4 DISCUSSION
In this study, four bacterial strains closely related to P. denitrificans MIC1-1T and P. bellariivoransJCM 13498T were newly isolated from crude oil- or corrosion-scale samples (Table S1, Table 1). Phylogenetically, these four strains formed a monophyletic lineage together with P. bellariivorans JCM 13498T and P. denitrificansMIC1-1T with a bootstrap value of 100 % (Fig. 1). The pairwise 16S rRNA sequence similarities among these four strains and two previously isolated strains, P. denitrificansMIC1-1T and P. bellariivorans JCM 13498T, were >95.8%, which was higher than the cutoff value for the delimitation of prokaryotic genera (95%) (Stackebrandt and Goebel, 1994; Tindall et al. , 2010). Thus, the four newly isolated strains belonged to the genus Prolixibacter . Among them, strains AT004 and KGS048 were considered to belong toP. denitrificans based on their 16S rRNA gene sequences and phenotypic traits.
Nitrate-reducing activity was observed in some but not all strains in the genus Prolixibacter . The three strains belonging to P. denitrificans , namely strains MIC1-1T, AT004, and KGS048, were all nitrate reducers, while Prolixibacter sp. NT017, a close relative of P. denitrificans , was nitrate non-reducer (Table 2). Although P. bellariivorans JCM 13498T and Prolixibacter sp. SD074 formed a second clade in the genus Prolixibacter (Fig. 1), the former was nitrate non-reducer and the latter was nitrate-reducer. Thus, there was no concordance between the phylogenetic relationships and the nitrate-reducing phenotype. The anaerobic growth of the nitrate-reducing strains was promoted by nitrate, while that of the nitrate-non-reducing strains was not (Fig. 2). The growth improvement with nitrate may be due to nitrate respiration, which provides more ATP than the fermentation, and due to nitrate assimilation, which provides more organic nitrogen to the hosts. The nitrogen assimilation in the nitrate-reducing strains was also supported by nitrogen stoichiometry analysis in Fig. 6.
Nitrate-reducing Prolixibacter strains enhanced Fe0 corrosion, which has been demonstrated by electron microscopic studies (Fig. 3 and 4), electrochemical studies (Fig. 5), and the biochemical analysis of corrosion products (Table 3, Fig. 6). On the other hand, the enhancement of Fe0-corroding activity was not observed in the nitrate-non-reducingProlixibacter strains.
MIC can be classified into two types, namely chemical MIC (CMIC) and electrical MIC (EMIC), according to Fe0-corrosion mechanisms (Enning et al. , 2012). We propose that the nitrate-reducing Prolixibacter strains enhanced Fe0 corrosion mainly through CMIC, as shown in Fig. S2, based on the results obtained between days 7 and 28, during which over 70% of the corrosion products were produced. One evidence for nitrite as the major causal agent of Fe0 corrosion came from the results presented in Fig. 6, which showed the reduction of nitrite in parallel to the Fe0 oxidation. The amount of nitrite consumed between days 7 and 28 was sufficient to produce the observed amounts of oxidized Fe0 (Fig. 7). This was further demonstrated by nitrite-induced chemical Fe0corrosion, as shown in Table S4. There was no evidence for biotic oxidation of Fe0 or Fe2+ coupled to the reduction of nitrate because the nitrate reduction by the nitrate-reducing strains was not enhanced by the presence of Fe0 and Fe2+ (Fig. 6). On the other hand, the nitrate-reducing strains may catalyze the oxidation of Fe2+ coupled to the reduction of nitrite which was observed previously (Schaedler et al ., 2017), because the oxidation of Fe0 by the nitrate-reducing strains formed both Fe2+ and Fe3+ in the average ratio of 3:2 (Table 3), while only a scarce amount of Fe3+ was formed in the abiotic oxidation of Fe0 (Table S4).
P. denitrificans MIC1-1T andProlixibacter sp. SD074 induced the corrosion of SS400 carbon steel in the presence of nitrate. The corrosion rates by these strains calculated by millimeters per year were 0.15 mm/year (Text S1). All of the iron-corrosive Prolixibacter strains were isolated from either a crude oil well or crude oil storage tanks. Nitrate is widely used to prevent MIC because it enhances the growth of nitrate-reducing bacteria, which competitively inhibit the growth of Fe0-corroding sulfate-reducing bacteria (Gittelet al. , 2009; Schwermer et al. , 2008; Telang et al. , 1997). However, sulfide-oxidizing, nitrate-reducing bacteria such as Sulfurimonas sp. strain CVO (Lahme et al. , 2019) have been suggested to increase corrosion after nitrate injection. This study demonstrated that nitrate-reducing Prolixibacter may be prevalent in oil fields, and thus has the potential to enhance steel corrosion in oil-fields subjected to nitrate injection.