[Show abstract][Hide abstract] ABSTRACT: Background:
Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg(-1), respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.
The quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 μM PMA, 5 min incubation and 56 min photoactivation for manure and 20 μM PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.
PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.
[Show abstract][Hide abstract] ABSTRACT: Anaerobic digestion is one way to manage organic wastes by converting wastes from agriculture, industries, wastewater treatment and other human processes into energy and fertilizers (i.e. digestates). The use of digestate as fertilizer is confronted with the problematic of health risks associated with the presence of pathogenic microorganisms. Pathogens from human and animal origin present in wastes can contaminate the digestate because of insufficient elimination of pathogens during anaerobic digestion and regrowth of surviving pathogens. Consequently, the valorisation of digestate is difficult, particularly regarding land spreading. Little is known on the survival of pathogens during storage and land spreading, especially with respect to biotic and abiotic factors determining the decay of pathogens. This study aims at investigating the factors that influence the survival of pathogens during the storage of digestates to improve the anaerobic digestion process and the agricultural valorisation of digestate. Digestates from agricultural wastes were analysed. The digestates originated from different digestion processes and are representative for the French anaerobic digestion sector. The digestates were inoculated with three pathogenic bacteria, which are among the most frequently reported in agricultural wastes: Salmonella enterica, Listeria monocytogenes and Campylobacter coli. The inoculated digestates were monitored in microcosms for 40 days under conditions simulating the storage (at 24°C, without shaking and in anaerobic conditions). The pathogens and E. coli, commonly used as indicator, were monitored using culture methods and quantitative PCR. The results showed a strong decay of C. coli (not detected anymore after 7 days) and S. enterica (survived up to 20 days) regardless the microcosm. In contrast, L. monocytogenes had not the same behaviour for all microcosms and was still detected after 40 days in some digestates. The survival of pathogens seemed to be different between digestates and between pathogenic strains, suggesting the important role of biotic and abiotic factors on the survival of pathogens. The survival of the studied pathogens has been analysed in regards to the bioavailability of organic matter and microbial activity and diversity. These results will permit to characterise the properties of digestates leading to favourable conditions for the reduction of the health risks associated with pathogens.
[Show abstract][Hide abstract] ABSTRACT: A number of prokaryotes actively contribute to lignin degradation in nature and their activity could be of interest for many applications including the production of biogas/biofuel from lignocellulosic biomass and biopulping. This review compares the reliability and efficiency of the culture-dependent screening methods currently used for the isolation of ligninolytic prokaryotes. Isolated prokaryotes exhibiting lignin-degrading potential are presented according to their phylogenetic groups. With the development of bioinformatics, culture-independent techniques are emerging that allow larger-scale data mining for ligninolytic prokaryotic functions but today, these techniques still have some limits. In this work, two phylogenetic affiliations of isolated prokaryotes exhibiting ligninolytic potential and laccase-encoding prokaryotes were determined on the basis of 16S rDNA sequences, providing a comparative view of results obtained by the two types of screening techniques. The combination of laboratory culture and bioinformatics approaches is a promising way to explore lignin-degrading prokaryotes.
[Show abstract][Hide abstract] ABSTRACT: The cultivation of microalgae with digestate supernatant is a promising process for the recovery of mineralized nutrients (P, N) from anaerobic digestion. Nevertheless, the variability of phosphorus concentration in the influent could limit this process. The impact of initial N:P ratios between 3 and 76 gN.gP-1 was studied and proved no growth limitation over 14-day batch experiments even when P was depleted. Nitrogen assimilation was not affected by phosphorus concentrations and reached 10.1 mgN.L-1.d-1 whereas phosphorus removal ranged from 0.6 to 2.0 mgP.L-1.d-1. The biomass N:P ratio was found to be a function of the influent N:P ratio. Phosphorus storage by microalgae was thus confirmed. Nitrification was found to be highly dependent on the initial phosphorus concentration. The evolution of microalgae communities was also monitored and revealed the advantage of Scenedesmus over Chlorella when the media was phosphorus-depleted.
