Fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor

Microbial products formed in biological wastewater treatment systems are closely related to system performance and status, and many of them have fluorescence spectral characteristics. In this work, the fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor were studied using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. Since the components of the microbial products are complex, the parallel factor analysis (PARAFAC) method was used to extract the real spectra from the overlapped spectra. Two principal components were identified from the EEM spectra. The peaks at excitation–emission maxima of 280/350 and 350/440 nm were, respectively, attributed to the fluorescence of proteins and NADH. Their real concentrations were quantified using the PARAFAC coupled with the second-order calibration method. Results show that the formation rate of proteins was correlated to the production rate of hydrogen and volatile fatty acids, as well as the substrate degradation rate. A close correlation between the hydrogen partial pressure and the two fluorophores was found out. This study provides a reliable and convenient approach, which could be potentially used for monitoring the wastewater treatment reactor performance through measuring the fluorescence spectra of the supernatant.     

SMP production by activated sludge in the presence of a metabolic uncoupler, 3,3′,4′,5-tetrachlorosalicylanilide (TCS)

3,3',4',5-Tetrachlorosalicylanilide (TCS) is an effective metabolic uncoupler utilized for microbial yield reduction. However, its potential impact, in particular on the soluble microbial products (SMP) formation, is unknown yet. Herein we study the effect of TCS on SMP production and analyze the related mechanism. The addition of TCS in activated sludge system led to an increased production of SMP, especially proteins. The SMP were produced in proportion to the substrate utilization at a low TCS concentration, while more non-substrate-associated SMP were released at a high TCS concentration. TCS simulated the production of extracellular polymeric substances (EPS) and enhanced cell lysis, which both contributed to SMP production. FTIR and EEM analyses show that the SMP, EPS, and cell lysis products have similar functional groups and fluorescence properties, indicating a similar origin of these substances. In addition, a dose of TCS increased the release of high molecular weight compounds due to cell lysis. This study might benefit for a better understanding of the response of activated sludge to metabolic uncouplers like TCS.     

Development of mechanistically based model for simulating soluble microbial products generation in an aerated/non-aerated SBR

Soluble microbial products (SMPs) are considered as the main organic components in wastewater treatment plant effluent from biological wastewater treatment systems. To investigate and explore SMP metabolism pathway for further treatment and control, two innovative mechanistically based activated sludge models were developed by extension of activated sludge model no.3 (ASM3). One was the model by combining SMP formation and degradation (ASM3-SMP model) processes with ASM3, and the other by combining both SMP and simultaneous substrate storage and growth (SSSG) mechanisms with ASM3 (SSSG-ASM3-SMP model). The detailed schematic modification and process supplements were introduced for comprehensively understanding all the mechanisms involved in the activated sludge process. The evaluations of these two models were demonstrated by a laboratory-scale sequencing batch reactor (SBR) operated under aerated/non-aerated conditions. The simulated and measured results indicated that SMP comprised about 83% of total soluble chemical oxygen demand (SCOD) in which biomass-associated products (BAPs) were predominant compared with utilization-associated products (UAPs). It also elucidated that there should be a minimum SMP value as the reactive time increases continuously and this conclusion could be used to optimize effluent SCOD in activated sludge processes. The comparative results among ASM3, ASM3-SMP and SSSG-ASM3-SMP models and the experimental measurements (SCOD, ammonia and nitrate nitrogen) showed clearly the best agreement with SSSG-ASM3-SMP simulation values (R = 0.993), strongly suggesting that both SMP formation and degradation and SSSG mechanisms are necessary in biologically activated sludge modeling for municipal wastewater treatment.     

Characterizing the photochemical degradation of aquatic humic substances from a dystrophic lake using excitation-emission matrix fluorescence spectroscopy and parallel factor analysis

Three-dimensional excitation-emission matrix (EEM) fluorescence and parallel factor analysis (PARAFAC) were used to monitor composition and reactivity changes caused by the photochemical degradation of aquatic humic substances (AHS) from a dystrophic lake in Kushiro Wetland, Japan. AHS-rich lake water was exposed to three treatments in summer and winter 2014: radiation with the full solar wavelength range, radiation with the >320-nm solar wavelength range, and no solar radiation. Irradiation caused AHS-like peaks to shift to shorter wavelengths in the EEM contour plots, implying that AHS photodegradation caused the formation of lower-molecular-weight fractions or more simply structured components. Three components were identified from PARAFAC analyses: AHS-1 (excitation/emission wavelengths of maxima: <252 and 315 nm/426 nm), AHS-2 (360 and 261 nm/489 nm), and AHS-3 (276 nm/403 nm). These components had different photosensitivities. AHS-1 was most sensitive to full solar radiation, while AHS-2 was most sensitive to >320-nm radiation. More photodegradation of these components occurred in the summer than in the winter, indicating that photodegradation depended on light intensity. AHS-3 was photoresistant. The different characteristics of the components reflected the in situ dynamics of the components. The AHS-3 fluorescence intensity was positively correlated with the dissolved organic carbon concentration but the AHS-1 and AHS-2 fluorescence intensities were not. The EEM–PARAFAC method was found to be a good tool for tracing AHS-like materials in situ and in the laboratory.     

