Experimental design
Monocultures of the residents (A. agilis , O. daejonense ,P. corrugata , S. rhizophila , and V. guangxiensis )
were grown statically in glass vials (microcosms) containing 6mL of
1/64th diluted TSB for 48 hours at
28oC. Their densities (number of colony forming units;
CFU) were standardized by their optical density measurements and the
monocultures were then combined to form an equal mixture of the five
species. 60µL of this mixture was added to each of 48 microcosms
containing 6mL of 1/64th TSB. Monocultures of the
invader, P. aeruginosa, were also grown statically for 48 hours
in 1/64th TSB, and then diluted 100-fold in M9 salt
buffer. Each invader inoculate was plated onto KB agar to confirm its
density.
Twelve microcosms received one of four mortality pulse disturbance
regimes: every 2, 4, or 8 days, or one disturbance only at initiation
(‘16 days’). This final treatment served as a control without repeated
pulse disturbance. Pulse disturbances involved transferring 60µL (1%)
of homogenised community into 6mL of fresh media, resulting in 99%
mortality. For each regime, 6 microcosms were challenged with 60µL of
the diluted invader culture and 6 microcosms were used as non-invaded
controls; invasions took place post-disturbance on days 4, 8, and 12,
and the invader inoculate was plated to quantify its density (Fig. 1).
Microcosms were kept static at 28oC with loose lids to
allow oxygen transfer.
After 16 days, all microcosms were thoroughly homogenised, mixed with
glycerol to a final concentration of 25%, and frozen at
-80oC. Thawed samples were plated on KB agar
containing X-gal to quantify their CFUs.