[Show abstract][Hide abstract] ABSTRACT: During anaerobic digestion, nutrients are mineralized and may require post-treatment for optimum valorization. The cultivation of autotrophic microalgae using the digestate supernatant is a promising solution; however the dark color of the influent poses a serious problem. First, the color of the digestates was studied and the results obtained using three different digestates demonstrated a strong heterogeneity although their color remained rather constant over time. The digestates absorbed light over the whole visible spectrum and remained colored even after a ten-fold dilution. Secondly, the impact of light and of substrate color on the growth of Scenedesmus sp. and on nitrogen removal were assessed. These experiments led to the construction of a model for predicting the impact of influent color and light intensity on N removal. Maximum N removal (8.5 mgN-NH4+.L-1.d-1) was observed with an initial optical density of 0.221 and 244 μmolE.m-².s-1 light and the model allows to determine N removal between 15.9 and 22.7 mgN-NH4+.L-1.d-1 in real conditions according to the dilution level of the influent and related color. Changes in the microalgae community were monitored and revealed the advantage of Chlorella over Scenedesmus under light-limitation. Additionally microalgae outcompeted nitrifying bacteria and experiments showed how microalgae become better competitors for nutrients when phosphorus is limiting. Furthermore, nitrification was limited by microalgae growth, even when P was not limiting.
Water Research 07/2014; 64:278-287. DOI:10.1016/j.watres.2014.07.012 · 5.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intensification of pig farming in Brittany has led to an increase flow of pollutants that can contaminate water. Effluent from farms on grates which represent about 90% of pig farms, cause chemical (nutrients, metals) and microbiological (pathogens) contamination. The transfer of nitrogen and phosphorus contribute to the eutrophication of surface waters. To apply the European directives on water quality, like the directive “nitrate”, a number of institutional and regulatory arrangements had been put in place to fight against the pollution of agricultural origin. Thus, it is possible to reduce nitrogen and phosphorus by adapting the feed to their physiological state. In areas with structural surpluses ofmanure treatment systems have been developed. It is more difficult to reduce concentrations of micro-organisms; they are able to survive through treatment. Treatments such as composting can significantly reduce the health risk.
L’intensification des élevages porcins en Bretagne a engendré un accroissement des flux polluants susceptibles de contaminer les eaux. Les effluents produits par les élevages sur caillebotis qui représentent environ 90% des élevages de porcs, engendrent des contaminations chimiques (nutriments, éléments métalliques) et microbiologiques (germes pathogènes). Le transfert de l’azote et du phosphore contribuent ainsi à l’eutrophisation des eaux de surface. Relevant de directives européennes, des dispositifs d’ordre réglementaire ont été mis en place pour lutter contre la pollution d’origine agricole, à l’exemple de la directive “nitrate”. De nombreuses études ont été réalisées pour limiter les teneurs en polluants présents dans le lisier. Ainsi, il est possible de réduire l’azote et le phosphore en adaptant l’alimentation des animaux à leur stade physiologique. Dans les zones d’excédents structurels, des filières de traitement des lisiers ont été développées. IL est cependant plus difficile de réduire les concentrations en micro)organismes pathogènes, ceux-ci étant capables de survivre au travers des traitements. Seuls les traitements thermiques tels que le compostage permettent de diminuer significativement le risque sanitaire.
Eau, milieux et aménagement, Une recherche au service des territoires, Edited by Aziz Ballouche, Aude Nuscia Taïbi, 03/2014: chapter Solutions mises en œuvre pour limiter la contamination des eaux par l’élevage porcin en Bretagne: pages p 135-148; Presses de l’Université d’Angers.
