Global Profiling of Metabolite and Lipid Soluble Microbial Products in Anaerobic Wastewater Reactor Supernatant Using UPLC-MSE.

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

Chemical Characterization of Low Molecular Weight Soluble Microbial Products in an Anaerobic Membrane Bioreactor.

Effluents from wastewater treatment systems contain a variety of organic compounds, including end products from the degradation of influent substrates, nonbiodegradable feed compounds, and soluble microbial products (SMPs) produced by microbial metabolism. It is important to identify the major components of these SMPs to understand what is in wastewater effluents. In this study, physical pretreatments to extract and concentrate low molecular weight SMPs (MW< 580 Da) from effluents were optimized. Liquid-liquid extraction (LLE) of a 200 mL effluent sample showed the best performance using a mixture of n-hexane, chloroform, and dichloromethane (70 mL) for extraction. For solid phase extraction (SPE), two OasisHLB cartridges were connected in-line to optimize recovery, and the eluted samples from each cartridge were analyzed separately to avoid overlapping peaks. Four solvents varying from polar to nonpolar (methanol, acetone, dichloromethane, and n-hexane) were selected to maximize the number of compound peaks eluted. A combination of SPE (OasisHLB) followed by LLE was shown to maximize compound identification and quantification. However, the compounds identified accounted for only 2.1 mg of chemical oxygen demand (COD)/L (16% of total SMP as COD) because many SMPs have considerably higher MWs. Finally, the method was validated by analyzing a variety of different reactor effluents and feeds.

"Protein" Measurement in Biological Wastewater Treatment Systems: A Critical Evaluation.

Five commercially available assay kits were tested on the same protein sample with the addition of 17 different types of interfering substances typically found in the biological wastewater treatment, and a comparison of the use of these assays with 22 different protein and peptide samples is also presented. It was shown that a wide variety of substances can interfere dramatically with these assays; the metachromatic response was also clearly influenced by different proteinaceous material. Measurement of the "protein" content in the effluent of an anaerobic membrane bioreactor was then carried out using these assay methods. Quantitative results of the "protein" concentration in the different effluent samples, with or without spiked additions of Bovine Serum Albumin (BSA), showed considerable disagreement. We concluded that the "protein" measured in wastewater samples using standard colorimetric assays often shows false positive results and has little correlation to their real value. A new analytical method needs to be developed in order to gain greater insight into the biological transformations occurring in anaerobic digestion, and how soluble microbial products (SMPs) are produced.

Characterization and biodegradability of sludge from a high rate A-stage contact tank and B-stage membrane bioreactor of a pilot-scale AB system treating municipal wastewaters.

In light of global warming mitigation efforts, increasing sludge disposal costs, and need for reduction in the carbon footprint of wastewater treatment plants, innovation in treatment technology has been tailored towards energy self-sufficiency. The AB process is a promising technology for achieving maximal energy recovery from wastewaters with minimum energy expenditure and therefore inherently reducing excess sludge production. Characterization of this novel sludge and its comparison with the more conventional B-stage sludge are necessary for a deeper understanding of AB treatment process design. This paper presents a case study of a pilot-scale AB system treating municipal wastewaters as well as a bio- (biochemical methane potential and adenosine tri-phosphate analysis) and physico-chemical properties (chemical oxygen demand, sludge volume index, dewaterability, calorific value, zeta potential and particle size distribution) comparison of the organic-rich A-stage against the B-stage activated sludge. Compared to the B-sludge, the A-sludge yielded 1.4 to 4.9 times more methane throughout the 62-week operation.

Worldwide surveys of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in water environment in recent years.

Recently, perfluorinated compounds (PFCs) have been noted as causes of some of the important environmental problems in recent years due to their occurrences and properties. The most commonly used PFCs are perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which have been used in many kinds of products. They have been found in surface water and tap water in both developed and developing countries around the world including in North America, Europe and Asia. In most countries, rivers are the source of tap water, which is one of the important pathways in which PFCs reach humans. It is essential to evaluate PFOS and PFOA contamination in the river basin. The purpose of this field study was to determine the presence of PFOS and PFOA in rivers around the world. The surveys were conducted in 15 countries during 2004 to 2010. In total, 539 samples were collected from the rivers in 41 cities. A solid phase extraction (SPE) coupled with HPLC-ESI-MS/MS were used for the analysis of these chemicals. PFOS and PFOA were detected in all 41 cities not only in industrialized areas but also in non-industrialized areas, representing that these compounds undergo long-range transportation in the environment. The average concentration of PFOS in each city ranged from not detected to 70.1 ng/L. The average concentration of PFOA in each city was in the range 0.2-1,630.2 ng/L. The industrialized areas show higher contamination in both PFOS and PFOA concentrations than non-industrialized areas. Industrial activities are some of the major sources of PFCs contamination in rivers.

