Comparison of biofilm cell quantification methods for drinking water distribution systems.

Drinking water quality typically degrades after treatment during conveyance through the distribution system. Potential causes include biofilm growth in distribution pipes which may result in pathogen retention, inhibited disinfectant diffusion, and proliferation of bad tastes and odors. However, there is no standard method for direct measurement of biofilms or quantification of biofilm cells in drinking water distribution systems. Three methods are compared here for quantification of biofilm cells grown in pipe loops samplers: biofilm heterotrophic plate count (HPC), biofilm biovolume by confocal laser scanning microscopy (CLSM) and biofilm total cell count by flow cytometry (FCM) paired with Syto 9. Both biofilm biovolume by CLSM and biofilm total cell count by FCM were evaluated for quantification of the whole biofilms (including non-viable cells and viable but not culturable cells). Signal-to-background ratios and overall performance of biofilm biovolume by CLSM and biofilm total cell count by FCM were found to vary with the pipe material. Biofilm total cell count by FCM had a low signal-to-background ratio on all materials, indicating that further development is recommended before application in drinking water environments. Biofilm biovolume by CLSM showed the highest signal-to-background ratio for cement and cast iron, which suggests promise for wider application in full-scale systems. Biofilm biovolume by CLSM and Syto 9 staining allowed in-situ biofilm cell quantification thus elimination variable associated with cell detachment for quantification but had limitations associated with non-specific staining of cement and, to a lesser degree, auto-fluorescence of both cement and polyvinyl chloride materials. Due to variability in results obtained from each method, multiple methods are recommended to assess biofilm growth in drinking water distribution systems. Of the methods investigated here, HPC and CLSM and recommended for further development towards application in full-scale systems. HPC is a sample and widely applied method that quantifies viable culturable cells. CLSM analysis allows the elimination of experimental variables associated with cell detachment and affords the opportunity to evaluate biofilm components such as extracellular polymeric substances through the addition of specific probes. These two methods can be applied together to assess biofilms known to degrade treated water quality during conveyance in full-scale drinking water treatment systems. The significance of improved biofilm assessment methods for drinking water distribution systems lies in advancing understanding of biofilm growth and control mechanisms that may lead to improved water quality during conveyance and at the tap for greater public health protection.

Short-chain fructo-oligosaccharide and inulin modulate inflammatory responses and microbial communities in Caco2-bbe cells and in a mouse model of intestinal injury.

BACKGROUND: Few studies have focused on the ability of prebiotics to prevent pathogen-induced cellular changes or alter the composition of the intestinal microbiota in complimentary relevant cell and animal models of inflammatory bowel disease. OBJECTIVE: The objective of this study was to determine if pretreatment with inulin and a short-chain fructo-oligosaccharide (sc-FOS) prevents enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in Caco2-bbe epithelial cells and what effect 10% wt:v sc-FOS or inulin has on C57BL/6 mice under sham conditions or pretreatment with prebiotics before Citrobacter rodentium infection (10(8) colony-forming units). METHODS: Actin rearrangement and tight junction protein (zona occludin-1) were examined with immunofluorescence. Barrier function was assessed by a fluorescent probe and by measuring transepithelial electrical resistance (TER). Alterations in cytokine gene expression and microbiome were assessed with quantitative reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. Short-chain fatty acids (SCFAs) were measured by GC. RESULTS: sc-FOS added to monolayers altered actin polymerization without affecting TER or permeability to a fluorescein isothiocyanate (FITC) probe, whereas inulin increased TER (P < 0.005) and altered actin arrangement without affecting FITC permeability. Neither prebiotic attenuated EHEC-induced decreases in barrier function. Prebiotics increased interleukin 10 (Il10) and transforming growth factor-ß (Tgfß) cytokine responses alone (P < 0.05) or with EHEC O157:H7 infection (P < 0.05) in vitro. Increases in tumor necrosis factor-α (Tnfα) (P < 0.05) and decreases in chemokine CXC motif ligand 8 (Cxcl8) (P < 0.05) expression were observed with prebiotic treatment prior to EHEC infection. No differences were noted in barrier function or cytokine responses in the absence or presence of C. rodentium in vivo. Alterations in microbiome were evident at 6 d and 10 d postinfection in treatment groups, but a change in C. rodentium load was not observed. Inulin and sc-FOS (P < 0.05) increased fecal SCFAs in the absence of infection. CONCLUSION: This study provides new insights as to how prebiotics act in complementary in vitro and in vivo models of intestinal injury.

Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

Interspecies interaction extends bacterial survival at solid-air interfaces.

Despite the ubiquity of biofilms in natural and man-made environments, research on surface-associated cells has focused primarily on solid-liquid interfaces. This study evaluated the extent to which bacterial cells persist on inanimate solid-air interfaces. The desiccation tolerance of bacterial strains isolated from indoor air, as well as of a test strain (Pseudomonas aeruginosa), was determined at different levels of relative humidity (RH) using the large droplet inoculation method in an aerosol chamber. The cells survived longer at lower (25 and 42%) than at high RH (95%). Four of the seven indoor strains selected for further study showed extended period of survival following deposition as 0.05-0.1 ml of washed culture followed by desiccation, each with different effects on the survival of the test strain, P. aeruginosa. A strain closely related to Arthrobacter species afforded the highest level of protection to the test strain. Even though the desiccation-tolerant strains survived when they were deposited as bioaerosols, the protective role towards the test strain was not observed when the latter was deposited as a bioaerosol. These, which are often-unculturable, bacteria may go undetected during routine monitoring of biofouling, thereby allowing them to act as reservoirs and extending the habitat range of undesired microorganisms.

A versatile and robust aerotolerant microbial community capable of cellulosic ethanol production.

The use of microbial communities in the conversion of cellulosic materials to bio-ethanol has the potential to improve the economic competitiveness of this biofuel and subsequently lessen our dependency on fossil fuel-based energy sources. Interactions between functionally different microbial groups within a community can expand habitat range, including the creation of anaerobic microenvironments. Currently, research focussing on exploring the nature of the interactions occurring during cellulose degradation and ethanol production within mixed microbial communities has been limited. The aim of this study was to enrich and characterize a cellulolytic bacterial community, and determine if ethanol is a major soluble end-product. Cellulolytic activity by the community was observed in both non-reduced and pre-reduced media, with ethanol and acetate being major fermentation products. Similar results were obtained when sterile wastewater extract was provided as nutrient. Several community members showed high similarity to Clostridium species with overlapping metabolic capabilities, suggesting clostridial functional redundancy.

Assessment of the working range and effect of sodium dichloroisocyanurate on Pseudomonas aeruginosa biofilms and planktonic cells.

Sodium dichloroisocyanurate (NaDCC) is a chemical agent that acts against microorganisms in a manner similar to that of sodium hypochlorite by releasing free available chlorine. NaDCC has been approved by the WHO for the emergency treatment of water and by the US EPA for routine treatment of water. Previous studies assessing the effectiveness of NaDCC for the treatment of water implied that NaDCC should have a wide array of disinfecting effects beyond the treatment of planktonic cells in potable water. In this study the biocidal effects of NaDCC against Pseudomonas aeruginosa cells in different growth modes including planktonic cells and biofilms were explored. The data showed that a 60% dilution of the standard NaDCC solution was effective in the treatment of both P. aeruginosa planktonic cells and biofilms.

A flow cell simulating a subsurface rock fracture for investigations of groundwater-derived biofilms.

Laboratory scale continuous-flow-through chambers (flow cells) facilitate the observation of microbes in a controlled, fully hydrated environment, although these systems often do not simulate the environmental conditions under which microorganisms are found. We developed a flow cell that mimics a subsurface groundwater-saturated rock fracture and is amenable to confocal laser scanning microscopy while allowing for the simple removal of the attached biomass. This flow cell was used to investigate the effect of toluene, a representative contaminant for non-aqueous phase liquids, on groundwater-derived biofilms. Reduced average biofilm biomass and thickness, and diminished diversity of amplifiable 16S rRNA sequences were observed for biofilms that developed in the presence of toluene, compared to the biofilms grown in the absence of toluene. The flow cell also allowed the detection of fluorescent protein-labelled cells.

