Accepted Manuscript Fate and biotransformation of phytosterols during treatment of pulp and paper wastewater in a simulated aerated stabilization basin Christy M. Dykstra, Hamilton D. Giles, Sujit Banerjee, Spyros G. Pavlostathis PII:
S0043-1354(14)00728-3
DOI:
10.1016/j.watres.2014.10.030
Reference:
WR 10949
To appear in:
Water Research
Received Date: 8 June 2014 Revised Date:
9 October 2014
Accepted Date: 13 October 2014
Please cite this article as: Dykstra, C.M., Giles, H.D., Banerjee, S., Pavlostathis, S.G., Fate and biotransformation of phytosterols during treatment of pulp and paper wastewater in a simulated aerated stabilization basin, Water Research (2014), doi: 10.1016/j.watres.2014.10.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fate and biotransformation of phytosterols during treatment of pulp and paper
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wastewater in a simulated aerated stabilization basin
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Christy M. Dykstraa, Hamilton D. Gilesa, Sujit Banerjeeb and Spyros G. Pavlostathisa,*
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School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0512, USA b School of Chemical and Biomolecular Engineering, Institute of Paper Science and Technology Georgia Institute of Technology, 500 10th Street, N.W., Atlanta, GA 30318, USA *Corresponding author. Tel.: +404-894-9367; fax: +404-894-8266; E-mail address:
[email protected] (S. G. Pavlostathis) Abstract
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Pulp and paper wastewater (PPW) contains significant concentrations of phytosterols, suspected of
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inducing endocrine disruption in aquatic species. Aerated stabilization basins (ASBs) are commonly
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used for the treatment of PPW, but phytosterol removal varies among treatment systems. The
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objective of this study was to better understand the removal processes and biotransformation of
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phytosterols within an ASB treatment system fed with untreated PPW. PPW settled solids and
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supernatant fractions showed that phytosterols are primarily associated with settleable solids, which
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carry phytosterols to ASB sediment where anoxic/anaerobic conditions prevail. Bioassays with
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supernatant and settled PPW fractions of the raw wastewater conducted under aerobic and anaerobic
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conditions, respectively, showed that solids disintegration and hydrolysis results in phytosterol
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release in ASBs. A simulated ASB, fed with PPW and operated for 2.4 years at three hydraulic
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retention times (HRTs; 22.2, 11.1 and 5.6 d) with total phytosterol and solids loading rates from 10
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to 42 µg/L-d and 44-178 mg/L-d, respectively, was used to determine the steady-state effluent
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quality and sediment characteristics. Although effluent COD and phytosterol concentrations were
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relatively low and stable (84-88% total COD removal; 82-94% total phytosterol removal) across the
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range of HRTs tested, sediment COD and phytosterol concentrations increased with increasing
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loading rate. On average, 51% of the phytosterols entering the ASB were removed via
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biotransformation, 40% were retained in the sediment, and the remaining 9% exited with the
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effluent. This study demonstrates the role of sediment as a source of phytosterol release in ASBs
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and highlights the importance of HRT and the PPW characteristics for predicting phytosterol fate in
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ASBs.
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Keywords: aerated stabilization basin; biotransformation; β-sitosterol; campesterol; phytosterols;
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stigmasterol
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1.
Introduction Phytosterols are naturally-occurring compounds produced by plants for a variety of
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biological purposes. Some phytosterols are associated with plant cell membranes and play a role in
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maintaining cell structure and membrane transport. Free phytosterols are not bound to cell materials
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and may serve as plant hormone precursors or act as phytosterol storage for cells. Phytosterols are
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comprised of a steroid ring structure, a hydroxyl group at C3 and a side chain with varying degrees
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of branching and/or saturation (Figure S1, Supplementary Material). The most abundant
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phytosterols are β-sitosterol, stigmasterol and campesterol (Cook et al., 1997; Mahmood-Khan and
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Hall, 2008, 2013), which differ only in the side chain structure (Figure S1). Phytosterols are highly
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hydrophobic, with an aqueous solubility of ≤ 2.0 μg/L (von Bonsdorff-Nikander et al., 2005;
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Dykstra et al., 2014a); thus, in aqueous systems, phytosterols are largely associated with solids and
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colloids (Cook et al., 1997; Mahmood-Khan and Hall, 2013). Pulp and paper wastewater (PPW)
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contains solid and colloidal matter that may carry sorbed phytosterols, as well as consisting, in part,
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of fibers and plant material that carry phytosterols as part of plant cell structures. Therefore, because
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of the solid and colloidal matter present in PPW, phytosterols may be present in PPW in
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concentrations well above their aqueous solubility (Xavier et al., 2009; Mahmood-Khan and Hall,
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2003, 2012).
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Exposure to elevated phytosterol concentrations can cause endocrine disruption in aquatic
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species, resulting in altered development of secondary sexual characteristics, decreased egg
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production, decreased spawning frequency and vitellogenin production in males (Gilman et al.,
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2003; Nakari and Erkomaa, 2003; Orrego et al., 2009; Miskelly, 2009). Moreover, there is evidence
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that phytosterol transformation products also have endocrine-disrupting effects (Howell and
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Denton, 1989; Christianson-Heiska et al., 2007). Thus, the fate and effect of phytosterols in both
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engineered and natural systems is of paramount importance relative to human and environmental
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health.
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The PPW contains significant phytosterol concentrations and pulp mill effluent discharges have been linked to observed changes in fish reproductive characteristics (Denton et al., 1985;
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Howell and Denton, 1989). During pulping and bleaching, phytosterols are released from plant
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material into wastewater, where concentrations of total phytosterols can range from 0.3 to 3.4 mg/L
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(Xavier et al., 2009; Mahmood-Khan and Hall, 2012). Prior to discharge, PPW is often treated
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biologically in aerated stabilization basins (ASBs). ASBs are lagoons with aeration tapered from
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influent to effluent to allow both oxidation and settling to occur within the same system. As such,
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ASBs are non-homogeneous systems with both spatial and temporal gradients of nutrients, oxygen
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and various wastewater components.
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ASBs are capable of removing phytosterols, although the extent of removal varies over time and among treatment systems (Mahmood-Khan and Hall, 2003, 2008). In a study of four U.S. pulp
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and paper mill ASBs, one had a net increase in phytosterols across the system, while the other ASBs
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removed 56-78% of the total sterols; stigmasterol increased across all ASBs (Cook et al., 1997).
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One ASB parameter that may affect phytosterol removal is the hydraulic retention time (HRT),
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which is directly related to the phytosterol loading rate. Indeed, HRT is an important parameter that
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affects phytosterol removal in activated sludge systems (Mahmood-Khan and Hall, 2013). Average
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ASB HRTs range from 3 to 10 days, but may be as long as 28 days in some cases (Sackellares et al.,
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1987; Lewis et al., 2012). A laboratory-scale ASB operated over 264 days achieved nearly complete
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removal of β-sitosterol and stigmasterol, thought to be due primarily to sorption, with only 9% of
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the stigmasterol removed through biodegradation and no appreciable biodegradation of β-sitosterol
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(Xavier et al., 2009). However, the wastewater used in the study contained low phytosterol
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concentrations, and was supplemented with exogenous phytosterols to reach 2 mg/L, which did not
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reflect actual ASB conditions where phytosterols primarily enter as plant cell-bound or solids-
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associated phytosterols. Therefore, it remains unclear how wastewater-associated phytosterols are
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removed in ASBs and to what extent biodegradation contributes to the overall phytosterol removal.
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Moreover, little is known about the microbial processes that occur within ASB systems and how the
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aerobic and anoxic/anaerobic zones interact.