Tracking variations in fluorescent-dissolved organic matter in an aerobic submerged membrane bioreactor using excitation–emission matrix spectra combined with parallel factor analysis

In this study, the variations in the fluorescent components of dissolved organic matter (DOM) were tracked for an aerobic submerged membrane bioreactor (MBR) at three different operation stages (cake layer formation, condensation, and after cleaning). The fluorescent DOM was characterized using excitation–emission matrix (EEM) spectroscopy combined with parallel factor analysis (PARAFAC). Non-aromatic carbon structures appear to be actively involved in the membrane fouling for the cake layer formation stage as revealed by much higher UV-absorbing DOM per organic carbon found in the effluent versus those inside the reactor. Four fluorescent components were successfully identified from the reactor and the effluent DOMs by EEM-PARAFAC modeling. Among those in the reactor, microbial humic-like fluorescence was the most abundant component at the cake layer formation stage and tryptophan-like fluorescence at the condensation stage. In contrast to the reactor, relatively similar composition of the PARAFAC components was exhibited for the effluent at all three stages. Tryptophan-like fluorescence displayed the largest difference between the reactor and the effluent, suggesting that this component could be a good tracer for membrane fouling. It appears that the fluorescent DOM was involved in membrane fouling by cake layer formation rather than by internal pore adsorption because its difference between the reactor and the effluent was the highest among all the four components, even after the membrane cleaning. Our study provided an insight into the fate and the behavior fluorescent DOM components for an MBR system, which could be an indicator of the membrane fouling.     

Fluorescence spectroscopy as a potential metabonomic tool for early detection of colorectal cancer

Fluorescence spectroscopy Excitation Emission Matrix (EEM) measurements were applied on human blood plasma samples from a case control study on colorectal cancer. Samples were collected before large bowel endoscopy and included patients with colorectal cancer or with adenomas, and from individuals with other non malignant findings or no findings (N = 308). The objective of the study was to explore the possibilities for applying fluorescence spectroscopy as a tool for detection of colorectal cancer. Parallel Factor Analysis (PARAFAC) was applied to decompose the fluorescence EEMs into estimates of the underlying fluorophores in the sample. Both the pooled score matrix from PARAFAC, holding the relative concentrations of the derived components, and the raw unfolded spectra were used as basis for discrimination models between cancer and the various controls. Both methods gave test set validated sensitivity and specificity values around 0.75 between cancer and controls, and poor discriminations between the various controls. The PARAFAC solution gave better options for analyzing the chemical mechanisms behind the discrimination, and revealed a blue shift in tryptophan emission in the cancer patients, a result that supports previous findings. The present findings show how fluorescence spectroscopy and chemometrics can help in cancer diagnostics, and with PARAFAC fluorescence spectroscopy can be a potential metabonomic tool.     

Formation of soluble microbial products by activated sludge under anoxic conditions

In this work, both experimental and modeling approaches are used to explore the formation of soluble microbial products (SMP) by activated sludge under anoxic conditions. With substrate consumption, the SMP concentration increases gradually. Utilization associated products (UAP) are the main fraction of SMP when substrate is present; whereas biomass associated products (BAP) are the major content of SMP as substrate is completely consumed. The fraction of the accumulated SMP accounts for 3-4% of initial organic substrate. Three dimensional excitation emission matrix analysis results indicate that the SMP concentration increases in the denitrification process. The accumulation of nitrite up to 22.6 mg/l under anoxic conditions has no significant effect on the SMP formation. With a consideration of SMP formation under anoxic conditions, an ASM3-based denitrification model is developed. The results show that the developed model is able to capture the relationship between the SMP formation and the substrate consumption by activated sludge in the denitrification process.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Erratum to: Fluorescence spectroscopy as a potential metabonomic tool for early detection of colorectal cancer

