Fractionation of proteins and carbohydrates of extracellular polymeric substances in a membrane bioreactor system

The major operational problem associated with membrane bioreactors (MBR) is membrane fouling, for which extracellular polymeric substances (EPS) are primarily responsible. In this work both the soluble and bound EPS (i.e. SMP and EPS) produced in an MBR system operating under sludge retention times (SRT) of 10, 15, 20 and 33 days were fractionized by means of membranes having variable molecular weight cutoffs (300 kDa, 100 kDa, 10 kDa & 1 kDa). The results show that increasing the SRT leads to a reduction of SMP and EPS and that these reductions are more pronounced for the SRTs in the range 10–20 days. This reduction is more significant for carbohydrates than for proteins. The decrease of SMP and EPS with increasing SRT from 10 to 20 days led to a significant decrease of the level of fouling. The further increase of SRT to 33 days did not significantly impact on the level of fouling as the SMP and EPS concentrations did not change much.     

Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge

This study extracted the soluble microbial products and loosely bound and tightly bound extracellular polymeric substances (EPS) from suspended sludge from a membrane bioreactor, original and aerobically/anaerobically digested, and compared their fouling potentials on a microfiltration membrane. The resistance of cake layer accounts for 95–98% of the total filtration resistances when filtering the whole sludges, with anaerobically digested sludge presenting the highest resistance among the three tested sludges. The tightly bound EPS has the highest potential to foul the membrane; however, the loosely bound EPS contribute most of the filtration resistances of the whole sludges. The foulants corresponding to the irreversible fouling have chemical fingerprints similar to those from loosely bound EPS, which have a greater predilection to proteins and humic substances than to polysaccharides.     

Impact of sludge retention time on MBR fouling: role of extracellular polymeric substances determined through membrane autopsy

The impact of sludge retention time (SRT) on the biofouling of a membrane bioreactor (MBR) by extracellular polymeric substances (EPS) was investigated. The MBR was operated at 60 and 20 d SRT. The gel layer (recovered through optimized membrane autopsy methods) and the cake layer were analyzed for their content and profile of EPS proteins and polysaccharides. The change to a shorter SRT led to decreased membrane filterability, concomitant with a higher expression of EPS proteins in the cake layer, which were identified as being mainly related with biosynthesis and stress functions. The gel layer was more substantial in internal fibers, with polysaccharides being the major component in this layer. With the decrease in SRT (and filterability decrease), the overall polysaccharide content and sugar variety increased. In conclusion, SRT impacted not only on the quantity but also the composition of EPS molecules, and both were shown to be important in biofouling.     

Soluble microbial products (SMP) and soluble extracellular polymeric substances (EPS) from wastewater sludge

Laspidou and Rittmann (Water Research 36:2711–2720, 2002) proposed that the soluble extracellular polymeric substances (EPS) are identical to soluble microbial products (SMP) in sludge liquor. In this paper, we compared the physicochemical characteristics of the SMP and soluble EPS from original and aerobically or anaerobically digested wastewater sludge. The surface charges, particle sizes, residual turbidities of polyaluminum chloride (PACl) coagulated supernatant, and chemical compositions of the SMP and soluble EPS containing suspensions were used as comparison index. Experimental results revealed that the particles in SMP and soluble EPS fractions extracted from original wastewater sludge, before and after digestion, were not identical in all physicochemical characteristics herein measured. The current test cannot support the proposal by Laspidou and Rittmann (Water Research 36:2711–2720, 2002) that SMP is identical to the soluble EPS from a wastewater sludge.     

Influence of solids retention time on membrane fouling: characterization of extracellular polymeric substances and soluble microbial products

The objective of this study was to investigate the influence of solids retention time (SRT) on membrane fouling and the characteristics of biomacromolecules. Four identical laboratory-scale membrane bioreactors (MBRs) were operated with SRTs for 10, 20, 40 and 80 days. The results indicated that membrane fouling occurred faster and more readily under short SRTs. Fouling resistance was the primary source of filtration resistance. The modified fouling index (MFI) results suggested that the more ready fouling at short SRTs could be attributed to higher concentrations of soluble microbial products (SMP). Fourier transform infrared (FTIR) spectra indicated that the SRT had a weak influence on the functional groups of the total extracellular polymeric substances (TEPS) and SMP. However, the MBR under a short SRT had more low-molecular-weight (MW) compounds (<1 kDa) and fewer high-MW compounds (>100 kDa). Aromatic protein and tryptophan protein-like substances were the dominant groups in the TEPS and SMP, respectively.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR)

