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     

Action of glutaraldehyde and nitrite against sulfate-reducing bacterial biofilms

A continuous flow reactor system was developed to evaluate the efficacy of antimicrobial treatments against sulfate-reducing bacterial biofilms. An annular reactor operating at a nominal dilution rate of 0.5 h⁻¹ was fed one-tenth strength Postgate C medium diluted in 1.5% NaCl and was inoculated with a mixed culture enriched from oilfield-produced water on the same medium. Thin biofilms developed in this reactor after 2 days of operation. The activity of these biofilms resulted in approximately 50 mg S l⁻¹ of sulfide at steady state prior to biocide treatment. Biocide efficacy was quantified by recording the time required for sulfide production to recover following an antimicrobial treatment. In a control experiment in which pure water was applied, the time required to reach 10 mg S l⁻¹ sulfide after the treatment was 1.7±1.2 h, whereas the time to reach this level of sulfide after a pulse dose of 500 mg l⁻¹ glutaraldehyde was delayed to 61±11 h. Nitrite treatment suppressed sulfide production as long as the nitrite concentration remained above 15 mg N l⁻¹. Sulfide production recovered more rapidly after nitrite treatment than it did after glutaraldehyde treatment. Received 01 February 2002/ Accepted in revised form 13 June 2002     

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。     

Planktonic nitrate-reducing bacteria and sulfate-reducing bacteria in some western Canadian oil field waters

Oil fields that use water flooding to enhance oil recovery may become sour because of the production of H₂S from the reduction of sulfate by sulfate-reducing bacteria (SRB). The addition of nitrate to produced waters can stimulate the activities of nitrate-reducing bacteria (NRB) and control sulfide production. Many previous studies have focused on chemolithotrophic bacteria that can use thiosulfate or sulfide as energy sources while reducing nitrate. Little attention has been given to heterotrophic NRB in oil field waters. Three different media were used in this study to enumerate various types of planktonic NRB present in waters from five oil fields in western Canada. The numbers of planktonic SRB and bacteria capable of growth under aerobic conditions were also determined. In general, microbial numbers in the produced waters were very low (<10 ml⁻¹) in samples taken near or at wellheads. However, the numbers increased in the aboveground facilities. No thiosulfate-oxidizing NRB were detected in the oil field waters, but other types of NRB were detected in 16 of 18 produced water samples. The numbers of heterotrophic NRB were equal to or greater than the number of sulfide-oxidizing, chemolithotrophic NRB in 12 of 15 samples. These results showed that each of the oil fields contained NRB, which might be stimulated by nitrate amendment to control H₂S production by SRB. Journal of Industrial Microbiology & Biotechnology (2002) 29, 83–92 doi:10.1038/sj.jim.7000274 Received 20 February 2002/ Accepted in revised form 14 May 2002     

Use of nitrate to control sulfide generation by sulfate-reducing bacteria associated with oily waste

Received 13 November 1998/ Accepted in revised form 03 March 1999     

Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system

District heating systems (DHS) are extreme aqueous environments characterized by high temperatures, high pH (9.5-10.0), and low nutrient availability. Culture-independent and culture-dependent techniques showed that DHS may nevertheless harbour geno- and phenotypically diverse bacterial biofilm communities. Approximately 50% of the cells in biofilms growing on mild steel coupons in rotortorque reactors connected to the return line (40 degrees C) of a Danish DHS were detectable by FISH analysis and thus were probably metabolically active. A bacterial 16S rRNA gene clone library generated from the biofilms was dominated by proteobacterial phylotypes (closely related to known aerobic species) and by phylotypes affiliated to the anaerobic class Clostridia. Anoxic enrichment cultures derived from biofilms primarily contained 16S rRNA gene and dsrAB (encoding major subunits of dissimilatory sulfite reductase) phylotypes affiliated to the latter class. Alkalitolerant and neutrophilic anaerobic bacteria were isolated from the DHS, including novel Gram-positive and deltaproteobacterial sulfate-reducers and sulfite-reducers constituting novel Gram-positive lineages. In total, 39 distinct 16S rRNA gene phylotypes representing ten classes were identified. The detection of several alkalitolerant, sulfide-producing, and, thus, potentially biocorrosive species underlines the need to maintain a high water quality in the DHS in order to prevent the proliferation of these species.     

Anaerobic oxidation of dimethylsulfide and methanethiol in mangrove sediments is dominated by sulfate-reducing bacteria

The oxidation of dimethylsulfide and methanethiol by sulfate-reducing bacteria (SRB) was investigated in Tanzanian mangrove sediments. The rate of dimethylsulfide and methanethiol accumulation in nonamended sediment slurry (control) incubations was very low while in the presence of the inhibitors tungstate and bromoethanesulfonic acid (BES), the accumulation rates ranged from 0.02–0.34 to 0.2–0.4 nmol g FW sediment⁻¹ h⁻¹, respectively. Degradation rates of methanethiol and dimethylsulfide added were 2–10-fold higher. These results point to a balance of production and degradation. Degradation was inhibited much stronger by tungstate than by BES, which implied that SRB were more important. In addition, a new species of SRB, designated strain SD1, was isolated. The isolate was a short rod able to utilize a narrow range of substrates including dimethylsulfide, methanethiol, pyruvate and butyrate. Strain SD1 oxidized dimethylsulfide and methanethiol to carbon dioxide and hydrogen sulfide with sulfate as the electron acceptor and exhibited a low specific growth rate of 0.010 ± 0.002 h⁻¹, but a high affinity for its substrates. The isolated microorganism could be placed in the genus Desulfosarcina (the most closely related cultured species was Desulfosarcina variabilis , 97% identity). Strain SD1 represents a member of the dimethylsulfide/methanethiol-consuming SRB population in mangrove sediments.     

