ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

Chapter 6: BACTERIAL COMMUNITY ANALYSIS
6.1 -
Introduction
To assess whether antifreeze protein (AFP) activity provided a selective
advantage to those bacterial isolates that were shown to have activity, and to identify the
spatial and temporal fluctuations in the lacustrine bacterial communities, a rudimentar\
molecular study of the eubacterial community was done.
The application of molecular biological techniques to microbial ecology- has
revolutionized our view of microbial diversity. It is now generally accepted that only a
small fraction of the actual bacterial diversity has been identified using traditional
culturing techniques (Muyzer et al., 1993; Ovreas et al., 1997: Hugenholtz et al., 1998:
Muyzer & Smalla, 1998; Bosshard et al., 2000). The apparent underestimation of
bacterial diversity and abundance within a community has been called "the great plate
count anomaly" whereby the use of viable plate counting and most-probable-number
techniques has proved inadequate to assess bacterial communities because current
culturing techniques fail to isolate the majority of environmental bacterial species
(Amann et al., 1995). Using molecular techniques the number of characterised bacterial
divisions has tripled to about 40 due to the identification of novel taxa without the need
for cultivation (Hugenholtz & Pace, 1996; Hugenholtz et al., 1998). Studying the
bacterial diversity of a community has therefore been dramatically advanced by the use
of environmental clone libraries (Bano & Hollibaugh, 2002) and techniques such as
fluorescent in situ hybridisation (FISH, Amann et al., 1995; Ekong & Wolfe. 1998;
Hugenholtz et al., 1998; Bano & Hollibaugh, 2002), which enable the study of un-
culturable bacterial species. Using techniques such as denaturing gradient gel
electrophoresis (DGGE. Muyzer et al., 1993) it is possible to rapidly analyse structural
and species composition change within a community over time and along
physicochemical gradients, such as those found within the lakes of the current stud)
(Chapter 3).
The use of DGGE in studying complex microbial populations was developed by
Muyzer et al. (1993) and has since been used to study microbial communities from many
different environments by separation of the variable V3 region of the 16S rRNA gene
159