Determination of perfluorinated compounds (PFCs) in solid and liquid phase river water samples in Chao Phraya River, Thailand.

Perfluorinated compounds (PFCs), especially perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are fully fluorinated organic compounds, which have been used in many industrial applications. These chemicals have contaminated surface water all over the world even in developing countries like Thailand. The previous study showed the contamination in Chao Phraya River in 2006 and 2007. The purposes of this field study were to determine the solid and liquid phase of PFCs contamination in Chao Phraya River and to compare the changes of PFC concentration in 2008. Surveys were conducted in the lower reach of Chao Phraya River in the industrialized area. A solid phase extraction (SPE) coupled with HPLC-ESI-MS/MS were used for the analysis for ten PFCs. Ten PFCs were analyzed to identify the contamination in both solid and liquid phases. PFCs were detected in both the solid and liquid phase in every sample. PFOA was the most dominant PFC while PFPA and PFOS were also highly detected in most samples. The average loadings of PFPA, PFOA and PFOS in Chao Phraya River were 94.3, 284.6 and 93.4 g/d, respectively. PFOS concentrations did not show differences between 2006 and 2008. However, PFOA concentrations were higher in 2008/5/26, while comparing other samplings. The ratio of solid:liquid PFPA (2.1:1.0) [(ng/g)/(ng/L)] was lower than PFOA (13.9:1.0) [(ng/g)/(ng/L)] and PFOS (17.6:1.0) [(ng/g)/(ng/L)]. The shorter chain (more hydrophilic) PFC was better to dissolve in water rather than adsorb onto suspended solids. PFOS also showed more potential to attach in the suspended solids than PFOA.

Perfluorinated compounds (PFCs) in Yodo River system, Japan.

There is increasing concern about occurrences of perfluorinated compounds (PFCs) in the environment due to their persistent, bioaccumulation, and potentially toxic effects. We investigated contamination of 11 PFCs (C4-C12) in the Yodo River system, which is a major source of drinking water for more than 11 million people of Kansai region in Japan. PFCs were detected in higher concentration even exceeding more than 1000 ng/L in some cases. Composition profile of total PFCs concentration showed that PFOS, PFHxA, PFOA, and PFNA were occurring dominantly in the river system. PFOS and PFOA concentration were nearly proportional to a previous study in the same river system, indicating continuous sink of the compounds into the river system. Although discharge of PFCs from wastewater effluents at upstream could be one of the major point sources, concentration did not exceed 29.4 ng/L at the downstream of main Yodo River, possibly, due to dilution effects. Total mass load of PFCs in mainstream of Yodo River was estimated 451.7 g/d based on observed river discharge and PFCs concentration. Results showed that Yodo River system could have a unique upstream-downstream linkage of PFCs contamination and the river could be a continuous pathway of PFCs exposure to the people of Kansai region.

Comparison of soluble microbial product (SMP) production in full-scale anaerobic/aerobic industrial wastewater treatment and a laboratory based synthetic feed anaerobic membrane system.

This study focused on the characterisation of soluble microbial products (SMPs) produced from a full-scale multi-stage (anaerobic/aerobic) industrial wastewater treatment plant, and contrasted them to the SMPs detected in the effluent of a lab-scale AnMBR treating synthetic wastewater to determine if there were any common solutes detected irrespective of the feed organics. Recently developed analytical methods using gas chromatography coupled mass spectrometry (GC-MS) and liquid chromatography coupled quadrupole-time-of-flight (LC-Q-ToF) for SMP characterisation in a wide molecular weight (MW) range of 30-2000 Da (Da) were applied. Samples collected from the Industrial Wastewater plant were the upflow anaerobic sludge blanket (UASB) influent and effluent, and aerobic membrane bioreactor (MBR) effluent before discharge. The GC-MS detected a spike in cyclooctasulphur in the UASB effluent, an indicator of shock-loading, which disappeared after the MBR process. Alkanes, acids and nitrogenous compounds were found to be the end-products from the GC-MS results, while LC-Q-ToF analysis revealed that eicosanoids, a group of cell-signalling molecules, were produced in the aerobic MBR, and made up 71% of its effluent. A comparison of the submerged anaerobic membrane bioreactor (SAMBR) and aerobic MBR effluents using GC-MS showed that there was only a small degree of similarity between the SMPs, comprising mainly long chain alkanes and phthalate. On the other hand, LC-Q-ToF showed a large contrast in compound composition, mostly having cell-signalling functions, which deepened our understanding of the different metabolic processes occurring in aerobic and anaerobic systems. These data could be useful for future work in various areas such as controlling quorum-sensing and biofilm formation, process optimisation and control, and microbial ecology.