[Show abstract][Hide abstract] ABSTRACT: This study evaluated the impact of storing chicken manure on the degradation of enrofloxacin (ENR) and ciprofloxacin (CIP), and on the survival of CIP-resistant Enterobacteriaceae. At 24 d of age, half of 8900 chickens received ENR for 5 d. After the animals departed, their manure was stored in two heaps for 63 d. Enterobacteriaceae were cultured on media containing 0 to 32 mg L⁻¹ of CIP. A total of 320 isolates were fingerprinted using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) to evaluate community structure. Initial concentrations of ENR and CIP in the heap were 22 and 1.8 mg kg⁻¹, respectively. Seventy-three percent of the two fluoroquinolones were eliminated during storage. The administration of ENR led to a 5.1 log₋₁₀ decrease in Enterobacteriaceae concentrations and emergence of CIP-resistant bacteria, which became dominant in the feces. concentrations decreased 1.2 to 2.3 log₋₁₀ 2 d after the heaps were made and continued to decline during storage. No resistant were found by Day 63. The highest CIP minimum inhibitory concentration (MIC) values observed among isolates of and of both and sp. were 128 and 4 mg L⁻¹, respectively. The dominant ERIC-PCR profiles changed over time. There was no relationship between genotype and resistance-isolated strains to CIP. Storing chicken manure in heaps appeared to be an effective way of limiting the entrance of CIP-resistant E. coli into the environment but did not prevent the dissemination of fluoroquinolones after land spreading.
[Show abstract][Hide abstract] ABSTRACT: La pollution organique issue des effluents d'élevage et des stations d'épuration urbaines conduit à un problème essentiel de santé publique lié à la contamination des eaux de surface où s'exercent des activités sensibles telles que la baignade. S'il est possible de déterminer les pollutions localisées liées à un dysfonctionnement des systèmes de traitement, il est beaucoup plus difficile d'identifier les pollutions organiques diffuses qui participent pourtant majoritairement à la dégradation de la qualité des eaux de surface. La problématique des pollutions diffuses est d'autant plus importante que la nouvelle réglementation européenne concernant les eaux de baignade (Directive 2006/7/CE) demande de constituer des profils de baignade qui nécessitent une identification et une hiérarchisation des sources de pollutions fécales. Le dénombrement de Escherichia coli et des entérocoques intestinaux stipulé par les textes réglementaires européens, représente actuellement le seul outil analytique permettant la mise en évidence d'une contamination fécale du milieu aquatique, sans toutefois différencier l'origine humaine ou animale de cette contamination. Il est donc nécessaire de développer de nouvelles méthodes de détection de la pollution fécale qui puissent non seulement mettre en évidence une contamination mais aussi en indiquer l'origine. C'est d'ailleurs dans cet objectif que s'est développé depuis quelques années, le concept de "Microbial Source Tracking" ("Traceurs de Sources Microbiennes") qui consiste à identifier à l'aide de marqueurs microbiologiques ou chimiques les sources de pollutions fécales. Dans ce contexte, six laboratoires de recherche se sont associés pour développer des techniques de traçage des contaminations fécales afin de proposer un outil opérationnel utilisable pour différencier les sources de pollution, de leur point d'émission jusqu'au milieu récepteur final que constituent les eaux de surface. Les marqueurs qui ont fait l'objet de cette étude sont des molécules chimiques naturelles (stéroïdes, caféine), des molécules de synthèse retrouvées dans les effluents de stations d'épuration ou des rapports de fluorescence de la matière organique ainsi que des micro-organismes (bactériophages, bactéries). A la suite des développements méthodologiques, plusieurs marqueurs ont été sélectionnés : - bactéries appartenant aux groupes bactériens dominants du tractus intestinal humain (Bifidobacterium adolescentis) et porcin (Lactobacillus amylovorus) ; - Bacteroidales spécifiques des humains, porcins et bovins (HF183, Pig-2-Bac, Rum-2-Bac); - génogroupes humains des bactériophages F ARN spécifiques; - rapports de stéroïdes : coprostanol/(24ethylcoprostanol+coprostanol) (R1) et sitostanol/coprostanol (R2); - caféine, benzophénone et tri(2-chloroethyl)phosphate (TCEP).
[Show abstract][Hide abstract] ABSTRACT: A wastewater treatment system including a screen, a vermifilter, macrophytes ponds, and constructed wetlands has been built after a pig housing on slatted-floor. The aims were, all at once, to recycle water for excretion washing and to produce, from the nutrients contained in the effluent, organic matter and plants that can be either sold or reused on the farm to reduce inputs. Analyses, made on the effluent at different steps of the treatment plant, show that the concentrations of the nitrogen, microorganisms and endocrine disruptors are drastically reduced, while the phosphorus and potassium removal go through the byproducts harvesting.
