Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection.

Evaluation of acidity estimation methods for mine drainage, Pennsylvania, USA.

Eighteen sites impacted by abandoned mine drainage (AMD) in Pennsylvania were sampled and measured for pH, acidity, alkalinity, metal ions, and sulfate. This study compared the accuracy of four acidity calculation methods with measured hot peroxide acidity and identified the most accurate calculation method for each site as a function of pH and sulfate concentration. Method E1 was the sum of proton and acidity based on total metal concentrations; method E2 added alkalinity; method E3 also accounted for aluminum speciation and temperature effects; and method E4 accounted for sulfate speciation. To evaluate errors between measured and predicted acidity, the Nash-Sutcliffe efficiency (NSE), the coefficient of determination (R (2)), and the root mean square error to standard deviation ratio (RSR) methods were applied. The error evaluation results show that E1, E2, E3, and E4 sites were most accurate at 0, 9, 4, and 5 of the sites, respectively. Sites where E2 was most accurate had pH greater than 4.0 and less than 400 mg/L of sulfate. Sites where E3 was most accurate had pH greater than 4.0 and sulfate greater than 400 mg/L with two exceptions. Sites where E4 was most accurate had pH less than 4.0 and more than 400 mg/L sulfate with one exception. The results indicate that acidity in AMD-affected streams can be accurately predicted by using pH, alkalinity, sulfate, Fe(II), Mn(II), and Al(III) concentrations in one or more of the identified equations, and that the appropriate equation for prediction can be selected based on pH and sulfate concentration.

Identification of the factors that control synthesis and accumulation of a therapeutic protein, human immune-regulatory interleukin-10, in Arabidopsis thaliana.

Plants are one of the most economical platforms for large-scale production of recombinant proteins for biopharmaceutical and industrial uses. A large number of human recombinant proteins of therapeutic value have been successfully produced in plant systems. One of the main technical challenges of producing recombinant proteins in plants is to obtain sufficient level of protein. This research aims to identify the factors that control synthesis and accumulation of recombinant proteins in stable transgenic plants. A stepwise dissection of human immune-regulatory interleukin-10 (IL-10) protein production was carried out using Arabidopsis thaliana as a model system. EMS-mutagenized transgenic Arabidopsis IL-10 lines, at2762 and at3262, produced significantly higher amount of IL-10 protein than the non-mutagenized IL-10 line (WT-IL-10). The fates of trans-gene in these sets of plants were compared in detail by measuring synthesis and accumulation of IL-10 transcript, transcript stability, protein synthesis and IL-10 protein accumulation. The IL-10 transcripts were more stable in at2762 and at3262 lines than WT-IL-10, which may contribute to higher protein synthesis in these lines. To evaluate whether translational regulation of IL-10 controls its synthesis in non-mutagenized WT-IL-10 and higher IL-10 accumulating mutant lines, we measured the efficiency of the translational machinery. Our results indicate that mutant lines with higher trans-gene expression contain more robust and efficient translational machinery compared with the control line.

Identification of calcium-independent and calcium-enhanced binding between S100B and the dopamine D2 receptor.

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100-300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. (1)H-(15)N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca(2+)-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca(2+)-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.

Nitrite reduction with hydrous ferric oxide and Fe(II): stoichiometry, rate, and mechanism.

Fe(II)/Fe(III) oxide is an important redox couple in environmental systems. Recent studies have revealed unique characteristics of Fe(II)/Fe(III) oxide and reactions with oxidizing or reducing agents. Nitrite was used as an oxidizing agent in this study in order to probe details of these reactions and hydrous ferric oxide (HFO) was used as the Fe(III) oxide phase. Abiotic nitrite reduction is a significant global producer of nitric oxide (a catalyst for production of tropospheric ozone) and nitrous oxide (a greenhouse gas and contributor to stratospheric ozone depletion). All experiments were conducted at pH 6.8 using a strictly anoxic environment with mass-balance measurements for Fe(II). Oxidation of Fe(II) was negligible in the absence of HFO. The reaction was fast in the presence of HFO and was described by d[Fe(II)]/dt=-k(overall)[Fe(II)(diss)][Fe(II)(solid-bound)][NO(2)(-)] (k(overall)=2.59x10(-7)microM(-2)min(-1)) for Fe(II)/Fe(III) molar ratios less than 0.30. The reaction was inhibited for higher Fe(II)/HFO ratios. The concentration of solid-bound Fe(II) was constant after an initial equilibration period and the reaction stopped when dissolved Fe(II) was depleted even though substantial solid-bound Fe(II) and nitrite remained. The results regarding rate-dependence and conservation of solid-bound Fe(II) and inhibition of reaction at high Fe(II)/Fe(III) ratios were similar to our earlier results for the Fe(II)/HFO/O(2) system [Park, B., Dempsey, B.A., 2005. Heterogeneous oxidation of Fe(II) on ferric oxide at neutral pH and a low partial pressure of O(2). Environmental Science and Technology 39(17), 6494-6500.].

