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Biological removal of phyto-sterols in pulp mill effluents
Journal of Environmental Management 131 (2013) 407e414
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Biological removal of phyto-sterols in pulp mill effluents Zahid Mahmood-Khan a, *, Eric R. Hall b a b
Agricultural Engineering Department, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Punjab 60800, Pakistan Department of Civil Engineering and The Pulp and Paper Centre, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2012 Received in revised form 3 September 2013 Accepted 23 September 2013 Available online 8 November 2013
Phyto-sterols and extractives found in pulp mill effluents are suspected to cause endocrine abnormalities in receiving water fish. The control of sterols in pulp mill effluents through biological secondary wastewater treatment was studied using two lab-scale bioreactor systems. After achieving a stable performance, both bioreactor systems successfully removed (>90%) sterols and the estimated biodegradation was up to 80%. Reactor 1 system operating at 6.7 0.2 pH effectively treated pulp mill effluent sterols spiked up to 4500 mg/L in 11 h HRT and 11 day SRT. However, Reactor 2 system operating at 7.6 0.2 pH performed relatively poorly. Retention time reductions beyond critical values deteriorated the performance of treatment systems and quickly reduced the sterols biodegradation. The biodegradation loss was indicated by mixed liquor sterols content that started increasing. This biodegradation loss was compensated by the increased role of bio-adsorption and the overall sterols removal remained relatively high. Hence, a relatively small (20e30%) loss in the overall sterols removal efficiency did not fully reflect the associated major (60e70%) loss in the sterols biodegradation because the amount of sterols accumulated in the sludge due to adsorption increased so the estimate of sterols removal through adsorption increased from 30e40% to 70e80% keeping the overall sterols removal still high. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Keywords: Phyto-sterols Biodegradation Bio-adsorption Pulp mill effluent Secondary wastewater treatment
1. Introduction Currently, there are concerns over the observed estrogenic, and in some cases, androgenic effects in fish and aquatic life in water bodies receiving pulp mill effluents (Basu et al., 2012; Cody and Bortone, 1997; Lehtinen et al., 1999). These effects have been attributed to certain chemicals, including plant sterols and wood extractives (Tremblay and Van Der Kraak, 1999; Vidal et al., 2007; Landman et al., 2008; Orrego et al., 2010, 2011; Hou et al., 2011). If not treated and controlled properly, plant sterols or phytosterols in pulp mill effluents may contaminate receiving waters (Mahmood-Khan and Hall, 2003, 2008), where they may act as endocrine disrupting chemicals (EDCs) or hormonally active agents (HAAs). A popular form of suspended growth systems is activated sludge treatment (AST) which is one of the typical treatment options employed for treating pulp mill effluents. Most of the AST systems are effectively removing majority of the organic pollutants thereby reducing biochemical oxygen demand (BOD) and acute toxicity
* Corresponding author. Tel.: þ92 61 9210298, þ92 302 5447015 (mobile); fax: þ92 61 9210098. E-mail addresses: [email protected], [email protected] (Z. MahmoodKhan), [email protected] (E.R. Hall).
(Springer, 1993) from pulp mill effluents. The activated sludge process has been proven to be both cost effective and reliable, in general. However, AST systems have shown instability in reducing sterols and other organic pollutants in pulp mill effluents (Cook et al., 1997; Van Ginkel et al., 1999; Mahmood-Khan and Hall, 2003). Other studies have shown a strong tendency of plant sterols to accumulate in secondary sludge during secondary biological treatment indicating adsorption as a major mechanism of sterols removal (Chandra and Singh, 2012; Magnus et al., 2000; McKague and Reeve, 2003; Mahmood-Khan and Hall, 2008). Many details about the behavior and fate of pulp mill effluent sterols entering biological wastewater treatment systems are not clear. Selective microbial degradation of sterols like Cholesterol and b-Sitosterol has also been reported (Arima et al., 1969; Niven et al., 2001; Kostamo and Kukkonen, 2003). However, there are questions about sterols removal mechanisms particularly about their biodegradation as the observed sterols removal from the effluents may be mainly due to their accumulation in secondary sludge rather than biodegradation. In Finland, a survey of elemental chlorine free kraft pulp mill, a paper mill and an integrated kraft pulp and paper mill, indicated 35e99% degradation of wood extractives and sterols from pulp mill effluents across secondary systems including AST (Kostamo et al., 2004). Vidal et al. (2007) also suggested a high (77e95%) removal of sterols from kraft mill
0301-4797/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.09.031
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effluents across a continuously fed anaerobic filter, with only 10% reduction in the methanogenic activity of the bioreactor. Besides adsorption, biodegradation may probably play an important role in sterols removal and control during secondary treatment of pulp mill effluents. Can sterols biodegradation play a significant role in sterols removal or just adsorption is the main mechanism of sterols removal during secondary treatment of pulp mill effluents? These are important questions to understand and improve the treatment and control of plant sterols in pulp industry wastewaters. Therefore, the objective of the present study was to examine the extent of sterols removal and the role of biodegradation during laboratory scale biological treatment of pulp mill effluents. For this purpose, sterols containing pulp mill effluents were treated using two labscale biological reactors equipped with secondary clarifiers, and sterols removal was monitored at various influent sterol concentrations and different operating conditions. 2. Materials and methods
a
Heated Water V
P
Air Supply
Air M
M N
M
C
H
M
H E
S
S
F D
R
S
D
D
R
S D
F
Feed
T
Recycle
Recycle Heated Water
Air
b
2.1. Lab-scale suspended growth activated sludge systems In the present study, AST was used in the form of two identical lab-scale bioreactors as shown in Fig. 1a and the dimensions are shown in Fig. 1b. Each bioreactor was made of PlexiglasÔ, with a 4 L aeration basin working capacity. The bioreactors were fitted with double-jacketed walls to provide the passage of circulating hot water for process temperature control. A double-jacketed secondary clarifier made with PlexiglasÔ was also provided for each bioreactor (Fig. 1c), so the two systems could be operated separately. Other mechanisms included influent and nutrients feeding through peristaltic pumps, pH controllers, mechanical mixing, air supply diffusers, arrangements for return and waste secondary sludge to allow desired operational conditions. The influent to the lab-scale bioreactors consisted of primary treated effluent from a Canadian Pulp Mill in British Columbia. The effluent was collected in 25 L NalgeneÔ containers and was refrigerated at 4 C before use. The bioreactors were inoculated with biomass obtained from the full-scale high purity oxygen (UNOX) AST system at the Mill, to grow acclimated biomass for treating pulp mill effluent sterols. The start-up process took six weeks. Air was supplied using two Aqua FizzzzÔ (A-962) fine bubble diffusers in each bioreactor. Sufficient air was supplied to keep the dissolved oxygen level around 5e6 mg/L for achieving oxygen transfer conditions similar to those of the full-scale UNOX-AST system. Additional mixing was provided using Electric ArrowÔ (Model # 1750) variable speed mechanical mixers rotating at 160e 180 RPM. 2.2. Nutrients, pH control and process temperature Nutrients were supplied in an approximate proportion of 100:3.5:1 for COD:N:P. NH4Cl was used for nitrogen, and a mixture of K2HPO4 and KH2PO4 was used for P. The pH control solutions were 0.2 N H2SO4 and 0.2 N NaOH. After the start-up phase, 0.2 N NaOH was mostly required to keep the reactor pH at the desired level. There were three phases of the biological treatment study and Reactor 1 was operated at a pH of 6.6e6.8 (near neutral conditions) throughout the three phases, as AST systems are usually operated. Reactor 2 was started at a pH of 7.0 0.2 in the 1st phase, but later in the 2nd and 3rd phase its pH was increased to 7.6e7.8 (slightly alkaline conditions) to cover the normal range of process pH at the full-scale UNOX-AST system. Slightly alkaline conditions may favor the treatment of organic pollutants in pulp mill effluents (Van Ginkel et al., 1999; Werker and Hall, 1999). The process pH was
15.24
Hot Water
Inflow 29.2
4L
21.6
Hot Water
Outflow 21.6
c 15.24
Hot Water
22.23
12-15
Overflow Hot Water
2.54 21.6 Mixed Liqour
Fig. 1. (a). Schematic of lab-scale suspended growth AST system: C pH control, D diffuser aerator, E final effluent, F influent feed, H heated water, M stirrer motor, N Nutrients, P air supply pump, R recycle, T temperature control, S mechanical stirrer, V control valve. (b). Dimensions (cm) of lab-scale bioreactors. (c). Dimensions (cm) of the lab-scale secondary clarifiers.
measured using pH/mV ChemCadet Cole ParmerÔ (Model # 598450) meter and maintained by using Cole ParmerÔ pH/pump systems (Model # 7142-60). Process temperature was maintained at 38e39 C using a VWRÔ Constant Temperature Circulator (Model # 1130 A) water bath for both bioreactors. This is the temperature at which the fullscale UNOX-AST plant was typically operated at the Mill. 2.3. Sterols spiking The primary-treated effluent was screened through a coarse strainer to remove fiber fines, into a 60 L holding tank and then used as influent to the bioreactors. The primary-treated influents were spiked when required, with recovered sterols (from wood waste) supplied by BC Chemicals, 2711 Pulp Mill Road, Prince George, BC, Canada, V2N 2K3. The sterols received in fluffy powdered solid state were dissolved in pulp mill effluent in a stepwise procedure as explained below.
