Environmental Engineering Reference
In-Depth Information
a
b
c
Fig. 2.6 Crystal shape assay. Ice crystals grown in the presence of (a) control solutions such as
buffer (shown), E. coli or medium, (b) Type I fish AFP and (c) P. borealis cultures were
examined under a microscope (40x) ([50] reproduced by permission of The Royal Society
of Chemistry)
Microscopic analysis of ice crystal morphology is another method for exam-
ining lysates and cultured samples, but it has not yet been adapted for the
screening of large sample sets. Typically it is done by freezing a microscopic
volume of solution in an oil droplet placed in a nanoliter osmometer. After
freezing, the temperature is slowly increased until a single crystal remains. Only
then is the temperature lowered slightly, and subsequently held constant, so that
the morphology of the ice crystal shape can be noted [43]. For example, when ice
crystals formed in the presence of the Chryseobacterium isolate, obtained after
selection in the cryocycler, the morphology was not disc-like as is seen in the
presence of E. coli, but rather more oblong [39]. Crystals formed in the presence of
the ice-affinity selected P. borealis were not bipyramidal as seen with many fish
AFPs, but more hexagonal or rectangular (Fig. 2.6). These different crystal shapes
likely reflect the preferential adsorption of these proteins to different ice faces.
2.4 Prospects for Applications to the Energy Sector and Elsewhere
The development of these new selective methods has allowed us to imitate the
rigors of high latitude environments in the laboratory and has resulted in the
selection of a group of microorganisms with a high proportion of ice-associating
properties. Although the analysis of the selectedculturesisstillin progress, a total
of 70% (9/13) of the recovered, identified genera have some type of ice-associating
activity. Four of 13 genera were recovered using both the cryocycler and ice affinity
methods (Wilson and Walker, unpublished). In addition, of the 13, almost all have
been previously described as associating with ice and have been recovered by others
from samples obtained from glacial cores, Antarctic lakes or sea ice [7, 44]. As
mentioned previously, ice-associating activities do not appear to be very common
properties amongst bacteria. To date, AFPs have been described in half a dozen
genera, but the corresponding sequences, which appear to be dissimilar from one
another, have been characterized or cloned and expressed from only a few of these
[12, 22, 28, 45]. In contrast, INPs, which have been described in a few plant-
associating bacteria, appear to be homologous proteins [35]. Thus methods to
select and recover ice-active microorganisms from a variety of habitats, including
Search WWH ::




Custom Search