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.