Z. Mahmood-Khan, E.R. Hall / Journal of Environmental Management 131 (2013) 407e414
The sterols were first dissolved in iso-propanol (2-propanol) after examining their solubility in different solvents: methanol, ethanol, acetone, hexane and 2-propanol. Each of these five solvents were separately added to conical-bottom penny-head stopper PyrexÔ glass centrifuge tubes (Corning 8084 15) calibrated to contain a specific volume (10e15 mL) and graduated to 0.1 mL. The centrifuge tubes contained a given mass of sterols to which solvent was added slowly, mixed with a vortex mixer and heated to about 40 C for 5e6 min. The solvent was added until the sterols were completely dissolved. Final volume was noted after adding the last drop and considered to represent sterols solubility in the solvent under consideration. The solubility of sterols at 40 C in methanol, ethanol, hexane, acetone and iso-propanol was tested to be 1.8e2, 6e7, 13.6e15, 28e30 and 32e36 mg sterols/mL solvent respectively. Relative to other solvents, 2-propanol was required in the least amount to dissolve a given mass of sterols. Therefore, 2-propanol was selected to pre-dissolve the semi-crystalline sterols. The sterols solution in 2-propanol was then introduced first in to a small volume of primary effluent (heated to 40 C) and then mixed with larger volume of primary effluent. The whole process was completed in the following three steps: I. A sterols stock-solution was prepared by dissolving 200 mg of sterols in 5.5 mL of iso-propanol (2-propanol) at about 40 C. II. The 5.5 mL sterols stock-solution was introduced to about 4 L of primary effluent at about 40 C and continuously mixed on a stirrer plate heater for 4e6 h to get an intermediate pulp mill effluent solution. III. The intermediate sterol solution was then introduced to a given volume (40e60 L, as required) of bioreactor influent and mixed for 2 h at room temperature to produce the desired concentration of sterols in the laboratory bioreactor influent. The spiked influent was continuously stirred and fed to the laboratory bioreactors, the general characteristics of the bioreactor influent are given in Table 1. The fraction of pulp mill effluent sterols that passed through WhatmanÔ AH-934 fiberglass filters was considered to be dissolved in this study. The particulate fraction was determined by the difference in the analyzed amount of sterols in the whole sample and the filtrate. Typically, 70e130 mg/L of COD was added as 2-propanol to the spiked primary-treated influent. 2.4. Chemical analyses Total suspended and volatile solids (TSS& VSS) were analyzed in the influent, effluent and mixed liquor and secondary sludge, according to the Standard Methods for the Examination of Water and Wastewater 2540 D and 2540 E (Clesceri et al., 1989). Chemical oxygen demand (COD) was measured through a closed reflux Table 1 Characteristics of primary effluent obtained from mill. Characteristic
Low
High
Typical
COD BOD TSS pH Temperature Native sterols Dissolved Spiked sterols Dissolved
1100 mg/L 300 mg/L 70 mg/L 6.8 37 C 600 mg/L 38% 1000 mg/L 35%
1500 mg/L 380 mg/L 120 mg/L 7.8 41 C 2500 mg/L 65% 4500 mg/L 70%
1300 mg/L 340 mg/L 80 mg/L 7.2 39 C 1400 mg/L 46% 2500 mg/L 50%
409
Table 2 Different phases of lab-scale bioreactor operation. Experimental phase (duration) Reactor 1 I (3 months)a II (3 months) III (2.5 months) Reactor 2 I (3 months)a II (3 months) III (2 months)
Sterol concentration (mg/L)
HRT (h)
SRT (d)
pH
1000e1500 2000e3500 3500e4500
28e20 20e13 13e8
27e23 23e10 10e7
6.6 0.2 6.7 0.2 6.7 0.2
1000e1500 2000e3500 3500e4500
24e20 20e11 11e9
25e22 22e12 12e8
7.0 0.2 7.6 0.2 7.6 0.2
a Results for 1st 40 days were not reported due to reactor start-up and sterol spiking problems.
colorimetric Standard Method 5220 D (Clesceri et al., 1989), using HachÔ DR/2000 Spectrophotometer by reading absorbance values at 600 nm. Total organic carbon (TOC) measurements were made through combustion-infrared Standard Method 5310 B (Clesceri et al., 1989), using a ShimadzuÔ TOC-500 (Shimadzu Corporation Kyoto, Japan) analyzer, calibrated with each batch of samples. Plant sterols were analyzed in the bioreactor influent, effluent and mixed liquor according to the analytical procedure described by Mahmood-Khan and Hall (2008) and is briefly given below. 2.4.1. Sterol extraction Depending upon the type of PPME (primary or secondary), 25e 100 mL of effluent was extracted three times with 15e40 mL of methyl-t-butyl ether (MTBE) using prefired, clean Pyrex TM centrifuge glass tubes. The mixture of PPME and MTBE was shaken for about 15 min by wrist-action mechanical shaker, allowed to settle for about 10 min, and centrifuged at an average relative centrifugal force of 1600 g for separation. The extracted sterols were incubated at 70 C for 4 h, for silylating the sterols as the trimethylsilyl ethers using BSTFA (N, O-Bis tri-methylsilyltriflouroacetamide). 2.4.2. Gas chromatographemass spectrometer analysis A HewlettePackard Model HP-6890 Series Gas ChromatographeMass Spectrometer (GCeMS) System was used with a J&W DB-5MS 30 m long, 0.25 mm coating, 0.25 mm I.D. capillary column. The GC oven conditions were: 130 C, 1 min hold, ramp_1 to 285 C@15 C/min, 3 min hold; ramp _2 to 310 C @ 2 C/min, 1 min hold, post run at 315 C, 2.17 min hold; inlet 290 C, carrier gas helium at 11.2 psi (1 psi ¼ 6.89 kPa) and 53.6 mL/min; mass selective detector at 280 C. The MS instrument HP 5973 Mass Spectrometer Detector coupled with the GC system provided satisfactory detection and identification of phytosterols and other test chemicals along with the GC system. Dotriacontane was used as an internal standard to account for gas chromatograph instrument performance and response. 2.5. Sterols removal experiments Both lab-scale bioreactors were started by inoculation using 300e400 mL of recycle activated sludge (RAS) resulting in about 1500 mg/L final MLSS concentration. The RAS was freshly obtained from Mill in 1 L plastic containers. The containers allowed for some headspace air for biomass respiration during travel. Additional RAS was also added to the bioreactors for re-seeding after the 2nd and the 3rd week. Primary effluent was fed to the bioreactors at a low flow rate resulting in about 24 h hydraulic retention time (HRT). The sterols removal from pulp mill effluent was assessed across the continuous-flow lab-systems in three phases, of 2e3 months each. Samples were taken after 2e3 HRT intervals, after the effluent
Z. Mahmood-Khan, E.R. Hall / Journal of Environmental Management 131 (2013) 407e414
3.2. Sterols in the effluent from the bioreactor systems
sterol concentration had stabilized. Each study phase was continued for about 3 times the average solids retention time (SRT) of the system. The SRT was controlled by wasting mixed liquor directly from the bioreactors. The influent sterols concentrations and the process control variables (HRT, SRT and pH) are given in Table 2.
The overall sterols removal in all the three phases is shown in Fig. 2b and c for Reactor 1 and Reactor 2 respectively. The loss/ removal of sterols through volatilization, stripping and chemical degradation may be neglected as the sterols are chemically stable, non-volatile and moderately non-polar in nature with melting points in 100e180 C range e.g. Sitosterol melts at about 136 C (Chapman and Hall, 1996), and Campesterol at 157 C. The observed sterols removal was therefore considered to be a combined result of adsorption to the suspended solids and biodegradation in the mixed culture environment of activated sludge as suggested by Mahmood-Khan and Hall (2008) and Kostamo et al. (2004).
3. Results and discussions 3.1. Sterols in the influent to the bioreactors The bioreactors were started with a relatively low flow rate (3.3e3.5 L/d). The flow rate and sterol concentrations were gradually increased after the operation of the bioreactors had stabilized (Fig. 2a). The composition individual sterol fractions in the influent remained similar throughout the study. b-Sitosterol (b-Sito), bSitostanol (b-Sitosta) and Campesterol (Campe) were the dominant fractions entering the bioreactors with Stigmasterol (Stigma) as a relatively minor fraction. The average composition of influent sterols was: b-Sito 46%, b-Sitosta 31%, Campe 19% and Stigma 4% (Fig. 2a). This is in agreement with the results obtained by other researchers (Cook et al., 1997; Mahmood-Khan and Hall, 2008; Magnus et al., 2000; Rydholm, 1965; Shoppee 1964). Ergosterol and Cholesterol mostly remained non-detectable.
a
3.3. Sterols removal and the bioreactor systems pH The main difference in the operation of the bioreactors was pH and the results show different performance for each unit (Fig. 2d). Reactor 2 was operated at a pH slightly higher than that of Reactor 1. The differences in reactor behavior may relate to a spectrum of interacting factors. Firstly, the pH differences within the typical range used for biological treatment may cause variations in the biobasin mixed cultures (Werker, 1998). Secondly, pH-associated changes in the solubility of sterols and/or other organics present in pulp mill effluent may alter their availability for microbial
b
5000
5000
Phase 2
4000 3500
4000
Phase 1
Phase 3
Total Sterols (µg/L)
Concentration (µg/L)
100
4500
4500
3000 2500 2000 1500
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60 2500 2000
40
Removal (%)
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90
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Influent
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Effluent_1
210
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% Removal_1
9.0
100 90
3000 60
2500 2000
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Removal (%)
3500
Process pH (pH units)
8.5 80
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8.0
60 7.5
50 40
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4000 Total Sterols (µg/L)
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0 60
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180
210
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% Removal_2
6.0 30
0 60 pH_1
90
120 pH_2
150 Day
180
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% Removal_1
240 % Removal_2
Fig. 2. (a). Plant sterols in the influent to the lab-scale bioreactors. (b). Total-sterols removal efficiency Reactor 1. (c). Total-sterols removal efficiency Reactor 2. (d). Process pH and total-sterols removal performance of Reactor 1 and Reactor 2 (indicated by suffix_1 and _2).
