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Insight into the short- and long-term effects of inorganic phosphate on anammox granule property
Accepted Manuscript Insight into the short- and long-term effects of inorganic phosphate on anammox granule property Zheng-Zhe Zhang, Jia-Jia Xu, Hai-Yan Hu, Zhi-Jian Shi, Zheng-Quan Ji, Rui Deng, Man-Ling Shi, Ren-Cun Jin PII: DOI: Reference:
S0960-8524(16)30234-6 http://dx.doi.org/10.1016/j.biortech.2016.02.097 BITE 16147
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 December 2015 19 February 2016 22 February 2016
Please cite this article as: Zhang, Z-Z., Xu, J-J., Hu, H-Y., Shi, Z-J., Ji, Z-Q., Deng, R., Shi, M-L., Jin, R-C., Insight into the short- and long-term effects of inorganic phosphate on anammox granule property, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.02.097
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Insight into the short- and long-term effects of inorganic phosphate on anammox granule property Zheng-Zhe Zhanga,b, Jia-Jia Xua,b, Hai-Yan Hua,b, Zhi-Jian Shia,b, Zheng-Quan Jia,b, Rui Deng a,b, Man-Ling Shia,b, Ren-Cun Jina,b* a
College of Life and Environmental Sciences, Hangzhou Normal University,
Hangzhou 310036, China b
Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration,
Hangzhou Normal University, Hangzhou 310036, China *
Corresponding author: Ren-Cun Jin
Tel.: +86-571-88062061; Fax: +86-571-28865333 E-mail address: [email protected]
Abstract The short- and long-term effects of inorganic phosphate on property of anaerobic ammonium oxidation (anammox) granule were investigated in this study. Acute exposure to high-level phosphate (≥ 50 mM) induced the cytoplasm leakage. During a 195-day continuous-flow operation, the gradually increasing phosphate (up to 500 mgP L-1) slightly affected the specific anammox activity, hardly impacted the heme c content, remarkably decreased the extracellular polymeric substances production and significantly stimulated the dehydrogenase activity of anammox granules. Microbial community analysis showed no shift in the dominant anammox strain and higher population but lower relative abundance of anaerobic ammonium-oxidizing bacteria
compared to the control granules. Interestingly, novel anammox granules with a hydroxyapatite core were cultivated, which possessed excellent settleability, huge granule diameter and superior mechanical strength. This study supported the application of granule-based anammox process as a pre-processing treatment in phosphate-containing and ammonia-rich wastewaters.
Keywords: anammox; phosphate; sludge characteristics; hydroxyapatite
1. Introduction Due to remarkable advantages, such as high nitrogen removal rates, low operational costs and small footprints, anaerobic ammonium oxidation (anammox) -based processes have been successfully implemented for the treatment of high-strength ammonium wastewater with low C:N ratios under mesophilic conditions, e.g., centrates from anaerobic digestion sludge (sidestream) and effluents from the production of nitrogenous fertilizers (Jin et al., 2012; Lotti et al., 2015). With the worldwide expansion to 100 full-scale installations by early 2015, the anammox process is entering a new stage of widespread use and development. The majority (approximately 75%) of installations are applied toward the sidestream treatment of municipal wastewater (Lackner et al., 2014). In particular, with recent advances in mainstream treatment, a net energy-producing sewage treatment plant providing effective nutrient removal is nearing feasibility (Gilbert et al., 2014; Lotti et al., 2014).
Phosphorus is an essential nutrient and is a major contributor to agricultural and industrial development. In 2000, 19.7 Mt of P was mined as phosphate rock. The majority of this, 15.3 Mt P, was used to produce fertilizers (Wilfert et al., 2015); thus, phosphates are abundant in wastewater from fertilizer production. Moreover, phosphates are commonly found in municipal wastewaters (mainstream and sidestream) due to their use in detergents or introduction from black water (de Graaff et al., 2011). Regarding P recovery, enhanced biological phosphorus removal (EBPR) is not yet widely applied, and the recovery potential is limited. Other phosphorus recovery methods, including sludge application to agricultural land or recovering phosphorus from sludge ash, also have limitations. Energy-producing wastewater treatment plants increasingly rely on phosphorus removal using iron (Wilfert et al., 2015). Generally, in flow line this procedure was after nitrogen removal process. Therefore, considerable amounts of phosphates might be introduced to anammox-based reactors. Due to the slow growth rate, low cell yield and high susceptibility of anaerobic ammonium-oxidizing bacteria (AnAOB) to variations in environmental conditions, the industrialization of anammox-based processes is restricted by inhibitory substances, such as phosphate in nitrogen-rich wastewater (Jin et al., 2012). However, disagreements have been reported in the literature in terms of the effects of phosphate on anammox biomass. van de Graaf et al. (1996) firstly observed a loss of activity for anammox culture (dominated by Ca. Brocadia anammoxidans) at phosphate concentrations above 5 mM. Thereafter, Egli et al. (2001) found no inhibitory effect
of phosphate at 20 mM for Ca. Kuenenia stuttgartiensis from a rotating biological contactor, while in the same system with the same genus, Pynart et al. (2003) observed that anammox activity decreased to 63% of the normal activity at 1.8 mM and further to 20% at 3.6 mM, but no further decrease (80% inhibition) at 9.2 mM in batch tests. Dapena-Mora et al. (2007) reported a higher tolerance of anammox biofilms (dominated by Ca. Kuenenia stuttgartiensis) from a gas-lift reactor to phosphate that IC50 was 20 mM. Recently, Carvajal-Arroyo et al. (2013) found that the exposure of suspended anammox enrichments (dominated by Ca. Brocadia) to phosphates caused a modest decrease in the specific anammox activity (SAA) with increasing phosphates concentrations (IC50 = 25.3 ± 5.9 mM), whereas phosphates stimulated the SAA of granular anammox enrichment (dominated by Ca. Brocadia) by 60% at concentrations ranging from 10-50 mM. However, these data were based on the results of batch experiments and lacked long-term evaluation in continuous-flow experiments. Lin et al. (2013) noted that apatite accumulation enhances the mechanical properties of anammox granules during the long-term addition of phosphate in continuous-flow experiments. Nevertheless, the influent phosphates concentration was maintained at 29.7 ± 9.7 mgP L-1. Given the prospective application of anammox-based processes in phosphate-containing and ammonium-rich wastewaters, clarifying the impacts of phosphates in various levels on anammox biomass is urgently required. To date, there have been no comprehensive investigations regarding the impacts of phosphate on anammox granule property. Therefore, the objectives of this study were to investigate (i) the short-term effects
of phosphate on anammox granules; (ii) the evolution of physiological characteristics and physic-chemical properties under different phosphate loads; and (iii) the compositions and formation mechanisms of the inorganic cores in anammox granules. 2. Materials and Methods 2.1 Synthetic wastewater and inoculums Inorganic synthetic wastewater containing substrates, mineral and trace elements was introduced into the reactors. Equimolar amounts of ammonium and nitrite in the forms of (NH4)2SO4 and NaNO2, respectively, were supplied as needed. The mineral medium composition was 10 mg L-1 NaH2PO4, 73.5 mg L-1 CaCl2•2H2O, 58.6 mg L-1 MgSO4•7H2O, and 840 mg L-1 NaHCO3. 1.25 ml of trace element solutions I and II were added per liter of wastewater. The composition of trace element solution I was 5 g L-1 EDTA and 9.14 g L-1 FeSO4•7H2O, and trace element solution II was composed of 15 g L-1 EDTA, 0.014 g L-1 H3BO4, 0.99 g L-1 MnCl2•4H2O, 0.25 g L-1 CuSO4•5H2O, 0.43 g L-1 ZnSO4•7H2O, 0.21 g L-1 NiCl2•6H2O, 0.22 g L-1 NaMoO4•2H2O and 0.24 g L-1 CoCl2•6H2O. Anammox seed granules were harvested from a high-loaded laboratory-scale up-flow anaerobic sludge blanket (UASB) reactor, which was fed with synthetic medium (as above) at a stable nitrogen removal rate (NRR) of approximately 10 kg N m-3 d -1 for over one year. 2.2 Batch experiments Batch exposure assays were performed in serum flasks with a total volume of 160 mL and a liquid phase volume of 120 mL. Equimolar amounts of ammonium and nitrite were added to the mineral medium. A total of 100 mL of basal mineral medium