Multiple redox zones exist within ASBs, which may be broadly divided into aerobic, anoxic,
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sulfate-reducing and fermentative/methanogenic zones. In each zone, different biological processes
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occur that are important to the fate of phytosterols. Under aerobic conditions, phytosterols are
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degradable (Malaviya and Gomes, 2008; Chamorro et al., 2009; Donova and Ergorova, 2012), but
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some evidence suggests the degradation rate may be linked to the growth phase of the requisite
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microbes, with faster kinetics under stationary phase conditions (Dykstra et al., 2014a). Under
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anoxic, nitrate-reducing conditions, cholesterol is known to be biodegraded (Harder and Probian,
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1997; Chiang et al., 2008), and indeed, phytosterol removal has been observed (Dykstra et al.,
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2014b). The biotransformation of phytosterols under sulfate-reducing conditions has also been
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observed, although only following a significant lag period. In the same study, phytosterol removal
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was not observed under fermentative/methanogenic conditions over the course of a 112 d assay
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(Dykstra et al., 2014b). From the results of these studies, it is clear that the biodegradability of
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phytosterols varies significantly between redox zones. The complexity of the biological activity and
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incomplete mixing conditions makes it difficult to predict the fate of PPW-associated phytosterols
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during ASB treatment.
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The objective of this study was to better understand the removal processes and biotransformation of phytosterols within an ASB treatment system by: i) determining the
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characteristics of PPW as a whole and as fractions separated through settling; ii) comparing the
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steady-state performance and phytosterol removal of a simulated ASB operated at three HRTs; and
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iii) investigating the microbial activity within the ASB aerobic and anoxic/anaerobic zones.
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2.
Materials and Methods
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2.1
Pulp and paper wastewater fractionation and characterization
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Untreated wastewater was obtained twice from a pulp and paper mill located in the southeastern
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US, stored at 4°C with minimum headspace for the duration of the study. At the time of collection,
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the pulp mill used the Kraft process to produce newsprint from a mixture of hardwood and
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softwood pulp. The raw wastewater from the second collection was analyzed for pH, total solids
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(TS), volatile solids (VS), total and soluble chemical oxygen demand (COD), ions, and
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phytosterols. To identify the PPW components that might be expected within ASB aerobic and
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anoxic/anaerobic zones, primarily affected by solids settling, the raw wastewater was separated by
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settling in a supernatant and a settled fraction using two 1000 mL glass graduated cylinders. Raw
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wastewater was thoroughly mixed and 1 L was poured into each of the graduated cylinders kept at
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room temperature (20-22°C). During settling, samples were removed from the center of each
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graduated cylinder at a depth of about 8 cm, where a clear zone was created, and absorbance was
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measured at 590 nm, the wavelength of maximum absorbance obtained from a preliminary scan.
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Settling was terminated at 15.5 h, when the absorbance measurements approached a stable value.
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Following settling, the supernatant was removed by and both wastewater fractions were analyzed
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for pH, TS, VS, total and soluble COD, ions, and phytosterols as described in Section 2.5, below.
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Measurable changes of these parameters were not detected during the 15.5 h settling.
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2.2
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ASB development and maintenance
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A simulated ASB was developed using ASB mixed liquor from the same pulp and paper mill where
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the PPW was collected. The ASB comprised a 7 L rectangular (21.6 cm length x 12.1 cm width x
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40.6 cm height) acrylic reactor with an influent line entering one end at the top of the water column
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and an effluent line exiting at the opposite end over a weir. The ASB was maintained at room
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temperature (20-22°C), fed with raw PPW amended with nitrogen (NH4Cl 30 mg N/L) and
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phosphorus (KH2PO4 10 mg P/L), which was adjusted to pH 6.5 with 2 N HCl. The feed was
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refrigerated at 3°C, constantly magnetically mixed, delivered to the ASB system with a positive
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displacement pump. The ASB upper zone was aerated with pre-humidified, compressed air
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delivered through glass pipettes at a depth of 4 cm from the water column surface, maintaining a
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dissolved oxygen (DO) concentration ≥7 mg/L throughout the water column. Settled solids were
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allowed to accumulate at the bottom of the ASB to simulate the various redox zones present in full-
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scale systems. DO was depleted within 1 cm of the water/sediment interface, indicating
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anoxic/anaerobic conditions within the sediment (Giles, 2012). The ASB was initially developed
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using a 22.2 d HRT over a period of 14 months. Subsequently, and in order to investigate the effect
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of the HRT on the system performance, the ASB was operated at two, lower HRTs. The ASB was
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operated for a total of 2.4 years as follows: 500 d at 22.2 d HRT (315 mL/d), 189 d at 11.1 d HRT
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(630 mL/d) and 175 d at 5.6 d HRT (1,260 mL/d). It should be noted that the raw PPW from the
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second collection, fed to the ASB while operated with the latter two HRTs, was 3-fold diluted to
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match the composition of the PPW from the first collection. At each HRT, the ASB was allowed to
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reach steady state, as determined by monitoring effluent pH, COD and nitrogen species. The ASB
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effluent and sediment at steady state were analyzed for pH, total suspended solids (TSS), volatile
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suspended solids (VSS), total and soluble COD, ions, and phytosterols.
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2.3
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ASB aerobic and anoxic/anaerobic activity
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To better understand the microbial activity occurring in the ASB aerobic and anoxic/anaerobic
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zones, a sample was removed from each zone while the ASB was operating at 5.6 d HRT. To obtain
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a relatively homogeneous sample from the aerobic zone of the ASB, the entire water column was
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slowly siphoned, excluding a 2.5 cm layer of liquid above the sediment, which was a transition zone
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between fully aerobic and anoxic/anaerobic, determined by DO measurements. The siphoned liquid
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was magnetically mixed and then 2 L was placed in a 4 L glass reactor vessel. To obtain the
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anoxic/anaerobic sample, the entire volume of sediment located below the transition zone was
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siphoned from the ASB into a helium-flushed vessel, where it was magnetically mixed before
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transferring 1.5 L to a 2.5 L helium-flushed, glass reactor. Both reactors were maintained at room
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temperature (20-22°C), constantly mixed magnetically and incubated for 96 h to provide a brief
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snapshot of the microbial activity in each zone. The aerobic reactor with the water column sample
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was aerated with pre-humidified, compressed air, and over time analyzed for pH, ammonia, nitrate,
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nitrite, total and soluble COD, TSS, VSS and phytosterols. The ASB sediment activity was assessed
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under anoxic/anaerobic conditions. Sediment located below the interfacial zone was transferred by
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siphoning to a 2.5 L helium-flushed, glass reactor. Gas production and composition were measured
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periodically throughout the incubation as mentioned in Section 2.5, below. Initial and periodic
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samples were taken and analyzed for pH, ammonia, nitrate, nitrite, sulfate, total and soluble COD,
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TSS, VSS, volatile fatty acids (VFAs) and phytosterols.
