Fermentative chemoorganoheterotrophic bacteria (FCB) and purple photoorganoheterotrophic bacteria (PPB) are two interesting microbial guilds to process carbohydrate-rich wastewaters. Their metabolic interactions have been studied in pure cultures or co-cultures, but little is known about mixed cultures. We studied the effect of reactor regimes (batch/chemostat) and illumination modes (continuous infrared light, dark, or light/dark cycles) on glucose conversions and process ecology of the interactions between FCB and PPB in mixed cultures. In batch, FCB (>80 % of sequencing read counts) outcompeted PPB, under any light conditions. In chemostat under continuous and alternating irradiance, three FCB populations were enriched (>70 %), while Rhodobacteraceae (PPB) made 30 % of the community. Glucose fermentation products were linked to the dominant FCB. Continuous culturing helped maintaining FCB and PPB in syntrophy: PPB grew on glucose metabolites produced by FCB. Engineering the association between FCB and PPB in mixed-culture processes can help to treat and valorize carbohydrate-rich aqueous waste.

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A transition to a circular economy is necessary to mitigate the negative effects on the environment of the exploitation and disposal of materials and to achieve society-wide benefits. The current produce-waste-dispose model is slowly changing toward a more sustainable produce-userecycle- upcycle model. In this context, bio-based processes using microbial mixed cultures are crucial to develop waste-to-resource valorization processes. Purple phototrophic bacteria (PPB) form a guild of hyper-versatile organisms found in almost all aqueous environments, thriving on infrared light energy, capturing organics by photoorganoheterotrophy, and even recycling CO2 by photolithoautotrophy. Due to their outstanding metabolic versatility, their organic and nutrient capture ability, and their biomass yields over substrate approaching 1 g CODx g-1 CODs, PPB are dedicated organisms to study and use for the development of water resource recovery applications. Despite already 80 years of research on PPB, their physiology still needs to get deciphered, and their environmental biotechnological exploitation is at its infancy. The aim of this thesis was to study and harness the metabolic versatility of PPB at different levels, from the elucidation of light-driven physiologies in pure cultures to the management of selection phenomena, population dynamics, and distributed metabolic functionalities in mixed cultures. The findings were aggregated to derive to mixed-culture bioprocess application perspectives for capturing organics and nutrients from municipal sewage and agri-food wastewater and producing valuable products, as bioplastics, biohydrogen or photopigments. In this thesis, a comprehensive overview of the potential of PPB for water resource recovery is given. The molecular principles and ecological dynamics governing the PPB metabolism were elucidated with the goal to demonstrate the potential of PPB-based biotechnologies.@en

Purple non-sulphur bacteria (PNSB) are an emerging group of microbes attractive for applied microbiology applications such as wastewater treatment, plant biostimulants, microbial protein, polyhydroxyalkanoates and H₂ production. These photoorganoheterotrophic microbes have the unique ability to grow selectively on organic carbon in anaerobic photobioreactors. This so-called selectivity implies that the microbial community will have a low diversity and a high abundance of a particular PNSB species. Recently, it has been shown that certain PNSB strains can produce antimicrobials, yet it remains unclear whether these contribute to competitive inhibition. This research aimed to understand which type of antimicrobial PNSB produce and identify whether these compounds contribute to their selective growth. Mining 166 publicly-available PNSB genomes using the computational tool BAGEL showed that 59% contained antimicrobial encoding regions, more specifically biosynthetic clusters of bacteriocins and non-ribosomal peptide synthetases. Inter- and intra-species inhibition was observed in agar spot assays for Rhodobacter blasticus EBR2 and Rhodopseudomonas palustris EBE1 with inhibition zones of, respectively, 5.1 and 1.5–5.7 mm. Peptidomic analysis detected a peptide fragment in the supernatant (SVLQLLR) that had a 100% percentage identity match with a known non-ribosomal peptide synthetase with antimicrobial activity.

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Purple bacteria (PPB), anoxygenic photoorganoheterotrophic organisms with a hyper-versatile metabolism and high biomass yields over substrate, are promising candidates for the recovery of nutrient resources from wastewater. Infrared light is a pivotal parameter to control and design PPB-based resource recovery. However, the effects of light intensities on the physiology and selection of PPB in mixed cultures have not been studied to date. Here, we examined the effect of infrared irradiance on PPB physiology, enrichment, and growth over a large range of irradiance (0 to 350 W m−2) in an anaerobic mixed-culture sequencing batch photobioreactor. We developed an empirical mathematical model that suggests higher PPB growth rates as response to higher irradiance. Moreover, PPB adapted to light intensity by modulating the abundances of their phototrophic complexes. The obtained results provide an in-depth phylogenetic and metabolic insight the impact of irradiance on PPB. Our findings deliver the fundamental information for guiding the design of light-driven, anaerobic mixed-culture PPB processes for wastewater treatment and bioproduct valorization.@en