and 1.25 mL of trace element solutions I and II per liter of sterilized water were introduced, resulting in a composition similar to that of the synthetic wastewater. Then, phosphate was added to the serum flasks as needed along with 10 mL of anammox biomass. The VSS concentration was approximately 2.5 g L-1 in each serum flask. The initial pH was fixed at approximately 7.5 by injections of 1 M hydrochloric acid or sodium hydroxide. The serum vials were subsequently flushed with 99.99% pure argon for 10 min and immediately sealed with butyl rubber to avoid oxygen leakage. Then, the serum flasks were placed in a 35 ± 1 °C thermostatic shaker at 180 rpm. After 10 h exposure, the mixtures were centrifuged for 5 min at 3,000 g. The supernatants were removed for the determination of lactate dehydrogenase (LDH) activity, dissolved organic matter (DOM) and UV-vis spectrum scanning (SHIMADZU UV-2550, Japan), and the resulting biomass was used for the heme c extraction. 2.3 Anammox reactors and operational strategy Two UASB reactors (R0 and R1) with effective volumes of 1.0 L each were fabricated from Plexiglas, covered with black cloth to prevent light-related inhibition and then placed in a thermostatic room at 35 ± 1 °C. After inoculation, the initial volatile suspended solid (VSS) concentration in these reactors was approximately 21 g L-1. The influent pH was self-stabilizing at pH 7.9 ± 0.1 without the addition of an acid or base. The continuous-flow experiments featured a constant influent substrate level of 280 mg L-1. The initial hydraulic retention time (HRT) was maintained at 1.2 h and the sludge retention time was controlled by the spontaneous sludge loss via the
effluent. NaH2PO4 and Na2HPO4 were added to the influent of R1 at molar ratios of 0.19, and the total phosphate concentrations are detailed in Table 1. The influent phosphate concentration was set at 500 mg L-1 from Day 165 until the end. The trace amount of phosphate (2 mgP L-1) was added into the control reactor (R0-CK) for the normal growth of the microorganisms. -----------------------------------------------Table 1 ----------------------------------------------2.4 EPS extraction and 3D-EEM fluorescence spectroscopy A heat-extraction method was employed for EPS extraction (Yin et al., 2015). Carbohydrate measurements were acquired using the anthrone method with a glucose standard, and the protein levels were measured using the modified Lowry method and bovine serum albumin as a standard (Wu et al., 2009). The EEM fluorescence spectra were measured using a fluorescence spectrophotometer (F-4600, Hitachi Co., Japan). The EEM spectra were collected with subsequent scanning emission spectra from 200 to 600 nm at 5-nm increments. The excitation and emission slits were maintained at 10 nm, and the scanning speed was set at 12,000 nm/min. The voltage of the photomultiplier tube (PMT) was set to 500 V for low-level light detection. The spectrum of double-distilled water was recorded as the blank. Origin 8.0 software was used to process the EEM data. Contour lines are shown for each EEM spectra to represent the fluorescence intensity. 2.5 Dehydrogenase activity assay The procedures were as follows: (1) 10 mL of the sludge sample was washed with 10 mL of an isotonic salt solution followed by centrifugation at 3000 g for 5 min; (2)
5 g field-moist weight sludge was homogenized with 15 mL of an autoclave -sterilized 0.85% saline solution; (3) 2 mL 2,3,5-triphenyltetrazoilum chloride (TTC) (5 g L-1), 1.5 mL Tris-HCl buffer solution (0.1 M, pH = 7.4) and 0.5 ml Na2SO3 (0.36%) were added to 2 mL of sludge liquid; (4) the mixed solution was incubated at 37 ± 1 °C for 4 h in the dark; (5) two drops of concentrated sulfuric acid were added to terminate the deoxidization reaction in the test tubes, and 5 mL toluene was added to extract the color product of the TF from the cells; (6) the reaction mixture was centrifuged at 6000 g for 5 min, and the absorbance of the supernatant was measured at 486 nm with a UV spectrometer; and (7) the same volume of sludge liquid was collected for biomass determination. Each value of TTC-DHA is expressed in units of µgTF g-1VSS h-1 and presented as the average of three replicates. 2.6 Heme c extraction and determination Five-milliliter field-moist anammox granules were centrifuged at 3000 g at 4 °C for 5 min followed by two washes with a sodium phosphate buffer solution (10 mM, pH 7.5). The washed pellets were then resuspended in 25 mL of the same buffer and then disrupted by sonication (800 W, at 4 °C for 20 min, Ultrasonic processor CPX 750, USA). The cell masses were separated by centrifugation (15,000 g) at 4 °C for 15 min. The supernatant was used for heme c content determination according to Berry and Trumpower (Berry and Trumpower, 1987). Briefly, the nitrogen ligands from the protein-bound heme were replaced by pyridine in alkali, and the resultant heme c was quantified as the difference between the spectra of the reduced (sodium dithionite crystals) and oxidized (potassium ferricyanide) compounds. The heme c concentration
was calculated based on a millimolar extinction coefficient of 23.97 mM cm-1 for the difference in absorption between the peak at 550 nm and the trough at 535 nm. The heme c content values are expressed as µmol g-1 VSS. 2.7 X-ray powder diffraction (XRD) Anammox granules for XRD analysis were previously dried and calcined in an oven at 500 °C for 2 h to remove the organic fraction. XRD analysis was performed using a Multiflex diffractometer (Bruker D8, Germany) with monochromatic Cu Kα1 radiation in the 2θ region between 5° and 90°. X-ray profile fitting was carried out using the Jade 6.5 program. 2.8 Strength of granules The microbial granule strength is described as an abrasion as a result of exposure to different shear rates, which can be evaluated by an ultrasonic method (Wan et al., 2013). In this process, 10 mL of randomly selected anammox granules were placed into a 50-mL conical flask that contained with 40 mL of deionized water, and then the tube was placed in an ultrasonic bath at 28 kHz, 70 W. The ultrasonic was intermittently applied at 2.5 s (on) - 3 s (off) cycles. The suspension turbidity data under ultrasound were measured spectrophotometrically at 600 nm. In this abrasion experiment at a constant shear rate, the production of fines (XF) per unit of volume and time was a first-order process in the concentration of larger granules (XNF). ୢూ ௧
= Kܺ
(1)
where K is a constant for the strength of the granules under the conditions applied. 2.9 Analytical procedures
The ammonium, nitrite, nitrate, total phosphate, VSS and suspended solid concentrations, as well as the pH and settling velocity (VS), were determined using standard methods (APHA et al., 2005). Anammox granules were taken regularly from the lab reactors to measure the SAA in ex situ batch experiments (Zhang et al., 2016). The LDH activity was detected using the UV spectrometer according to the protocols of the LDH assay kit (Jiancheng, China).The mean granule diameter (MGD) was measured using an image analysis system (QCOLite) with a Leica DM2LB microscope and a digital camera (Canon S30). Scanning electron microscopy (SEM) was used to observe the granule morphology using the methods reported by Tang et al. 2011. The Visual MINTEQ (VMINTEQ.v3.1) model was used to calculate the chemical equilibrium in the reactor. The total genomic DNA of each sample was extracted using the Power Soil DNA Kit (Mo Bio Laboratories, Carlsbad, CA). PCR amplification of the 16S rRNA gene was carried out using a primer set: Pla46F (5’-GAG TTT GAT CMT GGC TCA G-3’) and 1545R (5’-ACG GYT ACC TTG TTA CGA CTT-3’). For the detection of total bacteria and anammox bacteria, the 16S rRNA gene and hydrazine synthase structural genes (hzsA) were quantified with primer sets 338F -518R and hzsA_1597F-hzsA_1857R, respectively (Harhangi et al., 2012). The copy numbers were determined in triplicate using a CFX96 Touch™ thermocycler and a real-time detection system (Bio-Rad, CA, USA). Details are presented in the Supplementary Materials. An analysis of variance (ANOVA) was used to test the significance of the results, and p < 0.05 was considered statistically significant.