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2.4
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The objective of this assay was to determine the biodegradability of PPW and associated
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phytosterols under aerobic and anoxic/anaerobic conditions in batch systems with inocula taken
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from the ASB. Raw PPW was separated into a settled and a supernatant fraction, as described
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above. Inoculum from the ASB aerobic zone and anoxic/anaerobic zone was used to assess the
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ultimate biodegradability of the supernatant and settled wastewater fractions, respectively. The
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aerobic inoculum was biomass in a 500 mL sample from the laboratory ASB aerobic zone,
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centrifuged at 4,000 rpm for 20 min. The biomass pellet was then mixed with PPW wastewater
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supernatant to a total volume of 2 L, amended with ammonium chloride (60 mg N/L) and phosphate
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buffer (372 mg P/L; pH 7.2). The reactor was aerated with pre-humidified, compressed air and
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maintained for 28 d at room temperature (20-22°C) while magnetically mixed. Samples were taken
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periodically and analyzed for pH, total and soluble COD, nitrate, nitrite, ammonia, TSS, VSS and
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phytosterols. Anoxic/anaerobic inoculum was taken from the ASB sediment. The settled PPW
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fraction, 40 mL of anoxic/anaerobic inoculum, ammonium chloride (60 mg N/L) and phosphate
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buffer (372 mg P/L; pH 7.2) were combined in a 2.5 L helium-flushed glass reactor, for a total
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liquid volume of 2 L. The reactor was maintained for 28 d at room temperature (20-22°C) while
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magnetically mixed. Gas production and composition were measured periodically throughout the
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incubation. Liquid samples were analyzed for pH, total and soluble COD, sulfate, ammonia, TSS,
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VSS and phytosterols.
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2.5
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COD (closed reflux/colorimetric method ), pH, TS, TSS, VS and VSS were measured as described
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in Standard Methods (APHA, 2012). Nitrate, nitrite and sulfate were quantified using a Dionex DX-
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100 Ion chromatography unit (Dionex Corporation, Sunnyvale, CA). DO was measured by the
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luminescent method using a HACH HQ40d digital multimeter with a LDO101 oxygen probe
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(HACH Company, Loveland, CO). Ions were measured using ion chromatography/conductivity
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detection with samples filtered through a 0.2 µm syringe filters (Tugtas and Pavlostathis, 2007).
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Total gas production was measured using a pressure transducer (resolution -1 to 1.974 atm with an
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accuracy of 0.002 atm). Gas composition and VFAs were determined by gas chromatography with
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thermal conductivity and flame ionization detection, respectively, as previously reported (Okutman
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Tas and Pavlostathis, 2005; Misiti et al., 2013). Absorbance was measured with a Hewlett-Packard
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Model 8453 UV/Visible spectrophotometer (Hewlett-Packard Co., Palo Alto, CA) equipped with a
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diode array detector, deuterium and tungsten lamps and a 1 cm path length.
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Phytosterol concentrations were measured by liquid-liquid extraction followed by gas chromatography/mass spectroscopy and flame ionization detection (GC/MS-FID). Details on the
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extraction methods used were previously reported (Dykstra et al., 2014a; Dykstra, 2014). The
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extraction efficiency of cholesterol, used as a surrogate, was between 80 and 83%. Analysis of
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phytosterols and cholesterol was performed using an Agilent 7890 GC unit with a Zebron ZB-5HT
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column (Phenomenex Inc., Torrance, CA) which terminated in a Dean’s switch for simultaneous
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collection of MS data for identification and FID data for quantification. MS analysis was conducted
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by electrospray ionization (ESI) with positive ion polarity at 70 eV fragmentation voltage and a
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mass scan range of 50-500 m/z. The GC-MS/FID was operated according to the method described
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by Dykstra et al. (2014a). MS spectra were compared to standards and those in the NIST library for
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identification of sterols. FID peak areas were used to quantify sterols using a standard curve
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prepared with phytosterols and cholesterol dissolved in ethanol. Cholesterol was used as the
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surrogate and/or internal standard in all liquid/liquid extraction and GC analyses. The method
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minimum detection limit was 0.5 mg/mL in the injected solvent sample.
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3.
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3.1
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The estimated standard Gibbs free energy of formation for β-sitosterol, stigmasterol, campesterol
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and cholesterol are 94.98, 173.64, 87.87 and 54.81 kJ/mol, respectively (Dykstra et al., 2014a).
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Considering complete mineralization and neglecting biomass growth, phytosterol degradation is
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Results and Discussion Bioenergetics
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theoretically possible under aerobic, nitrate-reducing, sulfate-reducing and methanogenic conditions
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(Table S1, Supplementary Material). Aerobic oxidation and nitrate reduction are the most
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energetically favorable conditions, yielding an average of -105.0 and -98.5 kJ/eeq, respectively.
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Sulfate reduction and methanogenesis yield far less energy with an average of -5.5 and -2.8 kJ/eeq,
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respectively. Although theoretically feasible, phytosterol biotransformation via methanogenesis has
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not been observed. An assay conducted under fermentative/methanogenic conditions with an active
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mixed culture acclimated to PPW solids did not remove any phytosterols over the course of 112
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days (Dykstra et al., 2014b). The fate of phytosterols is highly dependent on the redox conditions
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encountered. In complex treatment systems such as ASBs, phytosterols are exposed to a range of
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redox conditions, making it difficult to predict their overall fate.
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3.2
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The results from the characterization of the whole and PPW fractions are given in Table 1. The
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settling of the wastewater was monitored by absorbance measurements at 590 nm (Figure S2). As
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expected, the TS, VS and total COD were highest in the settled fraction. Because phytosterols are
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hydrophobic with low solubility and relatively high octanol/water coefficient values (Dykstra et al.,
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2014a), they are expected to be largely associated with solids and colloids. Indeed, in the present
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study, the settled fraction, which contained the greater amount of solids, had the higher
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concentration of phytosterols. The settled fraction represented only 6.5% of the total wastewater by
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volume but contained 54% of the total solids, 63% of the total COD and 58% of the total
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phytosterols. By contrast, the supernatant fraction (i.e., soluble and colloidal), which represented
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93.5% of the total wastewater volume, contained 46% of the total solids, 37% of the total COD and
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42% of the total phytosterols. These results illustrate the significant phytosterol partitioning to
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solids, which has been noted in other PPW (Cook et al., 1997; Mahmood-Khan and Hall, 2003), and
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is due to sorption and/or the incorporation of phytosterols within the cell structure of plant fibers.
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As the majority of solids in an effective ASB are either degraded or accumulate in the sediment,
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phytosterols are likely to be carried into anoxic or anaerobic sediment along with the solids.
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Sediments in ASBs may therefore act as a phytosterol reservoir and may release phytosterols during
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In both the supernatant and settled wastewater fractions, the phytosterol profile was similar,
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with β-sitosterol as the dominant phytosterol, followed by stigmasterol and campesterol. However,
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β-sitosterol showed a greater affinity towards the supernatant fraction, comprising 92.0% of the
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total phytosterols in this fraction. This result might be explained by the association of β-sitosterol
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with colloids, as was found by a study of untreated wastewater from a Finnish softwood and
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hardwood mill, in which β-sitosterol was primarily associated with colloids (66%) (Leiviska et al.,
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2009). Stigmasterol and campesterol only made up 5.8 and 2.3% of the total phytosterols in the
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supernatant fraction. In comparison, the total phytosterols in the settled fraction consisted of 73.4%
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β-sitosterol, 14.3% stigmasterol and 12.3% campesterol. The total mass of phytosterols per mass of
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total solids was 136.1 µg/g and 158.6 µg/g for the supernatant and settled fractions, respectively. In
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both the supernatant and settled fractions, β-sitosterol was the primary phytosterol, followed by
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stigmasterol and campesterol. These results show that the total phytosterol mass distribution
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between the PPW supernatant and settleable fractions is similar.
