Evaluation of Proteins and Organic Nitrogen in Wastewater Treatment

Jun 17, 2010 - ... [email protected]; phone: 413-545-9456; fax: 413-545-2202., † ... as a first step to elucidate the fate and role of proteins in ...
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Environ. Sci. Technol. 2010, 44, 5352–5357

Evaluation of Proteins and Organic Nitrogen in Wastewater Treatment Effluents PAMELA J. WESTGATE† AND CHUL PARK* Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, Massachusetts 01003

Received January 22, 2010. Revised manuscript received May 25, 2010. Accepted May 28, 2010.

Proteins represent a large portion of organic nitrogen and carbon in wastewater treatment effluents, but their detailed characteristics and their role and fate in receiving waters are virtually unknown. We used two protein fractionation techniques to characterize effluent proteins and proteolytic enzymes in three activated sludge plants, as a first step to elucidate the fate and role of proteins in receiving water environments. The quantitative data first showed that the protein concentration in primary and secondary effluents was significantly correlated with organic nitrogen and could comprise up to 60% of effluent organic nitrogen. Protein separation results showed that some proteins persisted through secondary treatment, while others were produced during biological treatment. Despite a high similarity of protein and enzyme profiles in primary effluent across three facilities, those in secondary effluent were consistently different, suggesting that effluent proteins could serve as markers of different wastewater treatment works. These profile fingerprints can be used to track effluent proteins in laboratory bioassays, or directly in receiving waters, and may permit the determination of the fate of effluent proteins, and thus a significant fraction of effluent organic nitrogen, in the environment.

Introduction Eutrophication and subsequent fish kills in estuaries and ocean bays have been attributed to nitrogen (N) loading, largely from domestic wastewater treatment plants (WWTPs) (1). In response, regulators are decreasing the levels of nitrogen that can be released by WWTPs into water bodies that flow into marine environments. While WWTPs can be upgraded for enhanced N removal through nitrification and denitrification, the process removes mostly inorganic N. It is believed that effluent organic N (organic-N) remains largely unchanged throughout wastewater treatment, presumably due to its recalcitrant nature, which leads to effluent organic-N becoming a substantial fraction of the N in the final effluent (2). Thus, one major issue with effluent organic-N is whether it degrades and becomes bioavailable in receiving waters. Recently, some studies (2, 3) and a review article (4) have reported that effluent organic-N is bioavailable and induces algal growth. The studies, however, derived the results from batch-type bioassays with inoculations of a single species of algae, conditions that are far different from actual * Corresponding author e-mail: [email protected]; phone: 413545-9456; fax: 413-545-2202. † Current address: Kleinfelder/SEA Consultants, 200 Corporate Place, Rocky Hill, CT 06067. 5352



receiving waters. The fate of effluent organic-N in receiving waters is an important element that needs to be considered when setting effluent limits for N-loading from WWTPs to receiving waters through advanced N removal, yet there is currently no standard analytical technique to track that material. To gain better insight of effluent organic-N and its impact on receiving waters, improved characterization of effluent organic-N is necessary. Proteins are of particular importance in this regard, because they comprise a significant fraction of effluent organic-N (and carbon), and as macromolecules, they are potentially well characterizable. In one study, protein-N, also often referred to as combined amino acids, comprised up to 13% of the dissolved organic-N (DON) in 0.25-µm filtrate of secondary effluent (5). In another, although not directly shown as organic-N, proteins comprised 26-76% of the total organic carbon in the liquid phase of settled activated sludge from a membrane bioreactor and a fullscale activated sludge plant (6). In many other studies proteins have been found to be a major component of organic matter in wastewater effluent (7–11). In spite of its significant quantity in wastewater treatment effluent, previous work characterizing effluent proteins, reviewed by Pehlivanoglu-Mantas and Sedlak in 2006 (12), has not been extensive due to the complex nature of protein structures, number of source organisms, and the technical difficulties associated with characterizing proteins from effluents. Direct characterization of wastewater effluent proteins has thus been limited to their quantification. Proteins have commonly been measured using colorimetry (e.g., the Lowry method) or using a combination of hydrolysis and high-pressure liquid chromatography (HPLC), which measures amino acids, the building block of proteins (13). While these methods are able to quantify the proteins, they do not provide any further information about the proteins, such as their source, identification, or possible enzymatic activity. Some studies have examined the activity of specific enzymes, such as aminopeptidase, β-galactosidase, and phosphatase, in surface waters downstream of WWTP discharges (14–16). The difficulty of this approach to understanding the effects of wastewater treatment effluents on receiving waters is that other sources can contribute to enhancements in enzymatic activity. For example, in one study it was determined that increases in aminopeptidase activity in river water were not simply due to WWTP discharges alone, but were likely contributed to by nonpoint inputs into the system (15). Also, while likely to be present in wastewater effluent, none of these enzymes have specifically been identified in the effluent itself. Recently, human-derived proteins, such as human pancreatic elastase, have been found in the extracellular matrix of activated sludge flocs (17) and from anaerobically digested sludge product (18). The presence of sewage-derived polypeptides in settled sludge is an indication that these proteins are resistant to degradation in secondary aerobic treatment, and raises the question of whether these protein enzymes may also be present in the dissolved phase of activated sludge that is discharged into receiving waters. The full effects of releasing active enzymes such as elastase into the environment are unknown, but it is likely that they contribute to increasing the bioavailability of natural organic matter in the receiving water (15). Effluent proteins comprise a significant fraction of N and carbon in wastewater effluents and cause unknown, but potentially important, ecological and environmental impacts. Due to the lack of direct scientific investigation of effluent 10.1021/es100244s

 2010 American Chemical Society

Published on Web 06/17/2010

proteins, their characteristics and fate in receiving waters are also unknown. The specific objectives of this study were to (1) characterize proteins present in various facility effluents using protein and enzyme fractionation techniques, and (2) find their relationship to N species, particularly organic-N, in wastewater effluent. It was expected that evaluation of effluent proteins at the molecular level could lead to the establishment of a fingerprinting protocol that will be useful for assessing the efficiency of upstream processes, as well as determining the fate of these identified effluent proteins, an important class of organic-N, in receiving waters. We believe that this is the first application of protein and enzyme fractionation techniques to wastewater effluents that are discharged into receiving water environments.

