Desulfovibrio vulgaris as a model microbe for the study of corrosion under sulfate‐reducing conditions

Abstract

Corrosion of iron‐containing metals under sulfate‐reducing conditions is an economically important problem. Microbial strains now known as Desulfovibrio vulgaris served as the model microbes in many of the foundational studies that developed existing models for the corrosion of iron‐containing metals under sulfate‐reducing conditions. Proposed mechanisms for corrosion by D. vulgaris include: (1) H₂ consumption to accelerate the oxidation of Fe⁰ coupled to the reduction of protons to H₂; (2) production of sulfide that combines with ferrous iron to form iron sulfide coatings that promote H₂ production; (3) moribund cells release hydrogenases that catalyze Fe⁰ oxidation with the production of H₂; (4) direct electron transfer from Fe⁰ to cells; and (5) flavins serving as an electron shuttle for electron transfer between Fe⁰ and cells. The demonstrated possibility of conducting transcriptomic and proteomic analysis of cells growing on metal surfaces suggests that similar studies on D. vulgaris corrosion biofilms can aid in identifying proteins that play an important role in corrosion. Tools for making targeted gene deletions in D. vulgaris are available for functional genetic studies. These approaches, coupled with instrumentation for the detection of low concentrations of H₂, and proven techniques for evaluating putative electron shuttle function, are expected to make it possible to determine which of the proposed mechanisms for D. vulgaris corrosion are most important.

INTRODUCTION

Understanding the mechanisms for the corrosion of metals is key to developing strategies for preventing this economically significant problem ¹ , ² . Following the first suggestion that microbes might be important catalysts for the corrosion of metals ³ , ⁴ , a thorough analysis of the available data led to the conclusion that sulfate‐reducing microorganisms play a key role in iron corrosion ⁵ . However, in the 1930s, Spirillium desulfuricans was the only microbe known to be capable of sulfate reduction ⁵ . The genus and species names of this and similar strains of sulfate‐reducing bacteria investigated in corrosion studies have changed over time ⁶ , ⁷ , but most are now generally recognized as strains of Desulfovibrio vulgaris (Table 1).

Table 1.

Iron corrosion studies with Desulfovibrio vulgaris.

