Mitigation of the corrosion-causing Desulfovibrio desulfuricans biofilm using an organic silicon quaternary ammonium salt in alkaline media simulated concrete pore solutions

Abstract

Organic silicon quaternary ammonium salt (OSA), an environmentally friendly naturally occurring chemical, was used as a bacteriostatic agent against sulphate-reducing bacteria (SRB) on a 20SiMn steel surface in simulated concrete pore solutions (SCP). Four different media were used: No SRB (NSRB), No SRB and OSA (NSRB?+?OSA), With SRB (WSRB), With SRB and OSA (WSRB?+?OSA). After biofilm growth for 28 days, optimized sessile SRB cells survived at the high pH of 11.35 and as a result these cells caused the breakdown of the passive film due to the metabolic activities of the SRB. Corrosion prevention results showed that the OSA was effective in mitigating the growth of the sessile SRB cells and reduced corrosion in the SCP. These results were further confirmed by scanning electron microscope images, energy dispersive X-ray analysis, confocal-laser scanning microscopy, X-ray photoelectron spectroscopy and corrosion testing using electrochemical analysis.     

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。     

Evaluation of the sulfate-reducing bacterial population associated with stored swine slurry

Hydrogen sulfide, produced by sulfate-reducing bacteria (SRB), is one of the most potent malodors emitted from anaerobic swine waste storage systems. However, little is known about the prevalence and diversity of SRB in those systems. The goals of this study were to evaluate the SRB population in swine manure storage systems and to develop quantitative, real-time PCR (QRT-PCR) assays to target four of the SRB groups. Dissimilatory sulfite reductase (DSR) gene sequences were obtained from swine slurry stored in underground pits (43 clones) or in lagoons (34 clones). QRT-PCR assays were designed to target the dsrA gene of four novel groups of SRB. Sequences of dsrA clones from slurry samples grouped with those from three different cultured SRB: Desulfobulbus sp. (46 clones), Desulfovibrio sp. (24 clones and 5 isolates), and Desulfobacterium sp. (7 clones). However, DsrA sequences from swine slurry clones were generally less than 85% similar to those of cultured organisms. SRB from all four targeted SRB groups were detected in underground waste storage pits (6.6 x 10(3)-8.5 x 10(7) dsrA copies mL(-1) slurry), while only two groups of SRB were detected in lagoons (3.2 x 10(5)-2.5 x 10(6) dsrA copies mL(-1) slurry). To date, this is the only study to evaluate the phylogeny and concentration of SRB in any livestock waste storage system. The new QRT-PCR assays should facilitate sensitive, specific detection of the four novel groups of SRB in livestock waste storage systems.     

AFM study of the effect of metronidazole on surface structures of sulfate-reducing bacteria

The effect of metronidazole (ME) on sulfate-reducing bacteria (SRB) was studied by atomic force microscopy (AFM) in this paper. Topography images of SRB cell show that after exposure to ME individual cell shape is sharply modified. Topography and phase images of SRB cell wall show that after exposure to ME not only the roughness of the cell wall increases but also the physical performance of SRB surface is changed to be uniform. AFM frictional loops show that after exposure to ME, SRB surface friction is increased remarkably.     

脱氮硫杆菌生长特性及其对SRB生长的影响

由土壤中分离得到一株自养型的脱氮硫杆菌(Thiobacillus denitrigioans,硫杆菌属,硫杆菌科,革兰氏阴性化能自养细菌),该菌株的最佳生长pH为7．0。将此菌株与硫酸盐还原菌(Sulfate Reducing Bacteria,SRB,脱硫弧菌属,革兰氏阴性厌氧细菌)混合培养,测定SRB的菌量变化,结果表明,脱氮硫杆菌的生长抑制了硫酸盐还原菌的生长,降低了SRB的腐蚀性的代谢产物硫化物的浓度,腐蚀速率降低,有利于防治SRB引起的微生物腐蚀。     