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3.3
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To better assess the ultimate fate of PPW in ASB treatment systems, aerobic and anaerobic batch
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bioassays were conducted with wastewater fractionated by settling, using ASB aerobic and
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anoxic/anaerobic zones samples as inocula. The initial pH of the aerobic assay, conducted with the
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wastewater supernatant fraction, was 7 and slightly increased during the 28 d incubation period to
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7.45. The initial total COD decreased steadily through day 10 before reaching a plateau, with a final
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total COD of 294 mg/L (Figure 1A). The initial soluble COD was 97% of the total COD, indicating
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that most of the degradable organic material was in a soluble form (Figure 1A). The soluble COD
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decreased throughout the first 10 days, reaching a final soluble COD of 203 mg/L (Figure 1A).
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Although ammonia was present throughout the incubation period, nitrification was not observed
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until day 18, after which the ammonia began to decline but nitrate only increased slightly (Figure
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1B and 1C). Nitrifiers are sensitive to a number of compounds and the delayed nitrification may
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have been due to the inhibitory effect of compounds in the PPW. Fatty acids, phenols and
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chlorinated compounds, commonly found in PPW, are known inhibitors of nitrification (Makris,
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2003; Svenson et al., 2000; Strotmann and Eglsaer, 1995). Typically, wastewater entering the ASB
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is diluted, thus reducing the effect of potentially inhibitory substances. However, in this bioassay,
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no dilution occurred, exposing the nitrifiers to full strength PPW. Towards the end of the
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incubation, nitrification began at a slow rate, perhaps as a result of degradation and removal of such
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organic inhibitors by heterotrophic bacteria. It should be pointed out that although the analytical
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techniques used in the present study were not targeted towards the detection of possible phytosterol
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biotransformation intermediates, within routine analyses by GC/MS, possible steroid bioconversion
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products were not detected.
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The total phytosterols in the aerobic assay declined by 42% over the course of the 28 d
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incubation period (Figure 1D). However, the total phytosterol concentration increased by 161%
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within the first 10 d, primarily due to a 217% (1.02 mg/L) increase in stigmasterol. By comparison,
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β-sitosterol increased by 23% (0.04 mg/L) and campesterol remained stable within the first 10 d.
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Between the 10th and 28th day, the total phytosterol concentration decreased by 78%, with a 34%
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decrease in stigmasterol and a 71% decrease in β-sitosterol. The campesterol concentration
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remained at 20 µg/L throughout the incubation period. These results show that stigmasterol may
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increase during the aerobic treatment of PPW. Indeed, Cook et al. (1997) found an increase in
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stigmasterol ranging from 192 to 377% across four full-scale ASBs examined, and one ASB had a
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25% increase in β-sitosterol across the treatment system. Another study found ASB removal of
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stigmasterol to be highly dependent on the stigmasterol loading rate. When the rate was relatively
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low (0.18-0.19 mg/L-d), stigmasterol increased across the treatment system by 29-37%. However,
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at higher rates (0.60-0.62 mg/L-d), 90% stigmasterol removal was observed (Chamorro et al.,
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2009). Although the mechanisms behind the observed increase in stigmasterol are unknown, it is
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possible that it is due in part to the release of membrane-bound and sorbed phytosterols from settled
294
solids during their disintegration and microbial hydrolysis. The observed increase and subsequent
295
decrease in stigmasterol in the present study, could explain the variability in effluent stigmasterol
296
concentrations of the ASB operated at various HRTs in the present study (Table 2) as discussed in
297
Section 3,4, below. The highest effluent stigmasterol concentration was observed at 11.1 d HRT,
298
while lower concentrations were found at both shorter and longer HRTs. A moderate HRT could
299
allow for the release of phytosterols from hydrolyzed solids without providing enough time for
300
significant phytosterol biotransformation in the water column.
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During the anaerobic assay, conducted with the settled wastewater fraction, microbial activity was observed throughout the incubation period. The pH declined from 7.7 to 6.5 over the
303
course of 13 d and then remained stable for the remainder of the incubation period. The soluble
304
COD increased from 741 mg/L to 3,185 mg/L over the course of the incubation (Figure 2A). The
305
increase in soluble COD reflected the production of VFAs, primarily acetate and propionate (Figure
306
2B). At the end of the incubation, VFAs comprised 80% of the soluble COD. Sulfate was detected
307
for the first 6 d and was then depleted (Figure 2C). Gas production was moderate for the first 6 d,
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after which was accelerated, reaching 680 mL (at 25°C, 1 atm) at the end of the incubation (Figure
309
2D). CO2 was the main gaseous product (71.5%), along with CH4 (14.4%), H2S (4.1%), and the
310
remaining gaseous products (10.0%) were undetected. The disappearance of sulfate corresponded to
311
the appearance of hydrogen sulfide, indicating sulfate reduction was active (Figure 2E). The
312
production of methane increased towards the end of incubation when sulfate was depleted and
313
conditions began to favor methanogenesis. The total phytosterols increased by 73% during
314
incubation, primarily due to an increase in β-sitosterol (Figure 2F). Stigmasterol and campesterol
315
increased by 78 and 69%, respectively, but their concentrations were relatively low in comparison
316
to β-sitosterol. The increase in phytosterols was likely due in part to the release of phytosterols from
317
solids. Unlike the aerobic assay, the phytosterol concentrations continued to increase throughout the
318
incubation period. Additionally, a disproportionate increase in stigmasterol was not observed, which
319
suggests that the production or release of significant amounts of stigmasterol in ASBs primarily
320
occurs in the aerobic zone.
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Given the conditions of this study, the supernatant PPW fraction was readily degraded under aerobic conditions. During the incubation period, 66 and 76% of the total and soluble COD,
323
respectively, was degraded. Additionally, 42% of total phytosterols were removed. The settled PPW
324
fraction incubated under anaerobic conditions was biodegraded less and the total COD declined by
325
1% over 28 d of incubation. The soluble COD increased by 330% during the incubation due to the
326
production of VFAs. When the soluble COD contribution from VFAs was not considered, the
327
soluble COD removal was 14% over the incubation period. Total phytosterols increased by 73%,
328
which is likely due to the release of solids-associated phytosterols during the disintegration and
329
hydrolysis of organic solids.
330
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ASB performance at various HRTs
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The ASB influent, and steady state effluent and sediment characteristics at HRTs of 22.2, 11.1 and
332
5.6 d are given in Tables 2 and 3, respectively. TSS loading rates at HRTs of 22.2, 11.1 and 5.6d
333
were 44, 89 and 178 mg/L-d, respectively; phytosterol loading rates were 10, 21 and 42 µg/L-d at
334
the respective HRTs. The pH of both the effluent and the sediment remained stable across all three
335
HRTs tested, an indication of internal buffering of the system. The effluent quality declined only
336
slightly at shorter HRTs; total and soluble COD removal declined from 88 to 84% and from 76 to
337
64% between an HRT of 22.2 d and 5.6 d. Nitrification occurred at each HRT; nitrate increased
338
slightly from 0.12 mg N/L in the influent to 1.7-13.6 mg N/L in the effluent, while ammonia was
339
completely consumed. TSS and VSS in the effluent also were similar across all HRTs (17-31 mg/L
340
TSS; 14-17 mg/L VSS). These results demonstrate the ability of an ASB system to accommodate a
341
wide range of loading rates while producing effluent with a relatively consistent high quality.
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Although shortening the HRT had only a moderate effect on the effluent quality, the
343
sediment characteristics changed significantly. The total COD of the sediment increased from about
344
8 g/L at the longest HRT of 22.2 d to about 19 g/L at the shortest HRT of 5.6 d and the sediment
345
soluble COD also increased from 86 mg/L to 284 mg/L. The total COD increase is indicative of the
346
accumulation of PPW solids due to the higher loading rates at lower HRTs.
