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International audience; In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNAyield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D = 0.023 h (1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

International audience; Microbial communities have a key role for the performance of engineered ecosystems such as waste gas biofilters. Maintaining constant performance despite fluctuating environmental conditions is of prime interest, but it is highly challenging because the mechanisms that drive the response of microbial communities to disturbances still have to be disentangled. Here we demonstrate that the bioprocess performance and stability can be improved and reinforced in the face of disturbances, through a rationally predefined strategy of microbial resource management (MRM). This strategy was experimentally validated in replicated pilot-scale nitrifying gas-biofilters, for the two steps of nitrification. The associated biological mechanisms were unraveled through analysis of functions, abundances and community compositions for the major actors of nitrification in these biofilters, that is, ammonia-oxidizing bacteria (AOB) and Nitrobacter-like nitrite-oxidizers (NOB). Our MRM strategy, based on the application of successive, transient perturbations of increasing intensity, enabled to steer the nitrifier community in a favorable way through the selection of more resistant AOB and NOB sharing functional gene sequences close to those of, respectively, Nitrosomonas eutropha and Nitrobacter hamburgensis that are well adapted to high N load. The induced community shifts resulted in significant enhancement of nitrification resilience capacity following the intense perturbation.

Lactococcus lactis subsp. cremoris AM2 strain was previously shown to lyse early and extensively during cheese ripening. This strain is lysogenic and contains a prophage named ΦAM2. Lysis of strain AM2 and its prophage-cured derivative AM2-C in Saint-Paulin pressed-type cheese was monitored by the following parameters: cell viability, morphological changes of bacteria observed by electron microscopy and release of intracytoplasmic peptidases. Proteolysis was quantified by measuring soluble nitrogen (SN), phosphotungstic acid soluble nitrogen (PTA-N) and free amino acids. By contrast to the wild type strain AM2 which lyses early and extensively, its prophage-cured derivative AM2-C lyses only slowly and to a limited extent in cheese. These results indicate that the prophage ΦAM2 is involved in the lytic behaviour of L. lactis AM2 during cheese ripening. In addition, the comparison of two isogenic strains with similar enzymatic potential but different ability to lyse demonstrates that starter strain lysis results in a higher free amino acids production rate and a decrease of bitter taste.

International audience; Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD(PHB)/gCOD(particulate)) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 165 rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.

International audience; h i g h l i g h t s " Four biofilters were exposed to repeated substrate pulses of increasing intensity. " New quantitative indices of resistance and resilience capacity were proposed. " We highlighted different robustness levels according to the contaminants. " There was an impact of the perturbation regime at the community level (DGGE). " Bacterial community structure partially coupled to removal efficiency robustness. a b s t r a c t Since full-scale biofilters are often operated under fluctuating conditions, it is critical to understand their response to transient states. Four pilot-scale biofilters treating a composting gas mixture and undergoing repeated substrate pulses of increasing intensity were studied. A systematic approach was proposed to quantify the resistance and resilience capacity of their removal efficiency, which enabled to distinguish between recalcitrant (ammonia, DMDS, ketones) and easily degradable (esters and aldehyde) compounds. The threshold of disturbing shock intensity and the influence of disturbance history depended on the contaminant considered. The spatial and temporal distribution of the bacterial community structure in response to the perturbation regime was analysed by Denaturing Gradient Gel Electrophoresis (DGGE). Even if the substrate-pulses acted as a driving force for some community characteristics (community stratification), the structure-function relationships were trickier to evidence: the distributions of resistance and composition were only partially coupled, with contradictory results depending on the contaminant considered.

International audience; The spatial and temporal dynamics of microbial community structure and function were surveyed in duplicated woodchip-biofilters operated under constant conditions for 231 days. The contaminated gaseous stream for treatment was representative of composting emissions, included ammonia, dimethyl disulfide and a mixture of five oxygenated volatile organic compounds. The community structure and diversity were investigated by denaturing gradient gel electrophoresis on 16S rRNA gene fragments. During the first 42 days, microbial acclimatization revealed the influence of operating conditions and contaminant loading on the biofiltration community structure and diversity, as well as the limited impact of inoculum compared to the greater persistence of the endogenous woodchip community. During long-term operation, a high and stable removal efficiency was maintained despite a highly dynamic microbial community, suggesting the probable functional redundancy of the community. Most of the contaminant removal occurred in the first compartment, near the gas inlet, where the microbial diversity was the highest. The stratification of the microbial structures along the filter bed was statistically correlated to the longitudinal distribution of environmental conditions (selective pressure imposed by contaminant concentrations) and function (contaminant elimination capacity), highlighting the central role of the bacterial community. The reproducibility of microbial succession in replicates suggests that the community changes were presumably driven by a deterministic process.

International audience; A synthetic contaminated gas was generated, representative of gaseous emissions from sludge composting. It was composed of six volatile organic compounds (aldehyde, ketones, esters, sulphur compound) in an ammoniacal matrix. The gaseous stream was purified by biofiltration, in pilot scale biofilters filled with pine bark woodchips as organic carrier for biomass colonization. After reaching a constant high efficiency, with complete removal, the system was disturbed by transient loading shocks. The impact of perturbations was assessed by both performance evaluation (i.e. contaminant removal) and microbial behaviour. The microbial community was analysed in terms of density. The resilience of functional component following a perturbation was evaluated. This work highlighted the longitudinal distribution of both biodegradation activities and biomass density.

A molecularly imprinted polymer (MIP) was synthesized with 17 beta-estradiol (E2) as template. It was then capable to recover this compound by 100 +/- 0.6% from a 2 mu g/L aqueous solution. By comparison, E2 recoveries of 77 +/- 5.2%, 87.1 +/- 2.3% and 19.1 +/- 7.8%, were achieved using a non-imprinted polymer (NIP) synthesized under the same conditions (but without template), a commercial C18 extraction phase and granular-activated carbon (GAC), respectively When fluoxetine hydrochloride and acenaphthene were added as interferences to the aqueous solution at 2 mu g/L each, E2 was recovered by 95.5 +/- 4.0% from the MIP, compared to 54.5 +/- 9.4%, 76.0 +/- 2% and 14.3 +/- 0.1% from the NIP, C18 and GAC phases, respectively Estrogenic activity equivalent to the effect caused by 22.4ng E2/L was recorded in the MIP extract from a wastewater sample whereas no activity was detected in the NIP extract. This suggested the imprinted polymers removed estrogenic compounds. This study therefore demonstrates the potential of MIPs for the selective removal of endocrine-disrupting compounds. By using a synthetic analogue to natural hormone receptors, adsorption is based on the same property that makes the contaminants harmful. Biological treatment of enriched E2 was also demonstrated. (c) 2007 Elsevier Ltd. All rights reserved. (Less)

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251–269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554–1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.