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. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules. These biofilms are crucial to the operational success of a range of widely applied engineered-ecosystems designed for wastewater treatment. The structure and function of granule microbiomes underpin their utility. Here, granules were separated into ten size fractions (proxies for age), hypothesizing that small granules are 'young' and larger ones are 'old'. Gradients were observed across size in terms of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and profiles of extracellular polymeric substances, suggesting ongoing development of physico-chemical characteristics as granules develop. Short-read amplicon sequencing indicated a negative relationship between granule size and community diversity. Furthermore, as size increased, the methanogenic archaea dominated the microbiome. Small granules were found to harbour a sub-group of highly specific taxa, and the identification of generalists and specialists may point to substantial resilience of the microbiome. The findings of this study indicate opportunities for precision management of wastewater treatment systems. They suggest that size is an important indicator for aggregate utility - size may, indeed, determine many of the characteristics of both the individual-granule microbiomes and the overall function of a wastewater treatment system.

Isotherms adsorptions were generated using calf thymus DNA (10–300 µg/mL), two types of clay minerals, namely kaolinite KGa-2 and montmorillonite STx-1b and two soil (Cambisols and Andosols with clearly different mineralogical features and organic matter contents). The adsorption of calf thymus DNA with or without humic acid (C = 60 µg/mL) by clay mineral was studied using a new detection method: the colorimetric method at 600 nm and is compared to the traditional method (absorption at 260 nm). The adsorption isotherms of calf thymus DNA on the clays minerals using the colorimetric method conform to the Langmuir equation. The results show that the colorimetric method is independent of organic matter contents or the type of soils. It allows a better understanding of the interactions between DNA and clay minerals. To sum up, this method is straightforward, insensitive and sufficient with a limit of detection to plot isotherms of DNA on minerals in complex matrices.

The main objective of the proposed work was to optimize an adsorbent gel from biological material with the substitution of PVA by biopolymer extracted from cactus while maintaining the mechanical and sorption properties. The porous composite was based on cactus extract (CE) and sodium alginate (SA) and the mechanical properties and adsorption capacities of these beads were then compared with those modified using polyvinyl alcohol (PVA) and calcium carbonate (CC), respectively. Twenty formulations, based on CE, SA, PVA and CC, have made it possible to prepare, in accordance with an experimental design conducted in Mode 6, four major classes of beads. The three responses used to assess the evolution in bead performance were: the breaking point of the beads, bead rigidity based on a rheological study and the adsorption capacity of pentachlorophenol (PCP). The analysis of these responses showed that CE exerted a positive effect on both the mechanical strength (400-2200 Pa) and rigidity (5%-11%) of the beads, similar to that of PVA with the conservation of bead adsorption capacity (80-120 mg g(-1) of dry matter). The beads, based on CE and SA, revealed their regular shape with a porous structure and good chemical and physical stability. The FTIR spectral analysis suggested that the interactions between SA and CE take place via hydrogen interactions and/or covalent bonds between the hydroxyl and carboxyl groups.

Extracellular polymeric substances (EPS) produced by microorganisms have a key role in the sedimentary compartment, e.g. promoting aggregation and biostabilisation of sediment particles and increasing chemical reactivity at the water/sediment interface. Therefore, proper extraction methods are needed to study this EPS matrix. In this work, nine extraction methods based on physical (centrifugation, sonication), chemical (sodium hydroxide, sodium pyrophosphate, sodium tetraborate), and both chemical and physical (cation exchange resins, i.e. CER) treatments and their combinations, as well as the solid:liquid ratio used for extraction, were compared based on the quantity and compositions of extracted EPS. The organic carbon extracted was quantified and the nature of biochemical macromolecules (proteins, polysaccharides, and humic-like compounds) was evaluated using colorimetric methods. The amount of ATP was used as an indicator of cell lysis and showed contamination with intracellular materials in EPS extracted with chemical methods. Moreover, chemical extraction presented a large quantity of impurities due to non-removal of reactant salts by ultracentrifugation. For the nine methods tested, humic-like substances represented the main fraction of the extracted EPS, but for chemical extraction, the presence of humic materials from the sediment organic fraction was due to non-specific extraction of the EPS fraction. Therefore, chemicals methods are not recommended to extract EPS from sediment. Despite their low extraction efficiency, physical methods and CER, i.e. ‘soft’ extraction methods, are preferred using a solid:liquid ratio 1:40.

