Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50–90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.     

Inhibiting sulfate-reducing bacteria in biofilms on steel with antimicrobial peptides generated in situ

In batch and continuous fermentations, the reduction in corrosion of SAE 1018 mild steel and 304 stainless steel caused by inhibition of the reference sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris by a protective, antimicrobial-producing Bacillus brevis biofilm was investigated. The presence of D. vulgaris produced a thick black precipitate on mild steel and a higher corrosion rate in batch cultures than that seen in a mono-culture of non-antimicrobial-producing Pseudomonas fragi K upon the addition of SRB to the aerobic P. fragi K biofilm. In continuous reactors, the polarization resistance R _p decreased for stainless steel and increased for mild steel upon the addition of SRB to a P. fragi K biofilm. Addition of either 200 μg/ml ampicillin, chloramphenicol, or ammonium molybdate to batch and continuous reactors after SRB had colonized the metal was ineffective in killing SRB, as inferred from the lack of change in both R _p and the impedance spectra. However, when ampicillin was added prior to SRB colonization, the growth of SRB was completely inhibited on stainless steel in continuous reactors. Prior addition of ampicillin was only able to delay the growth of SRB on mild steel in continuous reactors. External addition of the purified peptide antimicrobial agent gramicidin S prior to the addition of SRB also inhibited the growth of SRB on stainless steel in continuous reactors, and the SRB were also inhibited on stainless steel in both batch and continuous reactors by producing gramicidin S in situ in a protective biofilm when the gramicidin-S-overproducing strain Bacillus brevis 18 was used. Received: 29 October 1998 / Received revision: 18 February 1999 / Accepted: 26 February 1999     

Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides. The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25–100 μg ml⁻¹. To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilisBE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 μg ml⁻¹ decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures. B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed. This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion. Received 27 October 1998/ Accepted in revised form 21 February 1999     

Neutrophilic iron-oxidizing "zetaproteobacteria" and mild steel corrosion in nearshore marine environments

Microbiologically influenced corrosion (MIC) of mild steel in seawater is an expensive and enduring problem. Little attention has been paid to the role of neutrophilic, lithotrophic, iron-oxidizing bacteria (FeOB) in MIC. The goal of this study was to determine if marine FeOB related to Mariprofundus are involved in this process. To examine this, field incubations and laboratory microcosm experiments were conducted. Mild steel samples incubated in nearshore environments were colonized by marine FeOB, as evidenced by the presence of helical iron-encrusted stalks diagnostic of the FeOB Mariprofundus ferrooxydans, a member of the candidate class "Zetaproteobacteria." Furthermore, Mariprofundus-like cells were enriched from MIC biofilms. The presence of Zetaproteobacteria was confirmed using a Zetaproteobacteria-specific small-subunit (SSU) rRNA gene primer set to amplify sequences related to M. ferrooxydans from both enrichments and in situ samples of MIC biofilms. Temporal in situ incubation studies showed a qualitative increase in stalk distribution on mild steel, suggesting progressive colonization by stalk-forming FeOB. We also isolated a novel FeOB, designated Mariprofundus sp. strain GSB2, from an iron oxide mat in a salt marsh. Strain GSB2 enhanced uniform corrosion from mild steel in laboratory microcosm experiments conducted over 4 days. Iron concentrations (including precipitates) in the medium were used as a measure of corrosion. The corrosion in biotic samples (7.4 ± 0.1 mM) was significantly higher than that in abiotic controls (5.0 ± 0.1 mM). These results have important implications for the role of FeOB in corrosion of steel in nearshore and estuarine environments. In addition, this work shows that the global distribution of Zetaproteobacteria is far greater than previously thought.     

A green biocide enhancer for the treatment of sulfate-reducing bacteria (SRB) biofilms on carbon steel surfaces using glutaraldehyde

Generally speaking, a much higher concentration of biocide is needed to treat biofilms compared to the dosage used to for planktonic bacteria. With increasing restrictions of environmental regulations and safety concerns on large-scale biocide uses such as oil field applications, it is highly desirable to make more effective use of biocides. In this paper a green biocide enhancer ethylenediaminedisuccinate (EDDS) that is a biodegradable chelator, was found to enhance the efficacy of glutaraldehyde in its treatment of sulfate-reducing bacteria (SRB) biofilms. Experiments were carried out in 100 ml anaerobic vials with carbon steel coupons. The ATCC 14563 strain of Desulfovibrio desulfuricans was used. Biofilms on coupon surfaces were visualized using scanning electron microscopy (SEM). Experimental results showed that EDDS reduced the glutaraldehyde dosages considerably in the inhibition of SRB biofilm establishment and the treatment of established biofilms on carbon steel coupon surfaces.     

Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms.

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.     

Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms.

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.     