Z. Mahmood-Khan, E.R. Hall / Journal of Environmental Management 131 (2013) 407e414
a
35
100 90
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80 70
25
60 20
50 40
15
30 20
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10 5
0 30
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3.4. Effect of HRT and SRT on the biological removal of sterols HRT_1
3.5. The role of sterols biodegradation and bio-adsorption Both of the bioreactors showed similar trends i.e. decreasing SRTs increased the amount of sterols discharge with waste
b
150 Day
180
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SRT_ 1
240
% Removal_1
100
30
90 80
25
70 60
20
50 15
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% Removal
HRT (h); SRT (d)
The laboratory bioreactors were started with relatively long retention times (HRTs and SRTs) in the 1st phase. When the biological process stabilized, and about 80% removal of sterols was achieved, the influent concentration was increased and the retention times were decreased gradually to match the full scale conditions (10 h HRT, 10 d SRT) by the end of the 3rd phase (Fig. 3a and b). Fig. 4a and b present the sterols associated with lab-systems biomass for the 2nd and the 3rd phases of the study. The results suggest an SRT of 11e12 day and an HRT of about 11 h were critical for effective removal of sterols during secondary treatment under the conditions of this experiment. The removal efficiencies generally declined with detention times below these critical values. SRT is a variable of fundamental importance that is functionally related to the steady-state specific growth rate of biomass in a completely-mixed bioreactor (Grady et al., 1999; Metcalf and Eddy, 2003). The biomass growth results in pollutant removal that is used as substrate and is biodegraded. It is known that a decrease in SRT, within the operating range of values, increases the microbial specific growth rate as well as the observed yield. However, for a fixed reactor volume, the amount of biomass in the reactor also decreases. Hence, if the sterols were being biodegraded in the lab bioreactors, the overall sterols removal through biodegradation will decrease with reducing SRT. Under these conditions a reduction in HRT appears to further reduce the biomass available for pollutant degradation, resulting in an additional loss of reactor performance. The observed loss in sterols removal was recovered through a small incremental increase in the system SRT and HRT for both reactors. This was probably the result of improved biodegradation of sterols through the re-establishment of sterol-degrading microorganisms. Although use of HRT has diminished in importance for sizing suspended growth reactors (WEF and ASCE, 1998), the results of this study suggest HRT can be an important consideration for the design and operation of biological systems treating pulp mill effluent sterols. Therefore, both solids and hydraulic retention times can impact the performance of biological systems treating pulp mill effluent within the normal range usually observed in fullscale operation. This may possibly be a reason for the reported fluctuations in sterols removal during the full-scale operation (Kostamo et al., 2004). However, biodegradation is not the only mechanism of sterols removal, and fluctuations in sterol adsorption to secondary solids will also impact the overall sterols removal.
% Removal
HRT (h); SRT (d)
degradation (Werker and Hall, 1999). For example, the solubility of pulp mill effluent organics like resin acids and kraft lignin increases with increasing pH (Marton, 1964; Werker, 1998). Higher solubility tends to reduce the sorptive behavior of such compounds (Klimenko et al., 2002; Shaw, 1992). A reduction in sterols sorption and/or biodegradation will cause the bioreactor sterol removal performance to decline, until such capacity is re-gained. Therefore, the differences in the performance of the two reactors may be attributable to the operating pH of each reactor. With some differences, both of the laboratory reactors were able to remove sterols from pulp mill effluents. However, the overall operation of Reactor 1 appeared to be more successful relative to that of Reactor 2. These results showed that (w95%) removal of sterols was achieved and maintained through biological treatment and near neutral pH was favorable for sterols removal across such systems.
411
30 10
20 10
5
0 30
60
HRT_2
90
120
150 Day SRT_2
180
210
240
% Removal_2
Fig. 3. (a). Reactor 1 hydraulic and solids retention times and total-sterols removal. (b). Reactor 2 hydraulic and solids retention times and total-sterols removal.
activated sludge (WAS). This suggested a switching between the two main mechanisms of sterols removal at this point the role of biodegradation appeared to have decreased and that of bioadsorption appeared to have increased. Nonetheless, once the process retention times were increased back, the mixed liquor sterol concentrations decreased again indicating an increased biodegradation capacity probably through re-establishment of sterol-degrading biomass depending upon the process conditions suitable for their growth. Another contributing factor may be the influent sterol concentrations that can affect reactor biomass growth and hence biomass concentrations present in the bioreactors. However, the influent sterol concentrations were relatively constant past day 190 of operation in the 3rd phase of the study (Fig. 2a). The effect of SRT is probably greater than that of the HRT in regard to the sterols associated with biomass for two reasons. First, the HRT was changed in a relatively narrow range of operating values. Second, the SRT effectively controls the amount of biomass present in the biobasin and the pollutant concentration in the biobasin during the operation of suspended growth secondary treatment systems (Grady et al., 1999; Metcalf and Eddy, 2003).
412
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15 15000 10 10000 5
5000 0 80
Campe
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Stigma
140
170 Day
ß-Sito
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ß-Sitosta
0 260
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ML_1 Sterols
SRT_1
b 35000
30
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Sterol Concentration (µg/L)
SRT (d)
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25000 20 20000 15 15000
SRT (d)
Sterol Concentation (µg/L)
a
Z. Mahmood-Khan, E.R. Hall / Journal of Environmental Management 131 (2013) 407e414
10 10000 5
5000
0 80
Campe
100
120
Stigma
140
ß-Sito
160
180 Day
200
ß-Sitosta
220
240
0 260
ML_2 Sterols
SRT_2
Fig. 4. (a). Variation in mixed liquor plant sterols with SRT in Reactor 1. (b). Variation in mixed liquor plant sterols with SRT in Reactor 2.
The composition of mixed liquor sterol fractions was similar in both bioreactors i.e. 10e11% Campe, 4e6% Stigma, 48e53% b-Sito, and 31e34% b-Sitosta (Fig. 4a and b). The relative proportion of Campe seemed to have decreased and that of b-Sito increased as compared with those in the influent i.e. 23% Campe, 5% Stigma, 43% b-Sito, and 31% b-Sitosta (Fig. 2a). This suggested a relatively higher rate of biodegradation of Campe compared with other sterols present and reverse was true for b-Sito. 3.6. Sterol mass flows and the performance of bioreactors Sterol mass flows and accumulation were examined for conclusive evidence about the shifting roles of sterols biodegradation and bio-adsorption during the normal operation of the bioreactor systems. The daily mass flows entering and leaving the bioreactors with treated effluents and with secondary sludge along with removal efficiencies and the mass flow based biodegradation estimates are shown in Fig. 5a and b. Sterols removal (%) was estimated by the difference in influent and effluent sterol mass flows and the biodegradation (%) was estimated by subtracting the sum of effluent and WAS sterols mass flow from the influent sterols mass flow, considering the difference in sterol
mass flows between the influent and the effluent plus WAS to be biodegraded. Reactor 1 system successfully handled the increasing loads up to 25 mg/d of sterols in the 1st and the 2nd phase of the study (till day 180) sustaining 90e95% removal and most (80%) of the removed sterols appeared to have been biodegraded. During the 3rd phase (after day 180) WAS-sterol mass flows increased rapidly with increasing sterols load followed by an increase in the effluent sterols mass flows (Fig. 5a). This reduced the removal efficiency to < 80% by day 220 and the biodegradation estimates declined to 50% or less. Under these conditions, the loss of sterols removal was mainly due to reduction in biodegradation. The performance losses improved by day 240, when influent sterols load reduced and the system retention times increased again (Figs. 5a and 3a). Reactor 2 successfully treated the increasing sterol loads up to 20 mg/d by day 135 and sustained 90% sterols removal. Almost 90% of the observed removal was contribution of biodegradation. After day 150 the effluent sterol mass flows increased rapidly with increasing influent sterols (Fig. 5b). Thus the removal efficiency rapidly reduced to <60% by day 160, followed by an increase in WAS sterols mass flows. This reduced the biodegradation estimates to <50% by day 180 without further loss in removal efficiency i.e. removal efficiencies did not reflect reduced (<50%) biodegradation at this stage (day 180e210). The loss in the performance of Reactor 2 was quickly recovered by the end of the 3rd phase (day 220) when the sterols load were reduced by increasing the system retention times (Figs. 5b and 3b). These considerations suggested the end of the 2nd phase (day 180) presented a critical operational stage for both bioreactors. Before the critical stage, both bioreactors demonstrated effective removal as well as biodegradation of sterols. After this, the bioreactors performance started deteriorating with increasing loads and the biodegradation particularly reduced to <50% in both cases. The importance of treatment systems retention times and influent substrate concentrations do not need further emphasis. The results showed, at a certain critical stage, a relatively small change in the process variables can considerably impact treatment systems performance through changes in the amount and concentration of biomass present for substrate assimilation. It is to be noted firstly that only 30e40% of the sterols entered the bioreactors in soluble state. The remaining sterols entered in a colloidal or particulate state that may be too large to be transported across the microbial cell walls. Thus, it must be acted on by extracellular enzymes to be hydrolyzed to release soluble constituents before it is used as substrate by the biomass. While the soluble portion of the incoming sterols will be immediately available for biological attack. Secondly, the particular chemical nature of sterols favors their adsorption to secondary biomass (McKague and Reeve, 2003). Any adsorption to the biomass will also remove the sterols from the liquid effluent even if they are not immediately biodegraded. Further, a relatively small (20e30%) reduction in sterols removal associated to a considerable (50%) biodegradation loss indicated additional removal was achieved through sterols bioadsorption (Fig. 5a and b). The improved performance in the last phase of the study showed that the sterols removal and biodegradation capacity could be re-established through some operational changes. For Reactor 1 system the WAS sterols mass flows started increasing (day 160) before the effluent sterol flows increased (Fig. 5a), whereas for Reactor 2 the effluent sterol flows started increasing by day 130 i.e. before the WAS sterol flows increased (Fig. 5b). This indicated that Reactor 1 effluent sterols were relatively more attached to the biomass. At the onset of the reactor performance loss the WAS sterols first started to increase. While Reactor 2 effluent sterols appeared to be in a relatively more