Organic matter (OM) in reservoir sediment consists of a range of biomolecules, but their individual contribution to the biogeochemical cycling of carbon and nitrogen nutrients is not documented. This work proposes to investigate whether the nature of the OM determines its accessibility in lacustrine sediment matrix. We adapted an OM chemical sequential method developed for soils (particle size ≤ 20 μm) for use on sediments collected from four reservoirs (particle size ≤ 2 mm), coupled with a carbon and nitrogen elementary analysis and colorimetric quantification. This method allowed for the extraction of more than 70% of carbon and 61% of nitrogen. This OM includes exchangeable, extractable, free particulate, and residual OM, whose carbon content represented < 2%, 64 to 86%, 4 to 16%, and 9 to 24%, respectively. The sum of chemically extracted biochemical molecules that recover the extracted elementary carbon and nitrogen represents the chemically extracted OM. Phenols are the main carbon contributor (55 to 60%), followed by carbohydrates and proteins (14 to 18% and 21 to 29%, respectively). Nitrogen is mainly composed of proteins, amino acids, and ammonium (46 to 56%, 20 to 34%, and 8 to 28%, respectively). Among the four reservoir sediment samples, this same trend applies to the exchangeable and extractable phases: phenols, carbohydrates, and proteins are similarly distributed along the extracted phases, whereas nitrogen, forming as ammonium, amino acids, and nitrogen oxides, exhibits specific distributions. In reservoir sediments, the nature of the carbon does not impact its physicochemical accessibility; nitrogen material presents more varied profiles depending on its accessibility.

Feeding cadmium (II) and selenium (IV) simultaneously to anaerobic granular sludge with the aim to synthesize cadmium selenide (CdSe) nanoparticles induces compositional changes in the extracellular polymeric substances (EPS) matrix of this sludge. A methanogenic anaerobic granular sludge was repeatedly exposed to Cd(II) (10-50 mg L-1) and selenite (79 mg L-1) for 300 days at pH 7.3 and 30 °C in a fed-batch feeding regime for enrichment of Se reducing bacteria and synthesis of CdSe nanoparticles. EPS fingerprints of the granular sludge, obtained by size exclusion chromatography coupled to a fluorescence detector, showed a significant increase in the intensity of protein-like substances with >100 kDa apparent molecular weight (aMW) upon repeated exposure to Cd(II) and Se(VI). This was accompanied by a prominent decrease in protein-like substances of aMW <10 kDa. The fingerprint of the humic-like substances showed emergence of a new peak with aMW of 13 to 300 kDa in the EPS extracted from the Cd/Se fed granular sludge. Experiments on metal(loid)–EPS interactions showed that the CdSe nanoparticles interact mainly with loosely bound-EPS (LB-EPS). This study showed that the formation of Se(0) and CdSe nanoparticles occurs in the LB-EPS fraction of the granular sludge and repeated exposure to Cd and Se induces compositional changes in the EPS matrix.

This study investigates the reuse potential of olive mill wastewater (OMW) high in tannin content and whose complete treatment requires the use of complex and expensive processes. Tannins from OMW could serve as bioflocculant compounds according to the literature. The defatted OMW is tested in a coagulation-flocculation process on synthetic water, and the best turbidity removal rate derived is: 92% +/- 1% at pH 11 for 100 mg L-1. Water-solubilized material at pH 11 precipitates after acidification at pH 6. This precipitate fraction (PP), solubilized at pH 11, is found to contain more active constituents with a turbidity removal rate of 82% +/- 2% for a 60 mg L-1 dose yet reveals an increase in absorbance at 254 nm. This increase in absorbance can be corrected by using lime for a pH adjustment to 11 with an absorbance removal at 254 nm of 50% +/- 1%. The bioflocculant material is characterized by means of physicochemical methods. Acid-base titration, enzymatic treatment and colorimetric dosage all confirm that tannins and/or flavonoids and cellulose constitute the active groups involved in the coagulation-flocculation process. (C) 2019 Elsevier Ltd. All rights reserved.