3 Results and discussion 3.1 Short-term effects of phosphate on anammox granules Previous batch experiments showed that phosphate at low levels (≤ 5 mM), acting as a buffer medium, slightly stimulated the specific anammox activity of anammox granules. Conversely, 10 mM-phosphate inhibited the SAA by 10%, and the inhibitory effects were exacerbated as the phosphate concentrations increased further. The IC50 value was calculated as 49.6 ± 6.4 mM by model simulation. Moreover, based on the kinetic experiments with various substrate levels, the inhibitory effect of phosphate at 20 mM on anammox granules was considered as uncompetitive inhibition pattern that decreased the maximum substrate uptake rate but enhanced the affinity of biomass to substrates. Specifically, phosphate does not directly bond with the enzymes of anammox metabolism but bond with complexes of enzymes and substrates, thereby forming triple-complexes. These triple-complexes could not be converted to products, resulting in decreased enzyme activity. Accordingly, the inhibitory effect of phosphate on anammox granules can be defined as a reversible inhibition process. However, when the anammox granules were exposed to high level of phosphate (≥ 50 mM) for 10 hours, the liquid phase acquired an orange coloration, which could indicate the cell lysis. In order to confirm this fact, UV–vis absorption spectra of the liquid phase from the batch tests were analyzed (Fig. 1a). A maximum peak between 400 and 410 nm was observed, similar to the previous study in which anammox biomass was shocked by high temperature of 45 °C (Dosta et al., 2008). Therefore, the orange color of the liquid phase could be attributed to the segregation of cytochrome c (Fig. S1). Indeed,
abnormally higher heme c content was detected in the liquid phase (Fig. 1b). Heme is an indispensable part of key anammox enzymes, including hydrazine synthase (HZS) and hydrazine dehydrogenase (HDH) (Jetten et al., 2009). Furthermore, the significantly higher LDH release (Fig. 1c) indicated that high levels of phosphate increased the permeability or induced the rupture of the cell membrane, thereby releasing intracellular polymeric substances to extracellular locations. This can be used for explaining the accumulation of protein and polysaccharide in the liquid phase (Fig. 1d). In terms of these results, taken as a whole, we updated the understanding of the phosphate inhibition mode: phosphate at range of 10 mM to 20 mM (may be higher but in this study 20 mM was determined) caused uncompetitive inhibition pattern, while phosphate higher than 50 mM induced an irreversible loss of the activity. However, cytoplasm leakage usually occurs when cells are exposed to hypotonic media, thus how to explain the mechanism of high level phosphate interacting with anammox cells needs further research. The results will trigger a radical reevaluation of the application of phosphate buffer in the bio-system containing anammox cells. -----------------------------------------------Fig.1 ----------------------------------------------3.2 Evolution of physiological characteristics 3.2.1 Heme content and specific anammox activity To further assess the long-term effects of phosphates, two continuous-flow UASB reactors were operated as described above. The specific anammox activity slightly increased by 8.3% at 10.8 mgP L-1 (Fig. 2a), consistent with the acute exposure results.
When influent phosphate concentration increased to 52.9 mgP L-1, the specific anammox activity was decreased by 19.3 %, incompatible with the acute exposure results that low level (≤ 5 mM) of phosphate stimulated specific anammox activity. It seemed that the anammox granules were susceptible to phosphate as high as 52.9 mgP L-1. However, as the influent phosphate concentration of R1 increased stepwise to 500 mg L-1 afterwards, the anammox granules recovered and showed equal activity with that of R0, indicating the gradually increased tolerance of anammox granules during long-term acclimatization. Furthermore, heme plays an important role in AnAOB energy metabolism and cell synthesis and producing the characteristic carmine color of anammox sludge (Tang et al., 2011). In this study, no significant differences in heme c contents were observed between two anammox granules over the course of a 195-day operation (Fig. 2b, independent-samples t-test, p > 0.05). -----------------------------------------------Fig.2 ----------------------------------------------3.2.2 Dehydrogenase activity The vast majority of redox reactions in the organisms were catalyzed by dehydrogenase and oxidase; dehydrogenase, an intracellular enzyme, is intimately involved in the oxidative phosphorylation process inside the cell and is an important indicator of microbial activity (Zhang et al., 2015a). As shown in Fig.2c, the presence of phosphates at 52.9 mgP L-1 remarkably stimulated TTC-DHA levels. Further, the TTC-DHA of anammox granules in R1 was 3.8-fold higher than that in R0 on Day 175. Interestingly, the electron transport rate as indicated by TTC-DHA was stimulated rather than inhibited, which appears to conflict with the observation of acute exposure.