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dissolved form which first passed through the secondary clarification. Recalling that Reactor 2 was operated at slightly alkaline (7.6 0.2 pH) conditions during phase 2 and 3 (Fig. 2d). The small differences in process pH within the typical range used for secondary treatment may influence specific contaminant solubility (Werker and Hall, 1999) thereby affecting the role of physicochemical factors during the biological treatment. 3.7. Cumulative mass flows and the role of sterols biodegradation Daily mass flows were added together to get cumulative mass flows. The sterols removal (influent minus effluent), retention by the system (influent minus effluent þ WAS) and biodegradation (retained minus accumulated) were also calculated (Fig. 6a and b) and the mass accumulation was directly obtained through the analysis of sterols in the mixed liquor. Cumulative mass flows should reflect an average picture of sterols removal, retention and biodegradation besides compensating for variations in the biomass stored sterols that may occur due to varying adsorption equilibria in response to the operational adjustments. Reactor 1 cumulative mass flows confirmed 90% removal, 70e 85% retention and 65e85% biodegradation of the influent sterols (Fig. 6a). Reactor 2 system showed 50e80% removal, 45e70% retention and 40e70% biodegradation of the incoming sterols
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(Fig. 6b). A close look at this data reveals that most (w90%) of the retained sterols were undergoing biodegradation. Although, sterols adsorption to biomass (bio-adsorption) has been anticipated as the major mechanism of sterols removal during full-scale secondary treatment (McKague and Reeve, 2003) and the contribution of biodegradation could be underestimated due the unavailability of required data. Results from both bioreactor systems showed sterols biodegradation was actually responsible for most of the observed removal of sterols. Hence, the contribution of biodegradation was considerably greater than anticipated initially. It is important to note that the cumulative biodegradation capacity of biological reactors could be lost and gained as discussed earlier. In the 3rd phase, cumulative biodegradation decreased to 65% for Reactor 1, maintaining the cumulative removal at 90%. The biodegradation declined to 55% for Reactor 2, keeping the associated removal still at 75%. The lost biodegradation did not show a similar reduction in removal, because of the increased contribution of sterols bio-adsorption. A negative correlation between the mixed liquor sterols and biodegradation efficiency suggested an increase in mixed liquor sterols was a better indicator of sterols biodegradation loss instead of the effluent sterol concentrations. This was particularly true for Reactor 1 operating at near neutral pH, the
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mixed liquor sterols increased 2e4 orders of magnitude owing to decreased biodegradation (Fig. 6a and b). Therefore, an increase in the concentrations of a particular organic like sterols in mixed liquor solids would indicate a biodegradation loss for the specific pollutant, even if the effluent concentrations do not suggest so. Such sub-optimal operation of treatment systems will increase potential of sudden pollutant release due to a minor operational disturbance. Nonetheless, plant sterols can be effectively removed and degraded using biological treatment systems optimized through relatively smaller manipulations of operational parameters that allow and maintain growth of sterols-degrading biomass. The optimized process conditions are expected to fall within the typical operating range used for secondary systems. Therefore, the treatment and removal of conventional pollutants is not expected to be compromised while providing effective control of moderately nonpolar organic pollutants like plant sterols. For treatment systems normally working at SRTs lower than the recommended SRT (11e12 day) for effective sterols degradation, an increase in SRT may require additional oxygen increasing the operational cost. However, the benefits of lower excess sludge generation and greater degree of BOD, COD and toxicity removal can offset the increased cost of a longer SRT. Alternatively, a treatment plant can be designed to offer optimized removal of sterols or other organic pollutant of interest, by maximizing the role of bio-adsorption. Such process shall however, require further treatment and proper disposal of the pollutant-rich excess sludge. Eskelinen et al. (2010) suggest need of integrating physicochemical and biological processes to complement degradation and achieve high removal of recalcitrant compounds in pulp mill effluents. 4. Conclusions Laboratory bioreactors treating pulp mill effluents, achieved high (90%) sterols removal after attaining stable operating conditions. Most (80e90%) of the removed sterols were estimated to be biodegraded. Sterols biodegradation was found to be sensitive to small changes in process variables. Reactor-1 (6.7 0.2 pH) and Reactor-2 (7.6 0.2 pH) successfully treated spiked sterols up to 4500 mg/L in 11e12 h HRT and 11 day SRT at 38e39 C. Further reductions in (hydraulic and solids) retention times deteriorated system performance. A relatively small (20e30%) decrease in the overall sterols removal was related to a major (60e70%) decline in biodegradation. This also increased the amount of sterols removed through bio-adsorption mechanism during the secondary biological wastewater treatment. Therefore the design and operation of treatment systems can be optimized for efficient removal of pulp mill effluent sterols utilizing either optimized biodegradation (longer retention times) or optimized bio-adsorption (shorter retention times) process. The latter choice may require additional bio-solids treatment. References Arima, K., Nagasawa, M., Moo, B., Tamura, G., 1969. Microbial transformation of sterols I. Decomposition of cholesterol by microorganisms. Agri. Biol. Chem. 33 (11), 1636e1643. Basu, N., Waye, A., Trudeau, V.L., Arnason, J.T., 2012. Extracts from hardwood trees used in commercial paper mills contain biologically active neurochemical disruptors. Sci. Total Environ. 414, 205e209. Chandra, R., Singh, R., 2012. Decolourisation and detoxification of rayon grade pulp paper mill effluent by mixed bacterial culture isolated from pulp paper mill effluent polluted site. Biochem. Engg. J. 61, 49e58. Chapman, Hall, 1996. Dictionary of Organic Compounds, sixth ed. Chapman and Hall, New York, N. Y. pp. 3012 & 5679.
Clesceri, L.S., Greenburg, A.E., Trussell, R.R., Franson, M.A.H. (Eds.), 1989. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, American Water Works Association, Water Pollution Control Federation. Cody, R.P., Bortone, S.A., 1997. Masculinization of mosquito fish as an indicator of exposure to kraft mill effluents. Bull. Environ. Cont. Toxicol. 58, 429e436. Cook, D.L., LaFleur, L., Parrrish, A., Jones, J., Hoy, D., 1997. Characterization of plant sterols from 22 US pulp and paper mills. Water Sci. Technol. 35 (2e3), 297e303. Eskelinen, K., Särkkä, H., Kurniawan, T.A., Sillanpää, M.E.T., 2010. Removal of recalcitrant contaminants from bleaching effluents in pulp and paper mills using ultrasonic irradiation and Fenton-like oxidation, electrochemical treatment, and/or chemical precipitation. Desalination 255 (1e3), 179e187. Grady Jr., C.P.L., Daigger, G.T., Lim, H.C., 1999. Biological Wastewater Treatment, second ed. Marcel Dekker, Inc., New York, Basel, USA. Hou, L., Xie, Y., Ying, G., Fang, Z., 2011. Developmental and reproductive characteristics of western mosquitofish (Gambusiaaffinis) exposed to paper mill effluent in the Dengcun River, Sihui, South China. Aqua. Toxicol. 103 (3e4), 140e 149. Klimenko, N., Winther-Nielsen, M., Smolin, S., Nevynna, L., Sydorenko, J., 2002. Role of the physico-chemical factors in the purification process of water from surface-active matter by biosorption. Water Res. 36, 5132e5140. Kostamo, A., Holmbom, B., Kukkonen, J.V.K., 2004. Fate of wood extractives in wastewater treatment plants at kraft pulp mills and mechanical pulp mills. Water Res. 38 (4), 972e982. Kostamo, A., Kukkonen, J.V.K., 2003. Removal of resin acids and sterols from pulp mill effluents by activated sludge treatment. Water Res. 37 (12), 2813e2820. Landman, M.J., Taylor, S., Van den Heuvel, M.R., 2008. Carotenoid depletion and ovarian dysfunction in common bully exposed to a New Zealand pulp mill effluent. Aquat. Toxicol. 87 (4), 303e309. Lehtinen, K.J., Mattsson, K., Tana, J., Engstrom, C., Lerche, O., Hemming, J., 1999. Effects of wood-related sterols on the reproduction, egg survival, and offspring of brown trout (Salmo trutta lacustris L.). Ecotoxicol. Environ. Saf. 42 (1), 40e49. Magnus, E., Hoel, H., Carlberg, G.E., 2000. TMP wastewater treatment, including a biological high-efficiency compact reactor. Toxicity reduction and removal of extractives. Nordic Pulp Paper Res. J. 15 (1), 37e45. Mahmood-Khan, Z., Hall, E.R., 2003. Occurrence and removal of plant sterols in pulp and paper mill effluents. J. Environ. Eng. Sci. 2, 17e26. Mahmood-Khan, Z., Hall, Eric, 2008. Quantification of plant sterols in pulp and paper mill effluents. Water Qual. Res. J. Can. 2/3, 173e181. Marton, J., 1964. On the structure of kraft lignin. Tappi J. 47 (11), 713e719. McKague, A.B., Reeve, D.W., 2003. Analysis of plant sterols in pulp mill effluents and sludges. In: TAPPI International Environmental Conference & Exhibition. Portland, OR, United States, May 4e7, 2003. TAPPI Press, Atlanta, GA, USA, pp. 61e 67. Metcalf, Eddy, 2003. In: Tchobanoglous, G., Burton, F.L., Stensel, H.D. (Eds.), Wastewater Engineering, Treatment and Reuse, fourth ed. McGraw-Hill, Toronto, Canada. Niven, S.J., Snape, J., Hetheridge, M., Evans, M., McEvoy, J., Rowland, S.J., 2001. Investigations of cholesterol transformation during sewage treatment: relevance to estrogen formation pathways? Sci. Total Environ. 279, 75e86. Orrego, R., Guchardi, J., Beyger, L., Krause, R., Holdway, D., 2011. Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Aquat. Toxicol. 104 (3e4), 299e307. Orrego, R., Guchardi, J., Krause, R., Holdway, D., 2010. Estrogenic and anti-estrogenic effects of wood extractives present in pulp and paper mill effluents on rainbow trout. Aquat. Toxicol. 99 (2), 160e167. Rydholm, S.A., 1965. Pulping Processes. Willey and Sons, New York, N.Y. USA. Shaw, D.J., 1992. Introduction to Colloid and Surface Chemistry, fourth ed. Butterworth-Heinemann Ltd., Oxford, UK. Shoppee, C.W., 1964. Chemistry of the Steroids. Butterworth & Co. Ltd., London, UK. Springer, A.M., 1993. Industrial Environmental Control, Pulp and Paper Industry, second ed. TAAPI Press, Atlanta, Georgia. Tremblay, L., Van Der Kraak, G., 1999. Comparison between the effects of the phytosterol ß-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environ. Toxicol. Chem. 18 (2), 329e336. Van Ginkel, C.G., Virtapohja, J., Steyaert, J.A.G., Alen, R., 1999. Treatment of EDTAcontaining pulp and paper mill wastewaters in activated sludge plants. TAPPI J. 82 (2), 138e142. Vidal, G., Becerra, J., Hernández, V., Decap, J., Xavier, C.R., 2007. Anaerobic biodegradation of sterols contained in kraft mill effluents. J. Biosci. Bioeng. 104 (6), 476e480. Werker, A.G., 1998. The Effect of pH on Microbial Activity and Community Structure in the Biological Removal of Resin Acids from Wastewater. Ph.D. Dissertation. Department of Civil Engineering, University of British Columbia, Vancouver, B.C. Canada. Werker, A.G., Hall, E.R., 1999. Limitations for biological removal of resin acids from pulp mill effluents. Water Sci. Technol. 40 (11e12), 281e288. WEF, ASCE., 1998. Design of Wastewater Treatment Plants, WEF Manual of Practice No. 8, fourth ed. ASCE Manuals and Reports on Engineering Practice No. 76, Water Environment Federation (WEF), vol. II. American Society of Civil Engineers (ASCE), VA, USA.