Effects of wastewater effluent organic materials on fouling in ultrafiltration.

The objective was to determine the effects of wastewater effluent organic materials (EfOM) on fouling of ultrafilters (100kDa polyethersulfone (PES)). EfOM constituents were sequentially removed, first by removing particles down to the approximate ultrafilter pore size and then by removing dissolved EfOM based on functionality. Particles and colloids >20nm accounted for 19% of total organic carbon (TOC), including 96% of EfOM >100kDa. Removal of particles and colloids resulted in increased fouling, attributed to increased contact of dissolved EfOM with the membrane. Hydrophobic and hydrophilic (HPO/HPI) acids were 22% of total EfOM, and accounted for nearly all of the fouling. HPO/HPI base/neutrals were 59% of EfOM, but did not cause any significant fouling. Although HPO/HPI base/neutrals did not cause any fouling, they were the dominant EfOM constituent at the surface of fouled and then hydraulically cleaned membranes, as measured by attenuated reflectance infrared spectroscopy. Since the filtration runs were short, the effects of HPO/HPI base/neutrals on long-term fouling should be further investigated, but these results cast doubt on the presumption that organic materials that are identified during membrane autopsies are necessarily a primary cause of fouling. These results also indicate that wastewater EfOM should be treated to remove HPO/HPI acids prior to membrane filtration.

Reduction of U(VI) by Fe(II) in the presence of hydrous ferric oxide and hematite: effects of solid transformation, surface coverage, and humic acid.

Fe(II) was added to U(VI)-spiked suspensions of hydrous ferric oxide (HFO) or hematite to compare the redox behaviors of uranium in the presence of two different Fe(III) (oxyhydr)oxides. Experiments were conducted with low or high initial sorption density of U(VI) and in the presence or absence of humic acid (HA). About 80% of U(VI) was reduced within 3 days for low sorbed U(VI) conditions, with either hematite or HFO. The {Fe(3+)} in the low U(VI) experiments at 3 days, based on measured Fe(II) and U(VI) and the assumed presence of amorphous UO(2(s)), was consistent with control by HFO for either initial Fe(III) (oxyhydr)oxide. After about 1 day, partial re-oxidation to U(VI) was observed in the low sorbed U(VI) experiments in the absence of HA, without equivalent increase of dissolved U(VI). No reduction of U(VI) was observed in the high sorbed U(VI) experiments; it was hypothesized that the reduction required sorption proximity of U(VI) and Fe(II). Addition of 5mg/L HA slowed the reduction with HFO and had less effect with hematite. Mössbauer spectroscopy (MBS) of (57)Fe(II)-enriched samples identified the formation of goethite, hematite, and non-stoichiometric magnetite from HFO, and the formation of HFO, hydrated hematite, and non-stoichiometric magnetite from hematite.

Arsenic removal by iron-modified activated carbon.

Iron-impregnated activated carbons have been found to be very effective in arsenic removal. Oxyanionic arsenic species such as arsenate and arsenite adsorb at the iron oxyhydroxide surface by forming complexes with the surface sites. Our goal has been to load as much iron within the carbon pores as possible while also rendering as much of the iron to be available for sorbing arsenic. Surface oxidation of carbon by HNO3/H2SO4 or by HNO3/KMnO4 increased the amount of iron that could be loaded to 7.6-8.0%; arsenic stayed below 10 ppb until 12,000 bed volumes during rapid small-scale tests (RSSCTs) using Rutland, MA groundwater (40-60 ppb arsenic, and pH of 7.6-8.0). Boehm titrations showed that surface oxidation greatly increased the concentration of carboxylic and phenolic surface groups. Iron impregnation by precipitation or iron salt evaporation was also evaluated. Iron content was increased to 9-17% with internal iron-loading, and to 33.6% with both internal and external iron loading. These iron-tailored carbons reached 25,000-34,000 bed volumes to 10 ppb arsenic breakthrough during RSSCTs. With the 33.6% iron loading, some iron peeled off.

Solubility of schoepite: comparison and selection of complexation constants for U(VI).

