Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics. We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are ‘young’ and larger ones are ‘old’. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions. Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes

Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics. We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are ‘young’ and larger ones are ‘old’. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions. Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes

Caractérisation de la fraction organique et minérale de la matrice extracellulaire issue de boues biologiques

Les bioréacteurs permettent d effectuer un traitement biologique des eaux usées grâce aux boues biologiques. Les boues sont des agrégats formés par des microorganismes inclus dans une matrice extracellulaire (MEC). La MEC, formée d une fraction organique et minérale, est considérée comme la maison du biofilm. L étude de la MEC nécessite son extraction des boues. Nous avons analysé l impact de la méthode d extraction sur les PEC (polymères extracellulaires) issues de différentes boues biologiques par chromatographie d exclusion stérique (HPSEC) couplée à une détection UV. L impact de la méthode d extraction sur la fraction minérale des MEC issues de boues activées a aussi été mis en évidence. La microscopie électronique à balayage a montré la présence d une fraction minérale particulaire. Nous avons ensuite caractérisé la fraction protéique et substances humiques like des PEC par HPSEC couplée la fluorimètrie. Cette méthode départage les protéines-like (large gamme de masse moléculaire (MM)) des substances humiques-like (MM<6kDa). Nous avons caractérisé spécifiquement la fraction protéique des PEC par électrophorèse dénaturante (SDS-PAGE) couplée à des colorations. La coloration PAS (acide périodique et réactif de Schiff) a permis de montrer la présence de glycoprotéines (MM de 100kDa). Pour les PEC issus de boues granulaires anaérobies, la coloration au bleu alcian pH 2.5 et pH 1 a montré la présence de protéoglycane et de proéoglycane sulfaté. La présence de ces hétéroprotéines a été confirmée par une immunodétection des motifs glucidiques (via des lectines).Bioreactors allow making a wastewater biological treatment with biological sludge. Sludges are aggregates formed with microorganisms embbeded in an extracellular matrix (ECM). ECM which contains an organic and mineral fraction, is considered as the "house" of the biofilm. The study of ECM requires its extraction from sludge. We analyzed the impact of the extraction method on the PEC (extracellular polymers) from various biological sludge by steric exclusion chromatography (HPSEC) coupled with an UV detection. The impact of the extraction method on the mineral fraction of ECM from activated sludges was showed too. The scanning electronic microscopy showed the presence of a solid mineral fraction. We then characterized the protein fraction and the humic-like substances of the PEC by HPSEC coupled with fluorimetry. This method decides between proteins-like (wide range of molecular mass (MM)) and substances humiques-like (MM< 6kDa). We characterized specifically the protein fraction of the PEC with electrophoresis (SDS-PAGE) coupled with various stains. The PAS stain (periodic acid and Schiff reagent) allowed to show the presence of glycoproteins (MM : 100kDa). For the PEC from anaerobic granular sludge, the alcian blue staining at pH 2.5 and pH 1 showed the presence of proteoglycan and sulphated proeoglycane respectively. The presence of these heteroprotein was confirmed by an immunodetection test on the glucidic motives (using lectins).LIMOGES-BU Sciences (870852109) / SudocSudocFranceTogoFRT

Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods

International audienceThis work characterises the mineral fraction of EPS extracts obtained using eight different methods from two activated sludges by total mineral content determination, Fourier Transformed Infrared spectrometry and with scanning electron microscopy coupled with an EDX probe. Despite EPS dialysis, the EPS extracts displayed a mineral fraction between 2% and 40% of the EPS dry weight depending on the extraction method used. The main mineral elements found in the EPS extract were Ca, Mg, Na, K, Al, Fe, Mn, P, Si and S, but their contents were strongly affected by the extraction method used. Some of the minerals are associated with the organic molecules within the EPS. The presence of mineral particles of various compositions and structures (clays, quartz or carbonate) in the EPS extract with a wide range in size was clearly demonstrated. Moreover, the association of metallic elements with the mineral particles in the EPS extract was highlighted

Influence of extraction method on size exclusion chromatography fingerprints of EPS from wastewater sludges

International audienc

Qualitative characterization of the protein fraction of exopolymeric substances (EPS) extracted with EDTA from sludge.