Year	Straina	Iron source	Lactate	Mechanismb	Reference
1934	Spirillum desulfuricans	CI	+	Ha	5
1939	Vibrio desulfuricans	MS	+	N	8
1947	Vibrio desulfuricans	MS	−	N	9
1951	Vibrio desulfuricans	Armco ingot iron	+ −	Ha	10
1952	Vibrio desulfuricans	Armco ingot iron	+ −	S	11
1960	D. desulphuricans Hildenborough NCIB 8303	MS	+	Ha	12
1964	D. desulphuricans Benghazi NCIB 8401	MS	+?	Ha	13
	America NCIB 8372
	Teddington R NCIB 8312
	Hildenborough NCIB 8303
	Llanelly NCIB 8446
1968	D. desulfuricans Teddington R	MS	+	S	14
1968	Hildenborough NICB 8303	MS	+	Ha	15
1971	D. desulfuricans Teddington R NCIB 8312	MS	+ − (+F)	Hs	16
1974	Hildenborough NCIB 8303	MS	+	S	17
1982	Isolated from River Thames' sediment	MS (EN2)	+	N	18
1986	Hildenborough NCIB 8303	MS	− (+A)	Ha	19
1986	Marburg DSM 2119 (Postgate and Campbell)	Steel	+ − (+A)	Ha	20
1986	Madison	MS	− (+F)	Ha	21
1990	Hildenborough NCIB 8303	MS	+	Ha	22
1991	DSM 1744 (Postgate and Campbell)	SS (AISI 3161)	−	N	23
1991	Not specified	Iron (99%) SS (Fe–15Cr–10Ni)	+	Hs	24
1991	Not specified	SS (410)	+	S	25
1991	Isolated from cutting oil emulsions	CS (SAE 1020)	+	N	26
1992	Woolwich NCIMB 8457	MS (BS970)	+	N	27
1993	NCIB 8303	Iron	+	N	28
	(Postgate and Campbell)	CS (SAE 1090)
	(Hildenborough)	SS (18‐8)
	(DSM 644)
1994	Not specified	CS (X52)	−	N	29
1995	Isolated from cutting oil emulsions	MS	+	N	30
1995	Not specified	SS (304)	+	N	31
1995	ATCC 25979	SS (304)	+	N	32
1997	Not specified	SS (AISI 304L)	+	N	33
1997	Not specified	SS (316L)	+	N	34
1999	ATCC 29579	MS (SAE 1018)	+	N	35
	(Postgate and Campbell)	SS 304
	(Hildenborough)
	(NCIB 8303, DSM 644)
2002	LMG 7563	MS	+	N	36
2004	Not specified	Iron	− (+A)	D	37
2004	ATCC 29579	MS (1010)	+	N	38
2007	DSM 664	MS (BST 503‐2)	+	N	39
2008	Hildenborough NCIMB 8303	CS (ASTM A366)	+ − (+A)	Ha	40
2008	Isolated from an oil field separator	MS (AISI 1018)	−	N	41
2008	DSMZ 644	Iron	+	N	42
		CS (ST 37)
		SS (304)
2010	DSMZ 644	Alloyed steel (1.4301, UNS 304)	+	N	43
2013	ATCC 7757	CS (C1018)	+	N	44
	(Postgate and Campbell)
	(C‐6, CT1, IFO 13699, NCIB 8372)
2014	ATCC 7757	CS (C1018)	+	N	45
2014	ATCC 7757	CS (X70)	+	N	46
2014	ATCC 7757	CS (API5L X‐70)	+	N	47
2014	ATCC 7757	CS (API5L‐X70)	+	N	48
2015	ATCC 7757	CS (C1018)	+	F	49
2015	ATCC 7757	SS (304)	+	F	50
2016	ATCC 7757	CS (C1018)	+	N	51
2016	ATCC 7757	CS (UNS G10100)	+	N	52
2016	Hildenborough DSM 644	MS (BST 503‐2)	+	N	53
2017	Not specified	CI	+	N	54
2017	ATCC 7757	MS	+	N	55
2017	ATCC 7757	CS (C1018)	+	N	56
2018	ATCC 7757	CS (C1018)	+	S	57
2019	Not specified	CS (1018)	+	N	58
2019	ATCC 7757	CS (1018)	+	N	59
2019	ATCC 7757	PS (X80)	+	N	59
2020	Hildenborough	CS (1030)	+	N	60
2020	ATCC 7757	SS (2205)	+	N	61
2020	Hildenborough	CS	+	N	62
2020	ATCC 7757	CS (X65)	+	F	63
2021	ATCC 7757	GS	+	N	64
2021	ATCC 7757	SS (410, 420, 316, 2206)	+	N	65
2021	ATCC 7757	CS (C1018)	+	N	66
2021	ATCC 7757	SS (2205)	+	N	67
2021	ATCC 7757	SS (2205)	+	N	68

^a Strains now considered to be Desulfovibrio vulgaris were previously designated as Spirillium desulfuricans, Vibrio desulfuricans, Desulfovibrio desulfuricans, and Desulfovibrio desulphuricans ⁶ , ⁷ . Therefore, microbes that were later renamed D. vulgaris are listed by the name designated in the original text. Only strain designations are listed for strains designated as D. vulgaris in the original text. Alternative designations for these strains are described in parentheses.

^b Primary corrosion mechanism discussed. Ha, abiotic H₂ production from iron; Hs, H₂ production from iron‐catalyzed by FeS mineral deposits; S, sulfide promoting iron corrosion; D, direct electron transfer; F, electron transfer with a flavin shuttle; N, not applicable (the studies focused on biofilm formation, growth inhibition, corrosion inhibition, etc.). +, lactate included; −, no lactate; − (+A), no lactate but acetate added; − (+F), cells grown on fumarate; CI, cast iron; CS, carbon steel; GS, galvanized steel; MS, mild steel; PP, pipeline steel; SS, stainless steel.