Membrane bioreactors (MBR) technology for wastewater offers many advantages over conventional technologies such as high effluent quality, less footprint and others. The main disadvantage of membrane bioreactors (MBR) is related to membrane fouling, which is mainly caused by extracellular polymeric substance (EPS) and soluble microbial products (SMP). This research studied EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR). The SAMBR was operated under two organic loading rates (OLR) (0.79 and 1.56 kg/m³ d) and was fed with synthetic wastewater with glucose as the carbon source. The results showed percentages of chemical oxygen demand (COD) removal above 95% and the highest COD removal rates were observed at the bottom of the reactor (>83%) for both OLR. The EPS showed a stratification with highest quantities in the supernatant. For the SMP the highest concentration was in the bottom of SAMBR where utilization predominated associated products whereas in the SAMBR supernatant predominated biomass associated products. The OLR change led to a significant increase in SMP accumulation but not in EPS. These facts showed that EPS and SMP dynamic in the SAMBR seemed to be mainly influenced by biological activity, total suspended solids concentration and substrate composition.     

Effect of membrane bioreactor configurations on sludge structure and microbial activity

The aim of this paper was to determine the effect of two different membrane bioreactor (MBR) configurations (external/immersed) on sludge structure and microbial activity. Sludge structure was deduced from rheological measurements. The high shear stress induced by the recirculation pump in the external MBR was shown to result in decreasing viscosity due to activated sludge (AS) deflocculation. Besides, soluble microbial products (SMP) release was higher in the external MBR (5 mg_COD g_MLVSS⁻¹) than in the immersed configuration (2 mg_COD g_MLVSS⁻¹). Microbial activity was followed from respirometry tests by focusing on the distinction between heterotrophs and autotrophs. An easier autotrophic microbe development was then observed in the immersed MBR compared to the external one. However, the external MBR was shown to allow better heterotrophic microbe development.     

The effects on operation conditions of sludge retention time and carbon/nitrogen ratio in an intermittently aerated membrane bioreactor (IAMBR)

An intermittently aerated membrane bioreactor (IAMBR) system has been developed to improve the efficiency of nutrient removal, and for the stable treatment of organic matter which is contained as suspended solid (SS) in the influent. The important operating factors of an intermittently aerated bioreactor (IABR) are sludge retention times (SRTs) and carbon/nitrogen (C/N) ratios. Because research on IAMBR is young, this paper explores the effect of SRTs and C/N ratios on these systems. For SRTs of 20, 25, 30, and 40 days, there was little difference in the removal of COD, T–N, and T–P. In comparing C/N ratios of 4.5, 7, and 10, the COD concentration in permeate with a C/N ratio of 10 was most stable, although the concentration of organic matter in the influent was high. The removal efficiencies of T–N and T–P in permeate with a C/N ratio of 10 were the highest at 92.9% and 88.9%, respectively. This implies that a C/N ratio above 10 should be maintained for a nutrient removal efficiency of approximately 90%.     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.     

The impact of different operating conditions on membrane fouling and EPS production

The main goal of this research was to investigate how different factors influence membrane fouling. The impact of the different concentrations of activated sludge and the amount of extracellular polymer substances (EPS) were monitored. Two pilot plants with submerged membrane modules (hollow fiber and flat sheet) were operated and the raw wastewater was used.Humic substances were identified as the major components of EPS in the activated sludge (more than 34%) in both pilot plants. As the basic constituent in permeate, humic substances were identified as the most dominant components in the effluent (61%) in both pilot plants. Conversely, proteins were mostly analyzed in permeate and supernatant below the detection limit. The total amount of EPS [mg g⁻¹ (VSS)] was similar for concentrations of activated sludge 6, 10 and 14 g L⁻¹. Carbohydrates were identified as the component of EPS which tends most to clog membranes.     

Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs

Divalent cations act as bridges among extracellular polymeric substances (EPS) and form cross-linkage for the self-immobilization of microbial biomass. However, their effects on the nitrification performance during the biological nitrogen removal are still unclear. In the present study, the effects of Mg²⁺ on the nitrifying activity, EPS and floc characteristics were investigated using a lab-scale sequencing batch reactor. The distribution of Mg²⁺ was quantified at different level of sludge floc. The results indicated that the nitrification activity was significantly improved when influent Mg²⁺ was below 1.1 mmol/L, but suppressed at 3 mmol/L. The overall performance characterized by COD, NH₄⁺-N and TN, the particle size and sludge flocculation ability rapidly increased with the increase of Mg²⁺ concentration. Mg²⁺ was mainly distributed in the pellet and changed slightly in supernatant, LB-EPS and TB-EPS. The four fluorescence peaks detected by three-dimensional excitation-emission matrix spectra were attributed to PN-like substances and humic acid-like substances in the LB-EPS and TB-EPS. The results of XPS analysis demonstrated that LB-EPS and TB-EPS comprised similar elements. Therefore, the types of EPS functional groups was unchanged under varied Mg²⁺ concentrations, while their proportions changed and LB-EPS/EPS was key factor for the changes of bioflocculation.     

Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus

Sorption of Cu(II) and Cd(II) onto the extracellular polymeric substances (EPS) produced by Aspergillus fumigatus was investigated for the initial pH of the solution, EPS concentrations, contact time, NaCl concentration, initial metal ion concentration and the presence of other ions in the solution. The results showed that the adsorption of metal ions was significantly affected by pH, EPS concentrations, initial metal concentration, NaCl concentration and co-ions. The sorption of Cu(II) and Cd(II) increased with increasing pH and initial metal ion concentration but decreased with an increase in the NaCl concentration. The maximum sorption capacities of A. fumigatus EPS calculated from the Langmuir model were 40 mg g⁻¹ EPS and 85.5 mg g⁻¹ EPS for Cu(II) and Cd(II), respectively. The binary metal sorption experiments showed a selective metal binding affinity in the order of Cu(II) > Pb(II) > Cd(II). Both the Freundlich and Langmuir adsorption models described the sorption of Cu(II) and Cd(II) by the EPS of A. fumigatus adequately. Fourier transform infrared spectroscopy (FTIR) analysis revealed that carboxyl, amide and hydroxyl functional groups were mainly correlated with the sorption of Cu(II) and Cd(II). Energy dispersive X-ray (EDX) system analysis revealed that the ion-exchange was an important mechanism involved in the Cu(II) and Cd(II) sorption process taking place on EPS.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.     

MHR、sCD14-ST、ApoM与糖尿病足溃疡预后的关系及对预后的预测价值

摘要 目的：探讨单核细胞数/高密度脂蛋白胆固醇比值（MHR）、可溶性白细胞分化抗原14亚型（sCD14-ST）、载脂蛋白M（ApoM）与糖尿病足溃疡（DFU）患者预后的关系及对预后的预测价值。方法：选取2018年1月-2020年12月本院收治的100例DFU患者资料进行回顾性分析,记录其性别、年龄、病程、体重指数（BMI）等一般临床资料以及空腹血糖、MHR、sCD14-ST、ApoM等实验室指标,并对纳入的100例患者进行6个月的随访,获得患者DFU愈合、截肢及死亡例数。采用单因素分析以及二元Logistics回归分析MHR、sCD14-ST、ApoM与DFU患者预后的关系,并采用ROC曲线检测MHR、sCD14-ST、ApoM水平及三项指标联合检测对不良预后的预测价值。结果：预后不良患者DFU病程、Wagner分级、MHR、sCD14-ST、ApoM水平均与预后良好患者有统计学差异（P＜0.05）。随着MHR、sCD14-ST水平的升高,不良结局发生率呈上升趋势（P＜0.05）;随着ApoM水平升高,不良结局发生率呈下降趋势（P＜0.05）。二元Logistic回归分析结果显示,DFU病程、MHR、sCD14-ST为DFU患者不良结局发生的独立危险因素（P＜0.05）,而ApoM为保护因素（P＜0.05）。ROC曲线分析显示,MHR、sCD14-ST、ApoM以及三项指标联合检测对DFU患者不良结局发生的预测线下面积（AUC）分别为0.731、0.729、0.763和0.864,其中联合检测的预测效能最高,其预测敏感度为68.57%、特异度为89.23%。结论：MHR、sCD14-ST、ApoM均与DFU患者的预后存在相关性,三项指标联合检测具有一定的预后预测价值。     

The relationship between Rb+ influx and K+-stimulated Mg2+-ATPase activity in oat roots of different K+ status flexible coupling?

