Emergence of a Synergistic Diversity as a Response to Competition in Pseudomonas putida Biofilms.

Genetic diversification through the emergence of variants is one of the known mechanisms enabling the adaptation of bacterial communities. We focused in this work on the adaptation of the model strain Pseudomonas putida KT2440 in association with another P. putida strain (PCL1480) recently isolated from soil to investigate the potential role of bacterial interactions in the diversification process. On the basis of colony morphology, three variants of P. putida KT2440 were obtained from co-culture after 168 h of growth whereas no variant was identified from the axenic KT2440 biofilm. The variants exhibited distinct phenotypes and produced biofilms with specific architecture in comparison with the ancestor. The variants better competed with the P. putida PCL1480 strain in the dual-strain biofilms after 24 h of co-culture in comparison with the ancestor. Moreover, the synergistic interaction of KT2440 ancestor and the variants led to an improved biofilm production and to higher competitive ability versus the PCL1480 strain, highlighting the key role of diversification in the adaptation of P. putida KT2440 in the mixed community. Whole genome sequencing revealed mutations in polysaccharides biosynthesis protein, membrane transporter, or lipoprotein signal peptidase genes in variants.

[Being diagnosed with COPD: A qualitative study of real life experience]. / Annonce de la BPCO : étude qualitative sur le vécu des patients.

INTRODUCTION: This qualitative study aimed to explore the real life experience of the patients with chronic obstructive pulmonary disease (COPD) at the time they receive the diagnosis. METHODS: Data were collected using face to face interviews in general practice as well as focus groups in a pulmonary rehabilitation centre. RESULTS: Thirty-four patients participated in the study. Most of them were made aware of their disease by a pulmonologist during hospitalisation for an acute exacerbation. Several terms were used to name the disease including emphysema, asthma, chronic bronchitis and COPD (acronym often not explained). At the time of the announcement, patients expressed responses which included for some a sense of stupefaction associated with anxiety and for others guilt and an attitude of denial. If the need for smoking cessation was mentioned by doctors, a lack of information at the time of the announcement was general. The chronic and potentially serious aspects of COPD were not understood or rarely mentioned. CONCLUSION: The announcement of the disease did not always appear to have been of good quality. Ideally, the diagnosis of COPD should be conveyed to people after its identification in a dedicated consultation, combined with better information and a proposal for psychological support.

Effect of ammonia on methane production pathways and reaction rates in acetate-fed biogas processes.

In order to understand the correlation between ammonia and methanogenesis metabolism, methane production pathways and their specific rates were studied at total ammonium nitrogen (TAN) concentrations of 0.14-9 g/L in three methanogenic sludges fed with acetate, at both mesophilic and thermophilic conditions. Results showed that high levels of TAN had significant inhibition on methanogenesis; this could, however, be recovered via syntrophic acetate oxidation (SAO) coupled with Hydrogenotrophic Methanogenesis (HM) performed by acetate oxidizing syntrophs or through Acetoclastic Methanogenesis (AM) catalyzed by Methanosarcinaceae, after a long lag phase >50 d. Free ammonia (NH3) was the active component for this inhibition, of which 200 mg/L is suggested as the threshold for the pathway shift from AM to SAO-HM. Methane production rate via SAO-HM at TAN of 7-9 g/L was about 5-9-fold lower than that of AM at TAN of 0.14 g/L, which was also lower than the rate of AM pathway recovered at TAN of 7 g/L in the incubations with a French mesophilic inoculum. Thermophilic condition favored the establishment of the SAO-catalyzing microbial community, as indicated by the higher reaction rate and shorter lag phase. The operational strategy is thus suggested to be adjusted when NH3 exceeds 200 mg/L.

Direct and correlated responses to selection in two lines of rabbits selected for feed efficiency under ad libitum and restricted feeding: I. Production traits and gut microbiota characteristics.