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Biological removal of phyto-sterols in pulp mill effluents Zahid Mahmood-Khan a, *, Eric R. Hall b a b
Agricultural Engineering Department, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Punjab 60800, Pakistan Department of Civil Engineering and The Pulp and Paper Centre, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2012 Received in revised form 3 September 2013 Accepted 23 September 2013 Available online 8 November 2013
Phyto-sterols and extractives found in pulp mill effluents are suspected to cause endocrine abnormalities in receiving water fish. The control of sterols in pulp mill effluents through biological secondary wastewater treatment was studied using two lab-scale bioreactor systems. After achieving a stable performance, both bioreactor systems successfully removed (>90%) sterols and the estimated biodegradation was up to 80%. Reactor 1 system operating at 6.7 0.2 pH effectively treated pulp mill effluent sterols spiked up to 4500 mg/L in 11 h HRT and 11 day SRT. However, Reactor 2 system operating at 7.6 0.2 pH performed relatively poorly. Retention time reductions beyond critical values deteriorated the performance of treatment systems and quickly reduced the sterols biodegradation. The biodegradation loss was indicated by mixed liquor sterols content that started increasing. This biodegradation loss was compensated by the increased role of bio-adsorption and the overall sterols removal remained relatively high. Hence, a relatively small (20e30%) loss in the overall sterols removal efficiency did not fully reflect the associated major (60e70%) loss in the sterols biodegradation because the amount of sterols accumulated in the sludge due to adsorption increased so the estimate of sterols removal through adsorption increased from 30e40% to 70e80% keeping the overall sterols removal still high. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Keywords: Phyto-sterols Biodegradation Bio-adsorption Pulp mill effluent Secondary wastewater treatment
1. Introduction Currently, there are concerns over the observed estrogenic, and in some cases, androgenic effects in fish and aquatic life in water bodies receiving pulp mill effluents (Basu et al., 2012; Cody and Bortone, 1997; Lehtinen et al., 1999). These effects have been attributed to certain chemicals, including plant sterols and wood extractives (Tremblay and Van Der Kraak, 1999; Vidal et al., 2007; Landman et al., 2008; Orrego et al., 2010, 2011; Hou et al., 2011). If not treated and controlled properly, plant sterols or phytosterols in pulp mill effluents may contaminate receiving waters (Mahmood-Khan and Hall, 2003, 2008), where they may act as endocrine disrupting chemicals (EDCs) or hormonally active agents (HAAs). A popular form of suspended growth systems is activated sludge treatment (AST) which is one of the typical treatment options employed for treating pulp mill effluents. Most of the AST systems are effectively removing majority of the organic pollutants thereby reducing biochemical oxygen demand (BOD) and acute toxicity
* Corresponding author. Tel.: þ92 61 9210298, þ92 302 5447015 (mobile); fax: þ92 61 9210098. E-mail addresses: [email protected], [email protected] (Z. MahmoodKhan), [email protected] (E.R. Hall).
(Springer, 1993) from pulp mill effluents. The activated sludge process has been proven to be both cost effective and reliable, in general. However, AST systems have shown instability in reducing sterols and other organic pollutants in pulp mill effluents (Cook et al., 1997; Van Ginkel et al., 1999; Mahmood-Khan and Hall, 2003). Other studies have shown a strong tendency of plant sterols to accumulate in secondary sludge during secondary biological treatment indicating adsorption as a major mechanism of sterols removal (Chandra and Singh, 2012; Magnus et al., 2000; McKague and Reeve, 2003; Mahmood-Khan and Hall, 2008). Many details about the behavior and fate of pulp mill effluent sterols entering biological wastewater treatment systems are not clear. Selective microbial degradation of sterols like Cholesterol and b-Sitosterol has also been reported (Arima et al., 1969; Niven et al., 2001; Kostamo and Kukkonen, 2003). However, there are questions about sterols removal mechanisms particularly about their biodegradation as the observed sterols removal from the effluents may be mainly due to their accumulation in secondary sludge rather than biodegradation. In Finland, a survey of elemental chlorine free kraft pulp mill, a paper mill and an integrated kraft pulp and paper mill, indicated 35e99% degradation of wood extractives and sterols from pulp mill effluents across secondary systems including AST (Kostamo et al., 2004). Vidal et al. (2007) also suggested a high (77e95%) removal of sterols from kraft mill
0301-4797/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.09.031
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effluents across a continuously fed anaerobic filter, with only 10% reduction in the methanogenic activity of the bioreactor. Besides adsorption, biodegradation may probably play an important role in sterols removal and control during secondary treatment of pulp mill effluents. Can sterols biodegradation play a significant role in sterols removal or just adsorption is the main mechanism of sterols removal during secondary treatment of pulp mill effluents? These are important questions to understand and improve the treatment and control of plant sterols in pulp industry wastewaters. Therefore, the objective of the present study was to examine the extent of sterols removal and the role of biodegradation during laboratory scale biological treatment of pulp mill effluents. For this purpose, sterols containing pulp mill effluents were treated using two labscale biological reactors equipped with secondary clarifiers, and sterols removal was monitored at various influent sterol concentrations and different operating conditions. 2. Materials and methods
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2.1. Lab-scale suspended growth activated sludge systems In the present study, AST was used in the form of two identical lab-scale bioreactors as shown in Fig. 1a and the dimensions are shown in Fig. 1b. Each bioreactor was made of PlexiglasÔ, with a 4 L aeration basin working capacity. The bioreactors were fitted with double-jacketed walls to provide the passage of circulating hot water for process temperature control. A double-jacketed secondary clarifier made with PlexiglasÔ was also provided for each bioreactor (Fig. 1c), so the two systems could be operated separately. Other mechanisms included influent and nutrients feeding through peristaltic pumps, pH controllers, mechanical mixing, air supply diffusers, arrangements for return and waste secondary sludge to allow desired operational conditions. The influent to the lab-scale bioreactors consisted of primary treated effluent from a Canadian Pulp Mill in British Columbia. The effluent was collected in 25 L NalgeneÔ containers and was refrigerated at 4 C before use. The bioreactors were inoculated with biomass obtained from the full-scale high purity oxygen (UNOX) AST system at the Mill, to grow acclimated biomass for treating pulp mill effluent sterols. The start-up process took six weeks. Air was supplied using two Aqua FizzzzÔ (A-962) fine bubble diffusers in each bioreactor. Sufficient air was supplied to keep the dissolved oxygen level around 5e6 mg/L for achieving oxygen transfer conditions similar to those of the full-scale UNOX-AST system. Additional mixing was provided using Electric ArrowÔ (Model # 1750) variable speed mechanical mixers rotating at 160e 180 RPM. 2.2. Nutrients, pH control and process temperature Nutrients were supplied in an approximate proportion of 100:3.5:1 for COD:N:P. NH4Cl was used for nitrogen, and a mixture of K2HPO4 and KH2PO4 was used for P. The pH control solutions were 0.2 N H2SO4 and 0.2 N NaOH. After the start-up phase, 0.2 N NaOH was mostly required to keep the reactor pH at the desired level. There were three phases of the biological treatment study and Reactor 1 was operated at a pH of 6.6e6.8 (near neutral conditions) throughout the three phases, as AST systems are usually operated. Reactor 2 was started at a pH of 7.0 0.2 in the 1st phase, but later in the 2nd and 3rd phase its pH was increased to 7.6e7.8 (slightly alkaline conditions) to cover the normal range of process pH at the full-scale UNOX-AST system. Slightly alkaline conditions may favor the treatment of organic pollutants in pulp mill effluents (Van Ginkel et al., 1999; Werker and Hall, 1999). The process pH was
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measured using pH/mV ChemCadet Cole ParmerÔ (Model # 598450) meter and maintained by using Cole ParmerÔ pH/pump systems (Model # 7142-60). Process temperature was maintained at 38e39 C using a VWRÔ Constant Temperature Circulator (Model # 1130 A) water bath for both bioreactors. This is the temperature at which the fullscale UNOX-AST plant was typically operated at the Mill. 2.3. Sterols spiking The primary-treated effluent was screened through a coarse strainer to remove fiber fines, into a 60 L holding tank and then used as influent to the bioreactors. The primary-treated influents were spiked when required, with recovered sterols (from wood waste) supplied by BC Chemicals, 2711 Pulp Mill Road, Prince George, BC, Canada, V2N 2K3. The sterols received in fluffy powdered solid state were dissolved in pulp mill effluent in a stepwise procedure as explained below.
