Environmental Engineering Reference
In-Depth Information
are highly repetitive proteins that are located on the outer membrane [23, 31, 32]
and aggregates of INPs likely provide templates for ice nucleation [29, 33].
Because the INP sequences are very similar [34], yet the bacteria are evolutio-
narily divergent, it has been suggested that the genes have been horizontally
transferred to several members of the plant-associated microbial community
[35]. Since many of these microbes are considered plant pathogens, it has been
speculated that INPs may serve to deliberately cause frost injury to plants,
thereby increasing the fitness of the host bacteria [36]. A few of these proteins
have been recombinantly expressed and one strain of Pseudomonas syringae is
used commercially for 'snow making' (Snomax, York Snow Inc., Centennial
CO, USA). In another application, a genetically manipulated, INP-minus ver-
sion of these bacteria can be successfully used when warranted by a short
growing season [36]. The introduced microbes inhibit the formation of ice on
crops, reducing extensive frost damage at temperatures just below freezing.
The search for novel products from ice-associating microorganisms that sur-
vive in extreme locations can be expensive and bureaucratically cumbersome.
In contrast, our investigations are based on the assumption that ice-associating
products can be found in microbes that also inhabit more easily sampled envir-
onments, including our own backyards. Techniques for identification and
isolation are described and discussed in the following sections.
2.3 Proposed Technology for Microbe Selection
Although microbes with low temperature adaptations may not be abundant
if conditions are warm, presumably all that is required for their discovery is
a powerful method of selection in order to recover the rarer cells with these
properties. New selection regimes have been developed for this purpose.
2.3.1 Cryocycler Selection
We designed an instrument for automatically subjecting microbial cultures to
programmed freeze-thaw treatments and dubbed it a 'cryocycler' (Fig. 2.1).
This instrument switches between two fixed temperature baths containing
ethylene glycol by using solenoid-activated valves. In this way it can achieve
maximum heating and cooling rates. Only a relatively few freeze-resistant
microbes have been shown to survive such strong selection, even after starting
with tens of thousands of cells from a mixed consortium [27]. Routinely, soil
samples are obtained from easily accessible temperate locations and cultured in
dilute medium. Full strength, rich medium can also be used. However, our
observations suggest that when using rich medium there is less culturable
diversity in soils from regions, such as boreal outcrops, surmised to be more
nutrient limited. Vials containing the cultured consortium are then placed in a
