Extracellular polymeric substances (EPS) are natural compounds secreted by bacteria during their metabolic activity. These polymers fulfill various functions: they protect microorganisms from toxic substances and unfavorable environmental conditions, allow aggregation into biofilms or suspended granules, help accumulate and assimilate nutrients, and are consumed in the absence of substrate. EPS are present in granular sludge in reactors performing the anaerobic oxidation of ammonium (Anammox). The objective of this study is to analyze different types of granules from various wastewater treatment plants (WWTPs), compare the characteristics and composition of the extracted EPS, and find correlations between the EPS features and the size of the granules. During this work, three different types of anammox granules are examined, two from depuration plants treating digestate from municipal WWTPs located in Belgium and the Netherlands (Anammox Belgium - BL, and Anammox Netherlands - NL) and the latest from a lab-scale reactor treating industrial wastewater from a textile industry (Textile Anammox - TEX). Seven EPS samples were extracted from these granules and analyzed to measure the total concentration of proteins (PN), polysaccharides (PS) and cytochrome-c, and their polymeric size distribution at the molecular weight cut-off of 3, 30, and 100 kDa. The EPS were extracted using the Na2CO3-heating method and were previously subjected to a qualitative Ultraviolet-Visible (UV-Vis) and excitation/emission fluorescence matrix (EEM) analyses. The studied granules showed great variability in size, ranging from an average diameter of 1.8 to 6.1 mm. The PN content is similar in all the extracted EPS and most of these polymer chains are larger than 3 kDa, while the PS concentration increases as the granule size decreases and are mainly composed of long polymer chains with dimension >30 kDa. The fluorescence analysis shows the presence of humic-like substances inside the EPS. The heme-c content is lower in TEX than in BL and NL giving them a green color instead of the usual reddish color of anammox bacteria. The different characteristics of granules and EPS may be due to the various reactor configurations and operational parameters, the different nitrogen loading rates, and the different composition between the digestate and the industrial wastewater.
Le sostanze polimeriche extracellulari (EPS) sono composti naturali secreti dai batteri durante la loro attività metabolica. Questi polimeri svolgono diverse funzioni: essi proteggono i microrganismi da sostanze tossiche e condizioni ambientali sfavorevoli, consentono l'aggregazione in biofilm o granuli sospesi, favoriscono l'accumulo e l'assimilazione di nutrienti e vengono consumati in assenza di substrato. Gli EPS sono presenti in fanghi granulari nei reattori che effettuano l'ossidazione anaerobica dell'ammonio (Anammox). L'obiettivo di questo studio è quello di analizzare diversi tipi di granuli da vari impianti di trattamento delle acque reflue (WWTP), confrontare le caratteristiche e la composizione degli EPS estratti e definire le correlazioni tra le caratteristiche dell'EPS e la dimensione dei granuli. In questo studio vengono esaminati tre diversi tipi di granuli anammox, due provenienti da WWTP che trattano digestato da impianti di depurazione comunali con sede in Belgio e nei Paesi Bassi (Anammox Belgium - BL, e Anammox Netherlands - NL) e l'ultimo da un reattore su scala di laboratorio che tratta acqua reflua industriale proveniente da un’industria tessile (Textile Anammox - TEX). Sette campioni EPS sono stati estratti da questi granuli e analizzati per misurare la concentrazione totale di proteine (PN), polisaccaridi (PS) e citocromo-c e la loro distribuzione dimensionale polimerica al molecolar weight cut-off di 3, 30 e 100 kDa. Gli EPS sono stati estratti utilizzando il metodo di Na2CO3-heating e sono stati precedentemente sottoposti ad analisi qualitative di spettroscopia Ultravioletto-Visibile (UV-Vis) e matrice di fluorescenza di eccitazione/emissione (EEM). I granuli studiati hanno mostrato una grande variabilità di dimensioni, che si estendono da un diametro medio da 1,8 a 6,1 mm. Il contenuto PN è simile in tutti gli EPS estratti e per la maggior parte queste catene polimeriche sono più grandi di 3 kDa, mentre la concentrazione di PS aumenta al diminuire della dimensione dei granuli e questi composti sono costituiti principalmente da lunghe catene polimeriche con dimensione >30 kDa. L'analisi di fluorescenza mostra la presenza di sostanze umiche all'interno degli EPS. Il contenuto di eme-c è inferiore in TEX rispetto a BL e NL, dando loro un colore verde invece dell’usuale colore rossastro dei batteri anammox. Le diverse caratteristiche dei granuli e dell'EPS possono essere dovute alle configurazioni dei vari reattori e dai loro parametri operativi, dai diversi nitrogen loading rate e la diversa composizione dell’acqua reflua municipale e industriale.
Analysis and characterization of extracellular polymeric substances extracted from anammox granules
RIZZARDI, GIACOMO
2020/2021
Abstract
Extracellular polymeric substances (EPS) are natural compounds secreted by bacteria during their metabolic activity. These polymers fulfill various functions: they protect microorganisms from toxic substances and unfavorable environmental conditions, allow aggregation into biofilms or suspended granules, help accumulate and assimilate nutrients, and are consumed in the absence of substrate. EPS are present in granular sludge in reactors performing the anaerobic oxidation of ammonium (Anammox). The objective of this study is to analyze different types of granules from various wastewater treatment plants (WWTPs), compare the characteristics and composition of the extracted EPS, and find correlations between the EPS features and the size of the granules. During this work, three different types of anammox granules are examined, two from depuration plants treating digestate from municipal WWTPs located in Belgium and the Netherlands (Anammox Belgium - BL, and Anammox Netherlands - NL) and the latest from a lab-scale reactor treating industrial wastewater from a textile industry (Textile Anammox - TEX). Seven EPS samples were extracted from these granules and analyzed to measure the total concentration of proteins (PN), polysaccharides (PS) and cytochrome-c, and their polymeric size distribution at the molecular weight cut-off of 3, 30, and 100 kDa. The EPS were extracted using the Na2CO3-heating method and were previously subjected to a qualitative Ultraviolet-Visible (UV-Vis) and excitation/emission fluorescence matrix (EEM) analyses. The studied granules showed great variability in size, ranging from an average diameter of 1.8 to 6.1 mm. The PN content is similar in all the extracted EPS and most of these polymer chains are larger than 3 kDa, while the PS concentration increases as the granule size decreases and are mainly composed of long polymer chains with dimension >30 kDa. The fluorescence analysis shows the presence of humic-like substances inside the EPS. The heme-c content is lower in TEX than in BL and NL giving them a green color instead of the usual reddish color of anammox bacteria. The different characteristics of granules and EPS may be due to the various reactor configurations and operational parameters, the different nitrogen loading rates, and the different composition between the digestate and the industrial wastewater.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/178105