[Show abstract][Hide abstract] ABSTRACT: Fecal contaminations of inland and coastal waters induce risks to human
health and economic losses. In order to improve water management, it is
necessary to identify the sources of contamination, which implies the
development of specific markers. In order to be considered as a valuable
host-specific marker, one must (1) be source specific, (2) occur in high
concentration in polluting matrices, (3) exhibit extra-intestinal
persistence similar to fecal indicator bacteria (FIB) and (4) not grow
out of the host. However, up to day no single marker has fulfilled all
those criteria. Thus, it has been suggested to use a combination of
markers in order to generate more reliable data. This has lead to the
development of a Microbial Source Tracking (MST) toolbox including FIB
and microbial and chemical specific markers in order to differentiate
between human, bovine and porcine fecal contaminations. Those specific
markers are, (1) genotypes of F-specific RNA bacteriophages, (2)
bacterial markers belonging to the Bacteroidales (human-specific HF183,
ruminant-specific Rum-2-Bac and pig-specific Pig-2-Bac markers), to the
Bifidobacterium (Bifidobacterium adolescentis) and pig-specific
Lactobacillus amylovorus, (3) fecal stanols and (4) caffeine. The
development of this MST toolbox was composed of four steps, from the
molecular scale to the watershed scale. At the molecular scale, the
specificity and the concentration of those markers were studied in
cattle and pig manures and in waste water treatment plant (WWTP)
effluents and influents. At the microcosm scale, the transfer of bovine
and porcine specific markers was investigated by rainfall simulations on
agricultural plots amended with cattle or pig manure. Moreover, the
relative persistence of FIB and human, porcine and bovine specific
markers was investigated in freshwater and seawater microcosms
inoculated with a WWTP influent, pig manure and cow manure. Finally, the
aforementioned MST toolbox has been validated at the catchment scale by
analysing three rivers impacted by fecal contaminations. The development
and the application of this MST toolbox have highlighted (1) the
specificity of the aforementioned markers, (2) their conservative
transfer from soils to rivers and (3) their difference of persistence in
seawater and in freshwater. Those results provide useful data in order
to identify and manage fecal contaminations of superficial waters. In
the case of single source contaminations, the markers provide coherent
information: (1) the bovine or porcine markers were not detected in a
river impacted by a WWTP effluent; (2) the occurrence of Rum-2-Bac and
the distribution of stanols indicated a bovine contamination in a river
flowing through cattle pasture. In the case of multiple source
contaminations, the combination of markers is necessary to identify the
main sources and the statistical treatment of the distribution of
stanols could provide an approximation of their proportion.
[Show abstract][Hide abstract] ABSTRACT: Fecal contamination of water resources is evaluated by the enumeration of the fecal coliforms and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and ), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure.
[Show abstract][Hide abstract] ABSTRACT: Since 2001 the swine experimental station of Guernévez has studied biological treatment plants for nutrient recovery and water recycling, suited to the fresh liquid manure coming out of flushing systems. An integrated system with continuous recycling was set up in 2007, associated with a piggery of 30 pregnant sows. It includes a screen, a vermifilter, and macrophyte ponds alternating with constructed wetlands. The screen and the vermifilter had a lower removal efficiency than in previous studies on finishing pigs. A settling tank was then added between the vermifilter and the first lagoon to collect the worm casts. A second vermifilter was added to recover this particulate organic matter. A storage lagoon was added to compensate for evaporative losses and complete pollution abatement, with goldfish as a bioindicator of water quality. The removal efficiency of the whole system was over 90% for COD and nitrogen, over 70% for phosphorus and potassium, and more than 4 logarithmic units for pathogens (E. coli, enterococci, C perfringens). Plant production was about 20 T DM ha(-1) y(-1). Floating macrophytes (Azolla caroliniana, Eichhornia crassipes, Hydrocotyle vulgaris) were more concentrated in nutrients than helophytes (Phragmites australis, Glyceria aquatica,…). Azolla caroliniana was successfully added to feed finishing pigs.