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The sterols were first dissolved in iso-propanol (2-propanol) after examining their solubility in different solvents: methanol, ethanol, acetone, hexane and 2-propanol. Each of these five solvents were separately added to conical-bottom penny-head stopper PyrexÔ glass centrifuge tubes (Corning 8084 15) calibrated to contain a specific volume (10e15 mL) and graduated to 0.1 mL. The centrifuge tubes contained a given mass of sterols to which solvent was added slowly, mixed with a vortex mixer and heated to about 40 C for 5e6 min. The solvent was added until the sterols were completely dissolved. Final volume was noted after adding the last drop and considered to represent sterols solubility in the solvent under consideration. The solubility of sterols at 40 C in methanol, ethanol, hexane, acetone and iso-propanol was tested to be 1.8e2, 6e7, 13.6e15, 28e30 and 32e36 mg sterols/mL solvent respectively. Relative to other solvents, 2-propanol was required in the least amount to dissolve a given mass of sterols. Therefore, 2-propanol was selected to pre-dissolve the semi-crystalline sterols. The sterols solution in 2-propanol was then introduced first in to a small volume of primary effluent (heated to 40 C) and then mixed with larger volume of primary effluent. The whole process was completed in the following three steps: I. A sterols stock-solution was prepared by dissolving 200 mg of sterols in 5.5 mL of iso-propanol (2-propanol) at about 40 C. II. The 5.5 mL sterols stock-solution was introduced to about 4 L of primary effluent at about 40 C and continuously mixed on a stirrer plate heater for 4e6 h to get an intermediate pulp mill effluent solution. III. The intermediate sterol solution was then introduced to a given volume (40e60 L, as required) of bioreactor influent and mixed for 2 h at room temperature to produce the desired concentration of sterols in the laboratory bioreactor influent. The spiked influent was continuously stirred and fed to the laboratory bioreactors, the general characteristics of the bioreactor influent are given in Table 1. The fraction of pulp mill effluent sterols that passed through WhatmanÔ AH-934 fiberglass filters was considered to be dissolved in this study. The particulate fraction was determined by the difference in the analyzed amount of sterols in the whole sample and the filtrate. Typically, 70e130 mg/L of COD was added as 2-propanol to the spiked primary-treated influent. 2.4. Chemical analyses Total suspended and volatile solids (TSS& VSS) were analyzed in the influent, effluent and mixed liquor and secondary sludge, according to the Standard Methods for the Examination of Water and Wastewater 2540 D and 2540 E (Clesceri et al., 1989). Chemical oxygen demand (COD) was measured through a closed reflux Table 1 Characteristics of primary effluent obtained from mill. Characteristic
Low
High
Typical
COD BOD TSS pH Temperature Native sterols Dissolved Spiked sterols Dissolved
1100 mg/L 300 mg/L 70 mg/L 6.8 37 C 600 mg/L 38% 1000 mg/L 35%
1500 mg/L 380 mg/L 120 mg/L 7.8 41 C 2500 mg/L 65% 4500 mg/L 70%
1300 mg/L 340 mg/L 80 mg/L 7.2 39 C 1400 mg/L 46% 2500 mg/L 50%
409
Table 2 Different phases of lab-scale bioreactor operation. Experimental phase (duration) Reactor 1 I (3 months)a II (3 months) III (2.5 months) Reactor 2 I (3 months)a II (3 months) III (2 months)
Sterol concentration (mg/L)
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SRT (d)
pH
1000e1500 2000e3500 3500e4500
28e20 20e13 13e8
27e23 23e10 10e7
6.6 0.2 6.7 0.2 6.7 0.2
1000e1500 2000e3500 3500e4500
24e20 20e11 11e9
25e22 22e12 12e8
7.0 0.2 7.6 0.2 7.6 0.2
a Results for 1st 40 days were not reported due to reactor start-up and sterol spiking problems.
colorimetric Standard Method 5220 D (Clesceri et al., 1989), using HachÔ DR/2000 Spectrophotometer by reading absorbance values at 600 nm. Total organic carbon (TOC) measurements were made through combustion-infrared Standard Method 5310 B (Clesceri et al., 1989), using a ShimadzuÔ TOC-500 (Shimadzu Corporation Kyoto, Japan) analyzer, calibrated with each batch of samples. Plant sterols were analyzed in the bioreactor influent, effluent and mixed liquor according to the analytical procedure described by Mahmood-Khan and Hall (2008) and is briefly given below. 2.4.1. Sterol extraction Depending upon the type of PPME (primary or secondary), 25e 100 mL of effluent was extracted three times with 15e40 mL of methyl-t-butyl ether (MTBE) using prefired, clean Pyrex TM centrifuge glass tubes. The mixture of PPME and MTBE was shaken for about 15 min by wrist-action mechanical shaker, allowed to settle for about 10 min, and centrifuged at an average relative centrifugal force of 1600 g for separation. The extracted sterols were incubated at 70 C for 4 h, for silylating the sterols as the trimethylsilyl ethers using BSTFA (N, O-Bis tri-methylsilyltriflouroacetamide). 2.4.2. Gas chromatographemass spectrometer analysis A HewlettePackard Model HP-6890 Series Gas ChromatographeMass Spectrometer (GCeMS) System was used with a J&W DB-5MS 30 m long, 0.25 mm coating, 0.25 mm I.D. capillary column. The GC oven conditions were: 130 C, 1 min hold, ramp_1 to 285 C@15 C/min, 3 min hold; ramp _2 to 310 C @ 2 C/min, 1 min hold, post run at 315 C, 2.17 min hold; inlet 290 C, carrier gas helium at 11.2 psi (1 psi ¼ 6.89 kPa) and 53.6 mL/min; mass selective detector at 280 C. The MS instrument HP 5973 Mass Spectrometer Detector coupled with the GC system provided satisfactory detection and identification of phytosterols and other test chemicals along with the GC system. Dotriacontane was used as an internal standard to account for gas chromatograph instrument performance and response. 2.5. Sterols removal experiments Both lab-scale bioreactors were started by inoculation using 300e400 mL of recycle activated sludge (RAS) resulting in about 1500 mg/L final MLSS concentration. The RAS was freshly obtained from Mill in 1 L plastic containers. The containers allowed for some headspace air for biomass respiration during travel. Additional RAS was also added to the bioreactors for re-seeding after the 2nd and the 3rd week. Primary effluent was fed to the bioreactors at a low flow rate resulting in about 24 h hydraulic retention time (HRT). The sterols removal from pulp mill effluent was assessed across the continuous-flow lab-systems in three phases, of 2e3 months each. Samples were taken after 2e3 HRT intervals, after the effluent
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3.2. Sterols in the effluent from the bioreactor systems
sterol concentration had stabilized. Each study phase was continued for about 3 times the average solids retention time (SRT) of the system. The SRT was controlled by wasting mixed liquor directly from the bioreactors. The influent sterols concentrations and the process control variables (HRT, SRT and pH) are given in Table 2.
The overall sterols removal in all the three phases is shown in Fig. 2b and c for Reactor 1 and Reactor 2 respectively. The loss/ removal of sterols through volatilization, stripping and chemical degradation may be neglected as the sterols are chemically stable, non-volatile and moderately non-polar in nature with melting points in 100e180 C range e.g. Sitosterol melts at about 136 C (Chapman and Hall, 1996), and Campesterol at 157 C. The observed sterols removal was therefore considered to be a combined result of adsorption to the suspended solids and biodegradation in the mixed culture environment of activated sludge as suggested by Mahmood-Khan and Hall (2008) and Kostamo et al. (2004).
3. Results and discussions 3.1. Sterols in the influent to the bioreactors The bioreactors were started with a relatively low flow rate (3.3e3.5 L/d). The flow rate and sterol concentrations were gradually increased after the operation of the bioreactors had stabilized (Fig. 2a). The composition individual sterol fractions in the influent remained similar throughout the study. b-Sitosterol (b-Sito), bSitostanol (b-Sitosta) and Campesterol (Campe) were the dominant fractions entering the bioreactors with Stigmasterol (Stigma) as a relatively minor fraction. The average composition of influent sterols was: b-Sito 46%, b-Sitosta 31%, Campe 19% and Stigma 4% (Fig. 2a). This is in agreement with the results obtained by other researchers (Cook et al., 1997; Mahmood-Khan and Hall, 2008; Magnus et al., 2000; Rydholm, 1965; Shoppee 1964). Ergosterol and Cholesterol mostly remained non-detectable.
a
3.3. Sterols removal and the bioreactor systems pH The main difference in the operation of the bioreactors was pH and the results show different performance for each unit (Fig. 2d). Reactor 2 was operated at a pH slightly higher than that of Reactor 1. The differences in reactor behavior may relate to a spectrum of interacting factors. Firstly, the pH differences within the typical range used for biological treatment may cause variations in the biobasin mixed cultures (Werker, 1998). Secondly, pH-associated changes in the solubility of sterols and/or other organics present in pulp mill effluent may alter their availability for microbial
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Fig. 2. (a). Plant sterols in the influent to the lab-scale bioreactors. (b). Total-sterols removal efficiency Reactor 1. (c). Total-sterols removal efficiency Reactor 2. (d). Process pH and total-sterols removal performance of Reactor 1 and Reactor 2 (indicated by suffix_1 and _2).
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3.4. Effect of HRT and SRT on the biological removal of sterols HRT_1
3.5. The role of sterols biodegradation and bio-adsorption Both of the bioreactors showed similar trends i.e. decreasing SRTs increased the amount of sterols discharge with waste
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The laboratory bioreactors were started with relatively long retention times (HRTs and SRTs) in the 1st phase. When the biological process stabilized, and about 80% removal of sterols was achieved, the influent concentration was increased and the retention times were decreased gradually to match the full scale conditions (10 h HRT, 10 d SRT) by the end of the 3rd phase (Fig. 3a and b). Fig. 4a and b present the sterols associated with lab-systems biomass for the 2nd and the 3rd phases of the study. The results suggest an SRT of 11e12 day and an HRT of about 11 h were critical for effective removal of sterols during secondary treatment under the conditions of this experiment. The removal efficiencies generally declined with detention times below these critical values. SRT is a variable of fundamental importance that is functionally related to the steady-state specific growth rate of biomass in a completely-mixed bioreactor (Grady et al., 1999; Metcalf and Eddy, 2003). The biomass growth results in pollutant removal that is used as substrate and is biodegraded. It is known that a decrease in SRT, within the operating range of values, increases the microbial specific growth rate as well as the observed yield. However, for a fixed reactor volume, the amount of biomass in the reactor also decreases. Hence, if the sterols were being biodegraded in the lab bioreactors, the overall sterols removal through biodegradation will decrease with reducing SRT. Under these conditions a reduction in HRT appears to further reduce the biomass available for pollutant degradation, resulting in an additional loss of reactor performance. The observed loss in sterols removal was recovered through a small incremental increase in the system SRT and HRT for both reactors. This was probably the result of improved biodegradation of sterols through the re-establishment of sterol-degrading microorganisms. Although use of HRT has diminished in importance for sizing suspended growth reactors (WEF and ASCE, 1998), the results of this study suggest HRT can be an important consideration for the design and operation of biological systems treating pulp mill effluent sterols. Therefore, both solids and hydraulic retention times can impact the performance of biological systems treating pulp mill effluent within the normal range usually observed in fullscale operation. This may possibly be a reason for the reported fluctuations in sterols removal during the full-scale operation (Kostamo et al., 2004). However, biodegradation is not the only mechanism of sterols removal, and fluctuations in sterol adsorption to secondary solids will also impact the overall sterols removal.