Fluorescence spectroscopy Excitation Emission Matrix (EEM) measurements were applied on human blood plasma samples from a case control study on colorectal cancer. Samples were collected before large bowel endoscopy and included patients with colorectal cancer or with adenomas, and from individuals with other non malignant findings or no findings (N = 308). The objective of the study was to explore the possibilities for applying fluorescence spectroscopy as a tool for detection of colorectal cancer. Parallel Factor Analysis (PARAFAC) was applied to decompose the fluorescence EEMs into estimates of the underlying fluorophores in the sample. Both the pooled score matrix from PARAFAC, holding the relative concentrations of the derived components, and the raw unfolded spectra were used as basis for discrimination models between cancer and the various controls. Both methods gave test set validated sensitivity and specificity values around 0.75 between cancer and controls, and poor discriminations between the various controls. The PARAFAC solution gave better options for analyzing the chemical mechanisms behind the discrimination, and revealed a blue shift in tryptophan emission in the cancer patients, a result that supports previous findings. The present findings show how fluorescence spectroscopy and chemometrics can help in cancer diagnostics, and with PARAFAC fluorescence spectroscopy can be a potential metabonomic tool.

     

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.     

Microbial transformation of biomacromolecules in a membrane bioreactor: implications for membrane fouling investigation

Background

The complex characteristics and unclear biological fate of biomacromolecules (BMM), including colloidal and soluble microbial products (SMP), extracellular polymeric substances (EPS) and membrane surface foulants (MSF), are crucial factors that limit our understanding of membrane fouling in membrane bioreactors (MBRs).

Findings

In this study, the microbial transformation of BMM was investigated in a lab-scale MBR by well-controlled bioassay tests. The results of experimental measurements and mathematical modeling show that SMP, EPS, and MSF had different biodegradation behaviors and kinetic models. Based on the multi-exponential G models, SMP were mainly composed of slowly biodegradable polysaccharides (PS), proteins (PN), and non-biodegradable humic substances (HS). In contrast, EPS contained a large number of readily biodegradable PN, slowly biodegradable PS and HS. MSF were dominated by slowly biodegradable PS, which had a degradation rate constant similar to that of SMP-PS, while degradation behaviors of MSF-PN and MSF-HS were much more similar to those of EPS-PN and EPS-HS, respectively. In addition, the large-molecular weight (MW) compounds (>100 kDa) in BMM were found to have a faster microbial transformation rate compared to the small-MW compounds (<5 kDa). The parallel factor (PARAFAC) modeling of three-dimensional fluorescence excitation-emission matrix (EEM) spectra showed that the tryptophan-like PN were one of the major fractions in the BMM and they were more readily biodegradable than the HS. Besides microbial mineralization, humification and hydrolysis could be viewed as two important biotransformation mechanisms of large-MW compounds during the biodegradation process.

Significance

The results of this work can aid in tracking the origin of membrane foulants from the perspective of the biotransformation behaviors of SMP, EPS, and MSF.     

Membrane fouling properties under different filtration modes in a submerged membrane bioreactor

Four flat-sheet membrane modules, which were operated under four different filtration modes but with the same treatment capacity, were used to treat synthetic wastewater in a submerged membrane bioreactor (MBR). Particle size distribution (PSD), gel filtration chromatography (GFC), capillary suction time (CST), and three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy were used to characterize membrane fouling properties. The high instantaneous flux induced faster fouling rate and continuous filtration mode was the most applicable filtration mode in this study. The average particle size of all foulants was smaller than that of bulk sludge; and the higher the instantaneous flux was adopted, the larger the average particle size of foulants would be. Only macromolecule substances were detected in all the foulants. The macromolecule substances in the influent were degraded by microorganism and retained by membrane, and small molecular substances could pass through membrane pores to enter the effluent. The membrane foulants had poorer dewaterability compared to the mixed liquor confirmed by CST measurement. Although there were several peaks associated with protein-like fluorophores, fulvic acid-like substances and humic acid-like organics in soluble microbial products (SMP) and extracellular polymeric substances (EPS) sample, it was found that the dominant fluorescence substances in membrane foulants were protein-like substances.     