This may be attributed to the appropriate pH environment: the long-term presence of phosphates acts as a buffer providing favorable culture conditions for microbial metabolism. 3.2.3 EPS amounts and composition EPS, a complex high-molecular-weight mixture of polymers excreted by microorganisms, is a major component in microbial aggregates and maintains these aggregates together in a three-dimensional matrix (Sheng et al., 2010). The total EPS production of R0 increased from 162.5 to 192.2 mg g-1VSS in the initial phase and then maintained stable. The EPS production of R1 initially increased up to 256.4 mg g-1 VSS with the application of phosphate at 52.9 mgP L-1, and reversed as the phosphate concentrations increased (Fig.2c). Finally, the EPS production of R1 was decreased to and maintained at approximately 85% of that of R0 over a long period of time (Days 105-175). Three peaks were easily identified in the 3D-EEM fluorescence spectra (Fig. 3). The first main peak was located at the excitation/emission wavelengths (Ex/Em) of 275/340-350 nm (Peak A); the second was observed at the Ex/Em of 225/340-350 nm (Peak B); and the third was identified at the Ex/Em of 225/305 nm (Peak C). These three peaks have been reported as protein-like peaks, in which the fluorescence is associated with soluble microbial by-product-like materials or tryptophan-containing proteins(Peak A), aromatic proteins-like substances (Peak B) and tyrosine-containing proteins (Peak C) (Zhang et al., 2015b). These protein-like components excreted by microorganisms, were indicative of different intracellular metabolic regulation
mechanisms. Obviously, three peak intensities (on Day 195) were reduced due to the decreased content of EPS, indicating that the presence of phosphate at high level impacted the anammox metabolism or modified the co-metabolism microflora in granules. The locations of Peak A and Peak B were red-shifted to longer wavelengths (Table 2), which may be attributed to the presence of carbonyl-containing substituents, carboxyl constituents, hydroxyl, alkoxyl, and amino groups (Ren et al., 2015). Therefore, the presence of phosphate at high level resulted in the structural differences in these protein-like compounds. -----------------------------------------------Fig. 3 --------------------------------------------------------------------------------------------Table 2----------------------------------------------3.3 Variations of physic-chemical properties 3.3.1 Microbially induced HAP precipitation in anammox granules XRD, an efficient tool for distinguishing crystalline minerals from those of amorphous structures, was used to identify the species of P minerals in the granule samples. As illustrated in Fig. S2, a number of distinct peaks in the diffractogram reflect the presence of crystalline forms. By comparison with a portable document format standard card (2004) using Jade 6.5, most of the stronger peaks coincide well with those of the hydroxyapatite (HAP, Ca5(PO4)3(OH)) pattern (FOM = 3.1). This result agrees with a previous report that HAP is a major phosphate mineral in aerobic granules (Mañas et al., 2011). Although several different P minerals have been previously reported in granules, such as whitlockite (Ca3(PO4)2, Ca18Mg2H2(PO4)14) and struvite (NH4Mg PO4·6H2O) (Huang et al., 2015; Lin et al., 2013), they were not
detected in the anammox granules in this study. Moreover, the low saturation index of whitlockite implied that whitlockite was poorly or very temporarily formed. Among the calcium phosphate family, HAP is considered the most stable and insoluble (Huang et al., 2015). Still, whether other intermediates are present or not remains unknown due to the limitation of XRD for micro-amounts of minerals. The influent pH and composition play important roles in phosphate precipitation due to their influence on the saturation index of different minerals. The Visual MINTEQ (VMINTEQ.v3.1) model was used to estimate the possible minerals precipitated in the reactor. The struvite saturation index was close to zero throughout all the phase due to ammonia consumption by anammox, indicating that the ammonium and magnesium concentrations were too low to cause struvite precipitation under these conditions. The production of vivianite was negligible because of the trace Fe element in the influent. Concerning calcium phosphates, hydroxyapatite was the most stable phase among the calcium phosphates that showed oversaturation conditions throughout the experiment (Table S1). Although the turbidity slightly increased with increased phosphates concentrations, no visible-shaped precipitation was observed in the influent or effluent. The evidence indicates that HAP only precipitated within the anammox biomass matrix and not in other places in the reactor, meaning that confined conditions were more favorable for HAP formation or accumulation than the conditions in the bulk. Two explanations can be proposed: (i) higher local pH and (ii) a special combination pattern. First, the internal pH can be higher than the bulk pH because of the acidity reduction in the
anammox reaction. The higher pH increased HAP supersaturation, thereby resulting in formation or accumulation in the inner region of anammox granules. Moreover, the dense structure of the granules encouraged the accumulation of HAP in the granule core and repressed the solubilization of the crystals (Huang et al., 2015). Second, recent studies have also revealed the considerable accumulation of P in the EPS of sludge in the enhanced biological phosphorus removal process (Zhang et al., 2013), implying a non-negligible role for EPS. AnAOB cells are enclosed by a matrix of EPS, which contains abundant functional groups such as carboxyl, phosphoric, amine, and hydroxyl groups that are negatively charged at neutral pH and are able to form organometallic complexes with multivalent cations via electrostatic attraction or other interactions (Liu and Fang, 2002). Ca2+ is the main multivalent cation binding to extracellular PS molecules (Jiang et al., 2003), thereby acting as a bridge interconnecting these components. Experimental observations suggest a scenario involving correlation with core formation, as depicted in Fig. 4. Once the granules surface is covered with precipitates, fresh bacterial cells tended to attach to the formed core to develop a large aggregate, which may be a survival strategy to avoid being washed out. -----------------------------------------------Fig. 4----------------------------------------------3.3.2 Granule diameter, settling properties and mechanical properties The granulation of anammox microorganisms resulted in increased granule diameters from 1.64 to 2.35 mm in R0 (Table 3). Due to stable protein to polysaccharide ratio (PN/PS) and VSS/SS ratios, no visible variations in settling
velocity or mechanical strength were observed during the 175-day operation. However, the floating ratio (volume ratio of floating sludge and total sludge) increased up to 20.6% on Day 175, as induced by a high shear force during long-term high-loaded operation with high hydraulic loading. The floating granules were large particles with hollow inside. These hollows were likely formed by bacterial decay deep inside the granules due to starvation or to the accumulation of dinitrogen gas under the limitations of mass transfer, which can be trapped inside the granules and contribute to the formation of gas pockets (Chen et al., 2014). Regarding R1, the long-term addition of phosphate caused a lower VSS/SS ratio that was triggered by the formation of an HAP core. Generally, a low VSS/SS ratio resulted in an inadequate biomass of bioreactors; however, the VSS concentration of R1 was greater than R0. In addition, the gradual decline in the PN/PS indicated a higher strength and better settleability. Indeed, these novel anammox granules possessed excellent settleability (settling velocity of approximately 285 m h-1), superior granule diameter (7.08 mm in average, Fig. S2b) and remarkable mechanical strength (4.4-fold higher than R0). Moreover, there were almost no floating granules. -----------------------------------------------Table 3----------------------------------------------3.4 Bacterial community dynamics The phylogenetic analysis revealed that the dominant anammox strain present in the two reactors on Day 195 belonged to the same species (99% related to Ca. Kuenenia stuttgartiensis), indicating no shift in the dominant anammox strain throughout the experimental period (Fig. 5). However, Q-PCR results showed that the
