Biofilms are defined as communities of microbial cells growing inside a complex self-synthesized matrix composed of extracellular polymeric substances (EPS). Within various kinds of biofilms, microbial granules play an important role in the field of biological wastewater (ww) treatment. The process catalyzed by anaerobic ammonium oxidizing (anammox) bacteria granules allows N-removal with major savings but its mechanical stability is critical for its application.
The anammox granules have viscoelastic properties comparable to a solid hydrogel, but the chemical composition of EPS from granular sludge is contradictorily reported in literature with the complete lack of proteomic information and furthermore the structural components of anammox EPS responsible for biofilm formation and stability remain unknown.
The anammox granular sludge is an innovative EU technology, currently applied for the treatment of high strength wastewaters at mesophilic temperatures. Anammox biofilm stability is a prerequisite for the application to municipal ww which opens up concrete perspectives for a complete redesign of the present energy-consuming into an energy-yielding ww treatment. The excess sludge produced in biological processes is currently considered as a waste product and the related costs of handling/disposal represents up to 50% of the wastewater treatment operative costs. The recovery of EPS-based biomaterial from excess sludge to be applied in other industrial sectors would therefore substantially increase the sustainability and economics of wastewater treatment and would promote the development of a circular economy contributing to the transformation of the conventional wastewater treatment plant in an efficient biorefinery.
The main project goal was to study the structural components of anammox EPS responsible for the mechanical properties posing the basis for both the development of innovative processes and the production of viscoelastic biomaterial from excess. In the attempt of fulfilling this challenging overall goal, the following objectives have been pursued:
Definition of a standardized extraction method for the efficient recovery of anammox EPS including the structural component responsible for the mechanical properties of anammox granular sludge;
Comprehensive chemical characterization of anammox EPS by the use of a wide set of spectroscopic, microscopic, photometric and physicochemical analytical tools;
Characterization of the viscoelastic behavior of the hydrogel formed by the recovered EPS and the gain of structural information by the use of modern rheological methods and small-angle scattering techniques;
Comprehensive proteomic characterization of recovered EPS by the use of chromatographic and mass-spectrometry techniques.
Conclusions
Development of an EPS extraction method with an high yield of extraction including the structural components of the EPS matrix and suggesting a recovery potential of about 20 gEPS per kgNH4-N removed from wastewater.
The proteinaceous fraction was found as the most abundant and characterized by a rather regular secondary structure with high content of β-sheet elements, also supported by ThT analysis.
A viscoelastic biomaterial was produced with the recovered EPS. The extended 3D network with strong water binding capacity seems to be constituted by the physical interaction of askew fibrils. TEM and AFM analysis confirmed the presence of fibrillary structures in both the original anammox granules and in the hydrogel formed with the extracted EPS.
A thorough proteomic study was performed on extracted EPS. 251 proteins were identified, of which 158 using shotgun proteomics on the soluble EPS fraction and 93 using LC-MS/MS on the re-suspended insoluble fraction. Most proteins were classified as integral component of membrane and catalytic and binding activities were found as most prominent physiological functions.