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The total effluent phytosterol concentration increased as the ASB HRT decreased (Table 2). Operation at the longest HRT tested (22.2 d) was the most effective in terms of overall phytosterol
349
removal (94%), while the total phytosterol removal at HRT values of 11.1 d and 5.6 d was 82 and
350
83%, respectively. The total phytosterol concentration in the effluent was nearly the same at both
351
11.1 d and 5.6 d HRTs. In contrast, the total phytosterol concentration in the ASB sediment was
352
significantly greater at the shorter HRTs (Table 3). Thus, the HRT had a far more significant effect
353
on the sediment than on the effluent phytosterol concentration. At a relatively long HRT,
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phytosterols in the aerobic zone have a greater opportunity to be oxidized. Additionally, organic
355
solids stay longer in the anoxic/anaerobic zones and as a result of solids degradation, solids-
356
associated phytosterols are released. At the longest HRT, the released phytosterols could have had
357
time to enter the aerobic zone and be oxidized. At the shortest HRT, the rate of solids accumulation
358
in the sediment far exceeds their degradation rate, thus leading to a relatively lower rate of
359
phytosterols release from the sediment to the overlaying water column and a significantly increased
360
phytosterol sediment concentration.
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The phytosterol distribution in the ASB effluent and sediment were different at the three HRTs tested. In the 22.2 d HRT effluent, β-sitosterol made up 40% of the total phytosterols but in
363
the 11.1 and 5.6 d HRT effluent, β-sitosterol represented only 19% and 7% of the total phytosterols,
364
respectively. Campesterol made up 17% of the total phytosterols in the 22.2 d HRT effluent but
365
represented 56% in the 5.6 d HRT effluent. Stigmasterol represented 37-65% of the total
366
phytosterols in the effluent, and was higher at 11.1 d HRT. The variations in the fractions are likely
367
the result of a number of complex processes taking place within the ASB including sorption,
368
desorption and biotransformation. Compared to the effluent, the phytosterol distribution did not
369
vary as much within the sediment, although some differences were noted. β-sitosterol represented
370
71% of the total phytosterols in the sediment at 22.2 d HRT and made up 26 and 70% of the total
371
phytosterols at HRTs of 11.1 d and 5.6 d, respectively. The lowest concentration of β-sitosterol was
372
observed at a moderate HRT (11.1 d). Stigmasterol made up 16-24% of total phytosterols in the
373
sediment and was highest at 11.1 d HRT. The sediment campesterol was significantly higher at
374
HRT 11.1 d (9.2 mg/L) than at 22.2 d HRT (1.3 mg/L) or 5.6 d HRT (2.9 mg/L)(Table 3). These
375
results indicate that HRT has a significant effect on the processes that affect phytosterol release and
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removal in ASBs. In order to better assess phytosterol removal, it is necessary to understand the
377
processes involved in phytosterol appearance and disappearance within an ASB as discussed below.
378
3.5
379
To better understand the processes that take place in the aerobic and anoxic/anaerobic zones of an
380
ASB, a sample was taken from each zone of the simulated ASB and incubated over 96 h. The
381
aerobic sample’s pH increased sharply from 7.65 to 8.31 over the first 8 h and then leveled out at
382
8.63 by the end of incubation. The initial soluble COD was relatively low (114 mg/L) and was
383
degraded by only 27% at 96 h (Figure 3A), indicating a low level of readily degradable COD was
384
present in the aerobic zone. Trace amounts of ammonia were detected only at 4 h and nitrate
385
remained relatively stable (6.6-7.2 mg N/L) over the incubation period. In an ASB system, benthal
386
feedback typically contributes ammonium to the aerobic zone (Bryant, 2010). VSS increased by
387
27.5% over the first 24 h of incubation, then remained stable, with a final VSS of 51 mg/L at the
388
end of incubation (Figure 3B). Total phytosterols decreased by 28.2% over the incubation period
389
(Figure 3C). Initially, campesterol was the predominant phytosterol, possibly due to a more rapid
390
biotransformation of β-sitosterol and stigmasterol, and declined by 33.3% during incubation. In
391
comparison, stigmasterol and β-sitosterol declined by 21.9 and 16.7%, respectively. The rate of total
392
phytosterol removal increased from 0.670 µg/L-h during the first 24 h to 3.23 µg/L-h from 24-96 h,
393
possibly due to the release of bound cholesterol oxidase and/or other enzymes as the culture entered
394
stationary phase. A similar accelerated phytosterol removal was reported in an aerobic batch assay
395
when the culture reached stationary phase (Dykstra et al., 2014a). Thus, as cell turnover increased,
396
the release of bound enzymes may have contributed to the accelerated phytosterol removal.
397 398
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ASB aerobic and anoxic/anaerobic activity
The sample from the ASB anoxic/anaerobic zone displayed greater microbial activity than the aerobic sample. The initial pH (6.48) was lower than that of the aerobic zone (7.65) and
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remained relatively stable throughout the incubation, with a final pH of 6.62. Unlike the aerobic
400
zone, the anoxic/anaerobic zone contains a large amount of organic matter to fuel microbial activity;
401
thus, greater activity was observed during incubation under anoxic/anaerobic conditions. The total
402
COD steadily declined by 17.6% over 96 h (Figure 4A), while the soluble COD increased by
403
137.7% (Figure 4B). Sulfate was quickly consumed and depleted within 48 h (Figure 4C). The gas
404
production rate was relatively high in the first 8 h and then continued at a steady rate, indicating
405
steady biological activity.
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The concentration of total phytosterols increased within the first 24 h and then gradually declined, with a net 105.6% increase by the end of the 96-h incubation (Figure 4E). The initial
408
phytosterol increase is likely due to the release of sorbed and/or bound phytosterols associated with
409
solids, as well as a result of solids disintegration and hydrolysis. Phytosteryl esters, which are
410
sterols bound to other molecules via ester bonds, are hydrolyzed by lipases, producing free
411
phytosterols (Shimada et al., 2003). Because steryl esters are found in non-trivial amounts in plant
412
cells, the contribution of steryl esters to the release of free phytosterols may be significant. For
413
example, the sapwood of Scot’s pine has been found to contain more than 7 times the amount of
414
steryl esters than free β-sitosterol per gram oven dried wood (Martinez-Iñigo et al., 1999). The
415
release of both cell-bound and sorbed phytosterols likely caused the 126% increase in total free
416
phytosterols over the first 24 h. A slight decrease in total free phytosterols occurred following the
417
initial increase, indicating removal. Our previous work has shown that phytosterol removal is
418
possible under sulfate-reducing conditions, although removal was only observed following a
419
significant lag time (Dykstra et al., 2014b). In the present study, over the first 24 h, β-sitosterol,
420
stigmasterol and campesterol increased by 119, 123 and 198%, respectively, indicating that
421
campesterol may be more easily released during solids degradation. Between 24 and 96 h, β-
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sitosterol and campesterol decreased by 8 and 38%, respectively and stigmasterol increased by 12%.
423
During this period, removal of β-sitosterol and campesterol exceeded their release from the solids,
424
while stigmasterol did not. These results suggest that stigmasterol is either released more slowly
425
over time and/or that the rate of removal is less than with β-sitosterol or campesterol. Although little
426
is known about the substrate specificity of enzymes in the anoxic degradation pathway of
427
phytosterols, aerobic cholesterol oxidases preferentially degrade β-sitosterol over stigmasterol
428
(Doukyu, 2009; MacLachlan et al., 2000), which may also be true for enzymes in the anoxic
429
pathway. Two enzymes have been identified in the initial stages of the anoxic metabolism of
430
cholesterol by Sterolibacteriun denitrificans under denitrifying conditions. The first enzyme,
431
AcmA, catalyzed the oxidation of the hydroxyl group and the subsequent isomerization to cholest-
432
4-en-3-one. A second enzyme, AcmB, catalyzed the oxidation to cholesta-1,4-dien-3-one, followed
433
by the hydroxylation of C25 on the side chain using water as the oxygen donor (Chiang, et al.,
434
2008). Enzymes that catalyze reactions on the side chain of a sterol likely vary in substrate
435
specificity between the three phytosterols because of the differences in side chain structure.
