The water-soluble fraction of extracellular polymeric substances from a resource recovery demonstration plant: characterization and potential application as an adhesive.

Currently, there is a growing interest in transforming wastewater treatment plants (WWTPs) into resource recovery plants. Microorganisms in aerobic granular sludge produce extracellular polymeric substances (EPS), which are considered sustainable resources to be extracted and can be used in diverse applications. Exploring applications in other high-value materials, such as adhesives, will not only enhance the valorization potential of the EPS but also promote resource recovery. This study aimed to characterize a water-soluble fraction extracted from the EPS collected at the demonstration plant in the Netherlands based on its chemical composition (amino acids, sugar, and fatty acids) and propose a proof-of-concept for its use as an adhesive. This fraction comprises a mixture of biomolecules, such as proteins (26.6 ± 0.3%), sugars (21.8 ± 0.2%), and fatty acids (0.9%). The water-soluble fraction exhibited shear strength reaching 36-51 kPa across a pH range of 2-10 without additional chemical treatment, suggesting a potential application as an adhesive. The findings from this study provide insights into the concept of resource recovery and the valorization of excess sludge at WWTPs.

Coupling extracellular glycan composition with metagenomic data in papermill and brewery anaerobic granular sludges.

Glycans are crucial for the structure and function of anaerobic granular sludge in wastewater treatment. Yet, there is limited knowledge regarding the microorganisms and biosynthesis pathways responsible for glycan production. In this study, we analysed samples from anaerobic granular sludges treating papermill and brewery wastewater, examining glycans composition and using metagenome-assembled genomes (MAGs) to explore potential biochemical pathways associated with their production. Uronic acids were the predominant constituents of the glycans in extracellular polymeric substances (EPS) produced by the anaerobic granular sludges, comprising up to 60 % of the total polysaccharide content. MAGs affiliated with Anaerolineacae, Methanobacteriaceae and Methanosaetaceae represented the majority of the microbial community (30-50 % of total reads per MAG). Based on the analysis of MAGs, it appears that Anaerolinea sp. and members of the Methanobacteria class are involved in the production of exopolysaccharides within the analysed granular sludges. These findings shed light on the functional roles of microorganisms in glycan production in industrial anaerobic wastewater treatment systems.

Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions.

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge.

Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438- > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. KEY POINTS: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68-14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2.

Metaproteomics, metagenomics and 16S rRNA sequencing provide different perspectives on the aerobic granular sludge microbiome.

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

Sialylation and Sulfation of Anionic Glycoconjugates Are Common in the Extracellular Polymeric Substances of Both Aerobic and Anaerobic Granular Sludges.

Anaerobic and aerobic granular sludge processes are widely applied in wastewater treatment. In these systems, microorganisms grow in dense aggregates due to the production of extracellular polymeric substances (EPS). This study investigates the sialylation and sulfation of anionic glyconconjugates in anaerobic and aerobic granular sludges collected from full-scale wastewater treatment processes. Size exclusion chromatography revealed a wide molecular weight distribution (3.5 to >5500 kDa) of the alkaline-extracted EPS. The high-molecular weight fraction (>5500 kDa), comprising 16.9-27.4% of EPS, was dominant with glycoconjugates. Mass spectrometry analysis and quantification assays identified nonulosonic acids (NulOs, e.g., bacterial sialic acids) and sulfated groups contributing to the negative charge in all EPS fractions. NulOs were predominantly present in the high-molecular weight fraction (47.2-84.3% of all detected NulOs), while sulfated glycoconjugates were distributed across the molecular weight fractions. Microorganisms, closely related to genera found in the granular sludge communities, contained genes responsible for NulO and sulfate group synthesis or transfer. The similar distribution patterns of sialylation and sulfation of the anionic glycoconjugates in the EPS samples indicate that these two glycoconjugate modifications commonly occur in the EPS of aerobic and anaerobic granular sludges.

Highly efficient carbon assimilation and nitrogen/phosphorus removal facilitated by photosynthetic O2 from algal-bacterial aerobic granular sludge under controlled DO/pH operation.