A flow cell simulating a subsurface rock fracture for investigations of groundwater-derived biofilms

Laboratory scale continuous-flow-through chambers (flow cells) facilitate the observation of microbes in a controlled, fully hydrated environment, although these systems often do not simulate the environmental conditions under which microorganisms are found. We developed a flow cell that mimics a subsurface groundwater-saturated rock fracture and is amenable to confocal laser scanning microscopy while allowing for the simple removal of the attached biomass. This flow cell was used to investigate the effect of toluene, a representative contaminant for non-aqueous phase liquids, on groundwater-derived biofilms. Reduced average biofilm biomass and thickness, and diminished diversity of amplifiable 16S rRNA sequences were observed for biofilms that developed in the presence of toluene, compared to the biofilms grown in the absence of toluene. The flow cell also allowed the detection of fluorescent protein-labelled cells (AU)

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Plasmid-mediated bioaugmentation of wastewater microbial communities in a laboratory-scale bioreactor.

Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation - the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain. Here we discuss how the catabolic plasmid pNB2 can be evaluated towards its potential to facilitate the degradation of a xenobiotic compound, 3-chloroaniline, and demonstrate the applicability of this plasmid to accomplish 3-chloroaniline degradation in a bioreactor setting after in situ transfer to suitable recipient strains.

Bioaugmentation of activated sludge towards 3-chloroaniline removal with a mixed bacterial population carrying a degradative plasmid.

A bioaugmentation approach combining several strategies was applied to achieve degradation of 3-chloroaniline (3CA) in semicontinuous activated sludge reactors. In a first step, a 3CA-degrading Comamonas testosteroni strain carrying the degradative plasmid pNB2 was added to a biofilm reactor, and complete 3CA degradation together with spread of the plasmid within the indigenous biofilm population was achieved. A second set of reactors was then bioaugmented with either a suspension of biofilm cells removed from the carrier material or with biofilm-containing carrier material. 3CA degradation was established rapidly in all bioaugmented reactors, followed by a slow adaptation of the non-bioaugmented control reactors. In response to variations in 3CA concentration, all reactors exhibited temporary performance breakdowns. Whereas duplicates of the control reactors deviated in their behaviour, the bioaugmented reactors appeared more reproducible in their performance and population dynamics. Finally, the carrier-bioaugmented reactors showed an improved performance in the presence of high 3CA influent concentrations over the suspension-bioaugmented reactors. In contrast, degradation in one control reactor failed completely, but was rapidly established in the remaining control reactor.

Bioaugmentation of microbial communities in laboratory and pilot scale sequencing batch biofilm reactors using the TOL plasmid.

The aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmid pWWO (TOL plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch biofilm reactors (SBBRs) treating synthetic wastewater containing benzyl alcohol (BA) as a model xenobiotic. The plasmid donor was a Pseudomonas putida strain chromosomally tagged with the gene for the red fluorescent protein carrying a green fluorescent protein labeled TOL plasmid, which confers degradation capacity for several compounds including toluene and BA. In the pilot scale SBBR donor cells were disappeared 84 h after inoculation while transconjugants were not detected at all. In contrast, both donor and transconjugant cells were detected in the laboratory scale reactor where the ratio of transconjugants to donors fluctuated between 1.9 x 10(-1) and 8.9 x 10(-1) during an experimental period of 32 days. BA degradation rate was enhanced after donor inoculation from 0.98 mg BA/min prior to inoculation to 1.9 mg BA/min on the seventeenth day of operation. Survival of a bioaugmented strain, conjugative plasmid transfer and enhanced BA degradation was demonstrated in the laboratory scale SBBR but not in the pilot scale SBBR.

Intimate coupling of photocatalysis and biodegradation in a photocatalytic circulating-bed biofilm reactor.

