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Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
The contribution of suspended solids to municipal wastewater PHA-based denitrification a
a
a
a
Eli Krasnits , Michael Beliavski , Sheldon Tarre & Michal Green a
Faculty of Civil and Environmental Engineering, Technion, Haifa 32000, Israel Published online: 23 Sep 2013.
Click for updates To cite this article: Eli Krasnits, Michael Beliavski, Sheldon Tarre & Michal Green (2014) The contribution of suspended solids to municipal wastewater PHA-based denitrification, Environmental Technology, 35:3, 313-321, DOI: 10.1080/09593330.2013.827728 To link to this article: http://dx.doi.org/10.1080/09593330.2013.827728
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 3, 313–321, http://dx.doi.org/10.1080/09593330.2013.827728
The contribution of suspended solids to municipal wastewater PHA-based denitrification Eli Krasnits, Michael Beliavski, Sheldon Tarre and Michal Green∗ Faculty of Civil and Environmental Engineering, Technion, Haifa 32000, Israel
Downloaded by [Dalhousie University] at 21:49 25 December 2014
(Received 5 April 2013; accepted 29 June 2013 ) The role of wastewater suspended solids in denitrification based on intracellular carbon storage was investigated in a biofilm sequencing batch reactor performing alternately anaerobic carbon storage and denitrification. Municipal wastewater as the feeding was compared with filtered wastewater and with acetate. The results show that the amount of PHA (polyhydroxyalkanoates) stored during a cycle was quite similar, irrespective of the substrate type used as feeding (acetate, real wastewater and real wastewater after filtration). PHA storage was limited even under excess chemical oxygen demand (COD) conditions, with a reducing power capacity enough for denitrification of only 25–26 mg/L N. However, when non-filtered wastewater was used, the denitrification capacity was about 50% higher (38 mg/L N) due to the contribution of entrapped suspended solids as the electron donor. In addition, the involvement of the hydrolyzed wastewater suspended solids resulted in a different PHA composition containing a much higher poly-3-hydroxyvalerate content. Keywords: microbial storage, denitrification, PHA, wastewater
1. Introduction Denitrification which is based on anaerobically stored polymers is a potentially cost-effective option for simultaneous removal of organics, nitrogen and phosphorus from wastewater. The main potential advantages are low chemical oxygen demand (COD)/N requirement with the corresponding elimination of costs involved in external substrate addition, low energy requirement for aeration and liquor recycling, and low sludge production.[1] Intracellular carbon and energy storage as polyhydroxyalkanoates (PHA) is a major metabolic pathway in conventional wastewater treatment plants.[2] Previous studies, in which simple organic compounds such as acetate were used as the substrate for microbial carbon storage, have shown that intracellular polymers (mainly polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV)) can serve as potentially effective electron donors for denitrification. [1,3–5] Denitrification on storage polymers can occur under either constant anoxic conditions, due to fluctuations in external electron donor availability, or under alternating anaerobic/anoxic conditions where the presence of external electron donor and electron acceptor is separated. Under anoxic conditions the adenosine triphosphate (ATP) and reducing equivalents required for substrate uptake and storage are generated mainly through the anoxic TCA cycle, while under anaerobic conditions reducing equivalents are produced through glycolysis and the main source for ATP
∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
can be either cleavage of polyphosphate (by ‘polyphosphate accumulating organisms’ (PAOs)) or glycolysis (by ‘glycogen accumulating organisms’ (GAOs)). Denitrification on PHA stored under constant anoxic conditions has been previously investigated with synthetic COD sources such as acetate, methanol and glucose.[4–9] Storage of PHA under anaerobic conditions has been previously studied in two-sludge systems as a potentially feasible configuration of enhanced biological phosphorus removal treatment system. In such two-sludge systems the nitrifying sludge and the denitrifying/PHA storing sludge are separated, and organics, nitrogen and phosphorus are removed concurrently. The removal efficiencies of such systems have been studied previously with synthetic COD sources such as acetate and propionate and to some extent with real wastewaters from different sources such as piggery wastewater, municipal wastewater and abattoir wastewater after anaerobic treatment with the addition of VFA.[1,10–14] However, none of these studies investigated the composition and stoichiometry of the storage biopolymers and the nitrogen removal potential of two-sludge systems fed with municipal wastewater. In addition, since the amount of PHA stored, whether using acetate or wastewater as substrate, is usually not enough for complete denitrification of a typical nitrogen content in municipal, the additional COD contribution of the adsorbed and entrapped wastewater suspended solids during storage may play an important role in