海洋铁细菌对紫铜腐蚀行为的影响

【目的】研究海洋铁细菌(Iron-oxidizing bacteria,IOB)对铜的腐蚀影响。【方法】采用电化学极化曲线、电化学阻抗谱(EIS)研究紫铜在海洋环境铁细菌影响下的腐蚀行为。【结果】扫描电子显微镜SEM形貌分析结果表明,在紫铜上附着的铁细菌呈杆状。电化学测试结果表明紫铜在含IOB海水中的腐蚀过程主要受活化极化控制,有菌海水的活化电阻比无菌海水的小6 000.cm2,表明在含IOB的海水中铜发生溶解反应受到的阻滞较小。【结论】IOB的存在导致紫铜的自腐蚀电位变小,使得紫铜发生腐蚀的倾向变大;IOB也使紫铜的阳极极化曲线塔菲尔斜率和变小,从而加速了紫铜的腐蚀进程。     

Recovery of phosphorus from natural water bodies using iron-oxidizing bacteria and woody biomass

There is a global concern that phosphorus (P) resources will be depleted in the near future, but the P flux from water to land is extremely limited, whereas the reverse flux is substantial. A new method for the recovery of P from natural water bodies was proposed using iron-oxidizing bacteria and woody biomass (heartwood of conifer) as a carrier and a practical demonstration was presented. The woody carrier was immersed in water abundant in iron-oxidizing bacteria and removed 1–10 weeks later. Our results showed that the immersed carrier collected biogenic iron (Fe) oxides produced by iron-oxidizing bacteria, and contained about 0.2 mg g^?1 of P after 3 weeks; this amount was higher than that contained in some P fertile soils used for cultivating plants. The biogenic Fe oxides on the carrier acted as a source of P for plant cultivation, and they could adsorb P from P-rich solutions (10 mg L^?1 of PO₄-P). Although our study involved only a small-scale trial, the proposed method can potentially aid in the effective use of P in water and in water quality improvement if conducted on a large scale.     

高温油藏生物膜内硫酸盐还原菌的腐蚀机理及防治策略

硫酸盐还原菌(sulfate-reducing bacteria,SRB)广泛分布于高温、高压及高盐的石油油藏中,在油藏硫循环中起主导作用。SRB能在油藏生物膜内生长,有微量低分子有机酸时利用硫酸盐为电子受体并将其还原成硫化氢。硫化氢会腐蚀管道,导致原油泄露等其他安全问题,每年造成的经济损失超过7 000亿元。本文首先总结了油藏生物膜内微生物菌群多样性,分析了生物膜内SRB及其相关菌群的协同腐蚀机理;然后讨论了高温油藏SRB介导的硫氮氢生物地球化学循环过程、胞外电子传递机制及其腐蚀作用,并通过几个高温油藏SRB生物膜内腐蚀的现场案例进一步阐明了SRB的腐蚀机制。在此基础上,提出了应对高温油藏生物膜内SRB腐蚀的生物纳米防治策略,这为高温油藏管道防腐提供了新思路。     

油田硫酸盐还原菌酸化腐蚀机制及防治研究进展

硫酸盐还原菌(Sulfate reducing bacteria,SRB)是一些厌氧产硫化氢的细菌的统称,是以有机物为养料的厌氧菌。它们广泛分布于pH值6-9的土壤、海水、河水、淤泥、地下管道、油气井、港湾及锈层中,它们生存于好气性硫细菌产生的沉积物下,其最适宜的生长温度是20-30℃,可以在高达50-60℃的温度下生存,与腐蚀相关的最主要的是脱硫脱硫弧菌(Desulfovibrio desulfuricans)。 它们是许多腐蚀问题的主因,例如油田系统金属管路的腐蚀等。在海上油田生产中,海水常被注入油井用于进行2次采油。富含硫酸盐的海水能加速油藏中SRB的生长,随之H₂S大量产生,引起油田水的酸化,H₂S具有毒性和腐蚀性,增加石油和天然气中的硫含量,并可能引起油田堵塞。SRB引起的腐蚀问题是拭待解决的最主要问题。国内外治理该问题的途径主要有物理杀灭、添加化学杀菌剂等方法,但是这些方法成本高,持续效果不显著。近几年来国外学者开始重点关注利用生物竞争排斥技术(Bio-competitive inhibition technology,BCX)控制硫酸盐还原菌的生长代谢的方法,该方法的原理为通过加入特定的药剂,激活油藏中的本源微生物或加入外源微生物,使其与SRB竞争营养源或产生代谢物抑制SRB的生长代谢,进而抑制H₂S的产生。GMT-LATA的科学家对在厌氧油气储层和开采系统中硝酸盐还原菌的作用进行了最早的研究,认为该细菌可以抑制硫酸盐还原菌的代谢活动。随后BCX技术已经在国外部分油田得到了应用,国内还没有在海油生产中应用的报道,但是也有学者对该方法进行了研究。     