Purpose Soil provides important ecosystemic services, in particular through its biodiversity, which plays a major role for humans. It is therefore essential to detect and, above all, quantify soil DNA in order to better understand and conserve this biodiversity. However, the techniques commonly used are not specific and do not always allow an easy and reliable detection in complex matrices rich in organic matter. The aim of this article is to develop an effective method to quantify DNA whatever the type of soil matrix. Materials and methods Two reference clays (kaolinite and montmorillonite which are ubiquitous in soils but present very different physicochemical properties) and two soils (Cambisols and Andosols with different mineralogy, organic matter content, and properties) were used for this purpose. The developed method, based on the use of diphenylamine with colorimetric detection at 600 nm, was compared to the traditional method (absorption at 260 nm). Results and discussion The results highlight the independence of the method in terms of organic matter content or soil type, as well as its simplicity and low cost. It opens up important possibilities of application, such as a better understanding of the interactions between DNA and mineral supports, as well as the effects of mineral species. As an illustration, the method was applied to study the sorption of calf thymus DNA on various substrates with or without humic acid for a specific concentration. DNA sorption has been successfully adjusted by the Langmuir model. Conclusions The method is specific and can be easily used in complex matrices commonly found in soils, regardless of the different properties in terms of mineralogical content, presence of organic matter, or physicochemical properties.

Due to over-exploitation, river sands are less available in certain areas. Other materials are substituted for use as packing materials, e.g. crushed aggregate resulting from the quarrying of massive rocks is considered a potentially suitable material. However, the real impact of this substitution on water treatment efficiency are not mastered because all available works was carried on small scale pilot and only during the first step of colonization. The present work compares the purification efficiencies of two river sands (RS1, RS2) and two crushed aggregates (CA1, CA2) using backed bed bioreactors over a 360-day operating cycle. The total suspended solids (TSS), organic removals and ammonium removals display similar removal rates except for the coarse river sand. The total nitrogen removal is limited for all materials. Statistical analysis indicates that pollutant removals mainly depend on both grain size (effective size: D-10) and the size uniformity coefficient (UC). Furthermore, a low D-10 and UC also favor the presence of an anoxic environment and may improve denitrification. The crushed aggregate, as a potential substitution for river sands whose deposits are becoming depleted, may provide similar treatment functionality, namely suitable pollutant removals and biomass support.

Soluble 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.

Sugarcane distillery waste water (SDW) or vinasse is the residual liquid waste generated during sugarcane molasses fermentation and alcohol distillation. Worldwide, this effluent is responsible for serious environmental issues. In Reunion Island, between 100 and 200 thousand tons of SDW are produced each year by the three local distilleries. In this study, the potential of Aspergillus niger to reduce the pollution load of SDW and to produce interesting metabolites has been investigated.

Extracellular polymeric substances (EPS) from biofilters (packed with river sand (RS) and crushed aggregate (CA)) set in on-site wastewater treatment systems are characterized over 360 days within bed thickness to investigate its correlation with the enrichment process. Biochemical component contents are monitored. Moreover, humic and protein-like compounds are characterized by means of Size Exclusion Chromatography (SEC) coupled with fluorescence. During the biomass enrichment phase, EPS biochemical components increase at the top of the biofilter (protein enrichment factor >70%). The protein-like components exhibit a very high MW fraction (apparent molecular weight (aMW) >1,000kDa), which may contribute to cell aggregation. Humic-like substances show similar SEC fingerprints to those of the feed water (aMW<6kDa) and are perhaps being metabolized at around Day 210 (as evidenced by a lower aMW). Only the dynamic polysaccharide partition in EPS differs between biofilters, with an increase for CA and a decrease for RS. Within the filtration bed thickness, lower biomass with a higher EPS content is observed, and the polysaccharide fraction increases by a factor of 2. Protein-like components exhibit a very high MW fraction at a lower magnitude. The clogging risk due to the presence of polysaccharides or their combination with high aMW extracellular proteins should be considered for alternative materials for onsite wastewater filtration system.

