Development of a reliable extraction method for the recovery of total genomic DNA from woodchip colonizing biofilm involved in gas biofiltration

[Abstract] This preliminary study focused on a critical step for the characterization of microbial ecosystem involved in biofiltration. Two aspects of nucleic acid recovery were explored: (i) cell dispersion (three methods tested) and (ii) total DNA extraction (four methods tested). The objective is to select the optimal combination of desorption/extraction methods, allowing subsequent molecular investigations to be reliable. Three relevant criteria are used to assess extraction efficiency: DNA amount and purity, and subsequent amplification feasibility

Selective removal of 17 beta-estradiol at trace concentration using a molecularly imprinted polymer

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

Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting

International audienceIn 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

Bacterial dynamics in steady-state biofilters: beyond functional stability

International audienceThe 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

Resistance and resilience of removal efficiency and bacterial community structure of gas biofilters exposed to repeated shock loads

International audienceh 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

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture

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

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture

International audiencePolyhydroxybutyrate (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