International audienceExopolymeric substances (EPS) were extracted by EDTA from activated and anaerobic granular sludge. Due to the presence of EDTA in EPS extract, interferences were pointed out for the characterization of EPS by means of the colorimetric methods and fluorescence spectroscopy. Other methods have been investigated to characterize the EPS protein fraction. Size exclusion chromatography (SEC), performed at a fluorescence excitation-emission matrix of 221/360 nm (tryptophan protein-like substances) for detection, was suitable and allowed obtaining a fingerprint of the protein-like substance fractions and determining apparent molecular weight (MW). Polyacrylamide gel electrophoresis (PAGE) was performed under either native or denaturing conditions. Various staining applications after EPS migration are effective in obtaining a protein (silver staining) or glycoprotein (PAS staining) fingerprint or MW distribution. SEC and PAGE are both appropriate techniques for the qualitative characterization of protein fractions from EPS extracted by EDTA and moreover differentiate EPS according to sludge origin and type

The use of a new mobile phase, with no multivalent cation binding properties, to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment

International audienceThe fingerprints of extracellular polymeric substances (EPS) extracted from different types of biomass used for wastewater treatment (i.e., activated sludge, filamentous activated sludge, anaerobic granular sludge, anaerobic flocculated sludge) were studied by size exclusion chromatography (SEC) with Amersham Biosciences Superdex 200 10/300 GL column with a theoretical resolving range of 10-600 kDa. A new mobile phase, which does not display binding properties for multivalent cations, was previously optimized. This mobile phase contained 75 mM Hepes buffer at pH 7 with 15% acetonitrile (v/v) and was selected to minimize ionic and hydrophobic interactions between the molecules that make up the EPS and the column packing. When EPS extracted from similar sludges is analyzed using different mobile phases, the number of chromatographic peaks obtained is quite similar, and differences are mainly observed in the relative absorbance of the chromatographic peaks. However, very different chromatograms (number and relative absorbance of chromatographic peaks) are obtained for EPS extracted from different types of sludges. Furthermore, when dysfunctions, such as filamentous bulking in the activated sludge, occur in a bioreactor, they also induce strong variations in chromatographic profiles

Influence of proteins and humic-like substances from soluble microbial products on membrane bioreactor fouling under normal and stress conditions

International audienceSoluble microbial products are one of the major fouling agents in membrane bioreactor (MBR). It is accepted that high molecular weights polysaccharides are the main contributors to membrane fouling but the presence in bulk solution of proteins and humic-like substances make fouling layer more complex. To better understand the role of both components in fouling establishment, they were quantified and characterized in bioreactor and permeate under various operating conditions (sludge retention time (SRT), synthetic or real wastewater (SWW or RWW), rapid variation of food to microorganisms (F/M) ratio). With SWW at hydraulic retention time (HRT) of 24 h, a larger part of proteins possessing small molecular weights (< 1 kDa) were obtained with increasing SRT from 20 to 50 d. At 50 d, these proteins present better retention (93%) and could participate in lowering gel layer porosity. MBR operating at SRT of 20 d was then preferable. At respective SRT and HRT of 50 d and 24 h with SWW, F/M ratio decrease (from 0.2 to 0.1 kg (COD).kg(MLVSS)(-1).d(-1) during 24 h) provoked implementation of a compact fouling layer which provoked a high TMP increase (0.83 kPa.h(-1)). Biodegradation of proteins involved in bio clusters structures were implied in this phenomenon

Separation and Purification Technology

Opuntia ficus indica has been identified for its bioflocculant properties in water treatment; however, its underlying mechanism and active compounds have not been clearly identified. Flocculent molecules of cactus solid material (CSM) under alkaline conditions were extracted at pH 10 and then precipitated under neutral conditions (pH 7). The precipitate was fractionated by ultrafiltration systems and analyzed using inverted phase chromatography and enzymatic treatments. This approach revealed that quercetin and starch constitute the active agents found in the fractionated parts at ≤3,000 and ≥10,000 Da, respectively. The use of quercetin or (potato) starch alone at 18 mg/L yielded 72% ± 2% and 54% ± 3% of turbidity removal, respectively. With a combination of both these components, a higher flocculation activity (84% ± 2%) could be obtained. From these experimental results, a flocculation model based on identified active constituents is being proposed in order to improve process knowledge