The wastewater treatment sector embraces mixed-culture biotechnologies for sanitation, environmental protection, and resource recovery. Bioprocess design, monitoring and control thrive on microbial processes selected in complex microbial communities. Microbial ecology and systems microbiology help access microbiomes and characterize microorganisms, metabolisms and interactions at increased resolution and throughput. Big datasets are generated from the sequencing of informational molecules extracted from biomasses sampled across process schemes. However, they mostly remain on science benches and computing clusters, without reaching the industry in a clear engineering objective function. A bilateral bridge should actionize this information. As systems microbiologists, we miss that engineering designs and operations rely on stoichiometry and kinetics. The added-value provided by microbial ecology and systems microbiology to improve capital (CAPEX) and operating expenditures (OPEX) needs to be addressed. As engineers, we miss that microbiology can be provide powerful microbial information on top of physical-chemical measurements for quantitative process design (e.g., nutrient removal systems) with detailed scientific description of phenomena inside microbiomes. In this perspective article, we allied academia and industry to address the state of shared knowledge, successes and failures, and to establish joint investigation platforms. Our roadmap involves three milestones to (i) elaborate an essential list of microbiological information needed to implement methods at the process line; (ii) characterize microbiomes from microorganisms to metabolisms, and shape conceptual ecosystem models as primer for process ecology understanding; (iii) bridge engineering and mathematical models with an analytical toolbox for fast- vs. high-throughput analyses to discover new microbial processes and engineer assemblies. We praise for a harmonized "language of love"(incorporating common vocabulary, units, protocols) across the water and environmental biotechnology sector to team up mindsets for a sewer- and plant-wide integration of systems microbiology and engineering.

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Purple non-sulfur bacteria (PNSB) show potential for microbial protein production on wastewater as animal feed. They offer good selectivity (i.e., low microbial diversity and high abundance of one species) when grown anaerobically in the light. However, the cost of closed anaerobic photobioreactors is prohibitive for protein production. Although open raceway reactors are cheaper, their feasibility to selectively grow PNSB is thus far unexplored. This study developed operational strategies to boost PNSB abundance in the biomass of a raceway reactor fed with volatile fatty acids. For a flask reactor run at a 2 day sludge retention time (SRT), matching the chemical oxygen demand (COD) loading rate to the removal rate in the light period prevented substrate availability during the dark period and increased the PNSB abundance from 50-67 to 88-94%. A raceway reactor run at a 2 day SRT showed an increased PNSB abundance from 14 to 56% when oxygen supply was reduced (no stirring at night). The best performance was achieved at the highest surface-to-volume ratio (10 m2 m-3 increased light availability) showing productivities up to 0.2 g protein L-1 day-1 and a PNSB abundance of 78%. This study pioneered in PNSB-based microbial protein production in raceway reactors, yielding high selectivity while avoiding the combined availability of oxygen, COD, and darkness.

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Mixed-culture biotechnologies are widely used to capture nutrients from wastewater. Purple non-sulfur bacteria (PNSB), a guild of anoxygenic photomixotrophic organisms, rise interest for their ability to directly assimilate nutrients in the biomass. One challenge targets the aggregation and accumulation of PNSB biomass to separate it from the treated water. Our aim was to enrich and produce a concentrated, fast-settling PNSB biomass with high nutrient removal capacity in a 1.5-L, stirred-tank, anaerobic sequencing-batch photobioreactor (SBR). PNSB were rapidly enriched after inoculation with activated sludge at 0.1 gVSS L^–1 in a first batch of 24 h under continuous irradiance of infrared (IR) light (>700 nm) at 375 W m^–2, with Rhodobacter reaching 54% of amplicon sequencing read counts. SBR operations with decreasing hydraulic retention times (48 to 16 h, i.e., 1–3 cycles d^–1) and increasing volumetric organic loading rates (0.2–1.3 kg COD d^–1 m^–3) stimulated biomass aggregation, settling, and accumulation in the system, reaching as high as 3.8 g VSS L^–1. The sludge retention time (SRT) increased freely from 2.5 to 11 days. Acetate, ammonium, and orthophosphate were removed up to 96% at a rate of 1.1 kg COD d^–1 m^–3, 77% at 113 g N d^–1 m^–3, and 73% at 15 g P d^–1 m^–3, respectively, with COD:N:P assimilation ratio of 100:6.7:0.9 m/m/m. SBR regime shifts sequentially selected for Rhodobacter (90%) under shorter SRT and non-limiting concentration of acetate during reaction phases, for Rhodopseudomonas (70%) under longer SRT and acetate limitation during reaction, and Blastochloris (10%) under higher biomass concentrations, underlying competition for substrate and photons in the PNSB guild. With SBR operations we produced a fast-settling biomass, highly (>90%) enriched in PNSB. A high nutrient removal was achieved by biomass assimilation, reaching the European nutrient discharge limits. We opened further insights on the microbial ecology of PNSB-based processes for water resource recovery.

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Microorganisms in natural and engineered environments interact with surfaces and form aggregates consisting of cells and an extracellular matrix. The design of the process and appropriate operational conditions drive the formation of these biofilms, flocs, and granular structures. The application of granular sludge technologies for nutrient removal is relatively new. Although research and practice benefit from several decades of investigation of biofilm and anaerobic granular sludge systems, a thorough understanding of factors affecting granulation is only beginning to emerge from bench, pilot, and full-scale investigations. Challenges intrinsic to maintaining granular and biofilm structures include management of resistance to substrate transport, establishment of targeted microbial niches, role of extracellular polymeric substances, and impacts of toxic compounds, among others. There is increasing recognition of the potential value of hybrid process configurations that optimize interactions between flocs, granules, and/or biofilm features for process enhancement and robustness. While these structures appear distinct, it is not uncommon to find a mixture of these structures present in a single system and dynamics leading to a transition from one structure to another. The transitions are dependent on changes in the microbial community and properties of the extracellular matrix. This review focuses on the drivers affecting formation and stability of flocs, biofilms, and granules and conditions that support integrated technologies for biological wastewater treatment.

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