Coupling advanced oxidative pretreatment with subsequent biodegradation demonstrates potential for treating wastewaters containing biorecalcitrant and inhibitory organic constituents. However, advanced oxidation is indiscriminate, producing a range of products that can be too oxidized, unavailable for biodegradation, or toxic themselves. This problem could be overcome if advanced oxidation and biodegradation occurred together, an orientation called intimate coupling; then, biodegradable organics are removed as they are formed, focusing the chemical oxidant on the non-biodegradable fraction. Intimate coupling has seemed impossible because the conditions of advanced oxidation, for example, hydroxyl radicals and sometimes UV-light, are severely toxic to microorganisms. Here, we demonstrate that a novel photocatalytic circulating-bed biofilm reactor (PCBBR), which utilizes macro-porous carriers to protect biofilm from toxic reactants and UV light, achieves intimate coupling. We demonstrate the viability of the PCBBR system first with UV only and acetate, where the carriers grew biofilm and sustained acetate biodegradation despite continuous UV irradiation. Images obtained by scanning electron microscopy and confocal laser scanning microscopy show bacteria living behind the exposed surface of the cubes. Second, we used slurry-form Degussa P25 TiO2 to initiate photocatalysis of inhibitory 2,4,5-trichlorophenol (TCP) and acetate. With no bacterial carriers, photocatalysis and physical processes removed TCP and COD to 32% and 26% of their influent levels, but addition of biofilm carriers decreased residuals to 2% and 4%, respectively. Biodegradation alone could not remove TCP. Photomicrographs clearly show that biomass originally on the exterior of the carriers was oxidized (charred), but biofilm a short distance within the carriers was protected. Finally, we coated TiO2 directly onto the carrier surface, producing a hybrid photocatalytic-biological carrier. These carriers likewise demonstrated the concept of photocatalytic degradation of TCP coupled with biodegradation of acetate, but continued TCP degradation required augmentation with slurry-form TiO2.

Bioaugmentation of aerobic microbial granules with Pseudomonas putida carrying TOL plasmid.

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation.

Microbial populations associated with fixed- and floating-bed reactors during a two-stage anaerobic process

Microbial populations associated with methanogenic fixed- or floating-bed bioreactors used for anaerobic digestion of lignocellulosic waste were investigated. Fluorescent in situ hybridization (FISH) was used to characterize microorganisms in samples obtained from different heights in the reactors, which were operated in a semi-continuous manner (feeding and mixing once every 2 days). The FISH results showed that Methanosaeta concilii cells were most numerous at the bottom of both reactors. M. concilii cells were more abundant in the fixed-bed reactor (FXBR), which performed better than the floating-bed reactor (FLBR). Species of the Methanosarcina genera (mainly M. barkeri and M. mazei) were also observed in the FLBR but rarely in the FXBR. Methane production in each of the reactors ranged from 0.29 to 0.33 m3 CH(4)/kg COD(rem) (chemical oxygen demand removed). The removal of volatile fatty acids (VFA; 70-75 h) in the FXBR was more efficient than in the FLBR (AU)

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Influence of detachment on substrate removal and microbial ecology in a heterotrophic/autotrophic biofilm.

Competition between heterotrophic bacteria oxidizing organic substrate and autotrophic nitrifying bacteria in a biofilm was evaluated. The biofilm was grown in a tubular reactor under different shear and organic substrate loading conditions. The reactor was initially operated without organic substrate in the influent until stable ammonia oxidation rates of 2.1 gN/(m(2)d) were achieved. A rapid increase of fluid shear in the tubular reactor on day 156 resulted in biofilm sloughing, reducing the biofilm thickness from 330 to 190 microm. This sloughing event did not have a significant effect on ammonia oxidation rates. The addition of acetate to the influent of the reactor resulted in decreased ammonia oxidation rates (1.8 gN/(m(2)d)) for low influent acetate concentrations (17 mg COD/L) and the breakdown of nitrification at high influent acetate concentrations (55 mg COD/L). Rapidly increasing fluid shear triggered biofilm sloughing in some cases--but maintaining constant shear did not prevent sloughing events from occurring. With the addition of acetate to the influent of the reactor, the biofilm thickness increased up to 1350 microm and individual sloughing events removed up to 50% of the biofilm. Biofilm sloughing had no significant influence on organic substrate removal or ammonia oxidation. During 325 days of reactor operation, ammonia was oxidized only to nitrite; no nitrate production was observed. This lack of nitrite oxidation was confirmed by fluorescent in situ hybridization (FISH) analysis, which detected betaproteobacterial ammonia oxidizers but not nitrite oxidizers. Mathematical modeling correctly predicted breakdown of nitrification at high influent acetate concentrations. Model predictions deviated systematically from experimental results, however, for the case of low influent acetate concentrations.