There is substantial evidence that Desulfovibrio species are involved in the corrosion of iron‐containing metals in anaerobic environments ⁶⁹ , ⁷⁰ . Desulfovibrio species were abundant within the microbial community on metal surfaces exposed to oil field production waters ⁷¹ , ⁷² , corroded oil pipelines ⁷³ , corroding steel pipe carrying oily seawater ⁷⁴ , rust layers on steel plates immersed in seawater ⁷⁵ , and the inner rust layer on carbon steel ⁷⁶ . Desulfovibrio species were recovered in culture from corrosion sites ⁷⁷ , ⁷⁸ , ⁷⁹ , ⁸⁰ , including a D. vulgaris strain isolated from an oil field separator in the Gulf of Mexico that was damaged by corrosion ⁴¹ . Microbial activity on the cathodes of bioelectrochemical systems is thought to be related to microbial corrosion ⁸¹ and Desulfovibrio species are often enriched on cathodes from diverse microbial communities ⁸² , ⁸³ , ⁸⁴ .

Several mechanisms for D. vulgaris corrosion of iron‐containing metals have been proposed (Figure 1). These mechanisms may not be mutually exclusive. As detailed in this review, each of these models still requires rigorous examination. However, with the increasing availability of molecular tools to probe microbial activity and tools for genetic manipulation of D. vulgaris ⁸⁵ , ⁸⁶ , it now may be the time to either eliminate or confirm some of the existing mechanistic models for D. vulgaris corrosion or to develop new models. The purpose of this review is to summarize the previously proposed routes for Desulfovibrio species iron corrosion and to suggest experimental approaches to further advance the understanding of corrosion by this popular model microbe.

Figure 1.

Previously proposed mechanisms for Desulfovibrio vulgaris to promote the corrosion of iron‐containing metals. These include the consumption of abiotically produced H₂ (1); consumption of H₂ generated via catalysis by FeS (2) or hydrogenase (3); direct electron transfer from Fe⁰ to cells via outer‐surface electron transport components on the cell surface (4); and Fe⁰ oxidation via reduction of the oxidized form of soluble flavin electron shuttle (Flavin_ox) with reduced flavin (Flavin_red) serving as the electron donor for sulfate reduction (5). The studies proposing these mechanisms are cited in the main text.

IRON CORROSION VIA AN H₂ INTERMEDIATE

The corrosion of iron‐containing metals results from the oxidation of metallic iron to ferrous iron:

$${\text{Fe}}^0\to {\text{Fe}}^{2+}+2{\mathrm{e}}^{-}.$$ (1)

As recognized in the early analysis of corrosion by sulfate reducers ⁵ , protons are a likely electron acceptor for the electrons derived from Fe⁰. In early studies, the product of proton reduction is often referred to as “metallic hydrogen,” but in the absence of data demonstrating that this form of hydrogen exists on the surface of corroding iron or can serve as an electron donor for microbial respiration, we assume that proton reduction yields H₂, a known electron donor for diverse microbes:

$${\text{Fe}}^0+2{\mathrm{H}}^{+}\to {\text{Fe}}^{2+}+{\mathrm{H}}_2.$$ (2)

H₂ is an electron donor for D. vulgaris:

$$4{\mathrm{H}}_2+{{\text{SO}}_4}^{2-}\to {\mathrm{S}}^{2-}+4{\mathrm{H}}_2\mathrm{O},$$ (3)

and growth on H₂ is possible if acetate is provided as a carbon source ⁸⁷ .

Substantial abiotic H₂ production from Fe⁰ was discounted in the early version of the model in which H₂ serves as an electron carrier between Fe⁰ and cells ⁵ . However, the mechanism by which cells promoted the oxidation of Fe⁰ with the production of H₂ was not specified. It is now known that the extent of abiotic H₂ production depends upon the form of the iron‐containing metal. For example, pure Fe⁰ abiotically produces substantial H₂ when submerged in anoxic water at circumneutral pH whereas 316 stainless steel does not ⁸⁸ , ⁸⁹ .

