Investigating extremotolerant microbes in non-extreme environments and altering the salinity growth limits of halophiles

In this thesis three key questions were addressed: 1) what are the physiological limits of extremophilic/extremotolerant microbes enriched from non-extreme environments; 2) how does the source environment (permanently hypersaline versus variable salinity) influence the development of a community enriched at different salinities; and 3) to what extent can populations adapt to grow at higher or lower salinity in a laboratory-evolution experiment. Methods included high-throughput microbial culturing, determination of microbial growth windows, microbial community analysis using Illumina MiSeq analysis of phylogenetically informative genes, and comparative genomics of evolved versus original strains. In addressing question 1, it was found that freshwater- and marine-derived microbes enriched in extreme conditions (e.g. with partially inhibitory concentrations of ZnCl2, CuSO4, NaCl, MgCl2, sorbitol or HCl) had a significantly wider tolerance to stressors than microbes from the same samples enriched in control conditions with no stressor. However, the growth rate of nearly all stressor-enriched microbial communities was lower than the growth rate of the control-enriched communities. This suggests that being able to grow both in extreme and in non-extreme conditions comes with a trade-off for these extremotolerant microbes. For question 2, All enrichments were almost entirely overtaken by one genus by the end of the experiment. The haloarchaeon Halorhabdus sp. outcompeted every other microbe in enrichments from the permanently high-salt environment, while in the more variable salt-spring samples the bacterium Halomonas sp. dominated the low-salt enrichments, and the haloarchaeon Haloferax sp. outgrew other species in the mid-, and high-salt enrichments. In the laboratory evolution experiment, the bacterium Halomonas elongata 1H9 acquired more mutations in its genome in sub-optimal and super-optimal salinity cultures than the archaeon Halobacterium salinarum 91-R6, in part due to its faster growth rate and shorter generation time, and hence, higher probability of mutations. Halomonas elongata 1H9 also demonstrated changes in growth and adaptation to the salinity of the medium in which it was cultured. However, further research needs to be done to identify a connection between the mutations and the evolved growth patterns. These findings support the theory that extremophiles and extremotolerants can be found far outside of their physiologically optimal habitat, are able to evolve reasonably fast to changes in environmental conditions, and verifies the ubiquitous dispersal of halophilic and halotolerant ‘microbial weed’ species.