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, 74 (9), 2841-51

Impact of Nitrate on the Structure and Function of Bacterial Biofilm Communities in Pipelines Used for Injection of Seawater Into Oil Fields

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Impact of Nitrate on the Structure and Function of Bacterial Biofilm Communities in Pipelines Used for Injection of Seawater Into Oil Fields

Carsten U Schwermer et al. Appl Environ Microbiol.

Abstract

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.

Figures

FIG. 1.
FIG. 1.
(a) Positions of RD in the seawater deoxygenation (DO) system. O2 was removed from the water in a two-stage gas-stripping process after filtration. (b) Schematic diagram of the experimental treatments. Black and white bars indicate exposure to NO3-rich (1 mM; NA biofilms) and NO3-free (control biofilms) seawater, respectively. RD were exchanged to opposite conditions on day 123. 15N and 35S tracer incubations were done on days 123 and 128 (hatched bars).
FIG. 2.
FIG. 2.
NA (a to f) and control (g to l) biofilms visualized by different approaches. Fresh coupons are shown in panels m and n. Binocular images show complete carbon steel coupons before (a and g) and after (b and h) removal of biofilm cover and after exposure of biofilms to sulfide (c and i). The phase-contrast microscopy images of 5-μm-thick biofilm cross-sections show structurally distinct zones, indicated by the numbers (d and j). Epifluorescence microscopy was applied to the cross-sections from the phase-contrast microscopy (e and k). Positive DAPI signals appear in white. SEM (backscattered electron) images show vertical coupon cross-sections. The arrows indicate MnS inclusions associated with pits (l) and surrounded by organic material inside pits (f).
FIG. 3.
FIG. 3.
(a and b) Microprofiles of NO3, NO2, redox potential (RP), and pH in NA and control biofilms grown in situ on carbon steel coupons for 4 months. (c and d) The situation 4 days after biofilm exchange to opposite NO3 conditions, designated NA(−) and control(+) biofilms. H2S, H2, and N2O were not detected in any biofilm. Zero depth indicates the biofilm-water boundary; dashed lines represent the estimated metal coupon surface position. Averages and standard deviations were calculated from three profiles measured across the biofilm.
FIG. 4.
FIG. 4.
Redox potential dynamics on the coupon surface in NA (○) and control (▪) biofilms during the 4-day tests. Arrows indicate the time of NO3 removal from NA biofilms and the addition of NO3 to control biofilms. Averages and standard deviations from three values measured at spots across the metal surface are shown.
FIG. 5.
FIG. 5.
Test of carbon limitation as measured by N2O production in an NA biofilm using an N2O microsensor and applying the acetylene block technique. Data represent N2O formation before (0 min) and after (12, 18, and 37 min) the addition of 1 mM methanol in the presence of 1 mM NO3.
FIG. 6.
FIG. 6.
(Top) DGGE fingerprints (in triplicate) of PCR-amplified 16S rRNA gene fragments obtained from NA and control biofilms before (4 months) and after (4 days) exchange using bacterial primers GM5F and 907RC. Arrows indicate the visible dominant bands. (Bottom) 16S rRNA gene cloning showing differences in the bacterial community composition in NA and control biofilms before and after exchange to opposite NO3 conditions. The relative abundance (percent) of sequences in each clone library (i.e., treatment) with the closest relative and the taxonomic affiliation is given. α, Alphaproteobacteria; γ, Gammaproteobacteria; δ, Deltabateria; ɛ, Epsilonbacteria; Alterom, Alteromonas sp.; Arcob, Arcobacter sp.; ARK, environmental Artic pack ice clones; CFB, Cytophaga/Flavobacteria/Bacteriodes; Colwell, Colwellia sp.; Methyloph, Methylophaga sp.; Roseob, Roseobacter sp.; Oleispir, Oleispira sp.; uncult, uncultured; mar, marine.

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