This difference in H₂ production between Fe⁰ and stainless steel could provide one method for evaluating whether D. vulgaris relies on H₂ production to consume electrons from iron‐containing metals. The closely related sulfate reducer D. ferrophilus reduced sulfate when pure Fe⁰ was the electron donor, but not in a medium with stainless steel ⁹⁰ . In contrast, Geobacter species capable of direct electron uptake could use either iron form as an electron donor ⁸⁸ , ⁸⁹ , ⁹⁰ . These results indicated that D. ferrophilus was incapable of direct electron uptake and required the production of H₂ to mediate electron transfer between Fe⁰ and cells.

The most direct approach to evaluating whether H₂ is an important intermediate in electron uptake from extracellular electron donors may be to generate a mutant that is unable to consume H₂ ⁹¹ . D. vulgaris Hildenborough has multiple hydrogenases that have different localizations and metal constituents: the periplasmic [NiFe] HynAB‐1 and HynAB‐2, the periplasmic [Fe] HydAB, the periplasmic [NiFeSe] HysAB, the cytoplasmic [Fe] HydC, and the cytoplasmic membrane‐bound Coo and Ech hydrogenases ⁹² . Deletions of genes for HydAB, HynAB‐1, or HysAB negatively impacted the growth of D. vulgaris Hildenborough with H₂ as the electron donor ⁹³ , ⁹⁴ , ⁹⁵ . However, these single‐gene deletion mutants and a double deletion mutant of HynAB‐1 and HydAB ⁹⁵ still grew on H₂ as the electron donor, indicating redundant, complementary functions of the multiple hydrogenases. Thus, the construction of a strain with multiple hydrogenase gene deletions may be required to rigorously evaluate the role of H₂ in corrosion.

Transcriptomic analysis comparing growth on H₂ supplied from proton reduction with an iron electrode poised at −1.1 V versus growth on H₂ simply bubbled into medium revealed that the genes for HynAB‐1 and HydAB were more highly expressed during growth on the cathodic H₂ ⁴⁰ . Gene transcripts for HysAB were more abundant when H₂ was bubbled into the medium. Gene deletions that prevented the function of the HynAB‐1 and HydAB hydrogenases inhibited electron uptake from the iron cathodes ⁴⁰ , as might be expected for cathodes poised at a low potential to induce H₂ production. The impact of the hydrogenase gene deletions on corrosion of iron that was not artificially poised at a negative potential was not determined because wild‐type cells could not be grown under these conditions ⁴⁰ . Lack of growth on unpoised iron suggests an inability to use Fe⁰ as an electron donor. This difference between artificially poised iron cathodes and unpoised iron metal is an important consideration when evaluating other studies ¹⁹ that have concluded that H₂ is an important intermediate in iron corrosion by D. vulgaris based on experiments with electrochemically poised iron electrodes.

However, there is some indirect evidence for H₂ serving as an intermediary electron carrier between Fe⁰ and D. vulgaris, especially when H₂ is not the sole electron donor. D. vulgaris did not reduce sulfate when steel wool was provided as the sole electron donor, but when lactate was added as an additional electron donor, more sulfide was produced than was possible from lactate oxidation alone ²⁰ . In contrast, D. sapovorans, which cannot utilize H₂, did not produce substantially more sulfide when grown in the presence of lactate and steel wool, than when grown with lactate alone. These results suggested that H₂ was an intermediary electron carrier for D. vulgaris electron uptake from the steel wool during growth with lactate ²⁰ . The expression of one or more uptake hydrogenases is expected to be upregulated when H₂ is serving as an electron donor ⁹⁶ , ⁹⁷ , ⁹⁸ . Thus, transcriptional and/or proteomic studies may be useful in further assessing the role of H₂ as an electron donor during corrosion in the presence of lactate. Additional indirect evidence for the importance of H₂ as an electron carrier was the finding that D. vulgaris corroded “mild steel” faster than the gram‐positive D. orientis, which cannot consume H₂ ¹² , ¹³ .