The fungus Phanerochaete chrysosporium was incubated at five sub-toxic concentrations of Ni2+ (0.5, 1, 5, 10 and 25 mg/L, respectively), and its metal immobilization ability as well as the alteration of some characteristics regarding the extracellular polymeric substances (EPS) were investigated. With the increased Ni2+ concentrations in the broth, higher Ni2+ amounts were measured in both intact fungal cells (biomass before EPS extraction) and EPS-free biomass (biomass after EPS extraction). The Ni2+ immobilization ability of the extracted EPS displayed a similar level at Ni2+ concentrations higher than 1 mg/L. The presence of Ni2+ in the broth decreased the zeta-potential of the intact biomass and increased cell surface hydrophobicity (CSH). Fourier transform infrared spectroscopy (FT-IR) analyses identified the presence of some functional groups, such as carboxyl, phosphoryl and hydroxyl groups, in the extracted EPS. The high hydrophobicity (>60%) of the extracted EPS was decreased by the increased Ni2+ concentration. The protein (PN) and polysaccharide (PS) content in the EPS was also affected by the increased Ni2+ concentration, and the abundance of PN-like molecules ranging from 0.5 kDa to 14 kDa was enriched. However, the fluorescence characteristics and apparent molecular weight (aMW) of the extracted EPS were not affected by the Ni2+ concentration. Therefore, one possible defense mechanism developed by the fungus towards Ni2+ stress is the adjustment of its EPS composition.

ORIGINAL RESEARCH ARTICLE 1 DIVERSITY CONVERGES DURING COMMUNITY ASSEMBLY IN METHANOGENIC 2 GRANULES, SUGGESTING A BIOFILM LIFE-CYCLE 3 4 Anna Christine Trego1, Cristina Morabito1, Simon Mills1, Stephanie Connelly2, 5 Isabelle Bourven3, Giles Guibaud3, Christopher Quince4, Umer Zeeshan Ijaz2*, 6 Gavin Collins1,2* 7 School of Natural Sciences, National University of Ireland Galway, University Road, 8 Galway, H91 TK33, Ireland. 9 School of Engineering, University of Glasgow, Oakfield Avenue, Glasgow G128LT, 10 United Kingdom. 11 Groupement de Recherche Eau Sol Environnement, Faculté des Sciences 12 Techniques, Université de Limoges, 123 Avenue Albert Thomas, 87060 Limoges 13 Cedex, France. 14 4Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, 15

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.

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.

Opuntia ficus-indica that belongs to the Cactaceae family and is a member of Opuntia kind has received increasing research interest for wastewater treatment by flocculation. The objectives of this study were (i) to provide more information regarding the active constituents of Opuntia spp. and (ii) to improve the extracting and using conditions of the flocculant molecules for water treatment. A classic approach by jar test experiments was used with raw and extracted material by solubilization and precipitation. The surface properties of solid material were characterized by FTIR, SEM, zeta potential measurement, and surface titration. The splitting based on the solubility of the material with pH and the titration of functional groups completed the method. The optimal pH value for a coagulation–flocculation process using cactus solid material (CSM) was 10.0 and a processing rate of 35 mg L−1. The alkaline pH of flocculation suggests an adsorption mechanism with bridging effect between particles by water-soluble extracted molecules. To validate this mechanism, an extraction water was carried out at pH = 10 (optimum of flocculation) and the solution was acidified (pH = 7) to allow precipitation of so considered active flocculant molecules. The strong flocculant property of this extract was verified, and titration of this solution showed at least one specific pKa of 9.0 ± 0.6. This pKa corresponds to phenol groups, which could be assigned to lignin and tannin.

The release of dissolved organic matter (DOM) from wetland soils is an important pathway for the input of organic compounds into adjacent aquatic environments. In the present study we investigated, under controlled laboratory conditions, the quantity and quality of DOM released from a wetland soil subject to waterlogging and reducing conditions. Three soil redox conditions (oxic, moderately reducing and advanced reducing) were distinguished based on nitrate, ferrous ions and sulfate concentrations in soil solution. Under each redox condition, the quantity (dissolved organic carbon (DOC), humic substances and peptides plus proteins (P-PN) and quality (aromaticity; specific ultraviolet absorbance at 254 nm (SUVA254nm)) and apparent molecular weight (aMW) distribution) of DOM were investigated. The results showed that soil redox condition affects the amount and properties of mobilised DOM. The rate of DOM release and SUVA254 values were highest during the transition from oxic to moderately reducing conditions, whereas both stabilised during progression to advanced reducing conditions. In addition, the mobilised DOM is expected to be more reactive because of an increase in polar substituents in aromatic structures between oxic and moderately reducing conditions. During the development of moderately reducing conditions, dissolved humic substances increased significantly, whereas their aMW distribution (between 500 and 6000) remained constant for each of the three different redox conditions. In contrast, the quantity of dissolved P-PN remained low and steady under the three redox conditions, whereas the aMW distribution of protein-like and microbial by-product-like compounds decreased during the development of reducing conditions (aMW of compounds between 100 and > 100 000).