Maialen Barret

PAH fate during the anaerobic digestion of contaminated sludge: Do bioavailability and/or cometabolism limit their biodegradation?

The anaerobic removal of 13 Polycyclic Aromatic Hydrocarbons (PAHs) was measured in five continuous anaerobic digestors with different feed sludge, in which abiotic losses were neglected. These feeds were chosen to generate different levels of PAH bioavailability and cometabolism within the reactors. Based on the accurate modelling of PAH sorption in sludge, the aqueous fraction (including free and sorbed-to-dissolved-and-colloidal-matter PAHs) was demonstrated to be bioavailable, which validated a widespread assumption about micropollutants bioavailability in sludge. It was also demonstrated that bioavailability is not the only influencing factor. Indeed, PAHs biodegradation resulted from a combination of bioavailability and cometabolism. An equation adapted from Criddle (1993, The Kinetics of Cometabolism. Biotechnology and Bioengineering 41, 1048-1056) that takes into account both mechanisms was shown to fit the experimental data, with dry matter removal rate identified as the criteria for cometabolism. The existence of a threshold of dry matter cometabolism was suggested, below which PAHs removal would not be possible. The parameters of the Criddle equation were demonstrated to depend on PAH molecular structure, and the results suggest that they would also be influenced by substrate composition and microbial population. This research provided original outcomes for the assessment of micropollutants fate. Indeed, the understanding of the driving mechanisms was improved, which has implications for the optimization of micropollutants removal.

Micropollutant and sludge characterization for modeling sorption equilibria.

The sorption of hydrophobic micropollutants in sludge is one of the major mechanisms which drive their fate within wastewater treatment systems. The objective of this study was to investigate the influence of both sludge and micropollutant characteristics on the equilibria of sorption to particles and to dissolved and colloidal matter (DCM). For this purpose, the equilibrium constants were measured for 13 polycyclic aromatic hydrocarbons, 5 polychlorobiphenyls and the nonylphenol, and five different sludge types encountered in treatment systems: a primary sludge, a secondary sludge, the same secondary sludge after thermal treatment, after anaerobic digestion, and after both treatments. After thermal treatment, no more sorption to DCM was observed. Anaerobic biological treatment was shown to enhance micropollutants sorption to particles and to DCM of one logarithmic unit, due to matter transformation. Partial least-squares linear regressions of sorption data as a function of micropollutant and sludge properties revealed that sludge physical and chemical characteristics were more influential than micropollutant characteristics. Two models were provided to predict the sorption of such micropollutants in any sludge. To our knowledge, this is the first time that a three-compartment approach is used to accurately model micropollutant sorption in sludge and to understand the driving mechanisms.

A three-compartment model for micropollutants sorption in sludge: Methodological approach and insights

In sludge resulting from wastewater treatment, organic micropollutants sorb to particles and to dissolved/colloidal matter (DCM). Both interactions may influence their physical and biological fate throughout the wastewater treatment processes. To our knowledge, sludge has never been considered as a three-compartment matrix, in which micropollutants coexist in three states: freely dissolved, sorbed-to-particles and sorbed-to-DCM. A methodology is proposed to concomitantly determine equilibrium constants of sorption to particles (K(part)) and to DCM (K(DCM)). Polycyclic Aromatic Hydrocarbons (PAHs) were chosen as model compounds for the experiments. The logarithm of estimated equilibrium constants ranged from 3.1 to 4.3 and their usual correlation to PAH hydrophobicity was verified. Moreover, PAH affinities for particles and for DCM could be compared. Affinity for particles was found to be stronger, probably due to their physical and chemical characteristics. This work provided a useful tool to assess the freely dissolved, sorbed-to-particles and sorbed-to-DCM concentrations of contaminants, which are necessary to accurately predict their fate. Besides, guidelines to investigate the link between sorption and the fundamental concept of bioavailability were proposed.

