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.     

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     

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。     

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.     

海水养殖生境中硫酸盐还原菌活性的抑制机制

【目的】海水养殖生境中的硫化物(H₂S)严重损害养殖生物健康,控制该条件下硫酸盐还原菌(sulfate-reducing bacteria,SRB)的代谢活性是有效抑制H₂S产生的重要途径。【方法】本研究利用稀释涂布-叠皿夹法对海水养殖生境底泥中SRB进行富集筛选,获得SRB菌株,通过投加硝酸盐对菌株产H₂S的活性进行抑制。【结果】获得的2株SRB Desulfovibrio sp.NY-1和Clostridium sp.NH-1,能够在35℃、pH为7.0及盐度为20–30 mg/L条件下,分别积累高达435和150 mg/L H₂S。硝酸盐不能有效抑制NY-1产H₂S的活性,基因调控作用以及缺乏将硝酸盐作为电子受体的酶体系是其不能被抑制的主要原因。硝酸盐对NH-1 H₂S产生活性有可逆性抑制,其具有硝酸盐异化还原成铵(dissimilatory nitrate reduction to ammonium,DNRA)的能力,优先利用硝酸盐作为电子受体。DNRA作用下的中间代谢产物亚硝酸盐是有效抑制菌株NH-1产H₂S活性的主要原因,其抑制机理主要为抑制菌株的生长繁殖。【结论】硝酸盐对不同SRB菌株具有不同的抑制机制和效果,在进行硫化物污染控制前需要对产生硫化物的SRB菌群进行分析判别。     

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     

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

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.     

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.     

Temperature effect on acetate and propionate consumption by sulfate-reducing bacteria in saline wastewater

Seawater toilet flushing, seawater intrusion in the sewerage, and discharge of sulfate-rich industrial effluents elevates sulfate content in wastewater. The application of sulfate-reducing bacteria (SRB) in wastewater treatment is very beneficial; as for example, it improves the pathogen removal and reduces the volume of waste sludge, energy requirement and costs. This paper evaluates the potential to apply biological sulfate reduction using acetate and propionate to saline sewage treatment in moderate climates. Long-term biological sulfate reduction experiments at 10 and 20 °C were conducted in a sequencing batch reactor with synthetic saline domestic wastewater. Subsequently, acetate and propionate (soluble organic carbon) conversion rate were determined in both reactors, in the presence of either or both fatty acids. Both acetate and propionate consumption rates by SRB were 1.9 times lower at 10 °C than at 20 °C. At 10 °C, propionate was incompletely oxidized to acetate. At 10 °C, complete removal of soluble organic carbon requires a significantly increased hydraulic retention time as compared to 20 °C. The results of the study showed that biological sulfate reduction can be a feasible and promising process for saline wastewater treatment in moderate climate.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.     

Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.     

Generalized relationship between numbers of bacteria and their viability in biofilms

Bacterial biofilms are confined communities that are encapsulated in protective layers of extracellular polymeric substances. Microscopic evaluation of biofilms of diverse bacterial strains on various substrata reveals that, in general, the percentage of viable bacteria decreases with the total number of bacteria in a biofilm.     

Role of heterotrophic aerobic denitrifying bacteria in nitrate removal from wastewater

With the increase in industrial and agricultural activities, a large amount of nitrogenous compounds are released into the environment, leading to nitrate pollution. The perilous effects of nitrate present in the environment pose a major threat to human and animal health. Bioremediation provides a cost-effective and environmental friendly method to deal with this problem. The process of aerobic denitrification can reduce nitrate compounds to harmless dinitrogen gas. This review provides a brief view of the exhaustive role played by aerobic denitrifiers for tackling nitrate pollution under different ecological niches and their dependency on various environmental parameters. It also provides an understanding of the enzymes involved in aerobic denitrification. The role of aerobic denitrification to solve the issues faced by the conventional method (aerobic nitrification–anaerobic denitrification) in treating nitrogen-polluted wastewaters is elaborated.