Sulfate reduction from phosphogypsum using a mixed culture of sulfate-reducing bacteria

Phosphogypsum (CaSO₄), a primary by-product of phosphoric acid production, is accumulated in large stockpiles and occupies vast areas of land. It poses a severe threat to the quality of water and land in countries producing phosphoric acid. In this study, the potential of sulfate-reducing bacteria for biodegradation of this sulfur-rich industrial solid waste was assessed. The effect of phosphogypsum concentration, carbon and nitrogen sources, temperature, pH and stirring on the growth of sulfate-reducing bacteria was investigated. Growth of sulfate-reducing bacteria was monitored by measuring sulfide production. Phosphogypsum was shown to be a good source of sulfate, albeit that the addition of organic carbon was necessary for bacterial growth. Biogenic sulfide production occurred with phosphogypsum up to a concentration of 40 g L⁻¹, above which no growth of sulfate-reducing bacteria was observed. Optimal growth was obtained at 10 g L⁻¹ phosphogypsum. Both the gas mixture H₂/CO₂ and lactate supported high amounts of H₂S formation (19 and 11 mM, respectively). The best source of nitrogen for sulfate-reducing bacteria was yeast extract, followed by ammonium chloride. The presence of nitrate had an inhibitory effect on the process of sulfate reduction. Stirring the culture at 150 rpm slightly stimulated H₂S formation, probably by improving sulfate solubility.     

Sulfate reduction from phosphogypsum using a mixed culture of sulfate-reducing bacteria

Phosphogypsum (CaSO₄), a primary by-product of phosphoric acid production, is accumulated in large stockpiles and occupies vast areas of land. It poses a severe threat to the quality of water and land in countries producing phosphoric acid. In this study, the potential of sulfate-reducing bacteria for biodegradation of this sulfur-rich industrial solid waste was assessed. The effect of phosphogypsum concentration, carbon and nitrogen sources, temperature, pH and stirring on the growth of sulfate-reducing bacteria was investigated. Growth of sulfate-reducing bacteria was monitored by measuring sulfide production. Phosphogypsum was shown to be a good source of sulfate, albeit that the addition of organic carbon was necessary for bacterial growth. Biogenic sulfide production occurred with phosphogypsum up to a concentration of 40 g L⁻¹, above which no growth of sulfate-reducing bacteria was observed. Optimal growth was obtained at 10 g L⁻¹ phosphogypsum. Both the gas mixture H₂/CO₂ and lactate supported high amounts of H₂S formation (19 and 11 mM, respectively). The best source of nitrogen for sulfate-reducing bacteria was yeast extract, followed by ammonium chloride. The presence of nitrate had an inhibitory effect on the process of sulfate reduction. Stirring the culture at 150 rpm slightly stimulated H₂S formation, probably by improving sulfate solubility.     

Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms.

The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O(2), H(2)S, NO(2)(-), NO(3)(-), NH(4)(+), and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 10(9) to 10(10) cells per cm(3) of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained Desulfobulbus spp. were found to be numerically important members of SRB populations (approximately 10(8) to 10(9) cells per cm(3)). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 microm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S(0)) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 microm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate.     

Removal of selenate from sulfate-containing media by sulfate-reducing bacterial biofilms

A biofilm-selected strain of a Desulfomicrobium sp. removed selenate from solution to sub-micromolar concentrations during growth on lactate (or hydrogen) and sulfate. Under sulfate-limited growth conditions, selenium was enzymatically reduced to selenide. Under excess sulfate conditions, selenate removal was primarily by enzymatic reduction to elemental selenium. Sequestration by biofilms was greater under the latter condition. Experiments with washed cell suspensions showed that high sulfate concentrations inhibited cell-specific selenate reduction, but when growing cells were exposed to selenate, the biomass increase achieved during incubations with abundant sulfate resulted in more rapid selenate removal. The addition of small amounts of sulfite, or thiosulfate, ameliorated this inhibition. Nitrate also inhibited selenate reduction in washed cell suspensions, apparently due to a general oxidizing effect. These results suggest that where biofilm-based sulfate-reducing bacteria (SRB) bioreactors are considered for the treatment of mixed metalliferous wastes that contain selenium oxyanions, adequate selenate removal should be achievable under a range of environmental conditions. The form and fate of the precipitated product will, however, be influenced by the dominant reduction pathway, which is controlled by environmental variables.     