314
E. Krasnits et al. Table 1.
Characteristics of wastewaters used during the research period. CODT
CODF a
TSS
Feed
N.S. wastewater after primary sedimentation
F = COD after filtration through b Filtered through a series of disc filters
Downloaded by [Dalhousie University] at 21:49 25 December 2014
a COD
260 (11) 402 (71) 288 (34)
NH3 -N
pH
260 (11) 229 (28) 245 (35)
–
–
4.7 (0.3) 5.9 (0.8) 6.8 (0.8)
60 (1.2) 61 (6.5) 65 (7.8)
7.0 (0.1) 7.5 (0.3) 7.3 (0.3)
139 (37) 17 (7)
121 (36) 15 (6)
0.45 μm syringe-driven filter. with gradually decreasing pore diameters – 50, 20, 10, and 5 μm.
the overall denitrification efficiency in a two-sludge system performing PHA-based denitrification (PBD).[15] This research study investigates for the first time, the contribution of wastewater suspended solids to PBD in order to allow for a higher nitrate removal. In addition, the influence of suspended solids on storage composition and stoichiometry was studied. The research was carried out using biofilm reactors performing COD storage and PBD as a part of a potential two-sludge system. This research is focused on nitrogen removal with the assumption that residual phosphorous can easily be removed by chemical precipitation.[15]
2.
PO4 -P
mg/L
Acetate-based synthetic wastewater
Filtered N.S. wastewaterb
VSS
Materials and methods
2.1. The wastewater Municipal wastewater collected from Haifa suburb NeveSheanan (N.S.) was used in this research study. The experiments were carried out with either wastewater after primary sedimentation or with filtered wastewater. Filtration was carried out on raw wastewater after primary sedimentation using a series of disc filters with gradually decreasing pore diameters – 50, 20, 10 and 5 μm filters. The filters were replaced on a daily basis to prevent substantial growth of anaerobic bacteria within the filter media. The wastewater filtration resulted in removal of around 90% of the suspended solids. In addition, acetate-based synthetic wastewater was used for comparison purposes. The acetate-based synthetic wastewater consisted of tap water to which 200 mg/L acetate, 50 mg/L yeast extract and 5 mg/L PO4 -P were added. The composition of the synthetic wastewater was selected to give a filtered COD, as well as P concentration, similar to those of the real wastewater. The characteristics of all three types of wastewaters used in this study are presented in Table 1. Since an integrated nitrification reactor was not studied in this research project, at the end of the storage stage the reactors were drained and the effluent was discarded. At the beginning of the denitrification stage, the reactors were
fed with a nitrate solution simulating the nitrification reactor effluent. The nitrate solution consisted of tap water to which 40–50 mg/L NO3 -N and 10 mg/L PO4 -P were added. 2.2. The reactors The experimental setup consisted of two batch biofilm reactors: one treating acetate-based synthetic wastewater (R1) and the second treating N.S. wastewater (R2). The influent and effluent streams of the reactors were sampled (triplicates) every two to three days for COD, total suspended solids (TSS), volatile suspended solids (VSS), NO3 -N, NO2 -N, PO4 -P and pH. In addition, liquor samples as well as biomass samples were taken periodically during the cycle. Biomass samples taken from the reactors were used to determine the TSS, VSS, PHA and glycogen contents. TSS and VSS contents in the biofilm reactors were determined based on the amount of biomass removed from three random plastic carriers. The carriers were rearranged in the biofilm reactors in a random fashion after each sampling. 2.2.1. The PBD biofilm reactors Two cylindrical reactors – R1 and R2, with a working volume of 2.1 L – were seeded with biomass from the Haifa municipal activated sludge wastewater treatment plant and operated under alternating anaerobic/anoxic conditions. The reactors were operated in a batch-wise mode as described in Figure 1: at the beginning of the anaerobic storage stage, R1 was fed with acetate-based synthetic wastewater, while R2 was fed with N.S. wastewater. At the end of the anaerobic storage stage, the liquors were drained and both reactors were fed with the nitrate and phosphorous solution. At the end of the denitrification stage, the reactors were drained again. Each PBD biofilm reactor contained 283 highdensity polyethylene plastic carriers (Aqwise® L = 14 mm 600 m2 /m3 ). A removable plastic grid was placed 10 cm below water surface to prevent the carriers from floating to the surface. Plastic balls (d = 2 cm) were used to cover the
Environmental Technology
Figure 1.