% Removal
HRT (h); SRT (d)
degradation (Werker and Hall, 1999). For example, the solubility of pulp mill effluent organics like resin acids and kraft lignin increases with increasing pH (Marton, 1964; Werker, 1998). Higher solubility tends to reduce the sorptive behavior of such compounds (Klimenko et al., 2002; Shaw, 1992). A reduction in sterols sorption and/or biodegradation will cause the bioreactor sterol removal performance to decline, until such capacity is re-gained. Therefore, the differences in the performance of the two reactors may be attributable to the operating pH of each reactor. With some differences, both of the laboratory reactors were able to remove sterols from pulp mill effluents. However, the overall operation of Reactor 1 appeared to be more successful relative to that of Reactor 2. These results showed that (w95%) removal of sterols was achieved and maintained through biological treatment and near neutral pH was favorable for sterols removal across such systems.
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Fig. 3. (a). Reactor 1 hydraulic and solids retention times and total-sterols removal. (b). Reactor 2 hydraulic and solids retention times and total-sterols removal.
activated sludge (WAS). This suggested a switching between the two main mechanisms of sterols removal at this point the role of biodegradation appeared to have decreased and that of bioadsorption appeared to have increased. Nonetheless, once the process retention times were increased back, the mixed liquor sterol concentrations decreased again indicating an increased biodegradation capacity probably through re-establishment of sterol-degrading biomass depending upon the process conditions suitable for their growth. Another contributing factor may be the influent sterol concentrations that can affect reactor biomass growth and hence biomass concentrations present in the bioreactors. However, the influent sterol concentrations were relatively constant past day 190 of operation in the 3rd phase of the study (Fig. 2a). The effect of SRT is probably greater than that of the HRT in regard to the sterols associated with biomass for two reasons. First, the HRT was changed in a relatively narrow range of operating values. Second, the SRT effectively controls the amount of biomass present in the biobasin and the pollutant concentration in the biobasin during the operation of suspended growth secondary treatment systems (Grady et al., 1999; Metcalf and Eddy, 2003).
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Fig. 4. (a). Variation in mixed liquor plant sterols with SRT in Reactor 1. (b). Variation in mixed liquor plant sterols with SRT in Reactor 2.
The composition of mixed liquor sterol fractions was similar in both bioreactors i.e. 10e11% Campe, 4e6% Stigma, 48e53% b-Sito, and 31e34% b-Sitosta (Fig. 4a and b). The relative proportion of Campe seemed to have decreased and that of b-Sito increased as compared with those in the influent i.e. 23% Campe, 5% Stigma, 43% b-Sito, and 31% b-Sitosta (Fig. 2a). This suggested a relatively higher rate of biodegradation of Campe compared with other sterols present and reverse was true for b-Sito. 3.6. Sterol mass flows and the performance of bioreactors Sterol mass flows and accumulation were examined for conclusive evidence about the shifting roles of sterols biodegradation and bio-adsorption during the normal operation of the bioreactor systems. The daily mass flows entering and leaving the bioreactors with treated effluents and with secondary sludge along with removal efficiencies and the mass flow based biodegradation estimates are shown in Fig. 5a and b. Sterols removal (%) was estimated by the difference in influent and effluent sterol mass flows and the biodegradation (%) was estimated by subtracting the sum of effluent and WAS sterols mass flow from the influent sterols mass flow, considering the difference in sterol
mass flows between the influent and the effluent plus WAS to be biodegraded. Reactor 1 system successfully handled the increasing loads up to 25 mg/d of sterols in the 1st and the 2nd phase of the study (till day 180) sustaining 90e95% removal and most (80%) of the removed sterols appeared to have been biodegraded. During the 3rd phase (after day 180) WAS-sterol mass flows increased rapidly with increasing sterols load followed by an increase in the effluent sterols mass flows (Fig. 5a). This reduced the removal efficiency to < 80% by day 220 and the biodegradation estimates declined to 50% or less. Under these conditions, the loss of sterols removal was mainly due to reduction in biodegradation. The performance losses improved by day 240, when influent sterols load reduced and the system retention times increased again (Figs. 5a and 3a). Reactor 2 successfully treated the increasing sterol loads up to 20 mg/d by day 135 and sustained 90% sterols removal. Almost 90% of the observed removal was contribution of biodegradation. After day 150 the effluent sterol mass flows increased rapidly with increasing influent sterols (Fig. 5b). Thus the removal efficiency rapidly reduced to <60% by day 160, followed by an increase in WAS sterols mass flows. This reduced the biodegradation estimates to <50% by day 180 without further loss in removal efficiency i.e. removal efficiencies did not reflect reduced (<50%) biodegradation at this stage (day 180e210). The loss in the performance of Reactor 2 was quickly recovered by the end of the 3rd phase (day 220) when the sterols load were reduced by increasing the system retention times (Figs. 5b and 3b). These considerations suggested the end of the 2nd phase (day 180) presented a critical operational stage for both bioreactors. Before the critical stage, both bioreactors demonstrated effective removal as well as biodegradation of sterols. After this, the bioreactors performance started deteriorating with increasing loads and the biodegradation particularly reduced to <50% in both cases. The importance of treatment systems retention times and influent substrate concentrations do not need further emphasis. The results showed, at a certain critical stage, a relatively small change in the process variables can considerably impact treatment systems performance through changes in the amount and concentration of biomass present for substrate assimilation. It is to be noted firstly that only 30e40% of the sterols entered the bioreactors in soluble state. The remaining sterols entered in a colloidal or particulate state that may be too large to be transported across the microbial cell walls. Thus, it must be acted on by extracellular enzymes to be hydrolyzed to release soluble constituents before it is used as substrate by the biomass. While the soluble portion of the incoming sterols will be immediately available for biological attack. Secondly, the particular chemical nature of sterols favors their adsorption to secondary biomass (McKague and Reeve, 2003). Any adsorption to the biomass will also remove the sterols from the liquid effluent even if they are not immediately biodegraded. Further, a relatively small (20e30%) reduction in sterols removal associated to a considerable (50%) biodegradation loss indicated additional removal was achieved through sterols bioadsorption (Fig. 5a and b). The improved performance in the last phase of the study showed that the sterols removal and biodegradation capacity could be re-established through some operational changes. For Reactor 1 system the WAS sterols mass flows started increasing (day 160) before the effluent sterol flows increased (Fig. 5a), whereas for Reactor 2 the effluent sterol flows started increasing by day 130 i.e. before the WAS sterol flows increased (Fig. 5b). This indicated that Reactor 1 effluent sterols were relatively more attached to the biomass. At the onset of the reactor performance loss the WAS sterols first started to increase. While Reactor 2 effluent sterols appeared to be in a relatively more
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Fig. 5. (a). Daily mass flows of total sterols entering and leaving Reactor 1 System. (b). Daily mass flows of total sterols entering and leaving Reactor 2 System.
dissolved form which first passed through the secondary clarification. Recalling that Reactor 2 was operated at slightly alkaline (7.6 0.2 pH) conditions during phase 2 and 3 (Fig. 2d). The small differences in process pH within the typical range used for secondary treatment may influence specific contaminant solubility (Werker and Hall, 1999) thereby affecting the role of physicochemical factors during the biological treatment. 3.7. Cumulative mass flows and the role of sterols biodegradation Daily mass flows were added together to get cumulative mass flows. The sterols removal (influent minus effluent), retention by the system (influent minus effluent þ WAS) and biodegradation (retained minus accumulated) were also calculated (Fig. 6a and b) and the mass accumulation was directly obtained through the analysis of sterols in the mixed liquor. Cumulative mass flows should reflect an average picture of sterols removal, retention and biodegradation besides compensating for variations in the biomass stored sterols that may occur due to varying adsorption equilibria in response to the operational adjustments. Reactor 1 cumulative mass flows confirmed 90% removal, 70e 85% retention and 65e85% biodegradation of the influent sterols (Fig. 6a). Reactor 2 system showed 50e80% removal, 45e70% retention and 40e70% biodegradation of the incoming sterols
% Removal; ML Sterols (mg/L)
a
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Influent_2
Accumulated_2
Effluent_2
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% Removed_2
% Retained _2
% Biodegraded_2
ML Sterols_2
Fig. 6. (a). Reactor 1 System: Cumulative mass flows, average removal, retention, biodegradation, and ML (mixed liquor) total sterols. (b). Reactor 2 System: Cumulative mass flows, average removal, retention biodegradation, and ML (mixed liquor) total sterols.