Solubility of UO(3) x nH(2)O and sorption of U(VI) onto ferric (hydr)oxides were measured at pH 5.9, 6.8, and 7.8 at 10(-3.5)atm CO(2) using reaction times up to 48 days. Precipitation was fastest in the presence of hydrous ferric oxide and slower with hematite or without an initial solid phase. Solubility after 48 days was statistically similar for low to intermediate initial supersaturation conditions and increased for the highest initial supersaturation. Schoepite was identified for low-to-intermediate initial conditions of supersaturation and was not found for the highest initial supersaturation. Predicted concentrations of monomeric and polymeric species differed considerably with the different suites of complexation constants, resulting in significant differences in predicted oxidation-reduction potential and mobility of U(VI) in groundwater. Solubilities for low to intermediate initial supersaturation were best represented using complexation constants from Langmuir, D. [1978. Uranium solution-mineral equilibria at low temperatures with applications to sedimentary ore deposits. Geochim. Cosmochim. Acta 42, 547-569] and log*K(sp)=5.39 for schoepite, while solubilities for very high initial supersaturation were consistent with amorphous UO(3) x nH(2)O.

The ATPase domain of SecA can form a tetramer in solution.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=[monomer](4)/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.

Influence of chitosan characteristics on the coagulation and the flocculation of bentonite suspensions.

Ten chitosan preparations with different molecular weights (MW) and degrees of deacetylation (DD) were tested for coagulation of 5 g L(-1) bentonite suspensions at pH 5 and 7 in demineralized water (DW) and in tap water (TW). Coagulation was better in TW than in DW for every condition and lower doses of chitosan were required at pH 5 than at pH 7. More than 95% of residual turbidity (after sedimentation in the absence of chitosan) was removed using less than 0.10 mg L(-1) chitosan in either TW or DW at pH 5 or in TW at pH 7. Higher doses were required for removal of turbidity in DW at pH 7, but in all cases the effective concentrations of chitosan were much lower than required for complete neutralization of the negative charge on the bentonite particles. Removal of turbidity was best for the higher MW chitosans in either the B series (89.5% DD) or the C series (95% DD) of chitosans. Overall, the results were consistent with destabilization of bentonite by the combined mechanisms of electrostatic patch and bridging. The improved performance of chitosan in TW could have been due to improved attachment to bentonite due to the presence of sulfate and other counter-ions in TW.

Simultaneous removal of phosphorus and EfOM using MIEX, coagulation, and low-pressure membrane filtration.

The feasibility of using magnetic ion exchange (MIEX) treatment, in-line alum coagulation, and low-pressure membrane filtration was investigated for the simultaneous removal of total phosphorus (TP) and effluent organic matter (EfOM) from biologically treated wastewater. The focus was also placed on minimizing fouling of polyvinylidene fluoride and polyethersulfone membranes, which are the most commonly used low-pressure membranes in new and retrofit wastewater treatment plants. MIEX alone was effective for the removal of EfOM, and MIEX plus a small alum dose was very effective in removing both EfOM and TP. MIEX removed phosphorus, but organic acids in EfOM were preferentially removed, and the effects of competing anions on the removal of EfOM were insignificant. All the pretreatment strategies decreased the resistance to filtration. The greatest decrease in fouling was achieved by using MIEX (15 mL L⁻¹) plus a very low dose of alum (∼0.5 mg Al L⁻¹). Sweep floc coagulation using alum and without MIEX also significantly decreased fouling but did not effectively remove EfOM and produced high floc volume that could be problematic for inside-out hollow-fibre modules. The addition of these reagents into rapid mix followed by membrane filtration would provide operational simplicity and could be easily retrofitted at existing membrane filtration facilities.

In-line coagulation with low-pressure membrane filtration.

The objectives were to investigate the effects of in-line coagulation on removal of natural organic matter (NOM) and turbidity and on fouling of membranes during ultrafiltration (UF). Coagulants were added prior to UF, without intermediate use of conventional processes for removal of particles. Synthetic and natural raw waters were examined. Charge neutralization, sweep floc, and under-dose conditions (with respect to conventional treatment) were all effective for removal of NOM and turbidity by UF. Most coagulation conditions resulted in decreased resistance to filtration and improved hydraulic removal of filter cake, including conditions that would be ineffective prior to conventional settling and rapid-filtration. Flocs that were produced under charge neutralization conditions and acidic under-dose conditions produced lower hydraulic resistance and were less compressible than for sweep floc conditions.