Table 2.
Successive stages during a cycle (156 min) in the PBD biofilm reactors.
Characteristics of the PBD biofilm reactors. TSS
VSS
mg/Lreactor
Downloaded by [Dalhousie University] at 21:49 25 December 2014
315
V0
Vr L
R1 – Acetate-based wastewater 6410 5384 1.42 0.14 R2 period 1 – Non-filtered wastewater 7950 6330 1.56 0.10 R2 period 2 – Filtered wastewater 6619 5255 1.63 0.08 Note: V0 = initial exchanged liquid volume; volume/exchanged volume + residual volume.
Vr = residual
water surface in order to minimize CO2 stripping and exposure to atmospheric oxygen. For mixing purposes a liquor recycling pump was operated in each reactor with an average 1 L/min flow rate during the entire cycle. The reactors were kept in a temperature-controlled room at 20(±2)◦ C. The only difference between R1 and R2 was the influent composition of the storage stage: R1 was fed with acetate-based synthetic wastewater for 60 days, while R2 was fed with two types of wastewater according to two time periods: • Period 1: During 67 days the reactor received N.S. municipal wastewater after primary sedimentation. • Period 2: During the following 60 days, the reactor received N.S. filtered wastewater. The reactors’ characteristics are given in Table 2. The amount of biosolids and their water retention properties influenced the exchanged volume (V0 ) and the residual volume fraction (Vr ) in the biofilm reactors (R1 and R2). The differences between TSS and VSS concentrations of the three reactors are mainly due to the different influents’ composition of the storage stage with the corresponding accumulation of wastewater-originated suspended solids, as well as biomass growth.
2.3. Analyses Influent and effluent pH, COD, filtered COD (CODF ), TSS, VSS, NO3 -N, NO2 -N, PO4 -P, acetate and NH3 -N were tested regularly. Filtered COD was determined as the COD after filtration through a 0.45 μm syringe-driven filter. All analyses were carried out according to standard methods.[16] NO3 -N, NO2 -N, PO4 -P and acetate were
tested using a 761 Metrohm (Switzerland) ion chromatograph (suppressed anion method). Biomass PHA concentration was calculated as the sum of PHB, PHV and poly-3-hydroxy-2-methylvalerate PH2 MV. In order to determine the PHA and glycogen concentrations in the biofilm reactors, biomass was removed from three random carriers from each reactor and transferred into water containing sulphuric acid (pH < 2). The carriers were rearranged in a random fashion after each biomass sampling. All biomass samples were centrifuged and the supernatant was discarded. The pellets were kept in a freezer (−20◦ C). PHB, PHV and PH2 MV concentrations (w/w) were determined according to Oehmen et al. [17] with a slight modification: biomass pellets were dried at 105◦ C for 2 h and placed in Pyrex tubes containing 2 mL chloroform and 2 mL methanol with sulphuric acid. For PHB and PHV determination, methanol with 3% sulphuric acid was used and the tubes were heated at 100◦ C for 3.5 h. For peak calibration, R-3-hydroxybutyric acid (3HB) and R3-hydroxyvaleric acid (3HV) copolymer (88:12) (Sigma Aldrich) was used. For PH2 MV determination, methanol with 10% sulphuric acid was used, the tubes were heated for 20 h and hydroxyhexanoic acid was used for peak calibration. After cooling, 2.5 mL deionized water was added to each tube and the tubes were shaken vigorously. After phase separation, the chloroform phase was dried over NaSO4 and filtered through a 0.2 μm syringe-driven PTFE filter to remove cell debris. Benzoic acid was used as the internal standard. An amount of 1.5 μl of the chloroform phase was injected to HP 6890 gas chromatograph (GC) equipped with an flame ionization detector (FID) detector. A 30 meter DB5, 0.32 mm, 0.25 μm film column was