Reducing CO2 emission and energy consumption is crucial for the sustainable management of wastewater treatment plants (WWTPs). In this study, an algal-bacterial aerobic granular sludge (AGS) system was developed for efficient carbon (C) assimilation and nitrogen (N)/phosphorus (P) removal without the need for mechanical aeration. The photosynthetic O2 production by phototrophic organisms maintained the dissolved oxygen (DO) level at 3-4 mg/L in the bulk liquid, and an LED light control system reduced 10-30% of light energy consumption. Results showed that the biomass assimilated 52% of input dissolved total carbon (DTC), and the produced O2 simultaneously facilitated aerobic nitrification and P uptake with the coexisting phototrophs serving as a C fixer and O2 supplier. This resulted in a stably high total N removal of 81 ± 7% and an N assimilation rate of 7.55 mg/(g-MLVSS∙d) with enhanced microbial assimilation and simultaneous nitrification/denitrification. Good P removal of 92-98% was maintained during the test period at a molar ∆P/∆C ratio of 0.36 ± 0.03 and high P release and uptake rates of 10.84 ± 0.41 and 7.18 ± 0.24 mg/(g- MLVSS∙h), respectively. Photosynthetic O2 was more advantageous for N and P removal than mechanical aeration. This proposed system can contribute to a better design and sustainable operation of WWTPs using algal-bacterial AGS.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Enrichment and application of extracellular nonulosonic acids containing polymers of Accumulibacter.

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. KEY POINTS: • NulOs in "Ca. Accumulibacter" are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

A review on recovery of extracellular biopolymers from flocculent and granular activated sludges: Cognition, key influencing factors, applications, and challenges.

A reasonable recovery of excess sludge may shift the waste into wealth. Recently an increasing attention has been paid to the recycling of extracellular biopolymers from conventional and advanced biological wastewater treatment systems such as flocculent activated sludge (AS), bacterial aerobic granular sludge (AGS), and algal-bacterial AGS processes. This review provides the first overview of current research developments and future directions in the recovery and utilization of high value-added biopolymers from the three types of sludge. It details the discussion on the recent evolvement of cognition or updated knowledge on functional extracellular biopolymers, as well as a comprehensive summary of the operating conditions and wastewater parameters influencing the yield, quality, and functionality of alginate-like exopolymer (ALE). In addition, recent attempts for potential practical applications of extracellular biopolymers are discussed, suggesting research priorities for overcoming identification challenges and future prospects.

Catabolism of sialic acids in an environmental microbial community.

Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

Insight on how biopolymers recovered from aerobic granular wastewater sludge can reduce the flammability of synthetic polymers.

Eco-friendly flame retardants are greatly required to meet the expectations of low-toxicity, environmental compatibility and sustainability. Extracellular polymeric substances (EPS), the biopolymers recovered from excess granular wastewater sludge, have been successfully incorporated into poly(vinyl alcohol) (PVA) by a solution casting method. Self-extinguishment of EPS was observed in a vertical burn test. Positive effects of EPS on the reduction of heat release rate and CO emission of EPS/PVA composites were also demonstrated. The presence of various types of phosphates was detected in the EPS and a possible flame-retardant mechanism has been proposed. The investigation of using granular sludge EPS to reduce the flammability of synthetic polymers may open the possibility of converting wastewater sludge into bio phosphorus-based flame retardants.

Production of nonulosonic acids in the extracellular polymeric substances of "Candidatus Accumulibacter phosphatis".

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in "Candidatus Accumulibacter phosphatis", a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of "Ca. Accumulibacter" to produce different types of NulOs. Proteomic analysis showed the ability of "Ca. Accumulibacter" to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of "Ca. Accumulibacter" in particular, and biofilms in general. KEY POINTS: •"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

Efficacy and Safety of Opioids in Treating Cancer-Related Dyspnea: A Systematic Review and Meta-Analysis Based on Randomized Controlled Trials.

