The influence of the chemical composition of drinking water on cuprosolvency by biofilm bacteria

AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.     

Meiothermus timidus sp. nov., a new slightly thermophilic yellow-pigmented species

Several yellow-pigmented isolates, with optimum growth temperatures between 55 and 60 degrees C, were recovered from hot springs in Central Portugal and the Azores. Phylogenetic analysis of the 16S rDNA showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus by biochemical characteristics and the fatty acid composition because they had very high levels of iso C15:0 and iso C17:0 and very low levels of anteiso C17:0 and iso C16:0. On the basis of the results presented here we propose the name Meiothermus timidus for the new species represented by strains SPS-243(T) (=LMG 22897(T)=CIP 108604(T)), RQ-10 and RQ-12.     

Microbiologically influenced corrosion: looking to the future.

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).     

Anti-biofilm potential of a glycolipid surfactant produced by a tropical marine strain of Serratia marcescens

A tropical marine bacterium isolated from the hard coral, Symphyllia sp. was identified as Serratia marcescens on the basis of morphological, biochemical and 16S rDNA analysis. The bacterium showed antimicrobial activity towards the pathogens Candida albicans and Pseudomonas aeruginosa and the marine biofouling bacterium Bacillus pumilus. S. marcescens displayed biosurfactant activity as evidenced by drop collapse, blood hemolysis and surface tension reduction (52.0-27 mN m(-1)). The active compound was purified by solvent extraction and silicic acid chromatography. Characterization was by thin layer chromatography, gas chromatography mass spectroscopy (GC-MS), Fourier transform infrared (FTIR) spectroscopy and (1)H as well as (13)C nuclear magnetic resonance (NMR) analysis. The surfactant was found to be a glycolipid composed of glucose and palmitic acid. The glycolipid prevented adhesion of C. albicans BH, P. aeruginosa PAO1 and B. pumilus TiO1. The glycolipid also disrupted preformed biofilms of these cultures in microtitre plates. Confocal laser scanning microscopy and electron microscopy confirmed the effective removal of biofilms from glass surfaces. The glycolipid derived from S. marcescens could thus serve as a potential anti-biofilm agent.     

污损生物膜细菌Pseudoalteromonas issachenkonii YT1305-1的鉴定及其代谢物化学成分分析

【目的】对污损生物膜细菌YT1305-1进行菌种鉴定;研究其作为污损生物膜优势菌之一的代谢产物。【方法】通过16S rRNA基因序列分析,结合系统进化树和细菌生理生化实验对菌种进行鉴定,通过硅胶柱层析分离方法和核磁共振检测技术分析其代谢物的化学成分。【结果】发现生物膜中存在明显的优势菌株,假交替单胞菌属为优势菌属之一。16S rRNA序列比对分析表明Pseudoalteromonas issachenkonii为优势菌种之一,将目标菌种命名为Pseudoalteromonas issachenkonii YT1305-1,对其代谢物化学成分进行分析,共得到10个化合物,其中包括5个二酮哌嗪(DKPs)类信号分子,环(甘氨酸-丙氨酸)(1)、环(脯氨酸-甘氨酸)(2)、环(脯氨酸-酪氨酸)(3)、环(4-羟基-脯氨酸-亮氨酸)(4)和环(4-羟基-脯氨酸-丙氨酸)(5),以及尿嘧啶(6)、胸腺嘧啶(7)、胸腺嘧啶脱氧核苷(8)、己二酸二(2-乙基己)酯(9)和邻苯二甲酸二(2-乙基己)酯(10)。【结论】污损生物膜中存在明显的优势菌,其中之一为P.issachenkonii YT1305-1,在其代谢产物中发现了疑似信号分子的物质DKPs,有研究表明该物质能调控生物膜的形成,进而影响生物污损的形成,为探究生物污损现象奠定了物质基础。     

Performance of a pilot-scale submerged membrane bioreactor (MBR) in treating bathing wastewater