Bioleaching of copper- and zinc-bearing ore using consortia of indigenous iron-oxidizing bacteria

Indigenous iron-oxidizing bacteria were isolated on modified selective 9KFe²⁺ medium from Baiyin copper mine stope, China. Three distinct acidophilic bacteria were isolated and identified by analyzing the sequences of 16S rRNA gene. Based on published sequences of 16S rRNA gene in the GenBank, a phylogenetic tree was constructed. The sequence of isolate WG101 showed 99% homology with Acidithiobacillus ferrooxidans strain AS2. Isolate WG102 exhibited 98% similarity with Leptospirillum ferriphilum strain YSK. Similarly, isolate WG103 showed 98% similarity with Leptospirillum ferrooxidans strain L15. Furthermore, the biotechnological potential of these isolates in consortia form was evaluated to recover copper and zinc from their ore. Under optimized conditions, 77.68 ± 3.55% of copper and 70.58 ± 3.77% of zinc were dissolved. During the bioleaching process, analytical study of pH and oxidation–reduction potential fluctuations were monitored that reflected efficient activity of the bacterial consortia. The FTIR analysis confirmed the variation in bands after treatment with consortia. The impact of consortia on iron speciation within bioleached ore was analyzed using Mössbauer spectroscopy and clear changes in iron speciation was reported. The use of indigenous bacterial consortia is more efficient compared to pure inoculum. This study provided the basic essential conditions for further upscaling bioleaching application for metal extraction.

     

The corrosion behaviour of mild steel and type 304 stainless steel in media from an anaerobic biodigestor

Mild steel and AISI 304 stainless steel samples were exposed to the aqueous solutions from an anaerobic biodigestor of wastewater, showing quite different behaviours. Carbon steel presented generalized corrosion whereas stainless steel, as-received or sensitized, tended to show some localized corrosion. Both materials presented bacteria attachment, particularly sulphate reducing bacteria (SRB), forming biofilms which were abundant and mixed with corrosion products on the mild steel surface and thin and patchy on stainless steel surfaces. Different types of anaerobic and aerobic bacteria were detected in the medium. Experiments were carried out both at 1 and at 7 atmospheres pressure, in presence of a gaseous phase containing N₂, CO₂ and CH₄. Potentiodynamic experiments were conducted in order to have a better insight on the electrochemical behaviour of the material in this medium.     

Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust

Iron (Fe(0) ) corrosion in anoxic environments (e.g. inside pipelines), a process entailing considerable economic costs, is largely influenced by microorganisms, in particular sulfate-reducing bacteria (SRB). The process is characterized by formation of black crusts and metal pitting. The mechanism is usually explained by the corrosiveness of formed H(2) S, and scavenge of 'cathodic' H(2) from chemical reaction of Fe(0) with H(2) O. Here we studied peculiar marine SRB that grew lithotrophically with metallic iron as the only electron donor. They degraded up to 72% of iron coupons (10?mm?×?10?mm?×?1?mm) within five months, which is a technologically highly relevant corrosion rate (0.7?mm?Fe(0) year(-1) ), while conventional H(2) -scavenging control strains were not corrosive. The black, hard mineral crust (FeS, FeCO(3) , Mg/CaCO(3) ) deposited on the corroding metal exhibited electrical conductivity (50?S?m(-1) ). This was sufficient to explain the corrosion rate by electron flow from the metal (4Fe(0) →?4Fe(2+) +?8e(-) ) through semiconductive sulfides to the crust-colonizing cells reducing sulfate (8e(-) +?SO(4) (2-) +?9H(+) →?HS(-) +?4H(2) O). Hence, anaerobic microbial iron corrosion obviously bypasses H(2) rather than depends on it. SRB with such corrosive potential were revealed at naturally high numbers at a coastal marine sediment site. Iron coupons buried there were corroded and covered by the characteristic mineral crust. It is speculated that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from abiotic or biotic sources in sediments.     

Corrosion behavior of carbon steel in the presence of two novel iron-oxidizing bacteria isolated from sewage treatment plants

In this work, two novel iron oxidizing bacteria (IOB), namely Gordonia sp. MZ-89 and Enterobacter sp. M01101, were isolated from sewage treatment plants and identified by biochemical and molecular methods. Then, microbially influenced corrosion (MIC) of carbon steel in the presence of these bacteria was investigated. The electrochemical techniques such as potentiodynamic polarization measurements and electrochemical impedance spectroscopy (EIS) were used to measure the corrosion rate and observe the corrosion mechanism. The results showed that the existence of these microorganisms decreased the corrosion potential and enhanced the corrosion rate. Scanning electron microscopy (SEM) images revealed the ground boundary attacks and pitting on carbon steel samples in the presence of these bacteria after polarization. Corrosion scales were identified with X-ray diffraction (XRD). It was demonstrated that these bacteria can greatly affect the crystalline phase of corrosion products that also confirmed by SEM results. It was inferred that these bacteria were responsible for the corrosion of carbon steel, especially in the form of localized corrosion.