used. For verification purposes, a few samples were also injected to Thermo Scientific Focus GC equipped with single quadrupole mass spectrometer detector. A 30 m, 0.25 mm, 0.25 μm film column was used. Glycogen was determined using an enzymatic glucose assay kit (Sigma Aldrich® GHK20). Biomass samples were dried for 2 h at 105◦ C. The dried biomass samples were placed in sealed Pyrex tubes containing 0.6 M HCL solution and heated at 100◦ C for 3 h. The resulting solution was neutralized using 10 M KOH solution and centrifuged to remove particulate matter. The supernatant was used to
316
E. Krasnits et al.
determine the glucose concentration using the enzymatic assay kit.
Downloaded by [Dalhousie University] at 21:49 25 December 2014
2.4.
Calculation of storage compound concentration (PHA and glycogen) The major constituents of microbial PHA are considered to be PHB, PHV and poly-3-hydroxy-2-methylvalerate (PH2 MV).[18] Since the detected concentrations of microbial PH2 MV were insignificant (
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
The contribution of suspended solids to municipal wastewater PHA-based denitrification a
a
a
a
Eli Krasnits , Michael Beliavski , Sheldon Tarre & Michal Green a
Faculty of Civil and Environmental Engineering, Technion, Haifa 32000, Israel Published online: 23 Sep 2013.
Click for updates To cite this article: Eli Krasnits, Michael Beliavski, Sheldon Tarre & Michal Green (2014) The contribution of suspended solids to municipal wastewater PHA-based denitrification, Environmental Technology, 35:3, 313-321, DOI: 10.1080/09593330.2013.827728 To link to this article: http://dx.doi.org/10.1080/09593330.2013.827728
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 3, 313–321, http://dx.doi.org/10.1080/09593330.2013.827728
The contribution of suspended solids to municipal wastewater PHA-based denitrification Eli Krasnits, Michael Beliavski, Sheldon Tarre and Michal Green∗ Faculty of Civil and Environmental Engineering, Technion, Haifa 32000, Israel
Downloaded by [Dalhousie University] at 21:49 25 December 2014
(Received 5 April 2013; accepted 29 June 2013 ) The role of wastewater suspended solids in denitrification based on intracellular carbon storage was investigated in a biofilm sequencing batch reactor performing alternately anaerobic carbon storage and denitrification. Municipal wastewater as the feeding was compared with filtered wastewater and with acetate. The results show that the amount of PHA (polyhydroxyalkanoates) stored during a cycle was quite similar, irrespective of the substrate type used as feeding (acetate, real wastewater and real wastewater after filtration). PHA storage was limited even under excess chemical oxygen demand (COD) conditions, with a reducing power capacity enough for denitrification of only 25–26 mg/L N. However, when non-filtered wastewater was used, the denitrification capacity was about 50% higher (38 mg/L N) due to the contribution of entrapped suspended solids as the electron donor. In addition, the involvement of the hydrolyzed wastewater suspended solids resulted in a different PHA composition containing a much higher poly-3-hydroxyvalerate content. Keywords: microbial storage, denitrification, PHA, wastewater