Methanoculleus spp. as a biomarker of methanogenic activity in swine manure storage tanks

Greenhouse gas emissions represent a major problem associated with manure management in the livestock industry. A prerequisite to mitigate methane emissions occurring during manure storage is a clearer understanding of how the microbial consortia involved in methanogenesis function. Here, we have examined manure stored in outdoor tanks from two different farms, at different locations and depths. Physico-chemical and microbiological characterization of these samples indicated differences between each tank, as well as differences within each tank dependent on the depth of sampling. The dynamics of both the bacterial and archaeal communities within these samples were monitored over a 150-day period of anaerobic incubation to identify and track emerging microorganisms, which may be temporally important in the methanogenesis process. Analyses based on DNA fingerprinting of microbial communities identified trends common among all samples as well as trends specific to certain samples. All archaeal communities became enriched with Methanoculleus spp. over time, indicating that the hydrogenotrophic pathway of methanogenesis predominated. Although the emerging species differed in samples obtained from shallow depths compared to deep samples, the temporal enrichment of Methanoculleus suggests that this genus may represent a relevant indicator of methanogenic activity in swine manure storage tanks.

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

ABSTRACT Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-13C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of 13C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the 13C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Influence of feed characteristics on the removal of micropollutants during the anaerobic digestion of contaminated sludge.

The removal of 13 polycyclic aromatic hydrocarbons, 7 polychlorobiphenyls and nonylphenol was measured during the continuous anaerobic digestion of five different sludge samples. The reactors were fed with one of the following: primary/secondary sludge (PS/SS), thermally treated PS, cellulose-added SS, or SS augmented with dissolved and colloidal matter (DCM). These various feeding conditions induced variable levels of micropollutant bioavailability (assumed to limit their biodegradation) and overall metabolism (supposed to be linked to micropollutant metabolism throughout co-metabolism). On the one hand, overall metabolism was higher with secondary sludge than with primary and the same was observed for micropollutant removal. However, when overall metabolism was enhanced thanks to cellulose addition, a negative influence on micropollutant removal was observed. This suggests that either the co-metabolic synergy would be linked to a specific metabolism or co-metabolism was not the limiting factor in this case. On the other hand, micropollutant bioavailability was presumably diminished by thermal treatment and increased by DCM addition. In both cases, micropollutant removal was reduced. These results suggest that neither overall metabolism nor bioavailability would absolutely limit micropollutant removal. Each phenomenon might alternatively predominate depending on the feed characteristics.

A novel fingerprint method to assess the diversity of methanogens in microbial systems

Understanding the ecology of methanogens in natural and engineered environments is a prerequisite to predicting or managing methane emissions. In this study, a novel high-throughput fingerprint method was developed for determining methanogen diversity and relative abundance within environmental samples. The method described here, designated amplicon length heterogeneity PCR of the mcrA gene (LH-mcrA), is based on the natural length variation in the mcrA gene. The mcrA gene encodes the alpha-subunit of the methyl-coenzyme M reductase, which is involved in the terminal step of methane production by methanogens. The methanogenic communities from stored swine and dairy manures were distinct from each other. To validate the method, methanogenic communities in a plug flow-type bioreactor (PFBR) treating swine manure were characterized using LH-mcrA method and correlated to mcrA gene clone libraries. The diversity and relative abundance of the methanogenic groups were assessed. Methanobrevibacter, Methanosarcinaceae, Methanoculleus, Methanogenium, Methanocorpusculum and one unidentified group were assigned to particular LH-mcrA amplicons. Particular phylotypes related to Methanoculleus were predominant in the last compartment of the PFBR where the bulk of methane was produced. LH-mcrA method was found to be a reliable, fast and cost-effective alternative for diversity assessment of methanogenic communities in microbial systems.

Physico-chemical characteristics and methanogen communities in swine and dairy manure storage tanks: spatio-temporal variations and impact on methanogenic activity.