436
3.6
437
While it is clear that ASBs remove phytosterols, the mechanisms involved are difficult to determine
438
because of complex interactions between water column and sediment. When considering influent
439
whole PPW and ASB effluent, the overall phytosterol removal by the ASB used in this study was
440
82-94% (Table 4). However, batch assays with the ASB sediment and the settled PPW fraction
441
indicated release of phytosterols associated with solids disintegration and hydrolysis. As a
442
simplified approach, the ASB water column was considered as a well-mixed, isolated system (i.e.,
443
no interaction with settled solids) and a phytosterol mass balance was conducted. Under these
444
assumptions, the water column achieved a total phytosterol removal of 57 to 86%, with the highest
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Phytosterol release and biotransformation
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removal at the longest HRT (Table 4). As discussed in Section 3.2, above, the supernatant portion
446
of the wastewater, which is most likely to remain in the aerobic zone of an ASB, carries 42% of the
447
total phytosterols. Thus, when the influent phytosterols associated with settleable wastewater solids
448
were not taken into account, the phytosterol removal efficiency was lower than when the whole
449
PPW was taken as the influent.
450
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Although the total phytosterol removal was positive at each HRT, the relatively higher βsitosterol removal obscured the low or even negative removal of stigmasterol and campesterol
452
across all HRTs (Table 4). Considering that β-sitosterol was significantly removed from the water
453
column at all HRTs, biotransformation and/or sorption to solids must have occurred. The water
454
column contained a low solids concentration (< 32 mg/L), indicating aerobic biotransformation was
455
likely the primary removal mechanism in this zone. We have previously demonstrated the
456
biotransformation of all three phytosterols under aerobic conditions, with β-sitosterol preferentially
457
removed over stigmasterol and campesterol (Dykstra et al., 2014a). Preferential biodegradation of
458
β-sitosterol has also been observed in previous studies (MacLachlan et al., 2000; Mahmood-Khan
459
and Hall, 2008; Doukyu, 2009).
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Using the isolated water column analysis, at the longest HRT of 22.2 d, stigmasterol and
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campesterol were removed by 10 and 30%, respectively. However, at the two shortest HRTs,
462
corresponding to the highest solids and phytosterols loadings, the analysis showed an increase in
463
stigmasterol (3-fold increase at 11.1 d HRT) and campesterol (6-fold increase at 5.6 d HRT)
464
concentrations, leading to negative stigmasterol and campesterol removal from the water column
465
(Table 4). Thus, an unaccounted source of phytosterols, attributed to the release of phytosterols
466
from settled solids undergoing disintegration and microbial hydrolysis, leads to this imbalance.
467
Indeed, anaerobic incubation of an ASB sediment sample resulted in a nearly 2-fold increase in
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phytosterol concentration over a period of 24 h without a significant phytosterol removal (Figure 4).
469
Another way to view this foregoing analysis is to calculate the required mass rate of phytosterols
470
released from the sediment to achieve a mass balance, assuming no losses in the water column (e.g.,
471
no biodegradation). A positive mass rate would then indicate the minimum rate of phytosterol
472
release from the sediment. Indeed, at the two shortest HRTs of 11.1 and 5.6 d, a positive mass rate
473
from the sediment to the water column must occur for stigmasterol and campesterol to close the
474
mass balance in the water column. The calculated minimum rate of release of stigmasterol and
475
campesterol from sediment was 0.2 and 2.7 µg/d, respectively, at an HRT of 11.1 d, and 9.0 and
476
20.7 µg/d, respectively, at an HRT of 5.6 d. In comparison, the total influent, including settleable
477
solids, carries 14.2-28.5 µg/d of stigmasterol and 12.2-24.3 µg/d of campesterol into the ASB at
478
these HRTs. Thus, at the shortest HRTs, the release of stigmasterol and campesterol into the water
479
column is significant. When phytosterol release from sediment solids was considered in the
480
calculation of ASB phytosterol removal, the efficiency increased from 82-94% to 88-96%.
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481
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The observed overall removal represents the combined removal from both biodegradation and settling. To estimate the contribution of biodegradation to the overall phytosterol removal at the
483
three HRTs, it was assumed that biodegradation took place in the aerobic water column only, that
484
phytosterol release occurred from the sediment into the water column and that phytosterol sorption
485
to solids in the water column was negligible. It should be noted that biodegradation of phytosterols
486
under the anoxic/anaerobic conditions prevailing in the sediment was not significant as shown by
487
the batch assays (Section 3.5, above) and previously reported (Dykstra et al., 2014b). Given these
488
assumptions, across all HRTs, the average phytosterol removal due to aerobic biodegradation in the
489
water column, solids settling and accumulation in the sediment, and effluent discharge was
490
calculated as 51, 40 and 9%, respectively.
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4.
492
Approximately 58% of phytosterols in the PPW used in this study were associated with the settled
493
PWW fraction, which carries phytosterols into anoxic/anaerobic zones where biotransformation of
494
phytosterols is not the main removal mechanism. Thus, ASB sediment acts as a phytosterol
495
reservoir and as solids are disintegrated/degraded, phytosterols are released into the aerobic water
496
column where undergo aerobic biodegradation. Phytosterols entering the ASB in this study were
497
primarily removed via biotransformation, with the remaining phytosterols settling in the sediment
498
and a small portion leaving with the effluent. Settling of solids in an ASB is important for
499
maintaining a low, stable effluent phytosterol concentration across a range of HRTs, although
500
sediment characteristics are significantly affected by HRT and solids loading rate. The results of
501
this study suggest that a model can be developed using HRT, wastewater settling characteristics,
502
and biotransformation rates as key parameters to predict overall ASB phytosterol removal and
503
accumulation in the sediment. Further research is needed to develop and test such a model, which
504
can then guide the design and operation of pulp and paper wastewater ASB treatment systems.
505
Acknowledgements
506
Special thanks to the Institute of Paper Science and Technology for the Paper Science and
507
Engineering fellowships awarded to C. M. Dykstra and H. D. Giles.
508
Appendix. Supplementary data.
509
Supplementary data associated with this article can be found in the online version, at doi:…
510
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Orrego, R., Guchardi, J., Hernandez, V., Krause, R., Roti, L., Armour, J., Ganeshakumar, M., Holdway, D., 2009. Pulp and paper mill effluent treatments have differential endocrine-
587
disrupting effects on rainbow trout. Environ. Toxicol. Chem. 28 (1), 181-188.
591 592 593 594 595 596 597
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590
model. Water Pollut. Control Fed. 59 (10), 877-883.
Shimada, Y., Nagao, T., Watanabe, Y., Takagi, Y., Sugihara, A., 2003. Enzymatic conversion of steryl esters to free sterols. J. American Oil Chemists’ Soc. 80 (3), 243-247. Strotmann, U.J., Eglsaer, H., 1995. The toxicity of substituted phenols in the nitrification inhibition test and luminescent bacteria test. Ecotoxicol. Environ. Safety 30 (3), 269-273.