Other early studies suggested that H₂ production from iron was not a mechanism for corrosion ¹⁰ , ¹¹ . However, the medium for investigating the possibility for H₂ serving as an electron donor did not include acetate, which is required as a carbon source for growth on H₂. Therefore, no conclusion on the role of H₂ is possible from those studies.

FACTORS PROMOTING H₂ PRODUCTION

In a diversity of microbes, hydrogenases released from lysed cells, or specifically transported to the outer surface of living cells, facilitate the production of H₂ from Fe^{0 99} , ¹⁰⁰ , ¹⁰¹ , ¹⁰² . For example, methanogens highly effective in corrosion can produce an extracellular hydrogenase that enhances H₂ production from Fe^{0 99} . Such extracellular hydrogenases have not been reported in Desulfovibrio species, but moribund cells of D. vulgaris release periplasmic hydrogenases that can retain activity for months ¹⁰³ . Subjecting D. vulgaris to starvation, a condition likely to promote cell death and lysis, enhanced corrosion ⁴⁵ , ⁵⁸ . Therefore, studies to evaluate the role of extracellular hydrogenases in corrosion by D. vulgaris are warranted.

The iron sulfide that precipitates on iron‐containing metals during corrosion coupled to sulfate reduction may also increase H₂ production. The addition of FeS reduced the overpotential necessary to produce H₂ from iron cathodes, suggesting a role for FeS in promoting the formation of H₂ ¹⁶ . In studies in which the culture was grown on fumarate rather than via sulfate reduction, the current was generated at more positive potentials when FeS was deposited on either mild steel or platinum cathodes ¹⁴ . These results further indicate that FeS may serve as a catalyst for H₂ generation. However, in studies with carbon steel coupons, it appeared that higher accumulations of sulfide inhibited corrosion ⁵⁷ . The ability of D. vulgaris to corrode steel with either benzyl viologen as the electron acceptor ¹⁵ or when growing on fumarate ²¹ demonstrated that sulfide production was not essential for corrosion. Thus, a clear‐cut concept for the role of FeS in corrosion has yet to be established.

Technology for measuring H₂ concentrations at extremely low concentrations during corrosion is available ⁸⁹ . Thus, with the appropriate H₂ detector it should be possible to directly evaluate the role of FeS in facilitating H₂ production from iron‐containing metals, simply by monitoring H₂ generation in the presence or absence of different quantities of FeS precipitate.

ELECTRON SHUTTLES OTHER THAN H₂

Soluble redox‐active molecules promote extracellular electron exchange between microbes and minerals, electrodes, and other microbial species ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ . These electron shuttles typically accelerate extracellular electron exchange by alleviating the need for outer‐surface electron transfer components to establish direct electrical contact with particulate extracellular donors and acceptors. The addition of riboflavin and flavin adenine dinucleotide enhanced D. vulgaris corrosion of carbon steel and stainless steel ⁴⁹ , ⁵⁰ , ⁶³ . However, amendments of these cofactors, which are important for the function of numerous proteins, could influence D. vulgaris growth and metabolism in many ways. To determine whether flavins can serve as an electron shuttle for the corrosion of iron‐containing metals coupled to sulfate reduction it is necessary to demonstrate that: (1) the metals are capable of reducing the flavins; and (2) that the reduced flavins can serve as electron donors for sulfate reduction.

DIRECT ELECTRON UPTAKE

Direct electron uptake from iron‐containing metals has been demonstrated with Geobacter sulfurreducens and Geobacter metallireducens ⁸⁸ , ⁸⁹ , ⁹⁰ . Strains unable to utilize H₂ readily reduced fumarate, nitrate, or Fe(III) with pure Fe⁰ or stainless steel as the electron donor. Deletion of genes for outer‐surface, multiheme c‐type cytochromes previously shown to be involved in electron exchange with other extracellular donors/acceptors inhibited the corrosion.