Bathing wastewater was treated by a pilot-scale submerged membrane bioreactor (MBR) for more than 60 days. The results showed that the removal rates of main pollutants of wastewater such as COD(Cr), LAS, NH(4)(+)-N and total nitrogen (TN) were above 93%, 99%, 99%, and 90%, respectively. The results of denaturing gel gradient electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) indicated that the bacteria were stable. The abundant nitrobacteria intercepted by the membrane led to the high removal rate of ammonia and TN. FISH and 16S rDNA gene sequence analysis revealed that some specific phylogenetic group of bacteria, the Pseudomonas sp. Ochrobactrum anthropi sp. and Enterobacter sp. probably played a major role in the development of the mature biofilms, which led to the severe irreversible membrane biofouling.     

Efficient removal of hexavalent chromium by a tolerant Streptomyces sp. affected by the toxic effect of metal exposure

AIMS: To isolate and analyse chromium-resistant micro-organisms suitable for bioremediation. METHODS AND RESULTS: Strain CG252, with a minimal inhibitory concentration of 500 microg ml(-1), was isolated from contaminated soils and identified as a Streptomyces sp. by 16S rDNA sequence analysis. Assays carried out at various Cr(VI) concentrations indicated that chromium removal was more efficient at lower concentrations and that this activity resulted in accumulation of Cr(III). Atomic adsorption analysis indicated that the chromium removed was not associated with cell mass and activity assays showed that the capacity to reduce Cr(VI) was most probably due to a soluble cytosolic enzyme. Cells grown as biofilms showed enhanced removal of Cr(VI) with respect to planktonic cells, while analysis of growth and colony morphology indicated that Cr(VI) had a toxic effect on this strain. CONCLUSIONS: Streptomyces sp. CG252 tolerated heavy metals and elevated levels of chromium, despite its negative effect on growth and development, and was efficient at removing Cr(VI) by promoting reduction to Cr(III). SIGNIFICANCE AND IMPACT OF THE STUDY: Strain CG252's capacity to tolerate heavy metals and to reduce Cr(VI) to the less toxic Cr(III), especially when forming biofilms, makes it a promising candidate for detoxification of sites containing this heavy metal.     

The use of ultrasonic imaging to evaluate the effect of protozoan grazing and movement on the topography of bacterial biofilms

AIMS: This study evaluated the effect of protozoan movement and grazing on the topography of a dual-bacterial biofilm using both conventional light microscopy and a new ultrasonic technique. METHODS AND RESULTS: Coupons of dialysis membrane were incubated in Chalkley's medium for 3 days at 23 degrees C in the presence of bacteria (Pseudomonas aeruginosa and Klebsiella aerogenes) alone, or in co-culture with the flagellate Bodo designis, the ciliate Tetrahymena pyriformis or the amoeba Acanthamoeba castellanii. Amoebic presence resulted in a confluent biofilm similar to the bacteria-only biofilm while the flagellate and ciliate created more diverse biofilm topographies comprising bacterial microcolonies and cavities. CONCLUSIONS: The four distinct biofilm topographies were successfully discerned with ultrasonic imaging and the method yielded information similar to that obtained with conventional light microscopy. SIGNIFICANCE AND IMPACT OF THE STUDY: Ultrasonic imaging provides a potential way forward in the development of a portable, nondestructive technique for profiling the topography of biofilms in situ, which might aid in the future management of biofouling.     