1. Introduction Denitrification which is based on anaerobically stored polymers is a potentially cost-effective option for simultaneous removal of organics, nitrogen and phosphorus from wastewater. The main potential advantages are low chemical oxygen demand (COD)/N requirement with the corresponding elimination of costs involved in external substrate addition, low energy requirement for aeration and liquor recycling, and low sludge production.[1] Intracellular carbon and energy storage as polyhydroxyalkanoates (PHA) is a major metabolic pathway in conventional wastewater treatment plants.[2] Previous studies, in which simple organic compounds such as acetate were used as the substrate for microbial carbon storage, have shown that intracellular polymers (mainly polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV)) can serve as potentially effective electron donors for denitrification. [1,3–5] Denitrification on storage polymers can occur under either constant anoxic conditions, due to fluctuations in external electron donor availability, or under alternating anaerobic/anoxic conditions where the presence of external electron donor and electron acceptor is separated. Under anoxic conditions the adenosine triphosphate (ATP) and reducing equivalents required for substrate uptake and storage are generated mainly through the anoxic TCA cycle, while under anaerobic conditions reducing equivalents are produced through glycolysis and the main source for ATP
∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
can be either cleavage of polyphosphate (by ‘polyphosphate accumulating organisms’ (PAOs)) or glycolysis (by ‘glycogen accumulating organisms’ (GAOs)). Denitrification on PHA stored under constant anoxic conditions has been previously investigated with synthetic COD sources such as acetate, methanol and glucose.[4–9] Storage of PHA under anaerobic conditions has been previously studied in two-sludge systems as a potentially feasible configuration of enhanced biological phosphorus removal treatment system. In such two-sludge systems the nitrifying sludge and the denitrifying/PHA storing sludge are separated, and organics, nitrogen and phosphorus are removed concurrently. The removal efficiencies of such systems have been studied previously with synthetic COD sources such as acetate and propionate and to some extent with real wastewaters from different sources such as piggery wastewater, municipal wastewater and abattoir wastewater after anaerobic treatment with the addition of VFA.[1,10–14] However, none of these studies investigated the composition and stoichiometry of the storage biopolymers and the nitrogen removal potential of two-sludge systems fed with municipal wastewater. In addition, since the amount of PHA stored, whether using acetate or wastewater as substrate, is usually not enough for complete denitrification of a typical nitrogen content in municipal, the additional COD contribution of the adsorbed and entrapped wastewater suspended solids during storage may play an important role in
314
E. Krasnits et al. Table 1.
Characteristics of wastewaters used during the research period. CODT
CODF a
TSS
Feed
N.S. wastewater after primary sedimentation
F = COD after filtration through b Filtered through a series of disc filters
Downloaded by [Dalhousie University] at 21:49 25 December 2014
a COD
260 (11) 402 (71) 288 (34)
NH3 -N
pH
260 (11) 229 (28) 245 (35)
–
–
4.7 (0.3) 5.9 (0.8) 6.8 (0.8)
60 (1.2) 61 (6.5) 65 (7.8)
7.0 (0.1) 7.5 (0.3) 7.3 (0.3)
139 (37) 17 (7)
121 (36) 15 (6)
0.45 μm syringe-driven filter. with gradually decreasing pore diameters – 50, 20, 10, and 5 μm.
the overall denitrification efficiency in a two-sludge system performing PHA-based denitrification (PBD).[15] This research study investigates for the first time, the contribution of wastewater suspended solids to PBD in order to allow for a higher nitrate removal. In addition, the influence of suspended solids on storage composition and stoichiometry was studied. The research was carried out using biofilm reactors performing COD storage and PBD as a part of a potential two-sludge system. This research is focused on nitrogen removal with the assumption that residual phosphorous can easily be removed by chemical precipitation.[15]
2.
