Vibriosis is a major epizootic disease that impacts free-living and farmed fish species worldwide. Use of probiotics is a promising approach for prevention of Vibrio infections in aquaculture. A probiotic anti-Vibrio strain, Bacillus pumilus H2, was characterized, and the mechanism of its effect was investigated. All 29 Vibrio strains tested were growth-inhibited by H2. The anti-Vibrio substance present in cell-free supernatant of H2 was purified and characterized by reversed-phase HPLC. Minimum inhibitory concentrations of the purified substance, determined in liquid media for various Vibrio strains, ranged from 0.5 to 64 µg/ml. Addition of the purified substance to Vibrio vulnificus culture inhibited cell growth (estimated by OD600). Confocal microscopy and scanning electron microscopy analyses showed that surface structure of V. vulnificus cells was damaged by the purified substance, as reflected by presence of membrane holes, disappearance of cellular contents, and formation of cell cavities. The major mechanism of this anti-Vibrio activity appeared to involve disruption of cell membranes, and consequent cell lysis. The purified anti-Vibrio substance was shown to be structurally identical to amicoumacin A by MS and NMR analysis. Our findings indicate that B. pumilus H2 has strong potential for prevention or treatment of fish vibriosis in the aquaculture industry.

Nitrogen (N₂) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N₂. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of ¹⁵N-labeled hydroxylamine to ¹⁵N₂. This conversion did not happen under oxygen (O₂)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a K_m of 92.9 ± 3.0 μM for hydroxylamine and a k_cat of 0.028 ± 0.001 s⁻¹. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N₂.

Bacterial community dynamics of the ANAMMOX reactor of an integrated “UASB + SHARON + ANAMMOX” system for treating piggery wastewater were investigated using the Illumina MiSeq method with samples obtained at ~ 2-week intervals during a 314-day period. With aerobic activated sludge as seeds and low content artificial wastewater (NH4+–N 50 mg/L; NO2−–N 55 mg/L) as influent for the ANAMMOX reactor, nitrogen removal was initially observed on day 38 with a removal rate 1.3 mg N L−1 day−1, and increased to 90.4 mg N L−1 day−1 on day 55 with almost complete removal of ammonia and nitrite, indicating a successful startup of the reactor. Increasing influent load stepwise to NH4+–N 272.7 mg/L/NO2−–N 300 mg/L, nitrogen removal rate increased gradually to 470 mg N L−1 day−1 on day 228, and maintained a stable level (~ 420 mg N L−1 day−1) following introduction of SHARON effluent since day 229. Correlation between microbial community dynamics and nitrogen removal capability was significant (r = 0.489, p < 0.001). Microbial community composition was determined by influent ammonia, influent nitrite, effluent nitrate and some undefined factors. Anammox bacteria, accounting for ~ 98.7% of Planctomycetes, became detectable (0.03% relative abundance) since day 38 and increased to 0.9% on day 58, well consistent with nitrogen removal performance of the reactor. Relative abundance of anammox bacteria gradually increased to 38.4% on day 140 with stepwise increased influent load; decreased to 0.4% on day 169 because of nitrite inhibition; increased to 19.24% on day 233 when the influent load was dropped; kept at ~ 9.0% with SHARON effluent used as influent and dropped to 3.3% finally. Anammox bacteria, only Candidatus Brocadia and Ca. Kuenenia detected, were the most abundant at genus level. Ca. Brocadia related taxa were enriched firstly under low load and detectable during the entire experimental period. Three main groups represented by Ca. Brocadia related OTUs were enriched or eliminated at different loads, but Ca. Kuenenia related taxa were enriched only under high load (NO2−–N > 300 mg/L), suggesting their different niches and application for different loads. These findings improve the understanding of relationships among microbial community/functional taxa, running parameters and reactor performance, and will be useful in optimizing running parameters for rapid startup and high, stable efficiency.

A novel bacterium, designated TRM 415^T, belonging to the genus Brevibacterium, was isolated from a sediment sample from a salt lake in Xinjiang province, China. Comparative 16S rRNA gene sequence analysis indicated that strain TRM 415^T was phylogenetically most closely related to Brevibacterium album YIM 90718^T (98.4 % sequence similarity) and had low similarity (<95.5 %) to other species of the genus Brevibacterium; however, DNA–DNA hybridization studies between strain TRM 415^T and B. album YIM 90718^T showed only 41.3 % relatedness. Strain TRM 415^T possessed meso-diaminopimelic acid as the diagnostic cell-wall diamino acid, MK-8(H₂) as the major menaquinone and polar lipids including phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C_17 : 0 and anteiso-C_15 : 0. The genomic DNA G+C content was 69 mol%. Based on the evidence from this polyphasic study, strain TRM 415^T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium salitolerans sp. nov. is proposed. The type strain is TRM 415^T (=JCM 15900^T =CCTCC AB 208328^T =KCTC 19616^T).

Optimization of running parameters in a bioreactor requires detailed understanding of microbial community dynamics during the startup and running periods. Using a novel piggery wastewater treatment system termed “UASB + SHARON + ANAMMOX” constructed in our laboratory, we investigated microbial community dynamics using the Illumina MiSeq method, taking activated sludge samples at ~2-week intervals during a ~300-day period. Ammonia-oxidizing bacteria (AOB) were further investigated by quantification of AOB amoA genes and construction of gene clone libraries. Major changes in bacterial community composition and dynamics occurred when running manner was changed from continuous flow manner (CFM) to sequencing batch manner (SBM), and when effluent from an upflow anaerobic sludge blanket (UASB) reactor for practical treatment of real piggery wastewater was used as influent; differences among these three experimental groups were significant (R 2 = 0.94, p < 0.01). When running manner was changed from CFM to SBM, relative abundance of the genus Nitrospira decreased sharply from 18.1 % on day 116 to 1.5 % on day 130, and to undetectable level thereafter. Relative abundance of the genus Nitrosomonas increased from ~0.67 % during the CFM period to 8.0 % by day 220, and thereafter decreased to a near-constant ~1.6 %. Environmental factors such as load ammonia, effluent ammonia, effluent nitrite, UASB effluent, pH, and DO levels collectively drove bacterial community dynamics and contributed to maintenance of effluent NH4 +-N/NO2 −-N ratio ~1. Theses results might provide useful clues for the control of the startup processes and maintaining high efficiency of such bioreactors.