AnAOB population in R1 on Day 195 was significantly higher than that of R0. Indeed, the SEM images showed that the cell distribution in the granules of R1 was much denser than that of R0 (Fig. S3a). However, the relative abundance of AnAOB was declined due to the explosion of total bacteria. 3.5 Correlation between granule properties and reactor performance As the influent phosphates concentration of R1 increased stepwise from 2 mg L-1 to 500 mg L-1 during the 195-day operation, no significant differences were observed in the nitrogen removal performance of the two reactors in terms of NRR. Chronic response of anammox granules to phosphate seem to be incompatible with the acute response. The phosphates in the bulk reactor were close to the influent concentration; thus, this difference cannot be explained by the decreased phosphates due to the accumulation of HAP. The variations in SAA level and heme c content confirmed the adaptability of anammox granules with the aid of gradual acclimatization. Afterwards, the nitrogen loading rate (NLR) was gradually improved to 21 kgN m-3 d-1 in parallel with a shortening HRT. R1 showed higher potential of nitrogen removal than R0 (Table 4). Surprisingly, high level of phosphate appears to be completely compatible with anammox granules. Finally, novel anammox granules with HAP cores were generated that possessed excellent settleability, huge granule diameter and superior mechanical strength. A previous report suggested the accumulation of inorganic precipitates in aerobic granules, which enhanced granule strength but reduced bioactivity (Ren et al., 2008). In this study, the HAP core not only increased the mechanical properties of anammox granules without reducing bioactivity but also
provided a stable microbial carrier for anammox organisms to grow on, resulting in the accumulation of high concentrations of biomass in a reactor despite working at extremely high hydraulic up-flow velocity; this was the crucial factor contributing to the very high NRR. -----------------------------------------------Table 4----------------------------------------------3.6 Implications of this work With the recent advances in anammox-based processes in mainstream treatment, a net energy-producing sewage treatment plant providing effective nutrient removal is nearing feasibility (Gilbert et al., 2014; Lotti et al., 2014). The results of this study indicated that the presence of phosphate may be beneficial and facilitate the application of anammox in the pre-processing treatment of sewage. Additionally, in terms of the application of anammox-based processes in the mainstream, how to obtain sufficient biomass retention in the high flow rate is one of the three major challenges (Gao et al., 2014). These anammox granules that were immobilized by HAP cores with excellent settleability and superior biomass attachment may provide a useful reference. Future work will be necessary to determine the practical conditions to take advantage of these anammox granules during the treatment of real wastewater. 4. Conclusions Acute exposure to high-level phosphate (≥50 mM) induced the cytoplasm leakage. The presence of phosphate as high as 500 mgP L-1 showed no obvious adverse effects on the physiological characteristics of anammox granules during continuous-flow operation. Moreover, no shift in the dominant anammox strain was observed, as well
as higher population but lower relative abundance of AnAOB. Interestingly, novel anammox granules with a HAP core were cultivated, which possessed excellent settleability, huge granule diameter and superior mechanical strength. Acknowledgments The authors wish to thank the National Key Technologies R&D Program of China (No. 2012BAC13B02) and the Natural Science Foundation of China (Nos. 51278162 and 51578204) for their partial support of this study. References: [1] APHA, AWWA, AEF, 2005. Standard methods for the examination of water and wastewater, 21st ed. American Public Health Association: Washington, DC, USA. [2] Berry, E.A., Trumpower, B.L., 1987. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem., 161, 1-15. [3] Carvajal-Arroyo, J.M., Sun, W., Sierra-Alvarez, R., Field, J.A., 2013. Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents. Chemosphere, 91, 22-27. [4] Chen, H., Ma, C., Yang, G., Wang, H., Yu, Z., Jin, R., 2014. Floatation of flocculent and granular sludge in a high-loaded anammox reactor. Bioresource Technol., 169, 409-415. [5] Dapena-Mora, A., Fernández, I., Campos, J.L., Mosquera-Corral, A., Méndez, R., Jetten, M.S.M., 2007. Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme Microb. Tech., 40, 859-865. [6] de Graaff, M.S., Vieno, N.M., Kujawa-Roeleveld, K., Zeeman, G., Temmink, H., Buisman, C.J.N., 2011. Fate of hormones and pharmaceuticals during combined anaerobic treatment and nitrogen removal by partial nitritation-anammox in vacuum collected black water. Water Res., 45, 375-383.
[7] Dosta, J., Fernández, I., Vázquez-Padín, J.R., Mosquera-Corral, A., Campos, J.L., Mata-Álvarez, J., Méndez, R., 2008. Short- and long-term effects of temperature on the Anammox process. J. Hazard. Mater., 154, 688-693. [8] Egli, K., Fanger, U., Alvarez, P.J., Siegrist, H., van der Meer, J.R., Zehnder, A.J., 2001. Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch. Microbiol., 175, 198-207. [9] Gao, H., Scherson, Y.D., Wells, G.F., 2014. Towards energy neutral wastewater treatment: methodology and state of the art. Environmental Science: Processes & Impacts, 16, 1223-1246. [10] Gilbert, E.M., Agrawal, S., Karst, S.M., Horn, H., Nielsen, P.H., Lackner, S., 2014. Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating low strength wastewater. Environ. Sci. Technol., 48, 8784-8792. [11] Harhangi, H.R., Le Roy, M., van Alen, T., Hu, B., Groen, J., Kartal, B., Tringe, S.G., Quan, Z., Jetten, M.S.M., Op Den Camp, H.J.M., 2012. Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria. Appl. Environ. Microb., 78, 752 -758. [12] Huang, W., Cai, W., Huang, H., Lei, Z., Zhang, Z., Tay, J.H., Lee, D., 2015. Identification of inorganic and organic species of phosphorus and its bio-availability in nitrifying aerobic granular sludge. Water Res., 68, 423-431. [13] Jetten, M.S., Niftrik, L.V., Strous, M., Kartal, B., Keltjens, J.T., Op Den Camp, H.J., 2009. Biochemistry and molecular biology of anammox bacteria. Crit. Rev. Biochem. Mol., 44, 65-84. [14] Jiang, H., Tay, J., Liu, Y., Tay, S.T., 2003. Ca2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors. Biotechnol. Lett., 25, 95-99. [15] Jin, R., Yang, G., Yu, J., Zheng, P., 2012. The inhibition of the Anammox process: A review. Chem. Eng. J., 197, 67-79. [16] Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H., van Loosdrecht, M.C.M., 2014. Full-scale partial nitritation/anammox experiences-An application