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。     

Reduction of chromate by fixed films of sulfate-reducing bacteria using hydrogen as an electron source

The ability of sulfate-reducing bacteria (SRB) to reduce chromate, Cr(VI), was evaluated using fixed-film growth systems and H₂ as the electron source. A main objective of the experiment was to distinguish between direct enzymatic reduction and indirect reduction by hydrogen sulfide, in order to subsequently verify and control the synergy of these two mechanisms. In batch experiments with the sulfate-reducing consortium CH10 selected from a mining site, 50 mg l⁻¹ Cr(VI) was reduced in 15 min in the presence of 500 mg l⁻¹ hydrogen sulfide compared to 16 mg l⁻¹ reduced in 1 h without hydrogen sulfide. Fixed films of a CH10 population and Desulfomicrobium norvegicum were fed-batch grown in a column bioreactor. After development of the biofilm, hydrogen sulfide was removed and the column was fed continuously with a 13-mg l⁻¹ Cr(VI) solution. Specific Cr(VI) reduction rates on pozzolana were close to 90 mg Cr(VI) h⁻¹ per gram of protein. Exposure to Cr(VI) had a negative effect on the subsequent ability of CH10 to reduce sulfate, but the inhibited bacteria remained viable. Journal of Industrial Microbiology & Biotechnology (2002) 28, 154–159 DOI: 10.1038/sj/jim/7000226 Received 20 September 2000/ Accepted in revised form 13 November 2001     

硫酸盐还原细菌生物被膜的形成与调控研究进展

硫酸盐还原细菌(sulfate-reducing bacteria,SRB)形成的生物被膜是微生物导致金属锈蚀行为的主要原因,同时也是重金属污水微生物修复技术的关键因子。生物被膜形成及调控机制研究对SRB的防治和利用均十分重要。本文综述了近年来SRB生物被膜的研究进展,包括SRB生物被膜的胞外多聚物组成和控制因子,并着重阐述了目前已知的调控因子对SRB生物被膜形成的影响。     

Interaction of Desulfovibrio desulfuricans biofilms with stainless steel surface and its impact on bacterial metabolism

AIMS: To study the influence of some metallic elements of stainless steel 304 (SS 304) on the development and activity of a sulfate-reducing bacterial biofilm, using as comparison a reference nonmetallic material polymethylmethacrylate (PMMA). METHODS AND RESULTS: Desulfovibrio desulfuricans biofilms were developed on SS 304 and on a reference nonmetallic material, PMMA, in a flow cell system. Steady-state biofilms were metabolically more active on SS 304 than on PMMA. Activity tests with bacteria from both biofilms at steady state also showed that the doubling time was lower for bacteria from SS 304 biofilms. The influence of chromium and nickel, elements of SS 304 composition, was also tested on a cellular suspension of Des. desulfuricans. Nickel decreased the bacterial doubling time, while chromium had no significant effect. CONCLUSIONS: The following mechanism is hypothesized: a Des. desulfuricans biofilm grown on a SS 304 surface in anaerobic conditions leads to the weakening of the metal passive layer and to the dissolution in the bulk phase of nickel ions that have a positive influence on the sulfate-reducing bacteria metabolism. This phenomenon may enhance the biocorrosion process. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the interactions between metallic surfaces such as stainless steel and bacteria commonly implied in the corrosion phenomena which is primordial to fight biocorrosion.     

硫酸盐还原菌与肠道疾病相关性的研究进展

硫酸盐还原菌(sulfate-reducing bacteria,SRB)是肠道菌群的重要组成成员之一。SRB在其增殖与新陈代谢过程中会产生硫化氢。现有研究显示,SRB的过度繁殖与炎症性肠病(inflammatory bowel disease,IBD)、肠易激综合征(irritable bowel syndrome,IBS)、乳糜泻(celiac disease,CLD)和结直肠癌(colorectal cancer,CRC)等密切相关,但目前尚无相关文献对SRB在肠道疾病中扮演的角色、致病机理和肠道微生态等研究进行系统性的综述。SRB在肠道疾病中的分布特征及其致病机制值得进一步总结与探讨。本文收集过去10年来发表的关于SRB与肠道疾病的文献并进行详细分析与归纳,对SRB在宿主肠道内的分布与生理特征、SRB在不同肠道疾病的相关分布特点,以及SRB致病机制的研究进展等方面进行详细论述,以期增加本领域研究人员对SRB的重视。同时,本文对未来如何深化SRB在肠道相关疾病的研究方向进行探讨,以期为SRB相关肠道疾病的预防与治疗提供一定参考。     

