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Gram-negative  Pseudomonas  and Gram-positive  Bacillus  are the most common spoilage bacteria in raw and pasteurized milk, respectively. In previous studies, nitrogen (N 2 ) gas flushing treatments of raw and pasteurized milk at cold chain-temperatures inhibited bacterial spoilage and highlighted different susceptibilities to the N 2  treatment with the exclusion of certain bacterial types. Here, we investigated the effects of pure N 2  gas flushing on representative strains of these genera grown in mono- or co-cultures at 15 and 25°C.  Bacillus weihenstephanensis , a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain. Among  Pseudomonas, P. tolaasii  LMG 2342 T  and strain C1, a raw milk psychrotroph, were selected. The N 2  gas flushing treatment revealed: (1) temperature-dependent responses; (2) inhibition of the growth of both pseudomonads; (3) emergence of small colony variants (SCVs) for  B. weihenstephanensis  strain KBAB4 at 15°C induced by the N 2  treatment or when grown in co-culture with  Pseudomonas  strains; (4) N 2  gas flushing modulates (suppressed or stimulated) bacterial antagonistic reactions in co-cultures; (5) most importantly, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that at 25°C the majority of the KBAB4 cells were killed by pure N 2  gas flushing. This observation constitutes the first evidence that N 2  gas flushing has bactericidal effects.

Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N2 atmosphere