PO4 -P
mg/L
Acetate-based synthetic wastewater
Filtered N.S. wastewaterb
VSS
Materials and methods
2.1. The wastewater Municipal wastewater collected from Haifa suburb NeveSheanan (N.S.) was used in this research study. The experiments were carried out with either wastewater after primary sedimentation or with filtered wastewater. Filtration was carried out on raw wastewater after primary sedimentation using a series of disc filters with gradually decreasing pore diameters – 50, 20, 10 and 5 μm filters. The filters were replaced on a daily basis to prevent substantial growth of anaerobic bacteria within the filter media. The wastewater filtration resulted in removal of around 90% of the suspended solids. In addition, acetate-based synthetic wastewater was used for comparison purposes. The acetate-based synthetic wastewater consisted of tap water to which 200 mg/L acetate, 50 mg/L yeast extract and 5 mg/L PO4 -P were added. The composition of the synthetic wastewater was selected to give a filtered COD, as well as P concentration, similar to those of the real wastewater. The characteristics of all three types of wastewaters used in this study are presented in Table 1. Since an integrated nitrification reactor was not studied in this research project, at the end of the storage stage the reactors were drained and the effluent was discarded. At the beginning of the denitrification stage, the reactors were
fed with a nitrate solution simulating the nitrification reactor effluent. The nitrate solution consisted of tap water to which 40–50 mg/L NO3 -N and 10 mg/L PO4 -P were added. 2.2. The reactors The experimental setup consisted of two batch biofilm reactors: one treating acetate-based synthetic wastewater (R1) and the second treating N.S. wastewater (R2). The influent and effluent streams of the reactors were sampled (triplicates) every two to three days for COD, total suspended solids (TSS), volatile suspended solids (VSS), NO3 -N, NO2 -N, PO4 -P and pH. In addition, liquor samples as well as biomass samples were taken periodically during the cycle. Biomass samples taken from the reactors were used to determine the TSS, VSS, PHA and glycogen contents. TSS and VSS contents in the biofilm reactors were determined based on the amount of biomass removed from three random plastic carriers. The carriers were rearranged in the biofilm reactors in a random fashion after each sampling. 2.2.1. The PBD biofilm reactors Two cylindrical reactors – R1 and R2, with a working volume of 2.1 L – were seeded with biomass from the Haifa municipal activated sludge wastewater treatment plant and operated under alternating anaerobic/anoxic conditions. The reactors were operated in a batch-wise mode as described in Figure 1: at the beginning of the anaerobic storage stage, R1 was fed with acetate-based synthetic wastewater, while R2 was fed with N.S. wastewater. At the end of the anaerobic storage stage, the liquors were drained and both reactors were fed with the nitrate and phosphorous solution. At the end of the denitrification stage, the reactors were drained again. Each PBD biofilm reactor contained 283 highdensity polyethylene plastic carriers (Aqwise® L = 14 mm 600 m2 /m3 ). A removable plastic grid was placed 10 cm below water surface to prevent the carriers from floating to the surface. Plastic balls (d = 2 cm) were used to cover the
Environmental Technology
Figure 1.
Table 2.
Successive stages during a cycle (156 min) in the PBD biofilm reactors.
Characteristics of the PBD biofilm reactors. TSS
VSS
mg/Lreactor
Downloaded by [Dalhousie University] at 21:49 25 December 2014
315
V0
Vr L
R1 – Acetate-based wastewater 6410 5384 1.42 0.14 R2 period 1 – Non-filtered wastewater 7950 6330 1.56 0.10 R2 period 2 – Filtered wastewater 6619 5255 1.63 0.08 Note: V0 = initial exchanged liquid volume; volume/exchanged volume + residual volume.
