Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N ₂ atmosphere

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.