survey. Water Res., 55, 292-303. [17] Lin, Y.M., Lotti, T., Sharma, P.K., van Loosdrecht, M.C.M., 2013. Apatite accumulation enhances the mechanical property of anammox granules. Water Res., 47, 4556-4566. [18] Liu, H., Fang, H.H.P., 2002. Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data. Biotechnol. Bioeng., 80, 806-811. [19] Lotti, T., Kleerebezem, R., Abelleira-Pereira, J.M., Abbas, B., van Loosdrecht, M.C.M., 2015. Faster through training: The anammox case. Water Res., 81, 261 -268. [20] Lotti, T., Kleerebezem, R., van Erp Taalman Kip, C., Hendrickx, T.L.G., Kruit, J., Hoekstra, M., van Loosdrecht, M.C.M., 2014. Anammox growth on pretreated municipal wastewater. Environ. Sci. Technol., 48, 7874-7880. [21] Mañas, A., Biscans, B., Spérandio, M., 2011. Biologically induced phosphorus precipitation in aerobic granular sludge process. Water Res., 45, 3776-3786. [22] Pynaert, K., Smets, B.F., Wyffels, S., Beheydt, D., Siciliano, S.D., Verstraete, W., 2003. Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl. Environ. Microb., 69, 3626-3635. [23] Ren, L., Ni, S., Liu, C., Liang, S., Zhang, B., Kong, Q., Guo, N., 2015. Effect of zero-valent iron on the start-up performance of anaerobic ammonium oxidation (anammox) process. Environ. Sci. Pollut. R., 22, 2925-2934. [24] Ren, T., Liu, L., Sheng, G., Liu, X., Yu, H., Zhang, M., Zhu, J., 2008. Calcium spatial distribution in aerobic granules and its effects on granule structure, strength and bioactivity. Water Res., 42, 3343-3352. [25] Sheng, G., Yu, H., Li, X., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnol. Adv., 28, 882-894. [26] Tang, C., Zheng, P., Wang, C., Mahmood, Q., Zhang, J., Chen, X., Zhang, L., Chen, J., 2011. Performance of high-loaded ANAMMOX UASB reactors
containing granular sludge. Water Res., 45, 135-144. [27] van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology, 142, 2187-2196. [28] Wan, C., Zhang, P., Lee, D., Yang, X., Liu, X., Sun, S., Pan, X., 2013. Disintegration of aerobic granules: Role of second messenger cyclic di-GMP. Bioresource Technol., 146, 330-335. [29] Wilfert, P., Kumar, P.S., Korving, L., Witkamp, G., van Loosdrecht, M.C.M., 2015. The relevance of phosphorus and iron chemistry to the recovery of phosphorus from wastewater: A review. Environ. Sci. Technol., 16, 9400-9414. [30] Wu, J., Zhou, H., Li, H., Zhang, P., Jiang, J., 2009. Impacts of hydrodynamic shear force on nucleation of flocculent sludge in anaerobic reactor. Water Res., 43, 3029-3036. [31] Yin, C., Meng, F., Chen, G., 2015. Spectroscopic characterization of extracellular polymeric substances from a mixed culture dominated by ammonia -oxidizing bacteria. Water Res., 68, 740-749. [32] Zhang, H., Fang, W., Wang, Y., Sheng, G., Zeng, R.J., Li, W., Yu, H., 2013. Phosphorus removal in an enhanced biological phosphorus removal process: roles of extracellular polymeric substances. Environ. Sci. Technol., 47, 11482 -11489. [33] Zhang, Z., Zhang, Q., Xu, J., Deng, R., Ji, Z., Wu, Y., Jin, R., 2016. Evaluation of the inhibitory effects of heavy metals on anammox activity: A batch test study. Bioresource Technol., 200, 208-216. [34] Zhang, Z., Cheng, Y., Zhou, Y., Buayi, X., Jin, R., 2015a. A novel strategy for accelerating the recovery of an anammox reactor inhibited by copper(II): EDTA washing combined with biostimulation via low-intensity ultrasound. Chem. Eng. J., 279, 912-920. [35] Zhang, Z., Buayi, X., Cheng, Y., Zhou, Y., Wang, H., Jin, R., 2015b. Anammox endogenous metabolism during long-term starvation: Impacts of intermittent and persistent modes and phosphates. Sep. Purif. Technol., 151, 309-317.
Figure Captions Fig. 1 Short-term effects of phosphate on anammox granules. (a) UV–vis absorption spectra; (b) Heme c concentrations; (c) LDH release; (d) Dissolved organic matter compositions (DOM) and contents of the liquid phase after 10-h exposure. PN: protein; PS: polysaccharide.
Fig. 2 Long-term effects of phosphate on the physiological characteristics of anammox granules. (a) Specific anammox activity (SAA); (b) Heme c content; (c) TTC-Dehydrogenase activity (TTC-DHA); (d) Extracellular polymeric substance (EPS) amounts and compositions of the anammox granules during different phases. PN: protein; PS: polysaccharide.
Fig. 3 Three-dimensional EEM fluorescence spectra of EPS. Anammox granules sampled from R0 (a) and R1 (b) on Day 175. Peak A: tryptophan-containing proteins; Peak B: aromatic proteins-like substances; Peak C: tyrosine-containing proteins.
Fig. 4 Mechanism for the formation of anammox granules with a microbially induced hydroxyapatite (HAP) core.
Fig. 5 Phylogenetic tree of anammox bacteria showing the positions of the clones (a: CK-Pla: R0 and P-Pla: R1) and (b) 16S rRNA gene copy numbers of total bacteria and hzsA obtained from the anammox granules in the two reactors after operation for 195 days.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1 Total phosphate concentrations in the influent and effluent of R1 during different phases. Time (d)
Influent (mgP L-1)
Effluent (mgP L-1)
1-15
2.07 ± 0.23
1.95 ± 0.31
16-30
5.14 ± 0.23
4.93 ± 0.19
30-45
10.8 ± 0.6
10.4 ± 0.6
46-60
20.5 ± 0.8
19.8 ± 0.7
61-75
52.9 ± 5.0
50.2 ± 4.6
76-90
79.7 ± 6.9
74.0 ± 7.2
91-105
124.0 ± 4.7
116.5 ± 6.0
106-120
162.5 ± 3.8
152.2 ± 4.4
121-135
208.5 ± 6.4
197.5 ± 7.3
136-150
301.7 ± 19.0
291.5 ± 22.0
151-165
406.7 ± 6.8
383.2 ± 21.2
166-195
498.9 ± 22.8
481. 9 ± 34.2
Table 2 EEM spectra parameters of the EPS extracted from the anammox granules sampled from two reactors on Day 175.
Peak A
Peak B
Peak C
Sample EX/EM (nm) Intensity EX/EM (nm) Intensity EX/EM (nm) Intensity R0
275/340
598.8
225/345
585.8
225/305
575.8
R1
275/350
503.5
225/350
515.2
225/305
440.2
Table 3 Physic-chemical properties of anammox granules during different phases. R eactor
R 0
R 1
Tim e (d)
VS (m h )
Floatin g ratiob (%)
10
56.3±21.7
0
45
50.7±14.2
5.3
75
55.4±19.2
10.4
105
56.9±14.2
15.6
135
58.6±12.1
17.6
175
58.5±23.0
20.6
10
55.4±22.1
0
45
58.8±25.6
5.1
75
67.9±24.3
9.7
105
77.0±36.5
7.6
135 175 a
a
-1
168.6±69. 1 285.5±68. 7
3.1 ≈0
MGDc (mm) 1.64±1.0 5 1.69±0.9 6 1.73±1.3 2 1.96±1.2 5 2.21±0.7 8 2.35±1.2 3 1.61±1.1 1 1.67±1.2 3 2.13±0.8 5 3.10±1.3 2 5.06±1.3 8 7.08±2.3 1
VS S (g L-1) 22.4 25.4 26.5 26.4 28.6 29.5 21.2 24.5 26.9 28.7 32.0 35.8
SS (g L-1) 29. 9 32. 2 36. 8 34. 3 32. 9 34. 3 28. 3 33. 1 39. 0 43. 4 76. 2 99. 4
VSS/S S
Mechanica l strength (K)
0.75
0.1414
0.76
-
0.72
0.1394
0.77
-
0.87
-
0.86
0.1349
0.75
0.1421
0.74
-
0.69
0.1002
0.66
-
0.42
-
0.36
0.0305
: Settling velocity (VS); b: Volume ratio of floating sludge and total sludge; c:Mean
granule diameter (MGD)
33
Table 4 Operation parameters of anammox reactors during different phases. N LR a
ime (d)
T (k gN m3
R0 Q b
(L d1 )
H R T (h )
Vu p
c
( m h1 )
d-1 ) 1 11. 1. 0. 20 -15 2 2 29 1 11. 1. 0. 20 6-60 2 2 29 6 11. 1. 0. 1-15 20 2 2 29 0 1 11. 1. 0. 51-1 20 2 2 29 95 1 14. 0. 0. 96-2 25 1 96 36 02 2 16. 0. 0. 03-2 30 9 80 44 11 2 21. 37 0. 0. 12-2 0 .5 64 54 20 a
c
NR Rd
NRE e
pHinf
pHeff
(%)
NR Rd
NRE e
pHinf
pHeff
(%)
10.2 ±0.2 10.1 ±0.2
90.6 ±0.6 89.8 ±1.5
7.83± 8.32± 0.14 0.13 7.77± 8.34± 0.08 0.14
10.1 ±0.1 10.0 ±0.2
90.1 ±0.6 89.1 ±1.5
7.80± 8.38± 0.11 0.05 7.76± 8.37± 0.09 0.20
9.9± 0.2
85.5 ±1.3
7.91± 8.48± 0.10 0.09
9.9± 0.3
88.4 ±2.3
7.80± 8.20± 0.03 0.05
10.0 ±0.3
88.7 ±2.5
7.95± 8.42± 0.09 0.08
10.3 ±0.3
91.5 ±2.2
7.89± 7.95± 0.05 0.07
12.1 ±0.5
85.6 ±3.5
7.90± 8.53± 0.13 0.05
12.7 ±0.2
90.9 ±1.2
7.80± 7.81± 0.03 0.12
12.4 ±0.8
73.1 ±4.6
7.89± 8.66± 0.15 0.72
14.5 ±0.2
86.7 ±1.4
7.82± 7.82± 0.05 0.10
11.1 ±0.7
52.3 ±3.4
7.88± 8.76± 0.11 0.11
17.3 ±0.6
82.2 ±2.8
7.80± 7.81± 0.03 0.09
: Nitrogen loading rate (NLR)
b
R1
: Quantity of flow (Q)
: Hydraulic up-flow velocity (Vup)
d: Nitrogen removal rate (NRR) e: Nitrogen removal efficiency (NRE)
34
Highlights Acute exposure to high-level phosphate (≥ 50 mM) induced the cytoplasm leakage. Phosphate up to 500 mgP L-1 showed no obvious adverse effects on physiological characteristics. HAP core improved the physic-chemical properties of anammox granules.