Vr = residual
water surface in order to minimize CO2 stripping and exposure to atmospheric oxygen. For mixing purposes a liquor recycling pump was operated in each reactor with an average 1 L/min flow rate during the entire cycle. The reactors were kept in a temperature-controlled room at 20(±2)◦ C. The only difference between R1 and R2 was the influent composition of the storage stage: R1 was fed with acetate-based synthetic wastewater for 60 days, while R2 was fed with two types of wastewater according to two time periods: • Period 1: During 67 days the reactor received N.S. municipal wastewater after primary sedimentation. • Period 2: During the following 60 days, the reactor received N.S. filtered wastewater. The reactors’ characteristics are given in Table 2. The amount of biosolids and their water retention properties influenced the exchanged volume (V0 ) and the residual volume fraction (Vr ) in the biofilm reactors (R1 and R2). The differences between TSS and VSS concentrations of the three reactors are mainly due to the different influents’ composition of the storage stage with the corresponding accumulation of wastewater-originated suspended solids, as well as biomass growth.
2.3. Analyses Influent and effluent pH, COD, filtered COD (CODF ), TSS, VSS, NO3 -N, NO2 -N, PO4 -P, acetate and NH3 -N were tested regularly. Filtered COD was determined as the COD after filtration through a 0.45 μm syringe-driven filter. All analyses were carried out according to standard methods.[16] NO3 -N, NO2 -N, PO4 -P and acetate were
tested using a 761 Metrohm (Switzerland) ion chromatograph (suppressed anion method). Biomass PHA concentration was calculated as the sum of PHB, PHV and poly-3-hydroxy-2-methylvalerate PH2 MV. In order to determine the PHA and glycogen concentrations in the biofilm reactors, biomass was removed from three random carriers from each reactor and transferred into water containing sulphuric acid (pH < 2). The carriers were rearranged in a random fashion after each biomass sampling. All biomass samples were centrifuged and the supernatant was discarded. The pellets were kept in a freezer (−20◦ C). PHB, PHV and PH2 MV concentrations (w/w) were determined according to Oehmen et al. [17] with a slight modification: biomass pellets were dried at 105◦ C for 2 h and placed in Pyrex tubes containing 2 mL chloroform and 2 mL methanol with sulphuric acid. For PHB and PHV determination, methanol with 3% sulphuric acid was used and the tubes were heated at 100◦ C for 3.5 h. For peak calibration, R-3-hydroxybutyric acid (3HB) and R3-hydroxyvaleric acid (3HV) copolymer (88:12) (Sigma Aldrich) was used. For PH2 MV determination, methanol with 10% sulphuric acid was used, the tubes were heated for 20 h and hydroxyhexanoic acid was used for peak calibration. After cooling, 2.5 mL deionized water was added to each tube and the tubes were shaken vigorously. After phase separation, the chloroform phase was dried over NaSO4 and filtered through a 0.2 μm syringe-driven PTFE filter to remove cell debris. Benzoic acid was used as the internal standard. An amount of 1.5 μl of the chloroform phase was injected to HP 6890 gas chromatograph (GC) equipped with an flame ionization detector (FID) detector. A 30 meter DB5, 0.32 mm, 0.25 μm film column was used. For verification purposes, a few samples were also injected to Thermo Scientific Focus GC equipped with single quadrupole mass spectrometer detector. A 30 m, 0.25 mm, 0.25 μm film column was used. Glycogen was determined using an enzymatic glucose assay kit (Sigma Aldrich® GHK20). Biomass samples were dried for 2 h at 105◦ C. The dried biomass samples were placed in sealed Pyrex tubes containing 0.6 M HCL solution and heated at 100◦ C for 3 h. The resulting solution was neutralized using 10 M KOH solution and centrifuged to remove particulate matter. The supernatant was used to
316
E. Krasnits et al.
determine the glucose concentration using the enzymatic assay kit.
Downloaded by [Dalhousie University] at 21:49 25 December 2014
2.4.
Calculation of storage compound concentration (PHA and glycogen) The major constituents of microbial PHA are considered to be PHB, PHV and poly-3-hydroxy-2-methylvalerate (PH2 MV).[18] Since the detected concentrations of microbial PH2 MV were insignificant (