To get insights into selection criteria for feed efficiency, 2 rabbit lines have been created: the ConsoResidual line was selected for residual feed intake (RFI) with ad libitum feeding and the ADGrestrict line was selected for ADG under restricted feeding (-20% of voluntary intake). The first objective of this study was to evaluate, after 9 generations of selection, the direct and correlated responses to selection on production traits in the 2 lines for traits recorded during growth. Second, applying the 2 feeding conditions used for selection to both selected lines plus the control unselected line (generation 0, G0) in a 2 × 3 factorial trial, the line performances were compared and the gut microbiota of the lines was characterized. The correlated responses in feed conversion ratio (FCR) were remarkably equivalent in both selected lines (-2.74 genetic σ) but correlated responses in other traits were notably different. In the ConsoResidual line, selection for decreased RFI resulted in a small negative correlated response in BW at 63 d old (BW63) and in a null response in ADG. In the ADGrestrict line, on the contrary, the correlated response in BW63 was substantial (+1.59 σ). The 2 selected lines had a FCR reduced by 0.2 point compared with the G0 line, and the same difference was found in both feeding regimens ( < 0.001). Indeed, selection on ADG would lead to heavier animals with no significant reduction of feed costs, whereas selection on RFI leads to lower feed costs and no increase of animal BW under ad libitum feeding. Altogether, our results do not suggest any genotype × environment interaction in the response to feeding regimens. The intestinal microbial communities from efficient rabbits differed from their unselected counterparts in terms of fermentation end products and microbial phylotypes, suggesting a central role of these microbes in the better feed efficiency of the rabbits.

Fluorescence-based tools for single-cell approaches in food microbiology.

The better understanding of the functioning of microbial communities is a challenging and crucial issue in the field of food microbiology, as it constitutes a prerequisite to the optimization of positive and technological microbial population functioning, as well as for the better control of pathogen contamination of food. Heterogeneity appears now as an intrinsic and multi-origin feature of microbial populations and is a major determinant of their beneficial or detrimental functional properties. The understanding of the molecular and cellular mechanisms behind the behavior of bacteria in microbial communities requires therefore observations at the single-cell level in order to overcome "averaging" effects inherent to traditional global approaches. Recent advances in the development of fluorescence-based approaches dedicated to single-cell analysis provide the opportunity to study microbial communities with an unprecedented level of resolution and to obtain detailed insights on the cell structure, metabolism activity, multicellular behavior and bacterial interactions in complex communities. These methods are now increasingly applied in the field of food microbiology in different areas ranging from research laboratories to industry. In this perspective, we reviewed the main fluorescence-based tools used for single-cell approaches and their concrete applications with specific focus on food microbiology.

Successive bioanode regenerations to maintain efficient current production from biowaste.

The long-term operation of efficient bioanodes supplied with waste-derived organics is a key challenge for using bioelectrochemical systems as a waste valorization technology. Here, we describe a simple procedure that allowed maintaining highly efficient bioanodes supplied with biowaste. Current densities up to 14.8 A/m(2) were obtained with more than 40% of the electrons introduced as biowaste being recovered in the electrical circuit. Three fed-batch reactors were started at different biowaste loading rates. A decline of coulombic efficiencies between 22 and 31% was recorded depending on the reactor over the first 3 weeks of operation. A renewal procedure of the anode was thereafter implemented, which led to a recovery of initial performances. The second and the third renewal, allowed maintaining stable high level performances with coulombic efficiency of approximately 40% over at least 3 weeks. Electroactive biofilm dynamics were monitored using 16S rRNA-gene amplicon sequencing. Retrieved sequences were dominated by Geobacter sulfurreducens-related reads (37% of total sequences), which proportion however varied along the experiment. Interestingly, sequences affiliated to various Bacteroidetes groups were also abundant, suggesting an adaptation of the anodic biofilm to the degradation of biowaste through metabolic interactions between microbial community members.

Similar evolution in delta 13CH4 and model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes.

Similar evolution was obtained for the stable carbon isotope signatures delta (13)CH(4) and the model-predicted relative rate of aceticlastic methanogenesis during mesophilic methanization of municipal solid wastes. In batch incubations, the importance of aceticlastic and hydrogenotrophic methanogenesis changes in time. Initially, hydrogenotrophic methanogenesis dominated, but increasing population of Methanosarcina sp. enhances aceticlastic methanogenesis. Later, hydrogenotrophic methanogenesis intensified again. A mathematical model was developed to evaluate the relative contribution of hydrogenotrophic and aceticlastic pathways of methane generation during mesophilic batch anaerobic biodegradation of the French and the Chinese Municipal Solid Wastes (FMSW and CMSW). Taking into account molecular biology analysis reported earlier three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) were considered in the model. The total organic and inorganic carbon concentrations, methane production volume, methane and carbon dioxide partial pressures values were used for the model calibration and validation. Methane isotopic composition (delta (13)CH(4)) evolution during the incubations was used to independently validate the model results. The model demonstrated that only the putrescible solid waste was totally converted to methane.

Impact of nitrate-enhanced leachate recirculation on gaseous releases from a landfill bioreactor cell.