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Sackellares, R.W., Barkley, W.A., Hill, R.D., 1987. Development of a dynamic aerated lagoon
Svenson, A., Sanden, B., Dalhammar, G., Remberger, M., Kaj, L., 2000. Toxicity identification and evaluation of nitrification inhibitors in wastewaters. Environ. Toxicol. 15 (5), 527-532. Tugtas, A.E., Pavlostathis, S.G., 2007. Effect of sulfide on nitrate reduction in mixed methanogenic cultures. Biotechol. Bioeng. 97 (6), 1448-1459.
EP
588
SC
586
von Bonsdorff-Nikander, A., Christiansen, L., Huikko, L., Lampi, A-M., Piironen, V., Yliruusi, J.,
599
Kaukonen, A.M., 2005. A comparison of the effect of medium- vs. long-chain triglycerides
600 601 602
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on the in vitro solubilization of cholesterol and/or phytosterol into mixed micelles. Lipids 40 (2), 181-190.
Xavier, C., Mosquera-Corral, A., Becerra, J., Hernandez, V., Vidal, G., 2009. Activated sludge
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versus aerated lagoon treatment of Kraft mill effluents containing β-sitosterol and
604
stigmasterol. J. Environ. Sci. Health, Part A 44 (4), 327-335.
26
ACCEPTED MANUSCRIPT
Table 1 – Characteristics of whole, settled and supernatant wastewater fractions. Supernatant
Settled
Wastewater
fractiona
fractiona
8.95 ± 0.02b
9.91 ± 0.02
8.83 ± 0.02
TS (g/L)
3.6 ± 0.03
1.8 ± 0.02
30.2 ± 0.3
VS (g/L)
1.2 ± 0.01
0.43 ± 0.004
12.4 ± 0.1
Total COD (mg/L)
2,501 ± 66
1,001 ± 113
24,078 ± 125
865 ± 31
849 ± 6
741 ± 35
Nitrite (mg N/L)
NDc
ND
ND
Nitrate (mg N/L)
ND
0.09 ± 0.003
ND
Ammonia (mg N/L)
ND
ND
ND
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Soluble COD (mg/L)
Chloride (mg Cl/L) Phosphate (mg P/L) Sulfate (mg S/L) Total phytosterols (µg/L)
Campesterol (µg/L)
77.2 ± 0.4
80.1 ± 0.6
53.1 ± 0.1
18.6 ± 0.1
10.0 ± 0.1
22.1 ± 0.1
113.0 ± 0.4
122.9 ± 0.3
114.4 ± 0.2
541 ± 20
245 ± 26
4,790 ± 32
439 ± 20
226 ± 25
3,514 ± 32
58 ± 2
14 ± 4
685 ± 5
44 ± 1
6±1
590 ± 2
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β-sitosterol (µg/L) Stigmasterol (µg/L)
SC
pH
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Whole
Parameter
Supernatant and settled fractions: 93.5 and 6.5 % (v/v) of whole wastewater, respectively
b
Mean ± standard deviation (n = 3)
c
ND, not detected
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a
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Table 2 –ASB influent and steady state effluent characteristics at three HRTs. HRT 22.2 d
HRT 11.1 d
HRT 5.6 d
6.5 ± 0.02a
7.44 ± 0.02
7.48 ± 0.02
7.53 ± 0.02
TSS (mg/L)
846 ± 19
31 ± 1.5
29 ± 1.4
17 ± 0.3
VSS (mg/L)
321 ± 6
16 ± 0.1
Total COD (mg/L)
834 ± 22
99 ± 4
Soluble COD (mg/L)
288 ± 10
69 ± 7
Nitrite (mg N/L)
NDb
ND
Nitrate (mg N/L)
0.12 ± 0.01
8.7 ± 0.2
Ammonia (mg N/L)
30
Chloride (mg Cl/L)
101.7 ± 0.1
Phosphate (mg P/L)
16.21 ± 0.03 37.7 ± 0.1
Sulfate (mg S/L) Total phytosterols (µg/L) β-sitosterol (µg/L)
ND, not detected
64 ± 12
103 ± 15
ND
ND
13.6 ± 1.0
1.7 ± 0.04
ND
ND
ND
155.6 ± 1.6
246 ± 18.9
139.2 ± 1.5
19.9 ± 2.5
25.9 ± 3.0
50.6 ± 3.0
49.0 ± 0.9
66.5 ± 4.6
39.8 ± 0.7
c
35.6 ± 3.9
34.1 ± 3.7
156.1 ± 2.5
4.5 ± 1.2
6.7 ± 0.8
2.4 ± 0.2
4.9 ± 3.0
c
23.2 ± 3.8
12.6 ± 3.4
1.9 ± 0.7
c
5.7 ± 0.6
19.1 ± 1.4
22.6 ± 0.8 19.3 ± 0.7
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Values reported by Giles (2012).
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c
130 ± 3
c
Mean ± standard deviation (n = 3)
b
106 ± 10
11.3 ± 3.3
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a
14 ± 0.4
198.0 ± 2.7
Stigmasterol (µg/L) Campesterol (µg/L)
17 ± 0.7
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pH
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Influent
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Parameter
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Table 3 – Steady state ASB sediment characteristics at various HRTs. HRT 22.2 d
HRT 11.1 d
HRT 5.6 d
pH
6.42 ± 0.02a
6.76 ± 0.02
6.76 ± 0.02
Total COD (mg/L)
8,037 ± 92
14,591 ± 575
18,762 ± 920
86 ± 35
119 ± 10
284 ± 50
Nitrite (mg N/L)
NRb, c
NDd
ND
Nitrate (mg N/L)
NR
0.04 ± 0.01
6.8 ± 0.2
Ammonia (mg N/L)
NR
9.3 ± 8.1
14 ± 0
Chloride (mg Cl/L)
NR
119 ± 0.5
599 ± 0.6
Phosphate (mg P/L)
NR
47.6 ± 1.6
728 ± 15
Sulfate (mg S/L)
NR
3.3 ± 0.1
61 ± 1.3
7,634 ± 1,148
β-sitosterol (µg/L)
1,682 ± 370
Campesterol (µg/L)
c
1,372 ± 54
Giles (2012).
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ND, not detected
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d
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Mean ± standard deviation (n = 3) NR, not reported
c
c
Stigmasterol (µg/L)
b c
SC
10,688 ± 1,207c
Total phytosterols (µg/L)
a
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Soluble COD (mg/L)
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Parameter
29
18,157 ± 280
43,838 ± 1,977
4,649 ± 122
30,618 ± 871
4,304 ± 113
10,334 ± 1,530
9,204 ± 225
2,886 ± 900
ACCEPTED MANUSCRIPT
Table 4 – Steady state phytosterol removal (%) in ASB. Phytosterols
HRT 22.2 d
HRT 11.1 d
HRT 5.6 d
β-sitosterol
97
96
98
Stigmasterol
78
-3
Campesterol
90
70
Total phytosterols
94
82
β-sitosterol
94
92
Stigmasterol
10
-328
Campesterol
30
-111
-607
Total phytosterols
86
57
59
44 1
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83
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Isolated Water Column Removal
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Observed Overall Removal
30
97
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LIST OF FIGURES Figure 1. Time course of total and soluble COD (A), ammonia (B), nitrate (C), and
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phytosterols (D) during the aerobic wastewater batch assay. Error bars represent mean values ± one standard deviation, n = 3.
Figure 2. Time course of total and soluble COD (A), VFAs (B), sulfate (C), gas production (at
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25°C, 1 atm) (D), gas composition (E) and phytosterols (F) during the anaerobic wastewater
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batch assay. Error bars represent mean values ± one standard deviation, n = 3. Figure 3. Activity of the ASB aerobic zone monitored by total and soluble COD (A), VSS (B), and phytosterols (C) through a 96-h batch assay. Error bars represent mean values ± one standard deviation, n = 3.
