Periodic Reporting for period 1 - POLIS (Studying the bricks of microbial cities: characterization and structural properties of exopolysaccharides and their interaction with proteins and cations in anammox granular sludge) Reporting period: 2015-12-01 to 2017-11-30 Summary of the context and overall objectives of the project 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.ConclusionsDevelopment 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The work was performed by the PI Tommaso Lotti at the Civil & Environ. Eng. Department (DICA) of Politecnico di Milano in collaboration with several departments of the University of Florence. The NaOH method was selected as EPS extraction method among the 8 tested, for its high yield and the ability to extract also the structural fraction, resulting in an average EPS recovery yield of 21%. The proteins and carbohydrates equivalent content of the extracted EPS was measured by standard spectrophotometric methods. The chemical composition was evaluated by FT-IR and UV-Vis analysis confirming the abundance of proteins and marginal presence of DNA (cell lysis). The secondary structure of proteins was analyzed by Circular Dichroism and deconvolution of the FTIR spectra indicating high β-sheet content confirmed by the Thioflavin-T staining technique coupled with fluorescence spectroscopy. The EPS extracted from anammox granular sludge was used to form a viscoelastic biomaterial at varying weight fractions wt% (gEPS/g) and its physico-chemical characterization was performed by rheological methods indicating a solid-like behavior typical of solid-like fluids with infinite relaxation time. Differential Scannig Calorimetry analysis was used to determine the water binding capacity by means of the calculation of the free-water index as function of the total wt% of EPS. 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 proteomic study of the extracted EPS was performed with enzymatic deglycosylation treatment prior trypsin digestion. Overall 251 proteins were identified, of which 158 using shotgun proteomics on the soluble fractions and 93 using LC-MS/MS on the re-suspended precipitated fractions.Obtained results were disseminated by presentation at 6 international conferences and one public event, the organization of 2 specialized intern.workshops and 3 online and 2 press communication articles. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) As a result, information obtained from POLIS research project will facilitate and support EPS research in anammox granular sludge area and more in general in the area of biofilm based environmental biotechnological applications; knowledge obtained provides insight on EPS composition and function. The identification of the structural components of EPS as amyloid-like fibrils poses the basis for the improvement of anammox granular sludge formation and stability which is a key factor for the optimization of currently applied technologies and especially for the innovative implementation of anammox based processes in contests where the necessity of a substantial decoupling of HRT and SRT imposes nearly complete anammox biomass retention. In particular, the mechanical stability of anammox biofilm is considered a prerequisite for the treatment of municipal wastewaters, allowing for a complete redesign of the present energy-consuming into an energy-yielding wastewater treatment system.Finally, the observed physico-chemical characteristics of the hydrogel biomaterial formed by EPS recovered from excess sludge stimulate its application in other industrial sectors creating the background for further studies in this area and could therefore substantially increase the sustainability and economics of wastewater treatment by promoting the development of a circular economy contributing to the transformation of the conventional wastewater treatment plant in an efficient biorefinery. Conceptual scheme of biorefinery-WWTP wih focus on anammox EPS recovery/reuse in circular economy