This study evaluates the impact of nitrate injection on a full scale landfill bioreactor through the monitoring of gaseous releases and particularly N(2)O emissions. During several weeks, we monitored gas concentrations in the landfill gas collection system as well as surface gas releases with a series of seven static chambers. These devices were directly connected to a gas chromatograph coupled to a flame ionisation detector and an electron capture detector (GC-FID/ECD) placed directly on the field. Measurements were performed before, during and after recirculation of raw leachate and nitrate-enhanced leachate. Raw leachate recirculation did not have a significant effect on the biogas concentrations (CO(2), CH(4) and N(2)O) in the gas extraction network. However, nitrate-enhanced leachate recirculation induced a marked increase of the N(2)O concentrations in the gas collected from the recirculation trench (100-fold increase from 0.2 ppm to 23 ppm). In the common gas collection system however, this N(2)O increase was no more detectable because of dilution by gas coming from other cells or ambient air intrusion. Surface releases through the temporary cover were characterized by a large spatial and temporal variability. One automated chamber gave limited standard errors over each experimental period for N(2)O releases: 8.1 +/- 0.16 mg m(-2) d(-1) (n = 384), 4.2 +/- 0.14 mg m(-2) d(-1) (n = 132) and 1.9 +/- 0.10 mg m(-2) d(-1) (n = 49), during, after raw leachate and nitrate-enhanced leachate recirculation, respectively. No clear correlation between N(2)O gaseous surface releases and recirculation events were evidenced. Estimated N(2)O fluxes remained in the lower range of what is reported in the literature for landfill covers, even after nitrate injection.

Anaerobic biodegradation of cellulosic material: batch experiments and modelling based on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis.

Utilizing stable carbon isotope data to account for aceticlastic and non-aceticlastic pathways of methane generation, a model was created to describe laboratory batch anaerobic decomposition of cellulosic materials (office paper and cardboard). The total organic and inorganic carbon concentrations, methane production volume, and methane and CO(2) partial pressure values were used for the model calibration and validation. According to the fluorescent in situ hybridization observations, three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H(2)/H(2)CO(3) as well as acetate-oxidizing syntrophs, were considered. It was shown that temporary inhibition of aceticlastic methanogens by non-ionized volatile fatty acids or acidic pH was responsible for two-step methane production from office paper at 35 degrees C where during the first and second steps methane was generated mostly from H(2)/H(2)CO(3) and acetate, respectively. Water saturated and unsaturated cases were tested. According to the model, at the intermediate moisture (150%), much lower methane production occurred because of full-time inhibition of aceticlastic methanogens. At the lowest moisture, methane production was very low because most likely hydrolysis was seriously inhibited. Simulations showed that during cardboard and office paper biodegradation at 55 degrees C, non-aceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens were the dominant methanogenic pathways.

Successful bacterial incorporation into activated sludge flocs using alginate.

Bioaugmentation experiments with the aerobic denitrifier Microvirgula aerodenitrificans were performed in an aerobic continuous stirred tank reactor (CSTR) treating urban wastewater. The fate of the added bacteria was monitored by a specific fluorescent oligonucleotide probe targeting 16S rRNA. The first addition of the strain led to its rapid disappearance because of grazing. Bacteria were then embedded within an alginate matrix before inoculation. Alginate fragments adhered to the existing flocs and were progressively colonized by the indigenous flora. Thereafter, microcolonies of the exogenous bacterium were found to be incorporated into existing flocs.

Leachate pre-treatment strategies before recirculation in landfill bioreactors.

Nitrified leachate recirculation represents a promising strategy for a more sustainable landfill management. Our objective was to determine the reactions involved in nitrate reduction in municipal solid waste batch biodegradation tests. Anaerobic digestion of waste in the three control reactors showed a good reproducibility. In two test reactors, nitrate was added at various moments of the waste degradation process. We observed that: (1) H2S concentration controlled the nitrate reduction pathway: above a certain threshold of H2S, dissimilatory nitrate reduction to ammonium (DNRA) replaced denitrification. (2) N2O/N2 ratio varied with the organic carbon concentration: the lower the easily biodegradable carbon concentration, the higher the N2O/N2 ratio. (3) N2 was consumed after denitrification. The possibility of a nitrogen fixation reaction in the presence of NH4 is discussed. Nitrified leachate recirculation during acidogenesis should be avoided because of higher H2S production which could induce DNRA.

Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence.