Occurrences and behavior of perfluorinated compounds (PFCs) in several wastewater treatment plants (WWTPs) in Japan and Thailand.

This study examines occurrences of 11 perfluorinated compounds (PFCs) in several wastewater treatment plants in Japan and Thailand. Surveys are conducted in eight wastewater treatment plants (WWTPs) in Japan and central WWTPs of five industrial estates (IEs) in Thailand. Samples are collected from all major treatment processes in order to understand the behavior of PFCs in WWTPs. PFCs are detected in all WWTPs in Japan and Thailand. Concentrations of PFCs even exceed several thousands ng/L in some WWTPs. PFOS, PFOA, and PFNA are mainly detected in WWTPs in Japan, while PFBuS, PFOA, and PFHxA are mainly detected in WWTP of IEs in Thailand. Even though some of the investigated WWTPs utilize biological treatment processes coupled with chlorination, ozonation, or activated carbon adsorption, they are found ineffective to remove PFCs. During the treatment process, PFCs are found to accumulate at exceptionally high concentration levels in the activated sludge of an aeration tank and returned activated sludge. Overall, the estimated total daily mass of discharged PFCs is 124.95 g/d (PFASs: 49.81 g/d; PFCAs: 75.14 g/d) from eight WWTPs in Japan and 55.04 g/d (PFASs: 12 g/d; PFCAs: 43.04 g/d) from five WWTPs in Thailand. Although the presented data are from a single observation in each WWTP, the results indicate that certain industries using PFCs in manufacturing processes could be the principle point source, while domestic activities could be releasing PFCs at detectable levels causing environmental concern.

Identification of the production and biotransformational changes of soluble microbial products (SMP) in wastewater treatment processes: A short review.

While the definition of soluble microbial products (SMP) remains somewhat contentious, they have been widely accepted to be the pool of organic compounds which are released by cells into their surroundings (liquid or otherwise) due to substrate metabolism and biomass decay. SMPs are also potential precursors of disinfection by-products, and are known to be important in membrane fouling. With recent developments in analytical methodologies, many of the low molecular weight (MW) compounds can now be identified, although they are often incorrectly identified as recalcitrant compounds present in the influent. The old hypothesis of "microbial infallibility" suggested that all organic compounds produced by bacteria will eventually be degraded by microorganisms. However, there are some limitations to this hypothesis due to; the time available for degradation, the rate of activity of the microorganisms themselves, synergistic effects, as well as the degree of complexity of the chemical substance. Therefore, it is important to identify and characterise the SMPs involved in these processes, which can then in turn support the research and development of improving wastewater treatment efficiency and effectiveness, and eventually reduce environmental damage. In addition, it is still unclear what the evolutionary purpose of these compounds are. This paper reviews the work that has been done on the production and biotransformation of chemical compounds up to now and which were reported to be found in wastewater treatment systems.

Composition and biotransformational changes in soluble microbial products (SMPs) along an anaerobic baffled reactor (ABR).

This work examined the production and catabolism/biotransformation dynamics of SMPs down the length of an eight-compartment-anaerobic baffled reactor (ABR) which physically separates the biological processes, in contrast to completely mixed reactors which do not enable these dynamics to measured, and this is totally novel. SMPs were extracted and characterised by gas and liquid chromatography coupled mass spectrometry to determine their composition and production/catabolism. 60%-70% of the feed compounds decreased from the first to fourth compartment; the increase in SMPs after the fourth compartment suggested a mixture of degraded and biotransformed compounds, and microbial products. High concentrations of low MW alkanes and alkenes, and higher MW (up to 2000 Da) lipids and amino acid derivatives accumulate in the last compartment at pseudo-steady state, and past work identifying polysaccharides/peptides as major membrane biofoulants have excluded these lipids. In addition, lipids and changes detected during feed transients have not been noted before in previous work. Finally, feed step-increases also increased some amino acid derivatives used in cell-signalling. Interestingly, some natural products from plant and fungal extracts were also found in the fourth compartment, where methanogenesis was the dominant process.