35
Graphical abstract
36
S0960-8524(16)30234-6 http://dx.doi.org/10.1016/j.biortech.2016.02.097 BITE 16147
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 December 2015 19 February 2016 22 February 2016
Please cite this article as: Zhang, Z-Z., Xu, J-J., Hu, H-Y., Shi, Z-J., Ji, Z-Q., Deng, R., Shi, M-L., Jin, R-C., Insight into the short- and long-term effects of inorganic phosphate on anammox granule property, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.02.097
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Insight into the short- and long-term effects of inorganic phosphate on anammox granule property Zheng-Zhe Zhanga,b, Jia-Jia Xua,b, Hai-Yan Hua,b, Zhi-Jian Shia,b, Zheng-Quan Jia,b, Rui Deng a,b, Man-Ling Shia,b, Ren-Cun Jina,b* a
College of Life and Environmental Sciences, Hangzhou Normal University,
Hangzhou 310036, China b
Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration,
Hangzhou Normal University, Hangzhou 310036, China *
Corresponding author: Ren-Cun Jin
Tel.: +86-571-88062061; Fax: +86-571-28865333 E-mail address: [email protected]
Abstract The short- and long-term effects of inorganic phosphate on property of anaerobic ammonium oxidation (anammox) granule were investigated in this study. Acute exposure to high-level phosphate (≥ 50 mM) induced the cytoplasm leakage. During a 195-day continuous-flow operation, the gradually increasing phosphate (up to 500 mgP L-1) slightly affected the specific anammox activity, hardly impacted the heme c content, remarkably decreased the extracellular polymeric substances production and significantly stimulated the dehydrogenase activity of anammox granules. Microbial community analysis showed no shift in the dominant anammox strain and higher population but lower relative abundance of anaerobic ammonium-oxidizing bacteria
compared to the control granules. Interestingly, novel anammox granules with a hydroxyapatite core were cultivated, which possessed excellent settleability, huge granule diameter and superior mechanical strength. This study supported the application of granule-based anammox process as a pre-processing treatment in phosphate-containing and ammonia-rich wastewaters.
Keywords: anammox; phosphate; sludge characteristics; hydroxyapatite
1. Introduction Due to remarkable advantages, such as high nitrogen removal rates, low operational costs and small footprints, anaerobic ammonium oxidation (anammox) -based processes have been successfully implemented for the treatment of high-strength ammonium wastewater with low C:N ratios under mesophilic conditions, e.g., centrates from anaerobic digestion sludge (sidestream) and effluents from the production of nitrogenous fertilizers (Jin et al., 2012; Lotti et al., 2015). With the worldwide expansion to 100 full-scale installations by early 2015, the anammox process is entering a new stage of widespread use and development. The majority (approximately 75%) of installations are applied toward the sidestream treatment of municipal wastewater (Lackner et al., 2014). In particular, with recent advances in mainstream treatment, a net energy-producing sewage treatment plant providing effective nutrient removal is nearing feasibility (Gilbert et al., 2014; Lotti et al., 2014).
Phosphorus is an essential nutrient and is a major contributor to agricultural and industrial development. In 2000, 19.7 Mt of P was mined as phosphate rock. The majority of this, 15.3 Mt P, was used to produce fertilizers (Wilfert et al., 2015); thus, phosphates are abundant in wastewater from fertilizer production. Moreover, phosphates are commonly found in municipal wastewaters (mainstream and sidestream) due to their use in detergents or introduction from black water (de Graaff et al., 2011). Regarding P recovery, enhanced biological phosphorus removal (EBPR) is not yet widely applied, and the recovery potential is limited. Other phosphorus recovery methods, including sludge application to agricultural land or recovering phosphorus from sludge ash, also have limitations. Energy-producing wastewater treatment plants increasingly rely on phosphorus removal using iron (Wilfert et al., 2015). Generally, in flow line this procedure was after nitrogen removal process. Therefore, considerable amounts of phosphates might be introduced to anammox-based reactors. Due to the slow growth rate, low cell yield and high susceptibility of anaerobic ammonium-oxidizing bacteria (AnAOB) to variations in environmental conditions, the industrialization of anammox-based processes is restricted by inhibitory substances, such as phosphate in nitrogen-rich wastewater (Jin et al., 2012). However, disagreements have been reported in the literature in terms of the effects of phosphate on anammox biomass. van de Graaf et al. (1996) firstly observed a loss of activity for anammox culture (dominated by Ca. Brocadia anammoxidans) at phosphate concentrations above 5 mM. Thereafter, Egli et al. (2001) found no inhibitory effect
of phosphate at 20 mM for Ca. Kuenenia stuttgartiensis from a rotating biological contactor, while in the same system with the same genus, Pynart et al. (2003) observed that anammox activity decreased to 63% of the normal activity at 1.8 mM and further to 20% at 3.6 mM, but no further decrease (80% inhibition) at 9.2 mM in batch tests. Dapena-Mora et al. (2007) reported a higher tolerance of anammox biofilms (dominated by Ca. Kuenenia stuttgartiensis) from a gas-lift reactor to phosphate that IC50 was 20 mM. Recently, Carvajal-Arroyo et al. (2013) found that the exposure of suspended anammox enrichments (dominated by Ca. Brocadia) to phosphates caused a modest decrease in the specific anammox activity (SAA) with increasing phosphates concentrations (IC50 = 25.3 ± 5.9 mM), whereas phosphates stimulated the SAA of granular anammox enrichment (dominated by Ca. Brocadia) by 60% at concentrations ranging from 10-50 mM. However, these data were based on the results of batch experiments and lacked long-term evaluation in continuous-flow experiments. Lin et al. (2013) noted that apatite accumulation enhances the mechanical properties of anammox granules during the long-term addition of phosphate in continuous-flow experiments. Nevertheless, the influent phosphates concentration was maintained at 29.7 ± 9.7 mgP L-1. Given the prospective application of anammox-based processes in phosphate-containing and ammonium-rich wastewaters, clarifying the impacts of phosphates in various levels on anammox biomass is urgently required. To date, there have been no comprehensive investigations regarding the impacts of phosphate on anammox granule property. Therefore, the objectives of this study were to investigate (i) the short-term effects