Material and Methods Wastewater Treatment Facilities. Primary and secondary effluents were collected from three wastewater treatment facilities that discharge to the Connecticut River in Western Massachusetts (Table S1 in Supporting Information). Facility A treats on average 17 413 m3/d (4.6 million gallons per day, mgd) and Facility B treats approximately 170 344 m3/d (45 mgd) of mixed industrial and domestic wastewater. The activated sludge at both facilities is aerated using diffused aeration. Facility B operates with an anoxic section before the aerobic treatment (the Ludzack-Ettinger process). Facility C uses conventional activated sludge and mechanical aeration, treating 15 520 m3/d (4.1 mgd) of mostly domestic, with trace industrial, wastewater. Sample Collection and Processing. One liter or more of primary effluent, and eight or more liters of secondary effluent were collected from tank outfalls at each of the sampling sites. Samples were collected in plastic containers and kept on ice until processing later the same day. Total suspended solids (TSS), volatile suspended solids (VSS), and chemical oxygen demand (COD) measurements were taken the day of collection, while samples were frozen for later analysis of protein quantity, total N, ammonium, nitrate, and nitrite. Secondary effluent from each collection was size fractionated through a 0.45-µm nitrocellulose membrane and subjected to all tests except solids. In this study, the “dissolved” or “soluble” fraction is designated as the material passing through a 0.45-µm nitrocellulose membrane. Protein Quantification. The protein concentration in ammonium sulfate concentrated samples was measured using the Frølund adaptation of the Lowry method (19). Initial attempts to measure the protein concentration in unconcentrated samples were also made using the Frølund adaptation of the Lowry method, which accounts for the interference of humic compounds in the protein measurement. This method, however, overcompensated for the humic compounds and resulted in negative protein concentrations in the secondary effluent samples, thus the quantity of proteins in unconcentrated effluent were measured using the original Lowry method (20). Light absorbances were measured with a Thermospectronic Genesys 10 UV spectrophotometer (Thermo Spectronic, Madison, WI). Standard curves for concentration calculations were created from 0, 10, 25, and 50 mg/L bovine serum albumin (BSA) standards diluted from a concentrated stock of BSA (Fisherbrand Scientific, Pittsburgh, PA). Ammonium Sulfate Precipitation. To visualize proteins using SDS-PAGE, the proteins in primary effluent, secondary effluent, and secondary filtered effluent were concentrated with 50% ammonium sulfate. The appropriate mass of ammonium sulfate was combined with 150 mL of primary effluent, 1.2 L of secondary effluent, and 2.2 L of 0.45-µm filtered secondary effluent, in 500-mL centrifuge bottles. Precipitation reactions occurred on ice for more than 12 h at 4 °C, followed by centrifugation at 11 730g for 45 min. The

precipitate was resuspended and dialyzed according to the method shown by Park et al. (18). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The SDS-PAGE was performed according to the method of Laemmli (21). After samples were concentrated with ammonium sulfate, they were prepared for size separation on polyacrylamide gels. Samples were incubated at approximately 95 °C for at least 10 min with a 3.3× buffer consisting of XT Mops sample buffer and a reducing agent (Bio-Rad, Hercules, CA). Secondary effluent samples were heat concentrated for up to 1 h to further concentrate the proteins in the samples enough to be resolved on polyacrylamide gels. Following heat concentration samples were centrifuged at 12 000 rpm for 3 min and the supernatant was used for SDS-PAGE. Prepared samples were loaded onto precast Criterion XT 4-12% gradient gels (Bio-Rad, Hercules, CA) and separated on the gels by a current of 80 V for 20 min, followed by 100 V for 2 h. After electrophoresis, gels were stained with silver nitrate or coomassie brilliant blue using Bio-Rad’s Silver Stain Kit or Bio-Safe stain (Bio-Rad, Hercules, CA). Gel images were digitally recorded using a CanoScan 8800F desktop scanner (Canon, Tokyo, Japan). Zymogram Analysis. To determine if samples contained active proteolytic enzymes they were subjected to zymogram analysis. Enzyme activity was determined by separating proteins using electrophoresis in a casein infused zymogram gel (Bio-Rad, Hercules, CA). Before electrophoresis the samples were combined with zymogram buffer (Bio-Rad) and centrifuged at 12 000 rpm for 3 min; the supernatant was used for the zymogram analysis. One-tenth of a microgram of protein from primary effluent was loaded into the primary lanes, and 0.4 µg and 0.5 µg of protein from secondary and filtered secondary effluent, respectively. Gel images were recorded as described above. Chemical Analysis. COD, TSS, and VSS were measured for primary and secondary effluents according to Standard Methods (22). COD was measured for secondary effluent filtered through a 0.45-µm filter, as well. Light absorbances for COD tests were determined using the Thermospectronic Genesys 10 UV spectrophotometer. Nitrogen Species. Total N concentrations in primary and secondary effluent, and 0.45-µm filtered secondary effluent were determined using the persulfate method (Hach, Loveland, CO) and confirmed using a Shimadzu TN analyzer (Shimadzu TOC-VCPH withTNM-1, Shimadzu North America, SSI Inc., Columbia, MD). Ammonium, nitrate, and nitrite ions in the solution phase (