Sorption kinetics of Fe(II), Zn(II), Co(II), Ni(II), Cd(II), and Fe(II)/Me(II) onto hematite.

The reactions of Fe(II) and other divalent metal ions including Zn, Co, Ni, and Cd on hematite were studied in single and competitive binary systems with high sorbate/sorbent ratios in 10 mM PIPES (pH 6.8) solution under strict anoxic conditions. Adsorbed Me(II) was defined as extractable by 0.5 N HCl within 20 h, and fixed Me(II) was defined as the additional amount that was extracted by 3.0 N HCl within 7 days. Binary systems contained Fe(II) plus a second metal ion. The extent of uptake of divalent metal ions by hematite was in order of Fe> or =Zn>Co> or =Ni>Cd. For all metals tested, there was an instantaneous adsorption followed by a relatively slow stage that continued for the next 1-5 days. This sequence occurred in both single and binary systems, and could have been due to a variety of sorption site types or due to slow conversion from outer- to inner-sphere surface complexes due to increasing surface charge. Sorption competition was observed between Fe(II) and the other metal ions. The displacement of Fe(II) by Me(II) was in order of Ni approximately Zn>Cd, and the displacement of Me(II) by Fe(II) was in order of Cd>Zn approximately Ni>Co. Fixed Fe(II) was in order of Fe+Co (20%)>Fe+Cd (6%)>Fe approximately Zn (4%)>Fe approximately Ni (4%) after 30 days. There was no fixation for the other metals in single or binary systems.

Fate of hydrolyzed Al species in humic acid coagulation.

The hydrolysis of Al-based coagulants in acidic conditions is necessary for the removal of organic matter by the coagulation/sedimentation process. However, interactions between hydrolyzed Al species and organic matter are complicated and this makes it difficult to optimize coagulant dosing for organics removal. The goal of this study was to investigate the reactions of hydrolyzed Al species in the coagulation of organic matter. Two polyaluminum chloride (PACl) coagulants, a commercial product with sulfate (PACl-C) and lab-prepared material (PACl-Al13) containing 7% and 96% of total Al as Al13, respectively, have been applied to investigate the coagulation of humic acid (HA). At pH 6, a lower dosage of PACl-Al13 than of PACl-C was required for optimized HA removal through coagulation/sedimentation due to the strong complexation and charge neutralization by Al13. Observation of the coagulation process using wet scanning electron microscopy showed that PACl-C produced both clustered flocs and linear precipitates in the presence of sulfate while PACl-Al13 produced curled precipitates due to the formation of intermolecular complex, when both coagulants were added at the optimum doses. Investigation of Al-HA floc by (27)Al-NMR and Al 2p XPS suggested that monomeric Al (Alm) was hydrolyzed into Al(OH)3 with tetrahedron for PACl-C coagulation while a half of Al13 slowly decomposed into octahedral Al-HA precipitates for PACl-Al13 coagulation. Meanwhile, C ls XPS indicated that aromatic CC of HA was preferentially removed from solution to Al-HA flocs for both PACl-C and PACl-Al13 coagulation. It was concluded that Al-HA complexation strongly affects the reaction pathways for Al hydrolysis and the final nature of the precipitates during PACl coagulation of HA and that the hydrolysis products are also strongly affected by the characteristics of the PACl coagulant.

Electrochemical struvite precipitation from digestate with a fluidized bed cathode microbial electrolysis cell.

Microbial electrolysis cells (MECs) can be used to simultaneously convert wastewater organics to hydrogen and precipitate struvite, but scale formation at the cathode surface can block catalytic active sites and limit extended operation. To promote bulk phase struvite precipitation and minimize cathode scaling, a two-chamber MEC was designed with a fluidized bed to produce suspended particles and inhibit scale formation on the cathode surface. MEC operation elevated the cathode pH to between 8.3 and 8.7 under continuous flow conditions. Soluble phosphorus removal using digester effluent ranged from 70 to 85% with current generation, compared to 10-20% for the control (open circuit conditions). At low current densities (≤2 mA/m(2)), scouring of the cathode by fluidized particles prevented scale accumulation over a period of 8 days. There was nearly identical removal of soluble phosphorus and magnesium from solution, and an equimolar composition in the collected solids, supporting phosphorus removal by struvite formation. At an applied voltage of 1.0 V, energy consumption from the power supply and pumping (0.2 Wh/L, 7.5 Wh/g-P) was significantly less than that needed by other struvite formation methods based on pH adjustment such as aeration and NaOH addition. In the anode chamber, current generation led to COD oxidation (1.1-2.1 g-COD/L-d) and ammonium removal (7-12 mM) from digestate amended with 1 g/L of sodium acetate. These results indicate that a fluidized bed cathode MEC is a promising method of sustainable electrochemical nutrient and energy recovery method for nutrient rich wastewaters.