Structure and species composition of mercury-reducing biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Nitric Oxide Treatment for the Control of Reverse Osmosis Membrane Biofouling

Biofouling remains a key challenge for membrane-based water treatment systems. This study investigated the dispersal potential of the nitric oxide (NO) donor compound, PROLI NONOate, on single- and mixed-species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis (RO) membranes. The potential of PROLI NONOate to control RO membrane biofouling was also examined. Confocal microscopy revealed that PROLI NONOate exposure induced biofilm dispersal in all but two of the bacteria tested and successfully dispersed mixed-species biofilms. The addition of 40 μM PROLI NONOate at 24-h intervals to a laboratory-scale RO system led to a 92% reduction in the rate of biofouling (pressure rise over a given period) by a bacterial community cultured from an industrial RO membrane. Confocal microscopy and extracellular polymeric substances (EPS) extraction revealed that PROLI NONOate treatment led to a 48% reduction in polysaccharides, a 66% reduction in proteins, and a 29% reduction in microbial cells compared to the untreated control. A reduction in biofilm surface coverage (59% compared to 98%, treated compared to control) and average thickness (20 μm compared to 26 μm, treated compared to control) was also observed. The addition of PROLI NONOate led to a 22% increase in the time required for the RO module to reach its maximum transmembrane pressure (TMP), further indicating that NO treatment delayed fouling. Pyrosequencing analysis revealed that the NO treatment did not significantly alter the microbial community composition of the membrane biofilm. These results present strong evidence for the application of PROLI NONOate for prevention of RO biofouling.     

Complete genome sequence of Meiothermus silvanus type strain (VI-R2)

Meiothermus silvanus (Tenreiro et al. 1995) Nobre et al. 1996 belongs to a thermophilic genus whose members share relatively low degrees of 16S rRNA gene sequence similarity. Meiothermus constitutes an evolutionary lineage separate from members of the genus Thermus, from which they can generally be distinguished by their slightly lower temperature optima. M. silvanus is of special interest as it causes colored biofilms in the paper making industry and may thus be of economic importance as a biofouler. This is the second completed genome sequence of a member of the genus Meiothermus and only the third genome sequence to be published from a member of the family Thermaceae. The 3,721,669 bp long genome with its 3,667 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Structure and Species Composition of Mercury-Reducing Biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Ribonuclease P RNA gene sequencing as a tool for molecular dereplication of myxobacterial strain collections

AIMS: Efficient strain dereplication is of great value during the generation of bacterial strain collections for industrial screening. We evaluated the utilization of the RNase P RNA gene (rnpB) sequence as a tool for molecular dereplication of myxobacteria. METHODS AND RESULTS: 16S rDNA (approx. 1 x 5 kbp) and rnpB (approx. 0 x 3 kbp) sequences were obtained and aligned. From 50 strains, we obtained 20 different sequences for the 16S rDNA and 24 for rnpB. Intersequence similarity was lower for rnpB than for 16S rDNA. CONCLUSIONS: rnpB allows the rapid discrimination of similar strains, with a higher resolution power as compared with 16S rRNA gene sequencing. It not only gives better discrimination, but is also faster and cheaper than 16S rDNA sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: Myxobacteria isolation and cultivation require time and experience. The application of rnpB sequencing to early myxobacterial strain dereplication may help in the generation of diverse strain libraries of these bacteria.     

Effect of chlorination on the development of marine biofilms dominated by diatoms

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5?min, 1?min, 5?min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15?min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (～20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.     

Antifouling activity of enzyme‐functionalized silica nanobeads

The amelioration of biofouling in industrial processing equipment is critical for performance and reliability. While conventional biocides are effective in biofouling control, they are potentially hazardous to the environment and in some cases corrosive to materials. Enzymatic approaches have been shown to be effective and can overcome the disadvantages of traditional biocides, however they are typically uneconomic for routine biofouling control. The aim of this study was to design a robust and reusable enzyme‐functionalized nano‐bead system having biofilm dispersion properties. This work describes the biochemical covalent functionalization of silica‐based nanobeads (hereafter referred to as Si‐NanoB) with Proteinase K (PK). Results showed that PK‐functionalized Si‐NanoB are effective in dispersing both protein‐based model biofilms and structurally altering Pseudomonas fluorescens biofilms, with significant decreases in surface coverage and thickness of 30.1% and 38.85%, respectively, while increasing surface roughness by 19 % following 24 h treatments on bacterial biofilms. This study shows that enzyme‐functionalized nanobeads may potentially be an environmentally friendly and cost effective alternative to pure enzyme and chemical treatments. Biotechnol. Bioeng. 2016;113: 501–512. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Optimal dosing regimen of nitric oxide donor compounds for the reduction of Pseudomonas aeruginosa biofilm and isolates from wastewater membranes