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Figure 4. Activity of the ASB anoxic/anaerobic zone monitored by total COD (A), soluble COD (B), sulfate (C), gas production (at 25°C, 1 atm) (D), and phytosterols (E) through a 96-h
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batch assay. Error bars represent mean values ± one standard deviation, n = 3.
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800 600 400 200
0.2
0.1
0.0
PHYTOSTEROLS (µg/L)
(B)
80 60 40 20 0
(D)
3000
Total phytos β-sitosterol Stigmasterol Campesterol
2000
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AMMONIA (mg N/L)
0
(C)
0.3
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Total COD Soluble COD
SC
COD (mg/L)
NITRATE (mg N/L)
(A)
1000
1000
0
0
5
10
15
20
25
0
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Time (days)
30
5
10
15
20
25
30
Time (days)
Figure 1. Time course of total and soluble COD (A), ammonia (B), nitrate (C), and phytosterols (D) during the aerobic wastewater batch assay. Error bars represent mean
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values ± one standard deviation, n = 3.
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15 10
Total COD Soluble COD
5 0
400 200
Time (d) vs tCOD (g/L) Time (d) vs sCOD (g/L)
1000
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120 100 80 60 40 0 0
0.6
CH4 H2S
0.4
CO2
0.2 0.0
5
10
15
20
PHYTOSTEROLS (mg/L)
0 160 (C) 140
20
0.8
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GAS COMPOSITION
(B)
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VFAs (mg COD/L)
600
0
3000
SULFATE (mg S/L)
800 (D)
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GAS PRODUCTION (mL)
COD (g/L)
20 (A)
60 (F) 50 40
Total phytos β-sitosterol Stigmasterol Campesterol
30 20 10 0
25
30
0
10
15
20
25
30
Time (days)
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Time (days)
5
Figure 2. Time course of total and soluble COD (A), VFAs (B), sulfate (C), gas production (at 25°C, 1 atm) (D), gas composition (E) and phytosterols (F) during the anaerobic wastewater batch assay. Error bars represent mean values ± one standard deviation, n = 3.
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200 150
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COD (mg/L)
Total COD Soluble COD
(A)
250
100 50 0
40
0 (C)
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VSS (mg/L)
60
20
PHYTOSTEROLS (µg/L)
SC
(B)
80
Total phytos β-sitosterol Stigmasterol Campesterol
40
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20
0
AC C
0
20
40
60
80
100
TIME (hours)
Figure 3. Activity of the ASB aerobic zone monitored by total and soluble COD (A), VSS (B), and phytosterols (C) through a 96-h batch assay. Error bars represent mean values ± one standard deviation, n = 3.
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30 20
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TOTAL COD (g/L)
40 (A)
10
300
100
100
50 0
0 (C)
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0 0
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SULFATE (mg S/L)
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(D)
200
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GAS PRODUCTION (mL)
400 (B)
20
40
60
PHYTOSTEROLS (mg/L)
SOLUBLE COD (mg/L)
0
(E)
30
Total phytos β-sitosterol Stigmasterol Campesterol
20
10
0 80
100
0
20
40
60
80
100
TIME (hours)
AC C
TIME (hours)
Figure 4. Activity of the ASB anoxic/anaerobic zone monitored by total COD (A), soluble COD (B), sulfate (C), gas production (at 25°C, 1 atm) (D), and phytosterols (E) through a 96-h batch assay. Error bars represent mean values ± one standard deviation, n = 3.
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Highlights Pulp and paper wastewater settleable fraction carries ca. 58% of total phytosterols
•
A simulated aerated stabilization basin (ASB) was operated for 2.4 years
•
ASB total phytosterol removal was 82-94% at three hydraulic retention times
•
Breakdown of settled solids in ASB sediments releases phytosterols
•
Biotransformation/settling account for 51/40% phytosterols removal, respectively
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Supplementary Material
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Fate and biotransformation of phytosterols during treatment of pulp and paper wastewater in a simulated aerated stabilization basin Christy M. Dykstraa, Hamilton D. Gilesa, Sujit Banerjeeb and Spyros G. Pavlostathisa,* a
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School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0512, USA
b
School of Chemical and Biomolecular Engineering, Institute of Paper Science and Technology
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Georgia Institute of Technology, 500 10th Street, N.W., Atlanta, GA 30318, USA *Corresponding author. Tel.: +404-894-9367; fax: +404-894-8266;
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E-mail address:
[email protected] (S. G. Pavlostathis)
Water Research
Date Prepared:
7 June 2014
Text Sections:
None
S1 – S2
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Figures:
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Tables:
S1
Pages:
4
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Figure S1. Sterol structures: (a) numbered sterol skeleton, (b) cholesterol, (c) β-sitosterol, (d) stigmasterol, (e) campesterol.
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3
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2
1
0 0
5
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ABSORBANCE (AU at 590 nm)
4
10
15
20
TIME (hours)
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Figure S2. Absorbance measurements of wastewater supernatant during settling. Error bars represent mean values ± one standard deviation, n = 2.
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Table S1. Sterol degradation reactions under various redox conditions and standard Gibbs free energy of reaction (ΔGro′).
Conditions / Reaction Aerobic Oxidation C29H50O + 41O2 → 29CO2 + 25H2O
SS
C29H48O + 40.5O2 → 29CO2 + 24H2O
CS
C28H48O + 39.5O2 → 28CO2 + 24H2O
Ch
C27H46O + 38O2 → 27CO2 + 23H2O
Nitrate Reduction βS
SC
βS
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Sterol
ΔGro′ kJ/mol
C29H50O + 32.8NO3- + 32.8H+ → 29CO2 + 41.4H2O + 16.4N2
ΔGro′ kJ/eeq
-17,218
-104.99
-17,060
-105.31
-16,588
-104.99
-15,933
-104.82
-16,149
-98.47
SS
C29H48O + 32.4NO3 + 32.4H → 29CO2 + 40.2H2O + 16.2N2
-16,004
-98.79
CS
C28H48O + 31.6NO3- + 31.6H+ → 28CO2 + 39.8H2O + 15.8N2
-15,558
-98.47
Ch
C27H46O + 30.4NO3- + 30.4H+ → 27CO2 + 38.2H2O + 15.2N2
-14,942
-98.30
-888.9
-5.42
-929.9
-5.74
-856.4
-5.42
-798.0
-5.25
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+
-
Sulfate Reduction βS
C29H50O + 20.5SO42- + 30.8H+ → 29CO2 + 25H2O + 10.3 H2S + 10.3HS-
SS
C29H48O + 20.3SO4 + 30.4H → 29CO2 + 24H2O + 10.1H2S + 10.1HS
CS
C28H48O + 19.8SO42- + 29.6H+ → 28CO2 + 24H2O + 9.9H2S + 9.9HS-
Ch
C27H46O + 19.0SO42- + 28.5H+ → 27CO2 + 23H2O + 9.5H2S + 9.5HS-
+
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2-
-
Methanogenesis
C29H50O + 16H2O → 8.5CO2 + 20.5CH4
-449.4
-2.74
SS
C29H48O + 16.5H2O → 8.8CO2 + 20.3CH4
-495.7
-3.06
C28H48O + 15.5H2O → 8.3CO2 + 19.8CH4
-432.9
-2.74
C27H46O + 15.0H2O → 8.0CO2 + 19.0CH4
-390.6
-2.57
CS Ch
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βS
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βS = β-sitosterol; CS = campesterol; SS = stigmasterol; Ch = cholesterol
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