Photoautotrophic hydrogen production by eukaryotic microalgae under aerobic conditions.

Eukaryotic algae and cyanobacteria produce hydrogen under anaerobic and limited aerobic conditions. Here we show that novel microalgal strains (Chlorella vulgaris YSL01 and YSL16) upregulate the expression of the hydrogenase gene (HYDA) and simultaneously produce hydrogen through photosynthesis, using CO2 as the sole source of carbon under aerobic conditions with continuous illumination. We employ dissolved oxygen regimes that represent natural aquatic conditions for microalgae. The experimental expression of HYDA and the specific activity of hydrogenase demonstrate that C. vulgaris YSL01 and YSL16 enzymatically produce hydrogen, even under atmospheric conditions, which was previously considered infeasible. Photoautotrophic H2 production has important implications for assessing ecological and algae-based photolysis.

Ultrasonic disintegration of microalgal biomass and consequent improvement of bioaccessibility/bioavailability in microbial fermentation.

BACKGROUND: Microalgal biomass contains a high level of carbohydrates which can be biochemically converted to biofuels using state-of-the-art strategies that are almost always needed to employ a robust pretreatment on the biomass for enhanced energy production. In this study, we used an ultrasonic pretreatment to convert microalgal biomass (Scenedesmus obliquus YSW15) into feasible feedstock for microbial fermentation to produce ethanol and hydrogen. The effect of sonication condition was quantitatively evaluated with emphases on the characterization of carbohydrate components in microalgal suspension and on subsequent production of fermentative bioenergy. METHOD: Scenedesmus obliquus YSW15 was isolated from the effluent of a municipal wastewater treatment plant. The sonication durations of 0, 10, 15, and 60 min were examined under different temperatures at a fixed frequency and acoustic power resulted in morphologically different states of microalgal biomass lysis. Fermentation was performed to evaluate the bioenergy production from the non-sonicated and sonicated algal biomasses after pretreatment stage under both mesophilic (35°C) and thermophilic (55°C) conditions. RESULTS: A 15 min sonication treatment significantly increased the concentration of dissolved carbohydrates (0.12 g g(-1)), which resulted in an increase of hydrogen/ethanol production through microbial fermentation. The bioconvertibility of microalgal biomass sonicated for 15 min or longer was comparable to starch as a control, indicating a high feasibility of using microalgae for fermentative bioenergy production. Increasing the sonication duration resulted in increases in both algal surface hydrophilicity and electrostatic repulsion among algal debris dispersed in aqueous solution. Scanning electron microscope images supported that ruptured algal cell allowed fermentative bacteria to access the inner space of the cell, evidencing an enhanced bioaccessibility. Sonication for 15 min was the best for fermentative bioenergy (hydrogen/ethanol) production from microalga, and the productivity was relatively higher for thermophilic (55°C) than mesophilic (35°C) condition. CONCLUSION: These results demonstrate that more bioavailable carbohydrate components are produced through the ultrasonic degradation of microalgal biomass, and thus the process can provide a high quality source for fermentative bioenergy production.

Comparison of two fractionation strategies for characterization of wastewater effluent organic matter and diagnosis of membrane fouling.

Two fractionation strategies were compared for characterizing organic components in effluent organic matter (EfOM) and natural organic matter (NOM). The first method is widely used and requires sample acidification and then re-neutralization during sequential organic removals onto resins. The second method uses a different suite of separation methods, does not require pH manipulation, and sequentially removes particles, colloids, organic acids, and hydrophobic neutrals without the need for adjusting pH. The NOM samples were dominantly organic acids while EfOM contained a broader distribution of organic functionalities so further evaluation was focused on EfOM. The new method completely removed colloidal matter from EfOM while the conventional fractionation method resulted in an increase in the percentage of EfOM >100 kDa with each fractionation step after filtration. Organic acids were removed in one fractionation step using the new method instead of three steps with the conventional method. The conventional method resulted in increased fouling after the final separation step apparently caused by production of inorganic colloids. The new fractionation method provided a clearer diagnosis that organic acids were the primary cause of fouling even though they were only 14% of EfOM organic carbon. We suggest that the new fractionation method should be considered for diagnosing the effects of NOM or EfOM on the performance of membrane filtration.