AIMS: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system. METHODS AND RESULTS: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed. Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight LIVE/DEAD kit were used for planktonic and biofilm flora monitoring. Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of beta-proteobacteria. In the water and the biofilm of the loops, continuous application of chlorine dioxide (0.5 mg l(-1)), or chlorine (2.5 mg l(-1)) were very effective in reducing the microbial flora, including L. pneumophila. Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0.5 mg l(-1)), whereas with monochloramine (0.5 mg l(-1)) and copper-silver ionization (0.8/0.02 mg l(-1)), the contamination remained significantly higher. Monochloramine and copper-silver did not remove the biofilm. During copper-silver application, Legionella re-growth was observed. Only chlorine dioxide led to detectable effects in the dead leg. Amoebae could not be eliminated, and after interrupting the treatments, L. pneumophila quickly recovered their initial levels, in all cases. CONCLUSIONS: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L. pneumophila in a domestic water system. Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg. Amoebae resisted to all the treatments applied and probably acted as reservoirs for L. pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system. Treatment strategies targeting free-living amoebae should lead to improved control of L. pneumophila. Such treatment strategies still have to be investigated.

Combined phosphate and nitrogen removal in a sequencing batch reactor using the aerobic denitrifier, Microvirgula aerodenitrificans.

A phosphate removal sludge was bioaugmented with the aerobic denitrifier, Microvirgula aerodenitrificans in order to reduce the nitrate produced during the aerobic nitrifying-phosphate uptake phase. Fluorescent in situ hybridization (FISH) was used to follow the fate of the added strain. In order to maintain the pure strain in the complex ecosystem, diverse physiological and kinetic based strategies of bioaugmentation were tested under the sequencing batch reactor (SBR) type culture. The nature of the M. aerodenitrificans inoculum (adapted to nitrate-aerobic conditions or to anoxic one) had no influence on the SBR performances and did not enhance aerobic denitrifying performances. The optimum quantity of the added strain (10% of the total biomass) seemed to have much more positive influence on the long term maintenance of the pure strain than on the SBR performances. A small but daily supply of M. aerodenitrificans gave exactly the same result than a massive and 1-day supply, i.e. no enhancement of performances and no amelioration of the length of maintenance. A continuous supply of carbon during the first hour of the aerobic phase combined to a 10% supply of M. aerodenitrificans gave the best compromise in terms of phosphate removal, nitrification and aerobic denitrification performances. It was accompanied too by a decreased number of the ammonia and nitrite-oxidizing bacteria and a modification of the nitrite-oxidizing floc structure. FISH on M. aerodenitrificans revealed that (i) before bioaugmentation, the strain was already present in the phosphate removal sludge and (ii) the added bacteria almost disappeared from the reactor after 16 HRT. In a last experiment, M. aerodenitrificans embedded in alginate beads allowed enhancement of both aerobic denitrifying performances and length of strain maintenance.

Ecological study of a bioaugmentation failure.

A nitrifying sequencing batch reactor was inoculated twice with the aerobic denitrifying bacterium Microvirgula aerodenitrificans and fed with acetate. No improvement was obtained on nitrogen removal. The second more massive inoculation was even followed by a nitrification breakdown, while at the same time, nitrification remained stable in a second reactor operated under the same conditions without bioaugmentation. Fluorescent in situ hybridization with rRNA-targeted probes revealed that the added bacteria almost disappeared from the reactor within 2 days, and that digestive vacuoles of protozoa gave strong hybridization signals with the M. aerodenitrificans-specific probe. An overgrowth of protozoa, coincident with the disappearance of free-living bacteria, was monitored by radioactive dot-blot hybridization only in the bioaugmented reactor. Population dynamics were analysed with a newly developed in situ quantification procedure of the probe-targeted bacteria. The nitrifying groups of bacteria decreased in a similar way in the bioaugmented and non-bioaugmented reactors. Other bacterial groups evolved differently. The involvement of different ecological parameters are discussed separately for each reactor. These results underline the importance of predator-prey interaction and illustrate the undesirable effects of massive bioaugmentation.

Microvirgula aerodenitrificans gen. nov., sp. nov., a new gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions.

A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge. The cells were Gram-negative, catalase- and oxidase-positive curved rods and very motile. They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors. The G&C content was 65 mol%. Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems. However, a phylogenetic analysis based on the 165 rRNA sequence showed that the aerobic denitrifier could not be assigned to any currently recognized genus. For these reasons a new genus and species, Microvirgula aerodenitrificans gen. nov., sp. nov., is proposed, for which SGLY2T is the type strain.