There are no examples of similar studies with sulfate‐reducing microorganisms. It was suggested that c‐type cytochromes positioned in the outer membrane of D. vulgaris might be able to make an electrical connection with Fe^{0 108} . However, subsequent studies have indicated that D. vulgaris does not have outer‐surface cytochromes ⁹² . Clear next steps in this line of investigation would be to rigorously verify whether cytochromes are exposed on the outer surface of D. vulgaris, and if so, evaluate their role in iron corrosion with the appropriate gene deletion studies. Genetic, biochemical, biophysical, and immunological approaches previously employed for investigating the location and function of the outer‐surface cytochromes of Geobacter would be suitable ¹⁰⁹ , ¹¹⁰ , ¹¹¹ , ¹¹² .

Although it has been suggested that several of the c‐type cytochromes of D. ferrophilus may be localized in the outer membrane, no genetic studies have been conducted to determine whether they are involved in extracellular electron exchange ⁹⁰ , ¹¹³ , ¹¹⁴ , ¹¹⁵ . As noted above, experimental analysis of iron corrosion by D. ferrophilus has suggested that it relies on H₂ as an intermediary electron carrier rather than direct electron uptake ⁹⁰ .

CONCLUSIONS AND FUTURE DIRECTIONS

Sulfate‐reducing microorganisms are considered to be important agents for catalyzing the corrosion of iron‐containing metals and D. vulgaris has historically been the model microbe of choice for elucidating the mechanisms for corrosion by sulfate reducers. As summarized above (Figure 1), previous studies have suggested several mechanisms by which D. vulgaris may enhance corrosion, but each of the proposed mechanisms requires further experimental evaluation. The mechanisms for electron transfer between iron‐containing metals and Geobacter species were elucidated with genome‐scale transcriptomics coupled with phenotypic analysis of mutant strains in which the genes for proteins hypothesized to be involved in electron transfer were deleted ⁸⁸ , ⁸⁹ . Deletion of the genes for hydrogenases and hypothesized outer‐surface electrical contacts made it possible to determine the role of H₂ as an intermediary electron carrier and to identify likely electrical contacts on the outer surface of the cell. A similar approach seems possible for the study of D. vulgaris corrosion mechanisms. Transcriptomics of D. vulgaris biofilms is possible ³⁹ , ⁴⁰ to aid in identifying components that may have increased expression during corrosion of iron‐containing metals versus other growth modes. Other candidates for corrosion components may be identified from the known physiological roles of proteins or their cellular location. Methods for the targeted deletion of genes in D. vulgaris are available ⁸⁵ , ⁸⁶ making it possible to evaluate the function of proteins hypothesized to be of importance. In fact, an extensive library of D. vulgaris mutants is publicly available, potentially eliminating the substantial investment of time and resources required for mutant construction ⁸⁶ . Comparison of corrosion capabilities with Fe⁰, which readily reduces protons to generate H₂, versus stainless steel, which does not generate H₂, provides an additional tool to evaluate the role of H₂ as an intermediary electron carrier for corrosion ⁸⁹ , ⁹⁰ . Mechanisms for the corrosion of carbon steel, the most common form of iron in structural materials, should also be investigated. The hypothesis that FeS deposits stimulate H₂ production from iron‐containing metals should be readily addressable with highly sensitive H₂ detection systems ⁸⁸ , ⁸⁹ . Mechanistic and genetic ¹¹⁶ , ¹¹⁷ , ¹¹⁸ , ¹¹⁹ , ¹²⁰ approaches for the study of the role of electron shuttles in electron transfer to minerals and electrodes should be applicable to the study of the role of flavins as electron shuttles for corrosion. Therefore, it is expected that D. vulgaris will continue to serve as an important model microbe for the further elucidation of mechanisms for corrosion under sulfate‐reducing conditions.

Ueki T, Lovley DR. Desulfovibrio vulgaris as a model microbe for the study of corrosion under sulfate‐reducing conditions. mLife. 2022;1:13–20. 10.1002/mlf2.12018