). In fact
the picture of biofilms that I get from the literature, and the diverse
amount of real biofilms I have studied, is that they generally do
consist of clusters/stacks/fronds/microlonies and spaces/channels/voids
around them. I am certainly not trying to imply that this is the only
type of structure but I am trying to make a point that there is not
enough published evidence to discount its relevance. I would like to
point out that I do not consider myself well read on the dental /
medical literature and so may be leaving some gaps.

3) Mushroom structure - I did not mention mushrooms in my first message,
however, this term seems to have stuck with the "Bozeman biofilms". I
have pictures of mushrooms and pictures of fluorescent beads flowing
underneath mushrooms on multi stalked legs but I don’t see these
structures all of the time. They are merely another variation on the
theme of heterogeneity and give insight on what is possibly occurring in
the real world.

4) Ricks SEM’s  - Rick says "These biofilms are usually very densely
packed cells no more than a few microns thick (I believe a monolayer in
many cases), discontinuous, and buried in EPS so that just the "backs"
of the cells are visible". Again I am not well read on concrete grown
biofilms but the picture of the biofilm that Rick is describing is
possibly from artefacts in the dehydration process. Brenda Little has
made nice comparisons with SEM and ESEM (hydrated) and found that with
SEM EPS is dehydrated and the entire structure collapses on the surface.
The cells do poke out of the EPS, however, when imaged with ESEM cells
are more difficult to see and are immersed in the opaque slime layer.
Ricks evidence also appears to contradicts Julian’s model - he has an
apparently homogenous biofilm growing on low nutrients.

5) Model predictions for the homogenous biofilm - from what Julian wrote
- "At the lower concentrations a microcolony will deplete a zone around
it of nutrient. This becomes a no go (grow) area so that at the lowes(t)
possible substrate concentration only stacks can form. As food increases
so these fuse leaving voids or channels. At the highest concentrations
all the voids are filled with bacteria since substrate concentration is
not limiting". This model seems to me to be a batch culture with no
convection, if convection in the biofilm is added, and we have proven
that this can occur, nutrient replenishment even under low nutrient
conditions can occur. 

ref: deBeer, D., Stoodley, P., and Lewandowski, Z. 1996. "Liquid flow
and mass transport in heterogeneous biofilms." Water Research.

Additionally Jo Rayner, in our Exeter labs, growing E. faecium biofilms
on 100,000 ppm sucrose + 37 g/l brain heart infusion broth (high
nutrients I would say) in flow cells (50 ml/hr) does not find that
homogenous biofilms are formed (at least after 48 hours) but that they
consist of cell clusters and surrounding channels.

So before assumptions are made on the significance of the structure of
real (living, hydrated, flow condition) biofilms using a computer model,
the parameters included in the model should first be explained and its
validity established.

If anyone has references to substantiate the claim that biofilm channels
are not significant I would be pleased to recieve them. I would also be
interested in references that describe thick homogenous biofilms.

I don’t think we have answered Rauke Hangen's original question: "I
would like to know if there are general accepted and used terms to
describe the different three-dimensional structures of biofilms on solid
surfaces and where I can find these terms", but have opened up many
other cans of worms !

Paul Stoodley, Research Fellow / Ph.D. Candidate
Environmental Microbiology Research Group
Department of Biological Sciences, Hatherly Lab.
Exeter University
Exeter EX4 4PS

Tel:	01392 264348
Fax:	01392 263700
email:	P.Stoodley at exeter.ac.uk

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