BACKGROUND: Dyspnea is one of the most distressing symptoms encountered by advanced cancer patients. In this study, we aimed to evaluate the role of opioids in the management of cancer-related dyspnea. METHODS: A systematic review and meta-analysis based on Randomized Controlled Trials was conducted in the databases PUBMED, EMBASE, and Cochrane Central Register of Controlled Trials testing the effect of opioids in relieving cancer-related dyspnea. Subgroup and sensitivity analyses were performed to evaluate various types of opioids in dyspnea management and stabilization of the study respectively. RESULTS: Eleven RCTs fulfilled the eligibility criteria and had a total of 290 participants. Nine of these studies were included in meta-analyses. Compared with control, opioid therapy showed a small positive effect in dyspnea, SMD-0.82 (95%CI = -1.54 to -0.10) and Borg score, WMD-0.95 (95%CI = -1.83 to -0.06); Opioid therapy did not increase the risk of somnolence, OR0.93 (95%CI = 0.34 to 2.58), whereas a negative effect on respiratory rate was observed,WMD-1.89 (95%CI = -3.36 to -0.43); Also, there was no evidence to suggest improved performance of the 6MWT test, WMD6.49 (95%CI = -34.23 to 47.21), or the level of peripheral oxygen saturation, WMD0.33 (95%CI = -0.59 to 1.24) after opioid therapy. Subgroup analysis yielded a small positive effect for morphine on dyspnea, SMD-0.78 (95%CI = -1.45 to -0.10), whereas fentanyl showed no improvement in dyspnea, SMD-0.44 (95%CI = -0.89 to 0.02). Sensitivity analysis showed no changes in the direction of effect when any one study was excluded from the meta-analyses. CONCLUSIONS: Our systematic review and meta-analysis indicated low quality evidence for a small positive effect of opioids in cancer-related dyspnea. Evidence for safety is insufficient as comprehensive adverse events were not adequately reported in studies.

Extracellular biopolymers recovered as raw biomaterials from waste granular sludge and potential applications: A critical review.

Granular sludge (GS) is a special self-aggregation biofilm. Extracellular polymeric substances (EPS) are mainly associated with the architectural structure, rheological behaviour and functional stability of fine granules, given that their significance to the physicochemical features of the biomass catalysing the biological purification process. This review targets the EPS excretion from GS and introduces newly identified EPS components, EPS distribution in different granules, how to effectively extract and recover EPS from granules, key parameters affecting EPS production, and the potential applications of EPS-based biomaterials. GS-based EPS components are highly diverse and a series of new contents are highlighted. Due to high diversity, emerging extraction standards are proposed and recovery process is capturing particular attention. The major components of EPS are found to be polysaccharides and proteins, which manifest a larger diversity of relative abundance, structures, physical and chemical characteristics, leading to the possibility to sustainably recover raw materials. EPS-based biomaterials not only act as alternatives to synthetic polymers in several applications but also figure in innovative industrial/environmental applications, including gel-forming materials for paper industry, biosorbents, cement curing materials, and flame retardant materials. In the upcoming years, it is foreseen that productions of EPS-based biomaterials from renewable origins would make a significant contribution to the advancement of the circular economy.

Pontiella desulfatans gen. nov., sp. nov., and Pontiella sulfatireligans sp. nov., Two Marine Anaerobes of the Pontiellaceae fam. nov. Producing Sulfated Glycosaminoglycan-like Exopolymers.

Recently, we isolated two marine strains, F1T and F21T, which together with Kiritimatiella glycovorans L21-Fru-ABT are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1T, F21T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligans F21T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.

Structural extracellular polymeric substances determine the difference in digestibility between waste activated sludge and aerobic granules.