重度肥胖症患者膳食干预前后肠道内硫酸盐还原菌的定量检测

目的检测重度肥胖症患者膳食干预过程中,肠道内硫酸盐还原菌（SRB）的数量变化,为进一步研究SRB与肥胖的关系提供参考。方法以SRB基因组中的重要功能基因一腺苷酰硫酸（APS）还原酶基因作为指示基因,通过实时定量PCR的方法检测了12名重度肥胖症患者在进行膳食干预过程中随着体重的降低,肠道内SRB的数量变化,同时以16SrRNA基因对肠道内总菌进行定量来计算SRB占总菌的相对比例。结果膳食干预过程中,随着患者体重的显著降低,其肠道内SRB占总菌的比例也显著下降。在维持期,患者体重仍和干预前有显著差异,但较干预期有所回升;其体内SRB含量也显著低于干预前,但相比干预期,有所上升。结论研究结果提示肠道菌群中SRB细菌与肥胖的发展有密切关系,为后续研究SRB细菌的功能和作用机理奠定了基础。     

Development and comparison of SYBR Green quantitative real‐time PCR assays for detection and enumeration of sulfate‐reducing bacteria in stored swine manure

Aims: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real‐time PCR detection of sulfate‐reducing bacteria (SRB) in stored swine manure. Methods and Results: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR‐R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus‐like Group 1 SRB in manure. Conclusions: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.     

Molecular analysis of microbial communities in mobile deltaic muds of Southeastern Papua New Guinea

A PCR-based approach combined with microbiological cultivation methods was employed to determine the occurrence of sulfate-reducing bacteria (SRB) in colon biopsy samples from ulcerative colitis patients and from non-colitic controls. The detection of mucosa-associated SRB was carried out by digoxigenin-dUTP-labelled PCR amplification, in liquid Postgate medium B and in a new liquid medium, termed VM medium I. Using Postgate medium B, the growth of SRB was confirmed in 92% of the colitic specimens and in 52% of non-colitic samples. However, PCR analysis and incubation in VM medium I detected SRB in 100% of biopsy material indicating ubiquitous presence of SRB in human colon mucosa.     

一种肠道硫酸盐还原菌的增菌培养基

目的针对已经分离、纯化的肠道硫酸盐还原菌,建立一种能快速、高效地培养菌体的培养基。方法比较营养丰富的GAM肉汤与常用于培养硫酸盐还原菌的选择性培养基Postgate的培养效果,摸索在GAM肉汤中添加不同浓度的硫酸盐对两种肠道硫酸盐还原菌-Desulfovibrio desulfuricans和Desulfovibrio intestina—zis的培养效果。确定效果最佳的改良GAM培养基配方,并测定在该培养基中D．desulful＇icans的生长曲线。结果与Postgate培养基相比,GAM肉汤能在2d内快速培养D．desulfugicans,但培养至6d时细菌数量大幅降低。在GAM肉汤中添加Na2SO4与FeSO4,在实验浓度范围内,均显著地促进硫酸盐还原菌的生长。在此基础上改良GAM肉汤培养基,培养得到的细菌数量较GAM肉汤显著提高。D．desulfuricans的生长曲线显示,2d时细菌生长达到最高峰,数量可达3．5×10^7 CFU／mL;培养6d,细菌数量为7．3×10^6 CFU／mL。结论基于GAM肉汤改良而得到的增菌培养基,能快速、高效地培养肠道硫酸盐还原菌,为后续进一步研究肠道硫酸盐还原菌的生理功能提供了支持。     

Safety of Brucella abortus strain RB51 in black bears

In two studies conducted from October 1999 to March 2000 and December 2000 to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4-3.1 x 10(10) colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n=12) or 2 ml of 0.15 M NaCl solution (saline, n=11). We did not detect a difference (P>0.05) in antibody titers to SRB51 in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study 1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51 did not appear to influence (P>0.05) reproductive performance.     

Survival of sulfate-reducing bacteria in oxic surface sediment of a seawater lake

Abstract Microhabitats and survival of sulfate-reducing bacteria (SRB) in an oxic surface sediment of a seawater lake were examined. The size of fractionation of the sediment suspension showed that most of SRB were associated with sediment particles larger than 10 μm. The D values (time in h required to destroy 90% of the initial viable population) for SRB in the whole sediment suspension and for SRB i n the < μ m and the < 5 μ m fractions were, respectively, 23.7, 10 and 4 when the SRB were exposed to air. Survival of the FeS-associated Desulfovibrio desulfuricans ( D value, 9.3) was higher than that of the free-living ones ( D value, 1.8). These results show that particle-associated SRB are more protected against oxygen than free-living ones in oxic sediments.     

Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization is a valuable tool for a more realistic assessment of SRB abundance in the natural environment. The distribution of SRB was investigated in a coastal marine sediment by hybridization of membrane-immobilized rRNA with oligonucleotide probes. As represented by general probe-target groups, SRB rRNA contributed between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulphobacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates (SRR) measured with 35SO4(2-) tracer in whole-core incubations. While SRB abundance was highest near the surface, peaking at around 1.5 cm, measured sulphate reduction rates were lowest in this region. A second peak of SRB rRNA was observed at the transition zone from oxidized to reduced sediment, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis of these estimated cell numbers were between 0.01 and 0.09 fmol SO4(2-) cell(-1) day(-1), which is below the rates that have been determined for pure cultures (0.2-50 fmol SO4(2-) cell(-1) day(-1)) growing exponentially at nearoptimal temperature with a surplus of substrates.     

A Study of the Relative Dominance of Selected Anaerobic Sulfate-Reducing Bacteria in a Continuous Bioreactor by Fluorescence <Emphasis Type="Italic">in Situ</Emphasis> Hybridization

The diversity and the community structure of sulfate-reducing bacteria (SRB) in an anaerobic continuous bioreactor used for treatment of a sulfate-containing wastewater were investigated by fluorescence in situ hybridization. Hybridization to the 16S rRNA probe EUB338 for the domain Bacteria was performed, followed by a nonsense probe NON338 as a control for nonspecific staining. Sulfate-reducing consortia were identified by using five nominally genus-specific probes (SRB129 for Desulfobacter, SRB221 for Desulfobacterium, SRB228 for Desulfotomaculum, SRB660 for Desulfobulbus, and SRB657 for Desulfonema) and four group-specific probes (SRB385 as a general SRB probe, SRB687 for Desulfovibrioaceae, SRB814 for Desulfococcus group, and SRB804 for Desulfobacteriaceae). The total prokaryotic population was determined by 4′,6-diamidino-2-phenylindole staining. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes showed that, of those microbial groupings investigated, Desulfonema, Desulfobulbus, spp., and Desulfobacteriaceae group were the main sulfate-reducing bacteria in the bioreactor when operated at steady state at 35°C, pH 7.8, and a 2.5-day residence time with feed stream containing 2.5 kg m⁻³ sulfate as terminal electron acceptor and 2.3 kg m⁻³ acetate as carbon source and electron donor.     

Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers

Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.     

Methylmercury and methane production potentials in North Carolina Piedmont stream sediments

Methylated mercury (MeHg) can be produced by all microbes possessing the genes hgcA and hgcB, which can include sulfate-reducing bacteria (SRB), iron-reducing bacteria (FeRB), methane-producing archaea (MPA), and other anaerobic microbes. These microbial groups compete for substrates, including hydrogen and acetate. When sulfate is in excess, SRB can outcompete other anaerobic microbes. However, low concentrations of sulfate, which often occur in stream sediments, are thought to reduce the relative importance of SRB. Although SRB are regarded as the primary contributors of MeHg in many aquatic environments, their significance may not be universal, and stream sediments are poorly studied with respect to microbial Hg methylation. We evaluated suppression of methanogenesis by SRB and the potential contributions from SRB, MPA and other MeHg producing microbes (including FeRB) to the production of MeHg in stream sediments from the North Carolina Piedmont region. Lower methanogenesis rates were observed when SRB were not inhibited, however, application of a sulfate-reduction inhibitor stimulated methanogenesis. Greater MeHg production occurred when SRB were active. Other MeHg producing microbes (i.e., FeRB) contributed significantly less MeHg production than SRB. MPA produced MeHg in negligible amounts. Our results suggest that SRB are responsible for the majority of MeHg production and suppress methanogenesis in mid-order stream sediments, similar to other freshwater sediments. Further investigation is needed to evaluate the generality of these findings to streams in other regions, and to determine the mechanisms regulating sulfate and electron acceptor availability and other potential factors governing Hg methylation and methane production in stream sediments.     

Long-term surveillance of sulfate-reducing bacteria in highly saline industrial wastewater evaporation ponds

Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (10⁶–10⁸ targets ml^-1) and lower abundance from the autumn-spring (10³–10⁵ targets ml^-1). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12–14°C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H₂S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (≈16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR.     

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film

A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.     

Molecular phylogenetic and biogeochemical studies of sulfate-reducing bacteria in the rhizosphere of spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO42- reduction rates and geochemical parameters. SO42- reduction was rapid in marsh sediments with rates up to 3.5 &mgr;mol ml-1 day-1. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO42- reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10(7) ml-1. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.