Identification of sulfate-reducing bacteria in methylmercury-contaminated mine tailings by analysis of SSU rRNA genes

Sulfate-reducing bacteria (SRB) are often used in bioremediation of acid mine drainage because microbial sulfate reduction increases pH and produces sulfide that binds with metals. Mercury methylation has also been linked with sulfate reduction. Previous geochemical analysis indicated the occurrence of sulfate reduction in mine tailings, but no molecular characterization of the mine tailings-associated microbial community has determined which SRB are present. This study characterizes the bacterial communities of two geochemically contrasting, high-methylmercury mine tailing environments, with emphasis on SRB, by analyzing small subunit (SSU) rRNA genes present in the tailings sediments and in enrichment cultures inoculated with tailings. Novel Deltaproteobacteria and Firmicutes -related sequences were detected in both the pH-neutral gold mine tailings and the acidic high-sulfide base-metal tailings. At the subphylum level, the SRB communities differed between sites, suggesting that the community structure was dependent on local geochemistry. Clones obtained from the gold tailings and enrichment cultures were more similar to previously cultured isolates whereas clones from acidic tailings were more closely related to uncultured lineages identified from other acidic sediments worldwide. This study provides new insights into the novelty and diversity of bacteria colonizing mine tailings, and identifies specific organisms that warrant further investigation with regard to their roles in mercury methylation and sulfur cycling in these environments.     

象山港海域硫酸盐还原菌的时空分布及其影响因素

分别于2007年7月(夏季)、11月(秋季)与2008年1月(冬季)、4月(春季),调查了象山港海域的水样(表层海水和上覆水)及沉积物中的硫酸盐还原菌丰度的时空分布特征及主要影响因素.结果表明,水样及沉积物中硫酸盐还原菌实测值的变化范围为30～2300 cells·ml-1,沉积物中的硫酸盐还原菌数量高于上覆水及表层海水;硫酸盐还原菌丰度的平面分布均呈现不均匀状态,人类开发活动较多的地区明显高于其他站点.根据硫酸盐还原菌数量,对底质进行腐蚀性评价结果表明,象山港大部分范围底质具有中等强度腐蚀性.有机质污染、水温、pH是表层海水中硫酸盐还原菌分布的重要影响因素(P＜0.01);上覆水中的硫酸盐还原菌含量与上覆水的营养盐(NO2--N、NH4+-N)呈显著正相关;沉积物中的硫酸盐还原菌含量与沉积物中的石油类呈显著正相关(P＜0.05).     

Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

基因工程菌生产抗菌肽的研究进展

目前,利用基因工程菌生产抗菌肽,提供有效和稳定的方法是抗菌肽生产的热点之一。了解抗菌肽工程菌表达系统、生产量尤为重要,为此本文阐述了抗菌肽的生产方式,介绍了基因工程菌表达系统的种类、组成及特点,分析了基因工程菌生产抗菌肽的主要困难因素,并对近些年利用基因工程菌生产抗菌肽的进展进行了综述。     

Formation of biofilms as an example of the social behavior of bacteria

This paper is a brief review of data on bacterial biofilms that occur inside and outside of host organisms. Such biofilms are of great ecological and clinical importance. The role of interspecies communications in the development of bacterial biofilms and infectious diseases is particularly emphasized. Considerable attention is given to the electron microscopic study of biofilms formed by Salmonella typhimurium cells incubated as a broth culture in microtubes without aeration. Bacterial samples taken from the biofilm and planktonic culture grown in the same microtube were comparatively investigated by transmission electron microscopy.     

Effects of legacy nuclear waste on the compositional diversity and distributions of sulfate-reducing bacteria in a terrestrial subsurface aquifer

The impact of legacy nuclear waste on the compositional diversity and distribution of sulfate-reducing bacteria in a heavily contaminated subsurface aquifer was examined. dsrAB clone libraries were constructed and restriction fragment length polymorphism (RFLP) analysis used to evaluate genetic variation between sampling wells. Principal component analysis identified nickel, nitrate, technetium, and organic carbon as the primary variables contributing to well-to-well geochemical variability, although comparative sequence analysis showed the sulfate-reducing bacteria community structure to be consistent throughout contaminated and uncontaminated regions of the aquifer. Only 3% of recovered dsrAB gene sequences showed apparent membership to the Deltaproteobacteria. The remainder of recovered sequences may represent novel, deep-branching lineages that, to our knowledge, do not presently contain any cultivated members; although corresponding phylotypes have recently been reported from several different marine ecosystems. These findings imply resiliency and adaptability of sulfate-reducing bacteria to extremes in environmental conditions, although the possibility for horizontal transfer of dsrAB is also discussed.     

Methane production by the sulfate-reducing bacteriumDesulfosarcina variabilis

The sulfate-reducing bacteriumDesulfosarcina variabilis VKM B-1694 was found to produce up to 1.62 Ώmol methane per mg protein when grown on different substrates. The role of methanogenesis and the physicochemical factors determining this process in sulfate-reducing bacteria are discussed.