(Fig. 6b). A close look at this data reveals that most (w90%) of the retained sterols were undergoing biodegradation. Although, sterols adsorption to biomass (bio-adsorption) has been anticipated as the major mechanism of sterols removal during full-scale secondary treatment (McKague and Reeve, 2003) and the contribution of biodegradation could be underestimated due the unavailability of required data. Results from both bioreactor systems showed sterols biodegradation was actually responsible for most of the observed removal of sterols. Hence, the contribution of biodegradation was considerably greater than anticipated initially. It is important to note that the cumulative biodegradation capacity of biological reactors could be lost and gained as discussed earlier. In the 3rd phase, cumulative biodegradation decreased to 65% for Reactor 1, maintaining the cumulative removal at 90%. The biodegradation declined to 55% for Reactor 2, keeping the associated removal still at 75%. The lost biodegradation did not show a similar reduction in removal, because of the increased contribution of sterols bio-adsorption. A negative correlation between the mixed liquor sterols and biodegradation efficiency suggested an increase in mixed liquor sterols was a better indicator of sterols biodegradation loss instead of the effluent sterol concentrations. This was particularly true for Reactor 1 operating at near neutral pH, the
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mixed liquor sterols increased 2e4 orders of magnitude owing to decreased biodegradation (Fig. 6a and b). Therefore, an increase in the concentrations of a particular organic like sterols in mixed liquor solids would indicate a biodegradation loss for the specific pollutant, even if the effluent concentrations do not suggest so. Such sub-optimal operation of treatment systems will increase potential of sudden pollutant release due to a minor operational disturbance. Nonetheless, plant sterols can be effectively removed and degraded using biological treatment systems optimized through relatively smaller manipulations of operational parameters that allow and maintain growth of sterols-degrading biomass. The optimized process conditions are expected to fall within the typical operating range used for secondary systems. Therefore, the treatment and removal of conventional pollutants is not expected to be compromised while providing effective control of moderately nonpolar organic pollutants like plant sterols. For treatment systems normally working at SRTs lower than the recommended SRT (11e12 day) for effective sterols degradation, an increase in SRT may require additional oxygen increasing the operational cost. However, the benefits of lower excess sludge generation and greater degree of BOD, COD and toxicity removal can offset the increased cost of a longer SRT. Alternatively, a treatment plant can be designed to offer optimized removal of sterols or other organic pollutant of interest, by maximizing the role of bio-adsorption. Such process shall however, require further treatment and proper disposal of the pollutant-rich excess sludge. Eskelinen et al. (2010) suggest need of integrating physicochemical and biological processes to complement degradation and achieve high removal of recalcitrant compounds in pulp mill effluents. 4. Conclusions Laboratory bioreactors treating pulp mill effluents, achieved high (90%) sterols removal after attaining stable operating conditions. Most (80e90%) of the removed sterols were estimated to be biodegraded. Sterols biodegradation was found to be sensitive to small changes in process variables. Reactor-1 (6.7 0.2 pH) and Reactor-2 (7.6 0.2 pH) successfully treated spiked sterols up to 4500 mg/L in 11e12 h HRT and 11 day SRT at 38e39 C. Further reductions in (hydraulic and solids) retention times deteriorated system performance. A relatively small (20e30%) decrease in the overall sterols removal was related to a major (60e70%) decline in biodegradation. This also increased the amount of sterols removed through bio-adsorption mechanism during the secondary biological wastewater treatment. Therefore the design and operation of treatment systems can be optimized for efficient removal of pulp mill effluent sterols utilizing either optimized biodegradation (longer retention times) or optimized bio-adsorption (shorter retention times) process. The latter choice may require additional bio-solids treatment. References Arima, K., Nagasawa, M., Moo, B., Tamura, G., 1969. Microbial transformation of sterols I. Decomposition of cholesterol by microorganisms. Agri. Biol. Chem. 33 (11), 1636e1643. Basu, N., Waye, A., Trudeau, V.L., Arnason, J.T., 2012. Extracts from hardwood trees used in commercial paper mills contain biologically active neurochemical disruptors. Sci. Total Environ. 414, 205e209. Chandra, R., Singh, R., 2012. Decolourisation and detoxification of rayon grade pulp paper mill effluent by mixed bacterial culture isolated from pulp paper mill effluent polluted site. Biochem. Engg. J. 61, 49e58. Chapman, Hall, 1996. Dictionary of Organic Compounds, sixth ed. Chapman and Hall, New York, N. Y. pp. 3012 & 5679.
Clesceri, L.S., Greenburg, A.E., Trussell, R.R., Franson, M.A.H. (Eds.), 1989. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, American Water Works Association, Water Pollution Control Federation. Cody, R.P., Bortone, S.A., 1997. Masculinization of mosquito fish as an indicator of exposure to kraft mill effluents. Bull. Environ. Cont. Toxicol. 58, 429e436. Cook, D.L., LaFleur, L., Parrrish, A., Jones, J., Hoy, D., 1997. Characterization of plant sterols from 22 US pulp and paper mills. Water Sci. Technol. 35 (2e3), 297e303. Eskelinen, K., Särkkä, H., Kurniawan, T.A., Sillanpää, M.E.T., 2010. Removal of recalcitrant contaminants from bleaching effluents in pulp and paper mills using ultrasonic irradiation and Fenton-like oxidation, electrochemical treatment, and/or chemical precipitation. Desalination 255 (1e3), 179e187. Grady Jr., C.P.L., Daigger, G.T., Lim, H.C., 1999. Biological Wastewater Treatment, second ed. Marcel Dekker, Inc., New York, Basel, USA. Hou, L., Xie, Y., Ying, G., Fang, Z., 2011. Developmental and reproductive characteristics of western mosquitofish (Gambusiaaffinis) exposed to paper mill effluent in the Dengcun River, Sihui, South China. Aqua. Toxicol. 103 (3e4), 140e 149. Klimenko, N., Winther-Nielsen, M., Smolin, S., Nevynna, L., Sydorenko, J., 2002. Role of the physico-chemical factors in the purification process of water from surface-active matter by biosorption. Water Res. 36, 5132e5140. Kostamo, A., Holmbom, B., Kukkonen, J.V.K., 2004. Fate of wood extractives in wastewater treatment plants at kraft pulp mills and mechanical pulp mills. Water Res. 38 (4), 972e982. Kostamo, A., Kukkonen, J.V.K., 2003. Removal of resin acids and sterols from pulp mill effluents by activated sludge treatment. Water Res. 37 (12), 2813e2820. Landman, M.J., Taylor, S., Van den Heuvel, M.R., 2008. Carotenoid depletion and ovarian dysfunction in common bully exposed to a New Zealand pulp mill effluent. Aquat. Toxicol. 87 (4), 303e309. Lehtinen, K.J., Mattsson, K., Tana, J., Engstrom, C., Lerche, O., Hemming, J., 1999. Effects of wood-related sterols on the reproduction, egg survival, and offspring of brown trout (Salmo trutta lacustris L.). Ecotoxicol. Environ. Saf. 42 (1), 40e49. Magnus, E., Hoel, H., Carlberg, G.E., 2000. TMP wastewater treatment, including a biological high-efficiency compact reactor. Toxicity reduction and removal of extractives. Nordic Pulp Paper Res. J. 15 (1), 37e45. Mahmood-Khan, Z., Hall, E.R., 2003. Occurrence and removal of plant sterols in pulp and paper mill effluents. J. Environ. Eng. Sci. 2, 17e26. Mahmood-Khan, Z., Hall, Eric, 2008. Quantification of plant sterols in pulp and paper mill effluents. Water Qual. Res. J. Can. 2/3, 173e181. Marton, J., 1964. On the structure of kraft lignin. Tappi J. 47 (11), 713e719. McKague, A.B., Reeve, D.W., 2003. Analysis of plant sterols in pulp mill effluents and sludges. In: TAPPI International Environmental Conference & Exhibition. Portland, OR, United States, May 4e7, 2003. TAPPI Press, Atlanta, GA, USA, pp. 61e 67. Metcalf, Eddy, 2003. In: Tchobanoglous, G., Burton, F.L., Stensel, H.D. (Eds.), Wastewater Engineering, Treatment and Reuse, fourth ed. McGraw-Hill, Toronto, Canada. Niven, S.J., Snape, J., Hetheridge, M., Evans, M., McEvoy, J., Rowland, S.J., 2001. Investigations of cholesterol transformation during sewage treatment: relevance to estrogen formation pathways? Sci. Total Environ. 279, 75e86. Orrego, R., Guchardi, J., Beyger, L., Krause, R., Holdway, D., 2011. Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Aquat. Toxicol. 104 (3e4), 299e307. Orrego, R., Guchardi, J., Krause, R., Holdway, D., 2010. Estrogenic and anti-estrogenic effects of wood extractives present in pulp and paper mill effluents on rainbow trout. Aquat. Toxicol. 99 (2), 160e167. Rydholm, S.A., 1965. Pulping Processes. Willey and Sons, New York, N.Y. USA. Shaw, D.J., 1992. Introduction to Colloid and Surface Chemistry, fourth ed. Butterworth-Heinemann Ltd., Oxford, UK. Shoppee, C.W., 1964. Chemistry of the Steroids. Butterworth & Co. Ltd., London, UK. Springer, A.M., 1993. Industrial Environmental Control, Pulp and Paper Industry, second ed. TAAPI Press, Atlanta, Georgia. Tremblay, L., Van Der Kraak, G., 1999. Comparison between the effects of the phytosterol ß-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environ. Toxicol. Chem. 18 (2), 329e336. Van Ginkel, C.G., Virtapohja, J., Steyaert, J.A.G., Alen, R., 1999. Treatment of EDTAcontaining pulp and paper mill wastewaters in activated sludge plants. TAPPI J. 82 (2), 138e142. Vidal, G., Becerra, J., Hernández, V., Decap, J., Xavier, C.R., 2007. Anaerobic biodegradation of sterols contained in kraft mill effluents. J. Biosci. Bioeng. 104 (6), 476e480. Werker, A.G., 1998. The Effect of pH on Microbial Activity and Community Structure in the Biological Removal of Resin Acids from Wastewater. Ph.D. Dissertation. Department of Civil Engineering, University of British Columbia, Vancouver, B.C. Canada. Werker, A.G., Hall, E.R., 1999. Limitations for biological removal of resin acids from pulp mill effluents. Water Sci. Technol. 40 (11e12), 281e288. WEF, ASCE., 1998. Design of Wastewater Treatment Plants, WEF Manual of Practice No. 8, fourth ed. ASCE Manuals and Reports on Engineering Practice No. 76, Water Environment Federation (WEF), vol. II. American Society of Civil Engineers (ASCE), VA, USA.