Contamination of perfluorinated compounds (PFCs) in Chao Phraya River and Bangpakong River, Thailand.

Perfluorinated compounds (PFCs) have been used for many years, and are distributed all over the world. This study focused on occurrences of PFCs, especially perfluorooctane sulfonate (PFOS) and perfluorooctonoic acid (PFOA) in Thai rivers and industrial estate discharges, while comparing results with rivers of other Asian countries (Japan, China, and Malaysia). Surveys were conducted in Chao Phraya River, Bangpakong River and three industrial estates. A solid phase extraction (SPE) and HPLC-ESI-MS/MS were used for the analysis of these chemicals. The average concentrations of PFOS and PFOA were 1.9 and 4.7 ng/L, respectively in Chao Phraya River, while lower concentrations were detected in Bangpakong River with the averages of 0.7 ng/L for both PFOS and PFOA. Higher concentrations were detected in all industrial estate discharges with the averages of 64.3 ng/L for PFOA and 17.9 ng/L for PFOS., Total loadings from three industrial estates were 1.93 g/d for PFOS and 11.81 g/d for PFOA. The concentration levels in Thai rivers were less than rivers in Japan, China, and Malaysia. However, PFCs loading rate of Chao Phraya River was much higher than Yodo River (Japan), due to the higher flow rate. The other six PFCs were found above the Limit of Quantification (LOQ) in most samples. PFHxS and PFNA were also highly detected in some river samples.

Post-treatment of anaerobic membrane bioreactor (AnMBR) effluent using activated carbon.

Anaerobic membrane bioreactors (AnMBR) are very effective for wastewater treatment, however, with the antibiotic ciprofloxacin (CIP) (0-4.7 mg CIP/L) in the feed their performance decreases, the characteristics of the effluent changes, and further treatment is needed to recycle or discharge the treated effluent. Batch experiments using six activated carbons to treat AnMBR effluents resulting from the treatment of a synthetic wastewater containing ciprofloxacin were carried out at 35 °C. 22-82% COD was removed at a dose of 1 g activated carbon/L, while size characterization showed the 13.4 kDa and <1 kDa fractions were the most difficult to adsorb, while CIP was often removed with high efficiencies of mainly 100%. Significant removal of VFAs also occurred, up to 100%, and this contributed greatly to COD removal. Nitrogen containing compounds and phenols showed the highest removal (∼100%), whereas other groups such as esters, alkanes, and alkenes showed lower removal efficiency.

Identification of recalcitrant compounds in a pilot-scale AB system: An adsorption (A) stage followed by a biological (B) stage to treat municipal wastewater.

This manuscript presents a comparison of the A-stage and B-stage sludges in terms of anaerobic biodegradability and low molecular weight compounds present in the supernatant using Gas Chromatography-Mass Spectrometry (GC-MS). The GC-MS analysis of A-stage and B-stage supernatants identified respectively 43 and 19 organic compounds consisting mainly of aromatics (27.9% and 21%), alcohols (25.6% and 15%) and acids (30.2% and 15%). The methane potential was found to be 349±1 mL CH4/g VS and 238±12 mL CH4/g VS, respectively. After anaerobic digestion of these sludges, a greater proportion of aromatics (42% and 58%) and a lower proportion of acids (10% and 10%) and alcohols (16% and 10%) was observed.

Characterization of soluble microbial products (SMPs) in a membrane bioreactor (MBR) treating synthetic wastewater containing pharmaceutical compounds.