Aerobic granular sludge (AGS) technology is an alternative to conventional activated sludge to reduce the process footprint and energy consumption. Strategies for the efficient management of its produced biomass, that is grown in a granular morphology as well, need further development. Anaerobic digestion (AD) is commonly applied in waste activated sludge (WAS) treatment and is a potential option also for produced AGS treatment. In earlier studies, the biochemical methane potential of AGS was found lower than that of WAS both grown in full-scale municipal wastewater treatment systems. In order to understand this difference, this study aimed to investigate the anaerobic conversion of structural extracellular polymeric substances (SEPS), which is a type of gel-forming biopolymer, being responsible for the aggregation of sludge. Using WAS and AGS as substrates, a comparative AD batch experiment was performed for 44 days during which the SEPS fraction was extracted from both types of sludge. The changes in the SEPS chemical composition was analysed by Fourier transformed infrared spectroscopy and three-dimensional excitation and emission matrix analysis. In addition, the mechanical strength of hydrogels of extracted polymers cross-linked with Ca2+ ions was investigated by dynamic mechanical analysis. Results showed that the amount of SEPS was reduced by 26% in AGS (SEPSAGS) and by 41% in WAS (SEPSWAS), respectively. Polysaccharides and, to a lesser extent, the proteins in the SEPSAGS were more refractory compared to those in SEPSWAS. This resulted in a lower loss of the gel stiffness of SEPSAGS than that of SEPSWAS during the AD process. Moreover, the release of SEPS from tightly bound EPS to loosely bound EPS were observed in both types of sludge, but that in AGS exhibited a lower transition rate. The observed properties explain the distinct differences in anaerobic biodegradability, the slower decomposition of the sludge structure, as well as the better dewaterability of AGS as compared to WAS after the AD process.

Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

Extracellular protein isolation from the matrix of anammox biofilm using ionic liquid extraction.

Anaerobic ammonium oxidation (anammox)-performing bacteria self-assemble into compact biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion achieved 19.5 ± 0.9 and 41.4 ± 1.4% (w/w) more solubilization of laboratory enriched Candidatus Brocadia sinica anammox granules than DNase and amylase, respectively. Nuclear magnetic resonance profiling of the granules confirmed proteins as dominant biopolymer within the EPS. Ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and N,N-dimethylacetamide (EMIM-Ac/DMAc) mixture was applied to extract the major structural proteins. Further treatment by anion exchange chromatography isolated homologous serine (S)- and threonine (T)-rich proteins BROSI_A1236 and UZ01_01563, which were major components of the extracted proteins, and sequentially highly similar to putative anammox extracellular proteins KUSTD1514 and WP_070066018.1 of Ca. Kuenenia stuttgartiensis and Ca. Brocadia sapporoensis, respectively. Six monosaccharides (i.e., arabinose, xylose, rhamnose, fucose, galactose, and mannose) were enriched for BROSI_A1236 against all other major proteins. The sugars, however, contributed < 0.5% (w/w) of total granular biomass and were likely co-enriched as glycoprotein appendages. This study demonstrates that BROSI_A1236 is a major extracellular component of Ca. B. sinica anammox biofilms that is likely a common anammox extracellular polymer, and can be isolated from the matrix following ionic liquid extraction.

Flame retardant property of flax fabrics coated by extracellular polymeric substances recovered from both activated sludge and aerobic granular sludge.

In this research, extracellular polymeric substances (EPS), such as EPSflocs and EPSgranules, were successfully extracted from activated and aerobic granular sludge, respectively, and tested as bio-based flame retardant materials. Flax fabric was coated by the biopolymeric substances and its flammability was evaluated based on a vertical burning test defined in US Federal Aviation Regulation. Both EPSflocs and EPSgranules coated flax fabrics achieved the self-extinguishment due to effective char formation. In particular, the result of the EPSgranules coated sample met the aviation requirements for the aircraft interior. Moreover, the presence of carbonated hydroxyapatite was identified in EPSgranules char residue by using FTIR and XRD analysis. It can contribute to the self-extinguishing property of the fabric by enhancing char formation. Thermogravimetric analysis also demonstrated that EPSgranules coated flax was able to produce greater amount of char residue and its decomposition rate was significantly reduced. This research indicates that there is a great potential to use this biopolymer as a resource for developing high performance bio-inspired flame retardant materials and contribute to a circular economy.