The effect of hydraulic retention time on the performance and fouling characteristics of membrane sequencing batch reactors used for the treatment of synthetic petroleum refinery wastewater

The use of membrane sequencing batch reactors, operated at HRT of 8, 16 and 24 h, was considered for the treatment of a synthetic petroleum wastewater. Increase in HRT resulted in statistically significant decrease in MLSS. Removal efficiencies higher than 97% were found for the three model hydrocarbon pollutants at all HRTs, with air stripping making a small contribution to overall removal. Particle size distribution (PSD) and microscopic analysis showed reduction in the protozoan populations in the activated sludge with decreasing HRT. PSD analysis also showed a higher proportion of larger and smaller sized particles at the lowest HRT. The rate of membrane fouling was found to increase with decreasing HRT; SMP, especially carbohydrate SMP, and mixed liquor apparent viscosity also showed a pronounced increase with decreasing HRT, whereas the concentration of EPS and its components decreased. FTIR analysis identified organic compounds as the main component of membrane pore fouling.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

Effect of 24-epibrassinolide on pea protein tyrosine phosphorylation after salinity action

The effects of the brassinosteroid hormone 24-epibrassinolide (EPB) on growth indices and tyrosine phosphorylation of soluble proteins in pea (Pisum sativum L.) leaves after salinity action were studied. Treatment of plants subjected to salinity (150 mM NaCl for 2 days) with 100 nM EPB for 2 h resulted in the resumed shoot growth. The analysis of phytohormone-induced changes in the tyrosine phosphoproteome after NaCl treatment showed that EPB suppressed tyrosine phosphorylation of proteins functioning in the dark stage of photosynthesis. This is evidently related to the recovery of photosynthetic activity and, as a consequence, of plant growth indices.     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

The intrinsic fluorescence of isolated central-nervous-system myelin-sheath preparations.

The intrinsic fluorescence characteristics of tyrosine and tryptophan residues in the proteins of isolated central-nervous-system myelin were investigated to gain information concerning the location of these residues within the intact membrane system. Tryptophan fluorescence from isolated myelin has an emission maximum at 325 nm that appears to arise from at least two different populations of tryptophan residues. Further evidence for heterogeneity of tryptophan location in the membrane is obtained from quenching studies with chloroform and acrylamide. It is speculated that one tryptophan population is hydrophobically situated and may be derived from the proteolipid protein of myelin, whereas the other tryptophan population is located at the membrane surface and may arise from the extrinsic basic protein. A significant tyrosine fluorescence is detected from isolated myelin, indicating that some of these residues are not quenched by structural interactions within the lipid--protein membrane system. Studies with freeze-dried resuspended myelin suggest that the structural arrangement of protein components in the dried rehydrated membrane system differs significantly from that of the freshly isolated myelin membrane.     

A review towards finding a simplified approach for modelling the kinetics of the soluble microbial products (SMP) in an integrated mathematical model of membrane bioreactor (MBR)

Soluble microbial products (SMPs) tend to accumulate in the membrane bioreactor (MBR) systems as a consequence of high membrane rejection and apparently low biodegradability within the wastewater treatment system. The extension of the activated sludge models (ASMs) with SMPs, therefore, has received crucial importance in recent days, particularly considering their potential use as indicators of the membrane fouling propensity. This paper presents a critical review of the formation and degradation kinetics of SMP subdivisions that have so far been used for the mathematical modelling of MBR. The paper identified a simplified approach to incorporate the kinetics of the SMP formation and degradation in the general mathematical models of MBR. It suggested that the inclusion of only four additional linear differential equations in the ASM1-SMP integrated mathematical model could simulate well the effluent quality and membrane fouling prediction. The model would also serve as a useful tool in optimizing operation conditions for better treatability and fouling control.     

THE INTRINSIC FLUORESCENCE CHARACTERISTICS OF THE MYELIN BASIC PROTEIN

—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m -urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head-groups. The fluorescence results suggest that the basic protein can be used as a natural, non-perturbing probe which will report on its environment after it has reacted with other membrane components.     

Temporal variations of membrane foulants in the process of using flat-sheet membrane for simultaneous thickening and digestion of waste activated sludge

Membrane foulants were extracted at different operation time in simultaneous sludge thickening and digestion reactors using flat-sheet membranes. Temporal variations of foulants were analyzed by three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy, gel filtration chromatography (GFC), particle size distribution (PSD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Results showed that during the first 4 days fouling was mainly assigned to internal membrane foulants (IMFs), and afterwards external membrane foulants (EMFs) increased dramatically. EEM analysis showed that both IMFs and EMFs changed during the operation. Cluster analysis demonstrated that the characteristics of IMFs were relatively similar; however, both quantity and properties of EMFs were changed. GFC analysis showed that EMFs contained more molecules with large molecular weight compared to IMFs. PSD analysis illuminated that particle size of EMFs gradually increased and was larger than that of IMFs. ATR-FTIR analysis indicated that the foulants on membranes consisted of polysaccharides and proteins.