PREDOMINANCE OF ZOOGLOEA SP. DURING AEROBIC GRANULAR SLUDGE BIOFILMS DEVELOPMENT FOR WASTEWATER TREATMENT

Aerobic granular sludge (AGS) biofilm process is a sustainable technology for full biological nutrients removal (BNR) from municipal and agro-industrial wastewater, and for secondary clarification in single sequencing batch reactors (SBR). Fundamental research on the structure and dynamics of microbial communities inside AGS biofilms remains to be performed in addition to full-scale implementation, in order to link process conditions and reactor efficiency, and to tailor metabolic activities for stable treatment performance. Ten bubble column SBRs were run during three successive campaigns for (i) cultivating AGS, (ii) optimizing the AGS biofilm stability, and (iii) investigating process conditions favouring full BNR. Molecular ecology analyses on eubacterial communities performed during each first month of start-up revealed that optimally dense and fast-settling AGS biofilms were composed of 60-90% of Zoogloea sp. of the β-proteobacterial order Rhodocyclales, and of less than 20% of undesired Burkholderiales filamentous bulking heterotrophic bacteria. Zoogloea sp. was favourably selected by combining optimal process parameters: inoculum from a full BNR plant, 20°C, 60min- slow plug-flow anaerobic influent feeding regime, 0.018 m s-1 air superficial velocity, and settling time lower than 5 min. The application of a short settling phase induced a washout dynamics between the times of SBR inoculation and AGS nucleation. This low initial sludge retention time selected for a fast-settling biomass, but hampered the growth of relevant phosphorus-accumulating organisms, nitrifiers and denitrifiers. Organic carbon was fully removed, while phosphorus and nitrogen were not treated during the first 70 d. Zoogloea-like organisms can store soluble volatile fatty acids into intracellular polyhydroxyalcanoates (PHA). PHA-accumulating bacteria are expected to be denser and to settle faster than other species, and can stay in the AGS-SBR system during the initial washout dynamics. Zoogloea sp. can produce in addition extracellular polysaccharide polymers which could help to form and stabilize the early AGS biofilm structure.

Identification of microorganisms in Aerobic Granular Sludgeactively involved in biological phosphorus removal

Arnaud Michel Gelb

One of the aims of the treatment of domestic and industrial wastewater is the removal of phosphorus prior to discharge into the environment. Since phosphorus concentrations in wastewater exceed the requirement of bacterial growth, biological phosphorus removal is based on the ability of a group of microorganisms, named “Polyphosphate Accumulating Organisms” (PAO), to form large quantities of polyphosphate in their cells. In this study, we are focussing on the bacteria actively involved in the phosphorus removal in a lab scale bioreactor operated with aerobic granular sludge technology. This process based on dense microbial biofilms is a cost-effective and land-saving alternative to the conventional wastewater treatment with activated sludge. The identification of the microorganisms belonging to PAO relies on their distinct phenotype assessed with fluorescent probes targeting the intracellular polyphosphate structures. Sequencing of the 16S rRNA gene amplicons of PAO selected with flow-cytometry will be used to reveal the phylogenetic affiliation of the different actors involved in the phosphorus removal. This is of particular significance since low abundant microorganisms may play an important role in the phosphorus removal performance of aerobic granular sludge. We hypothesize that the maintenance of different populations involved in the same functional process is explained by multiple ecological niches present in such biofilm structures.

2016

Identification of microorganisms in Aerobic Granular Sludgeactively involved in biological phosphorus removal

Arnaud Michel Gelb

2016

Imaging the aerobic granular sludge microbial community using light-sheet fluorescence micros

Arnaud Michel Gelb, Karen Gemayel, Christof Holliger, Julien Maillard

One of the aims of wastewater treatment is the removal of phosphorus before the water is discharged into the environment. Since phosphorus concentrations in wastewater exceed the requirement of bacterial growth, biological phosphorus removal is based on the ability of a group of microorganisms, named “Polyphosphate Accumulating Organisms” (PAO), to store large quantities of intracellular phosphate in form of polyphosphate. In this study, we are focusing on the PAO actively involved in bioreactors operated with Aerobic Granular Sludge (AGS) technology. This process based on dense microbial biofilms is a cost-effective and landsaving alternative to the conventional biological wastewater treatment with activated sludge. This promising technology has received a significant commercial interest, but questions remain unanswered regarding the system performances in the context of full-scale applications. Like many natural microbial communities, the microbial structure of the AGS is complex. Its spatial organization is shaped by the diffusion of nutrients from the external environment and by the diffusion of microbial by-products formed in the biofilm. Understanding how the AGS microbial community works requires the elucidation of its spatial architecture and development. Light-sheet fluorescence microscopy is a powerful tool for examining microbial communities by overcoming the limitations of the classical three-dimensional confocal fluorescence microscopy. This technique shapes the excitation laser into a thin sheet providing an optical sectioning in order to illuminate the sample on a single plane. Then the emission signal is collected by a perpendicular lens. The large datasets generated are analyzed with a pipeline on the FIJI platform[1] to extract quantitative information. Preliminary results indicate that AGS are composed of heterogeneous biofilm aggregates. Interestingly, our observations are contrasting the AGS model structure previously described[2]. We would like to highlight the necessity of multiple observations when highly variable structures, like AGS, are analyzed in order to capture data which are representative of the studied system. [1] Schindelin et al. in Nat. Methods 28;9(7):676-82 (2012) [2] Winkler et al. in Water Res. 15;46(16):4973-80 (2012) Poster 3 Conférence: Swiss Microbial Ecology Meeting 2019 (Lausanne) Titre: Anaerobic Biodegradation of organohalide pollutants: a crucial step towards the elucidation of proteins involved Auteurs: Lorenzo Cimmino, Adrian Schmid, Christof Holliger, Julien Maillard Abstract: Halogenated organic compounds (so-called organohalides) represent one of the major class of groundwater pollutants. The exploration of how organohalides are used as energy source is important in terms of ecosystem remediation but is also essential for the complete understanding of microbial metabolic interactions in the environment. Organohalide respiration (OHR) is a bacterial anaerobic process in which chlorinated compound, e.g. tetrachloroethene (PCE), is used as terminal electron acceptor. In the present work, Desulfitobacterium hafniense TCE1 and Dehalobacter restrictus, our model organohalide-respiring bacteria (OHRB) harbouring the pceABCT gene cluster, will be considered for the study of PCE respiration. To date, the function of PceA, the key catalytic enzyme in the process, and PceT, the dedicated molecular chaperone for PceA maturation, are well defined. However, the roles of PceB and PceC are not yet elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis of a possible PceABC respiratory complex is tempting but the question remains largely unanswered. The present work represents an evaluation of the stoichiometry of PceA, PceB and PceC proteins via quantitative proteomics applied to the membranes fractions of our model organisms. In a second phase, the use of Blue-Native electrophoresis technology will be considered to investigate whether PceC participates in a membrane-bound protein complex toge.

2019

Imaging the aerobic granular sludge microbial community using light-sheet fluorescence micros

Arnaud Michel Gelb, Karen Gemayel, Christof Holliger, Julien Maillard

2019