The role of inorganic nitrogen in successful formation of granular biofilms for wastewater treatment that support cyanobacteria and bacteria

Recently, the use of phototrophs for wastewater treatment has been revisited because of new approaches to separate them from effluent streams. One manifestation uses oxygenic photogranules (OPGs) which are dense, easily-settleable granular biofilms of cyanobacteria, which surrounding populations of heterotrophs, autotrophs, and microalgae. OPGs can remove COD and nitrogenous compounds without external aeration. To better grow and maintain biomass in the proposed wastewater process, this study seeks to understand the factors that contribute to successful granulation. Availability of initial inorganic nitrogen, particularly ammonium, was associated with successful cultivation of OPGs. In the first days of granulation, a decrease in ammonium coupled with an increase in a cyanobacterial-specific 16S rRNA gene, may suggest that ammonium was assimilated in cyanobacteria offering a competitive environment for growth. Though both successful and unsuccessful OPG formation demonstrated a shift from non-phototrophic bacterial dominated communities on day 0 to cyanobacterial dominated communities on day 42, the successful community had a greater relative abundance (46%) of OTUs associated with genera Oscillatoria and Geitlernema than the unsuccessful community (27%), supporting that filamentous cyanobacteria are essential for successful OPG formation. A greater concentration of chlorophyll b in the unsuccessful OPG formation suggested a greater abundance of algal species. This study offers indicators of granulation success, notably availability of inorganic nitrogen and chlorophyll a and b concentrations for monitoring the health and growth of biomass for a potential OPG process

EXTRACELLULAR POLYMERIC SUBSTANCES IN OXYGENIC PHOTOGRANULES: INVESTIGATION OF THEIR ROLE IN PHOTOGRANULATION IN A HYDROSTATIC ENVIRONMENT

The purpose of this dissertation was to assess the critical role of extracellular polymeric substances (EPS) in the photogranulation of activated sludge, in a hydrostatic environment. The first section evaluates the fate and dynamics of different fractions of EPS in sludge-based photogranulation under hydrostatic conditions. The study shows that during the transformation of activated sludge into a photogranular biomass, sludge’s base-extractable proteins selectively degrade. Strong correlations between base-extracted proteins and the growth of chlorophyll a and chlorophyll a/b ratio suggest that the bioavailability of this organic nitrogen is linked with selection and enrichment of filamentous cyanobacteria under hydrostatic conditions. The results of soluble and sonication-extractable EPS and microscopy also show that the growth of filamentous cyanobacteria required large amounts of polysaccharide-based EPS for their motility and maintenance. With findings on the progression of photogranulation, the fate and dynamics of EPS, and microscopy on microstructures associated with EPS, potential mechanisms of photogranulation occurring under hydrostatic conditions are discussed. The second section evaluates and shows that multiple EPS extraction methods are required in order to characterize EPS during the transformation of activated sludge into a photogranule in a hydrostatic environment. The present study reveals why cyanobacteria are selected and how different fractions of EPS and their recycle leads to photogranulation in hydrostatic conditions. Despite differences in sludge inoculum, EPS extraction using five different methods, centrifugation, cation exchange resin (CER), base, sonication, and heat, show that trends are significantly similar, statistically, between two sludge sources (Amherst and Hadley). The results presented above show that different EPS extraction methods are required to capture different fractions of EPS with respect to protein, polysaccharide, and humic acid composition and organic carbon and nitrogen content. EPS extraction methods for polysaccharides was found to be the most biased, followed by humic acids, then proteins. This suggests that different methods target different EPS fractions (more associated with polysaccharides), but may share overlap between the methods (proteins and humic acids). All methods had statistically significant moderate to strong correlations with one or more constituents, chlorophylls, nitrogen species, and select cations and anions, which have been previously established as strong indicators of successful granule formation. These results suggest that different EPS fractions are linked to multiple processes during hydrostatic photogranulation, including the enrichment of filamentous cyanobacteria, nitrogen metabolism and recycle of organic nitrogen, assimilation and biofilm incorporation of ammonium (NH4+-N), and biofilm structure, further suggesting that the role of EPS is a complex process with multiple courses of action. The final section focuses on the addition and role of cations for the enhancement of activated sludge photogranulation in a hydrostatic environment. This study observed that the addition of monovalent (sodium-Na+) and divalent cations (Calcium-Ca2+ and Magnesium- Mg2+), at specific concentrations 10-40 meq/L, leads to a higher percentage of total and spherical granules, in comparison to light control (no cation amendment) and dark control cultivations. Based on crude EPS, ammonium sulfate precipitation, and sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results, Light+Ca2+ treatments show greater recovery of CER protein after ASP, and different recovery patterns in comparison to light and dark control, suggesting that more hydrophobic protein is available. This further infers that the addition of Ca2+ may influence the hydrophobicity of EPS proteins during hydrostatic photogranulation. After ASP, SDS-PAGE was applied and banding pattern across the treatments showed same the EPS protein on a molecular level which did not change with the addition of Ca2+ (in comparison to control). This may further imply that enhancement is more likely due to Ca2+ cation bridging, versus changes on a molecular level influenced by the microbial community

Filamentous cyanobacteria in granular biofilms containing microalgae and bacteria

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The importance of filamentous cyanobacteria in the development of oxygenic photogranules

Microorganisms often respond to their environment by growing as densely packed communities in biofilms, flocs or granules. One major advantage of life in these aggregates is the retention of its community in an ecosystem despite flowing water. We describe here a novel type of granule dominated by filamentous and motile cyanobacteria of the order Oscillatoriales. These bacteria form a mat-like photoactive outer layer around an otherwise unconsolidated core. The spatial organization of the phototrophic layer resembles microbial mats growing on sediments but is spherical. We describe the production of these oxygenic photogranules under static batch conditions, as well as in turbulently mixed bioreactors. Photogranulation defies typically postulated requirements for granulation in biotechnology, i.e., the need for hydrodynamic shear and selective washout. Photogranulation as described here is a robust phenomenon with respect to inoculum characteristics and environmental parameters like carbon sources. A bioprocess using oxygenic photogranules is an attractive candidate for energy-positive wastewater treatment as it biologically couples CO2 and O-2 fluxes. As a result, the external supply of oxygen may become obsolete and otherwise released CO2 is fixed by photosynthesis for the production of an organic-rich biofeedstock as a renewable energy source

The Oxygenic Photogranule Process for Aeration-Free Wastewater Treatment

This study presents the oxygenic photogranule (OPG) process, a light-driven process for wastewater treatment, developed based on photogranulation of filamentous cyanobacteria, nonphototrophic bacteria, and microalgae. Unlike other biogranular processes requiring airlift or upflow-based mixing, the OPG process was operated in stirred-tank reactors without aeration. Reactors were seeded with hydrostatically grown photogranules and operated in a sequencing-batch mode for five months to treat wastewater. The new reactor biomass propagated with progression of photogranulation under periodic light/dark cycles. Due to effective biomass separation from water, the system was operated with short settling time (10 min) with effective decoupling of hydraulic and solids retention times (0.75 d vs 21–42 d). During quasi-steady state, the diameter of the OPGs ranged between 0.1 and 4.5 mm. The reactors produced effluents with average total chemical oxygen demand less than 30 mg/L. Nitrogen removal (28–71%) was achieved by bioassimilation and nitrification/denitrification pathways. Oxygen needed for the oxidation of organic matter and nitrification was produced by OPGs at a rate of 12.6 ± 2.4 mg O₂/g biomass-h. The OPG system presents a new biogranule process, which can potentially use simple mixing and natural light to treat wastewater