Greenhouse gas emissions represent a major environmental problem associated with the management of manure from the livestock industry. Methane is the primary GHG emitted during manure outdoor storage. In this paper, the variability of two swine and two dairy manure storage tanks was surveyed, in terms of physico-chemical and microbiological parameters. The impact of the inter-tank and spatio-temporal variations of these parameters on the methanogenic activity of manure was ascertained. A Partial Least Square regression was carried out, which demonstrated that physico-chemical as well as microbiological parameters had a major influence on the methanogenic activity. Among the 19 parameters included in the regression, the concentrations of VFAs had the strongest negative influence on the methane emission rate of manure, resulting from their well-known inhibitory effect. The relative abundance of two amplicons in archaeal fingerprints was found to positively influence the methanogenic activity, suggesting that Methanoculleus spp. and possibly Methanosarcina spp. are major contributors to methanogenesis in storage tanks. This work gave insights into the mechanisms, which drive methanogenesis in swine and dairy manure storage tanks.

Anaerobic Removal of Trace Organic Contaminants in Sewage Sludge: 15 Years of Experience

Trace organic contaminants (TOCs) correspond to a broad range of molecules generated either directly or indirectly by human activity. Even though TOCs are found at low concentrations in the environment, they often accumulate by biomagnification and bioaccumulation into biological organisms and cause irreversible damages in biological systems through direct or indirect toxic effects such as endocrine disruption and tumour initiation. This manuscript presents the main findings of over fifteen years of research focusing on biological removal of various TOCs found in sewage sludge from urban treatment plants. A special focus of the research was made on microbial processes in complex anaerobic ecosystems. Four families of compounds mostly retrieved in urban plants were studied: the polycyclic aromatic hydrocarbons (PAHs), the polychlorobiphenyls (PCBs), the phthalic acid esters (PAEs), and the nonylphenol ethoxylates (NPEs). It was observed that the microbial capability for removing low amounts of TOCs required a long adaptation time and was often limited by the bioavailability of these compounds. In fact, the overall biodegradation resulted from the numerous interactions existing between the matrix (organic matter) and the microbial ecosystems according to the physico-chemical sorption properties of these compounds. Mechanistic aspects were also tackled in depth and specific models were developed for better understanding the network of interactions between TOCs, microorganisms, and organic matter. These findings could be extrapolated to other ecosystems such as soils and sediments. Finally, it was shown that microbial cometabolism was essential for TOC removal, and the concept of bioavailability was not only dependent on the nature, the level, and the sorption properties of TOCs but was also strongly dependent on the nature and the concentration of the sludge organic matter. Specific parameters were proposed for better evaluating the fate of TOCs in microbial anaerobic processes and technological solutions for efficient removal of these compounds were also proposed.

Phylogenetic identification of methanogens assimilating acetate-derived carbon in dairy and swine manures.

In order to develop approaches for reducing the carbon footprint of the swine and dairy industries, it is important first to identify the methanogenic communities that drive methane emissions from stored manure. In this study, the metabolically active methanogens in substrate-starved manure samples taken from two dairy and one swine manure storage tanks were identified using [(13)C]-acetate and DNA stable-isotope probing (DNA-SIP). Molecular analysis of recovered genomic [(13)C]-DNA revealed that two distinct clusters of unclassified methanogen populations affiliated with the Methanoculleus genus, and the populations affiliated with Methanoculleus chikugoensis assimilated acetate-derived carbon (acetate-C) in swine and dairy starved manure samples, respectively. Furthermore, carbon flow calculations indicated that these populations were the primary contributors to methane emissions during these anoxic SIP incubations. Comparative analysis of mcrA gene abundance (coding for a key enzyme of methanogenesis) for Methanoculleus spp. in fresh feces and a wider range of stored dairy or swine manure samples, by real-time quantitative PCR using newly designed specific primers, demonstrated that the abundance of this genus significantly increased during storage. The findings supported the involvement of these particular methanogen populations as methane emitters from swine and dairy manure storage tanks. The study revealed that the ability to assimilate acetate-C for growth in manure differed within the Methanoculleus genus.