Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 94

Phospholipids PLFA analysis (gas chromatogra-
phy) can be used to estimate total biofilm biomass as
well as biomass levels of different taxonomic groups.
In the biofilm studies of Droppo et al. (2007),
for example, the pattern of PLFA accumulation
(expressed as pmol PLFA mm −2 surface area) was
similar to that of polysaccharides, showing that the
increase in biomass of biofilm organisms was closely
similar to the entire biofilm (including matrix).
PLFA analysis can also be used to assess changes
in the biomass of major groups of biofilm organisms,
including eukaryotes (determination of polyenoics),
gram-negative biota (monosaturated fatty acids) and
gram-positive bacteria (tertiary and branched fatty
acids). PLFA analysis was used to demonstrate a
major switch from prokaryote to eukaryote organ-
isms (Fig. 2.26) in the experimental biofilm studies
of Droppo et al . (2007), paralleling similar changes
in the plankton community.
The biomass of eukaryotic algae in epilithic
biofilms was monitored by Lyon and Ziegler (2009)
by determination of polyunsaturated PLFAs (16:2ω4,
18:2ω6, 18:3ω3, 20:4ω6, 20:5ω3). Other eukaryotes
may also contain these fatty acids, so it is important
to remove biofilm invertebrates prior to analysis and
to check that other eukaryotic microorganisms are
absent or present only at low levels.

prokaryotenucleotidesequencesprovidedataongen-
era of bacteria, blue-green algae and also eukaryote
algae via their chloroplast genomes (Table 2.9).
In some cases, the biofilm shows a major temporal
shift in dominant organisms. Within the cohort of
organisms identified by this method, Droppo et al.
(2007) demonstrated a clear transition from bacteria
(5 day biofilm) to algae, with Scenedesmus (green
alga) dominating at 9 days and Phormidium (blue-
green alga) at 15 days.

2.9.4 Matrix structure

The organic matrix of biofilms is secreted by dif-
ferent organisms within the community and is com-
posed largely of carbohydrate, with DNA, proteins
and uronic acids also present (Bura et al ., 1998). It
is colloidal in nature, with the 10-20 nm colloidal
particles being aggregated into a fibrillar network.
Various types of microscopy have been used to
visualise the structure of the biofilm matrix and the
microorganisms (including algae) in it. One major
problem with studying the biofilm matrix is that it
has a high water content, and the specimen dehydra-
tion associated with conventional light and electron
microscopy leads to complete loss of integrity. Alter-
native procedures for examination therefore need to
be used:
Detached globules of biofilm matrix (flocs) float-
ing in the planktonic phase can be sedimented and
examined by phase-contrast microscopy.

Nucleotide analysis Sequence analysis of 16s
RNA genes, using denaturing gradient gel elec-
trophoresis can lead to the identification of a wide
range of microbial species within biofilms. These

Table 2.9 Identification of Major Biofilm Genera by 16s RNA Gene Sequence Analysis.

Main Taxonomic Group DNA Analysed Genera

Blue-green algae Main genomic DNA Phormidium, Chroococcidiopsis, Anabaena,

Synechococcus

Eukaryote algae Chloroplast genome Scenedesmus, Chlorella

Bacteria
α Proteobacteria Main genomic DNA Caulobacter
Cytophaga / Flavobacterium / Bacteroides Group Main genomic DNA Flavobacterium
Proteobacterium

Main genomic DNA Sphingobacterium

Source: Droppo et al. , 2007. Limnology and Oceanography.
Genera identified in experimental (laboratory) biofilms, with a database (GenBank) sequence match of at least 90%.