3.1 Characterization of Prolixibacter strains isolated from crude-oil- and corrosion-scale samples
Previously, P. denitrificans MIC1-1T was isolated from a crude-oil-emulsion sample collected from an oil well in Akita Prefecture, Japan (Iino et al. , 2015a; Iino et al. , 2015b). In this study, a similar method was used to isolate pure bacterial cultures from three crude-oil-emulsion samples and one corrosion-scale sample collected from four different locations (Table S1). Bacteria capable of growing anaerobically in two different media, SPYSw and YPSw, were screened by repeated “dilution and growth” cycles as described in the Materials and Methods. A total of 76 pure cultures were obtained, which were characterized by 16S rRNA gene sequencing. As shown in Table S1, 16 Prolixibacter strains whose 16S rRNA gene sequences showed >95% identity to that of P. denitrificans MIC1-1T were isolated using SPYSw medium, while only one Prolixibacter strain was obtained when YPSw medium was used for the screening. In the latter medium, bacteria belonging to the genera Aeromonas , Arcobacter ,Marinilabilia , and Thiomicrospira were the predominant bacteria.
Only one Prolixibacter culture from a single source was used for further studies. Three pure cultures derived from three different crude oil samples were designated strains AT004, KGS048, and SD074, while one pure culture obtained from a corrosion-scale sample was designated strain NT017 (Table 1). Phylogenetic analysis based on 16S rRNA gene sequences showed that all four isolates formed a cluster with P. denitrificans MIC1-1T and P. bellariivoransJCM 13498T (original strain name is F2T) in the neighbor-joining (NJ) tree, which was supported by a bootstrap value of 100%. Thus, all the isolates were accommodated in the genus Prolixibacter , orderMarinilabiliales , and phylum Bacteroidetes (Fig. 1) . Among the four isolates, strains AT004, KGS048, and NT017 were closely related to P. denitrificansMIC1-1T, with 98.4–99.2% identity in their 16S rRNA gene sequences, whereas strain SD074 was phylogenetically distinct fromP. bellariivorans JCM 13498T and P. denitrificans MIC1-1T, with pairwise sequence identities of 97.3% and 95.6%, respectively (Table S2).
The cells of all the isolates were mainly rods with a width of approximately 0.3–0.5 µm and a length of approximately 1.2–6.5 µm and had rough cell surfaces (Fig. S1). Spherical cells with a size of 0.6–0.8 µm and long rod cells with a length of 15 µm or more were observed sometimes. Cells usually occurred singly or in pairs. Motility and spore formation were not observed during phase-contrast microscopy. The cell pellets of strains AT004 and KGS048 collected using centrifugation were salmon pink, while those of strains NT017 and SD074 were beige. The cells of strains AT004, KGS048, NT017, and SD074 were stained Gram-negatively by conventional Gram staining (Table S2).
All Prolixibacter strains grew anaerobically, with the same growth yields, in Sw medium supplemented with 0.1% (wt/vol) yeast extract (YSw medium) and YSw medium devoid of NH4Cl (ammonium-free YSw medium) (Fig. 2). Ammonium was formed upon the growth of these strains in ammonium-free YSm medium (Table 2), indicating that this compound was generated by the catabolism of amino acids and other nitrogen-containing compounds present in yeast extract. Thus, yeast extract served as sources of carbon, energy, and nitrogen for the growth of the Prolixibacter strains in ammonium-free YSw medium. The anaerobic growth of P. denitrificans MIC1-1Tand three newly isolated strains (AT004, KGS048, and SD074) was enhanced in the presence of nitrate (Fig. 2) showing that nitrate respiration improved the growth yield of these strains. On the other hand, neither growth stimulation by nitrate nor the reduction of nitrate was observed in strain NT017 and P. bellariivorans JCM 13498T (Fig. 2 and Table 2), indicating that these two strains did not respire nitrate. The ammonium concentrations in the nitrate-amended cultures of the nitrate-reducing strains were significantly higher than those in the nitrate-free cultures of the nitrate-reducing strains (P<0.05, Student’s t-test), while such trends were not observed in the nitrate-non-reducing strains (Table 2). Thus, it seems that the nitrate-reducing strains reduced nitrate not only to nitrite, but also to ammonium. The sum of the nitrite and ammonium concentrations formed during the cultivation of the nitrate-reducing strains were always smaller than the concentrations of nitrate metabolized by these strains. This stoichiometric anomaly could be interpreted as either that ammonium was assimilated by hosts, or that nitrate was also converted to other products than nitrite and ammonium,e.g. nitric oxide.
Yeast extract in YSm medium could be replaced by D-glucose, but not by simple organic acids, including lactate, pyruvate, and acetate (Table S2).
Based on the phylogenetic positions shown in Fig. 1, the phenotypic properties shown in Table S2, and the nitrate-reducing activities described above, strains AT004 and KGS048 are considered to belong toP. denitrificans . Strain NT017, whose 16S rRNA gene sequence was 98.9% identical to that of P. denitrificansMIC1-1T, differed from P. denitrificans by its cell color and the absence of nitrate respiration. Strain SD074 was considered to be a new species because of the low identity of its 16S rRNA gene sequence compared with those of P. bellariivorans andP. denitrificans .