Microbial populations associated with fixed- and floating-bed reactors during a two-stage anaerobic process.

Microbial populations associated with methanogenic fixed- or floating-bed bioreactors used for anaerobic digestion of lignocellulosic waste were investigated. Fluorescent in situ hybridization (FISH) was used to characterize microorganisms in samples obtained from different heights in the reactors, which were operated in a semi-continuous manner (feeding and mixing once every 2 days). The FISH results showed that Methanosaeta concilii cells were most numerous at the bottom of both reactors. M. concilii cells were more abundant in the fixed-bed reactor (FXBR), which performed better than the floating-bed reactor (FLBR). Species of the Methanosarcina genera (mainly M. barkeri and M. mazei) were also observed in the FLBR but rarely in the FXBR. Methane production in each of the reactors ranged from 0.29 to 0.33 m3 CH(4)/kg COD(rem) (chemical oxygen demand removed). The removal of volatile fatty acids (VFA; 70-75 h) in the FXBR was more efficient than in the FLBR.

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities.

BACKGROUND: The beta-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. RESULTS: We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. CONCLUSION: The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

Distribution of the Mycobacterium community and polycyclic aromatic hydrocarbons (PAHs) among different size fractions of a long-term PAH-contaminated soil.

Summary Mycobacterium is often isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil as degraders of PAHs. In model systems, Mycobacterium shows attachment to the PAH substrate source, which is considered to be a particular adaptation to low bioavailability as it results into increased substrate flux to the degraders. To examine whether PAH-degrading Mycobacterium in real PAH-contaminated soils, in analogy with model systems, are preferentially associated with PAH-enriched soil particles, the distribution of PAHs, of the PAH-mineralizing capacity and of Mycobacterium over different fractions of a soil with an aged PAH contamination was investigated. The clay fraction contained the majority of the PAHs and showed immediate pyrene- and phenanthrene-mineralizing activity upon addition of (14)C-labelled pyrene or phenanthrene. In contrast, the sand and silt fractions showed a lag time of 15-26 h for phenanthrene and 3-6 days for pyrene mineralization. The maximum pyrene and phenanthrene mineralization rates of the clay fraction expressed per gram fraction were three to six times higher than those of the sand and silt fractions. Most-probable-number (MPN)-polymerase chain reaction demonstrated that Mycobacterium represented about 10% of the eubacteria in the clay fraction, while this was only about 0.1% in the sand and silt fractions, indicating accumulation of Mycobacterium in the PAH-enriched clay fraction. The Mycobacterium community composition in the clay fraction represented all dominant Mycobacterium populations of the bulk soil and included especially species related to Mycobacterium pyrenivorans, which was also recovered as one of the dominant species in the eubacterial communities of the bulk soil and the clay fraction. Moreover, Mycobacterium could be identified among the major culturable PAH-degrading populations in both the bulk soil and the clay fraction. The results demonstrate that PAH-degrading mycobacteria are mainly associated with the PAH-enriched clay fraction of the examined PAH-contaminated soil and hence, that also in the environmental setting of a PAH-contaminated soil, Mycobacterium might experience advantages connected to substrate source attachment.

Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture.

The degradation of polycyclic aromatic hydrocarbons (PAHs) by an undefined culture obtained from a PAH-polluted soil and the same culture bioaugmented with three PAH-degrading strains was studied in carbon-limited chemostat cultures. The PAHs were degraded efficiently by the soil culture and bioaugmentation did not significantly improve the PAH degrading performance. The presence of PAHs did, however, influence the bacterial composition of the bioaugmented and non-bioaugmented soil cultures, resulting in the increase in cell concentration of sphingomonad strains. the initial enhancement of the degradation of the PAHs by biostimulation gradually disappeared and only the presence of salicylate in the additional carbon sources had a lasting slightly stimulating effect on the degradation of phenanthrene. The results suggest that bioaugmentation and biostimulation have limited potential to enhance PAH bioremediation by culture already proficient in the degradation of such contaminants.