Membrane fouling by bacterial biofilms remains a key challenge for membrane-based water purification systems. Here, the optimal biofilm dispersal potential of three nitric oxide (NO) donor compounds, viz. sodium nitroprusside, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (MAHMA NONOate) and 1-(hydroxy-NNO-azoxy)-L-proline, disodium salt, was investigated using Pseudomonas aeruginosa PAO1 as a model organism. Dispersal was quantitatively assessed by confocal microscopy [bacterial cells and the components of the extracellular polymeric substances (EPS) (polysaccharides and extracellular DNA)] and colony-forming unit counts. The three NO donor compounds had different optimal exposure times and concentrations, with MAHMA NONOate being the optimal NO donor compound. Biofilm dispersal correlated with a reduction in both bacterial cells and EPS. MAHMA NONOate also reduced single species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis membranes, as well as in isolates combined to generate mixed species biofilms. The data present strong evidence for the application of these NO donor compounds for prevention of biofouling in an industrial setting.     

Individual microflora beget unique oral microcosms

AIMS: To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter-individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs). METHODS AND RESULTS: Two sets of triplicate MSDs were established. One set was inoculated using fresh saliva from three separate volunteers; the second set was inoculated from one saliva donor. Both were incubated in an anaerobic cabinet. BF and PA were analysed at 24-h intervals by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Hierarchical dendrograms were constructed in order to sort community fingerprints over time, based on community relatedness. The MSD supported complex oral communities, as evidenced by DGGE (>20 distinct DGGE bands) and confocal scanning laser microscopy. DGGE band sequencing revealed bacteriological diversity and a high incidence of anaerobic species, including Prevotella sp. Dendrograms demonstrated marked inter-individual variation in the relative species abundance within salivary inocula from different volunteers (DV) and each associated MSD (all >45%, majority c. 85% concordance). Less variation was shown between triplicate models established using saliva from a single volunteer (SV) (all >58%; majority c. 95% concordance). PAs clustered together with the associated biofilms and inocula in the majority of cases for the DV MSDs whilst SV MSD community profiles clustered between replicate MSDs. CONCLUSIONS: Data indicate that marked inter-individual variations in human salivary composition can be partially replicated in individualized MSD microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the in vitro reproduction of individual oral microbiotas and suggests that taking inter-individual variability into account will increase the relevance of microcosm studies.     

Community structure of microbial biofilms associated with membrane-based water purification processes as revealed using a polyphasic approach

The microbial communities of membrane biofilms occurring in two full-scale water purification processes employing microfiltration (MF) and reverse osmosis (RO) membranes were characterized using a polyphasic approach that employed bacterial cultivation, 16S rDNA clone library and fluorescence in situ hybridization techniques. All methods showed that the alpha-Proteobacteria was the largest microbial fraction in the samples, followed by the gamma-Proteobacteria. This suggested that members of these two groups could be responsible for the biofouling on the membranes studied. Furthermore, the microbial community structures between the MF and RO samples were considerably different in composition of the most predominant 16S rDNA clones and bacterial isolates from the alpha-Proteobacteria and only shared two common groups ( Bradyrhizobium, Bosea) out of more than 17 different bacterial groups observed. The MF and RO samples further contained Planctomycetes and Fibroacter/ Acidobacteria as the second predominant bacterial clones, respectively, and differed in minor bacterial clones and isolates. The community structure differences were mainly attributed to differences in feed water, process configurations and operating environments, such as the pressure and hydrodynamic conditions present in the water purification systems.     

Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.     

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.