This study investigated the behaviour and characteristics of soluble microbial products (SMP) in two anoxic-aerobic membrane bioreactors (MBRs): MBRcontrol and MBRpharma, for treating municipal wastewater. Both protein and polysaccharides measured exhibited higher concentrations in the MBRpharma than the MBRcontrol. Molecular weight (MW) distribution analysis revealed that the presence of pharmaceuticals enhanced the accumulation of SMPs with macro- (13,091 kDa and 1587 kDa) and intermediate-MW (189 kDa) compounds in the anoxic MBRpharma, while a substantial decrease was observed in both MBR effluents. Excitation emission matrix (EEM) fluorescence contours indicated that the exposure to pharmaceuticals seemed to stimulate the production of aromatic proteins containing tyrosine (10.1-32.6%) and tryptophan (14.7-43.1%), compared to MBRcontrol (9.9-29.1% for tyrosine; 11.8-42.5% for tryptophan). Gas chromatography-mass spectrometry (GC-MS) analysis revealed aromatics, long-chain alkanes and esters were the predominant SMPs in the MBRs. More peaks were present in the aerobic MBRpharma (196) than anoxic MBRpharma (133). The SMPs identified exhibited both biodegradability and recalcitrance in the MBR treatment processes. Only 8 compounds in the MBRpharma were the same as in the MBRcontrol. Alkanes were the most dominant SMPs (51%) in the MBRcontrol, while aromatics were dominant (40%) in the MBRpharma. A significant decrease in aromatics (from 16 to 7) in the MBRpharma permeate was observed, compared to the aerobic MBRpharma. Approximately 21% of compounds in the aerobic MBRcontrol were rejected by membrane filtration, while this increased to 28% in the MBRpharma.

Analytical methods for soluble microbial products (SMP) and extracellular polymers (ECP) in wastewater treatment systems: a review.

Effluents from biological processes contain a wide range of complex organic compounds, including soluble microbial products (SMP) and extracellular polymers (ECP), released during bacteria metabolism in mixed culture in bioreactors. It is important to clearly identify the primary components of SMPs and ECPs in order to understand the fundamental mechanisms of biological activity that create these compounds, and how to reduce these compounds in the effluent. In addition, these compounds constitute the main foulants in membrane bioreactors which are being used more widely around the world. A review on the extraction of ECP, characterization, and identification of SMPs and ECPs is presented, and we summarize up-to-date pretreatments and analytical methods for SMPs. Most researchers have focused more on the overall properties of SMPs and ECPs such as their concentrations, molecular weight distribution, aromaticity, hydrophobic and hydrophilic properties, biodegradability, and toxicity characteristics. Many studies on the identification of effluent SMPs show that most of these compounds were not present in the influent, such as humic acids, polysaccharides, proteins, nucleic acids, organic acids, amino acids, exocellular enzymes, structural components of cells and products of energy metabolism. A few groups of researchers have been working on the identification of compounds in SMPs using advanced analytical techniques such as GC-MS, LC-IT-TOF-MS and MALDI-TOF-MS. However, there is still considerably more work needed to be done analytically to fully understand the chemical characteristics of SMPs and ECPs.

Mass flows of perfluorinated compounds (PFCs) in central wastewater treatment plants of industrial zones in Thailand.

Perfluorinated compounds (PFCs) are fully fluorinated organic compounds, which have been used in many industrial processes and have been detected in wastewater and sludge from municipal wastewater treatment plants (WWTPs) around the world. This study focused on the occurrences of PFCs and PFCs mass flows in the industrial wastewater treatment plants, which reported to be the important sources of PFCs. Surveys were conducted in central wastewater treatment plant in two industrial zones in Thailand. Samples were collected from influent, aeration tank, secondary clarifier effluent, effluent and sludge. The major purpose of this field study was to identify PFCs occurrences and mass flow during industrial WWTP. Solid-phase extraction (SPE) coupled with HPLC-ESI-MS/MS were used for the analysis. Total 10 PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluoropropanoic acid (PFPA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorohexane sulfonate (PFHxS), perfluoronanoic acid (PFNA), perfluordecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), and perfluorododecanoic acid (PFDoA) were measured to identify their occurrences. PFCs were detected in both liquid and solid phase in most samples. The exceptionally high level of PFCs was detected in the treatment plant of IZ1 and IZ2 ranging between 662-847ngL(-1) and 674-1383ngL(-1), respectively, which greater than PFCs found in most domestic wastewater. Due to PFCs non-biodegradable property, both WWTPs were found ineffective in removing PFCs using activated sludge processes. Bio-accumulation in sludge could be the major removal mechanism of PFCs in the process. The increasing amount of PFCs after activated sludge processes were identified which could be due to the degradation of PFCs precursors. PFCs concentration found in the effluent were very high comparing to those in river water of the area. Industrial activity could be the one of major sources of PFCs contamination in the water environment.