The relationship between Rb⁺ influx and microsomal ATPase activity stimulated by K⁺ and Mg²⁺+ K⁺ was investigated for roots of 7-day-old seedlings of oat (Avena sativa L., cv. Brighton). Different concentrations of K⁺ in the roots, K⁺_root were produced by cultivating plants in complete nutrient solutions of different dilutions and dFifferent K⁺ concentrations at various temperatures. Experiments were performed in both light and darkness. The range of the influx/ATPase ratios was large with a factor of 5 or more between the highest and lowest values. In most cases, the highest ratios were obtained at low K⁺_root and at high temperatures, and the lowest at high K⁺_root and at low temperatures. At high temperatures (20 and 25°C) in the light, the influx/ATPase ratio was constant, independently of K⁺_root, if K⁺ in the medium was kept constant but the bulk of the nutrient solution diluted. If K⁺ was varied and the other components of the medium kept constant, the normal relation of decreasing influx/ATPase ratio at increasing K⁺_root was found; thus, Rb⁺ influx appears regulated by both the internal and external potassium conditions. Also in darkness, at 15°C and with K ⁺ in the medium varied, the influx/ATPase ratio was independent of K⁺_root but in the corresponding light experiments, ratio and K⁺_root had the normal, inverse relationship. The difference between light and dark conditions appears to indicate that growth rate is of importance for the relationship between energy input and transport. Our data lead to the concept of “flexible coupling” between transducers) of energy and ion carrier. Without excluding other possibilities, this may be one of the mechanisms for ecological adaptation to variations in the root medium.     

Correlation between the activity of membrane-bound ATPase and the decay rate of flash-induced 515-nm absorbance change in chloroplasts in intact leaves,assayed by means of rapid isolation of chloroplasts

(1) The relationship between activation of the membrane-bound ATPase and the stimulation of dissipation of the flash-induced membrane potential by preillumination was studied in intact spinach leaves by measuring the ATPase activity of rapidly isolated chloroplasts and the decay of the flash-induced 515-nm absorbance change (ΔA₅₁₅) in intact leaves. (2) The decay of ΔA₅₁₅ was accelerated by preillumination. The ΔA₅₁₅ decay in leaves treated with N,N′-dicyclohexylcarbodiimide (DCCD) became slower and was not accelerated by preillumination. However, treatment with DCCD did not lower the intensity of delayed fluorescence. (3) Membrane-bound ATPase of chloroplasts which were rapidly isolated from the preilluminated leaves (90 s preparation time) showed a higher activity (over 200 μmol P_i/mg chlorophyll per h in the case of 2-min preillumination) than that of chloroplasts isolated from dark-adapted leaves. (4) The acceleration of ΔA₅₁₅ decay and the activation of ATPase showed similar dependences on illumination time in intact leaves. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea, carbonyl cyanide p-chlorophenylhydrazone and DCCD inhibited the activation of ATPase and the acceleration of the ΔA₅₁₅ decay by preillumination. (5) The ATPase activity of chloroplasts isolated from illuminated leaves showed a single exponential decay (‘dark inactivation in vitro’). The ATPase activity induced by illuminating the leaves became lower as the dark interval between illumination and the isolation of chloroplasts was increased (‘dark inactivation in vivo’). The time course of the decay of activity had a lag and showed a sigmoidal curve when plotted semilogarithmically. The decay had an apparent half-time of 25 min. (6) The recovery of the accelerated ΔA₅₁₅ decay in preilluminated leaves to the original slow rate showed a sigmoidal decay similar to that of the activity of ATPase in intact leaves with a half-time of about 23 min in the dark. (7) It was concluded that the decay rate of ΔA₅₁₅ reflected the chloroplast ATPase activity in intact leaves and that the ion conductance of thylakoid membrane was mainly determined by the H⁺ flux through the ATPase, the activity of which was increased after the formation of the high-energy state.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.