of phosphate on anammox granules; (ii) the evolution of physiological characteristics and physic-chemical properties under different phosphate loads; and (iii) the compositions and formation mechanisms of the inorganic cores in anammox granules. 2. Materials and Methods 2.1 Synthetic wastewater and inoculums Inorganic synthetic wastewater containing substrates, mineral and trace elements was introduced into the reactors. Equimolar amounts of ammonium and nitrite in the forms of (NH4)2SO4 and NaNO2, respectively, were supplied as needed. The mineral medium composition was 10 mg L-1 NaH2PO4, 73.5 mg L-1 CaCl2•2H2O, 58.6 mg L-1 MgSO4•7H2O, and 840 mg L-1 NaHCO3. 1.25 ml of trace element solutions I and II were added per liter of wastewater. The composition of trace element solution I was 5 g L-1 EDTA and 9.14 g L-1 FeSO4•7H2O, and trace element solution II was composed of 15 g L-1 EDTA, 0.014 g L-1 H3BO4, 0.99 g L-1 MnCl2•4H2O, 0.25 g L-1 CuSO4•5H2O, 0.43 g L-1 ZnSO4•7H2O, 0.21 g L-1 NiCl2•6H2O, 0.22 g L-1 NaMoO4•2H2O and 0.24 g L-1 CoCl2•6H2O. Anammox seed granules were harvested from a high-loaded laboratory-scale up-flow anaerobic sludge blanket (UASB) reactor, which was fed with synthetic medium (as above) at a stable nitrogen removal rate (NRR) of approximately 10 kg N m-3 d -1 for over one year. 2.2 Batch experiments Batch exposure assays were performed in serum flasks with a total volume of 160 mL and a liquid phase volume of 120 mL. Equimolar amounts of ammonium and nitrite were added to the mineral medium. A total of 100 mL of basal mineral medium
and 1.25 mL of trace element solutions I and II per liter of sterilized water were introduced, resulting in a composition similar to that of the synthetic wastewater. Then, phosphate was added to the serum flasks as needed along with 10 mL of anammox biomass. The VSS concentration was approximately 2.5 g L-1 in each serum flask. The initial pH was fixed at approximately 7.5 by injections of 1 M hydrochloric acid or sodium hydroxide. The serum vials were subsequently flushed with 99.99% pure argon for 10 min and immediately sealed with butyl rubber to avoid oxygen leakage. Then, the serum flasks were placed in a 35 ± 1 °C thermostatic shaker at 180 rpm. After 10 h exposure, the mixtures were centrifuged for 5 min at 3,000 g. The supernatants were removed for the determination of lactate dehydrogenase (LDH) activity, dissolved organic matter (DOM) and UV-vis spectrum scanning (SHIMADZU UV-2550, Japan), and the resulting biomass was used for the heme c extraction. 2.3 Anammox reactors and operational strategy Two UASB reactors (R0 and R1) with effective volumes of 1.0 L each were fabricated from Plexiglas, covered with black cloth to prevent light-related inhibition and then placed in a thermostatic room at 35 ± 1 °C. After inoculation, the initial volatile suspended solid (VSS) concentration in these reactors was approximately 21 g L-1. The influent pH was self-stabilizing at pH 7.9 ± 0.1 without the addition of an acid or base. The continuous-flow experiments featured a constant influent substrate level of 280 mg L-1. The initial hydraulic retention time (HRT) was maintained at 1.2 h and the sludge retention time was controlled by the spontaneous sludge loss via the
effluent. NaH2PO4 and Na2HPO4 were added to the influent of R1 at molar ratios of 0.19, and the total phosphate concentrations are detailed in Table 1. The influent phosphate concentration was set at 500 mg L-1 from Day 165 until the end. The trace amount of phosphate (2 mgP L-1) was added into the control reactor (R0-CK) for the normal growth of the microorganisms. -----------------------------------------------Table 1 ----------------------------------------------2.4 EPS extraction and 3D-EEM fluorescence spectroscopy A heat-extraction method was employed for EPS extraction (Yin et al., 2015). Carbohydrate measurements were acquired using the anthrone method with a glucose standard, and the protein levels were measured using the modified Lowry method and bovine serum albumin as a standard (Wu et al., 2009). The EEM fluorescence spectra were measured using a fluorescence spectrophotometer (F-4600, Hitachi Co., Japan). The EEM spectra were collected with subsequent scanning emission spectra from 200 to 600 nm at 5-nm increments. The excitation and emission slits were maintained at 10 nm, and the scanning speed was set at 12,000 nm/min. The voltage of the photomultiplier tube (PMT) was set to 500 V for low-level light detection. The spectrum of double-distilled water was recorded as the blank. Origin 8.0 software was used to process the EEM data. Contour lines are shown for each EEM spectra to represent the fluorescence intensity. 2.5 Dehydrogenase activity assay The procedures were as follows: (1) 10 mL of the sludge sample was washed with 10 mL of an isotonic salt solution followed by centrifugation at 3000 g for 5 min; (2)
5 g field-moist weight sludge was homogenized with 15 mL of an autoclave -sterilized 0.85% saline solution; (3) 2 mL 2,3,5-triphenyltetrazoilum chloride (TTC) (5 g L-1), 1.5 mL Tris-HCl buffer solution (0.1 M, pH = 7.4) and 0.5 ml Na2SO3 (0.36%) were added to 2 mL of sludge liquid; (4) the mixed solution was incubated at 37 ± 1 °C for 4 h in the dark; (5) two drops of concentrated sulfuric acid were added to terminate the deoxidization reaction in the test tubes, and 5 mL toluene was added to extract the color product of the TF from the cells; (6) the reaction mixture was centrifuged at 6000 g for 5 min, and the absorbance of the supernatant was measured at 486 nm with a UV spectrometer; and (7) the same volume of sludge liquid was collected for biomass determination. Each value of TTC-DHA is expressed in units of µgTF g-1VSS h-1 and presented as the average of three replicates. 2.6 Heme c extraction and determination Five-milliliter field-moist anammox granules were centrifuged at 3000 g at 4 °C for 5 min followed by two washes with a sodium phosphate buffer solution (10 mM, pH 7.5). The washed pellets were then resuspended in 25 mL of the same buffer and then disrupted by sonication (800 W, at 4 °C for 20 min, Ultrasonic processor CPX 750, USA). The cell masses were separated by centrifugation (15,000 g) at 4 °C for 15 min. The supernatant was used for heme c content determination according to Berry and Trumpower (Berry and Trumpower, 1987). Briefly, the nitrogen ligands from the protein-bound heme were replaced by pyridine in alkali, and the resultant heme c was quantified as the difference between the spectra of the reduced (sodium dithionite crystals) and oxidized (potassium ferricyanide) compounds. The heme c concentration
was calculated based on a millimolar extinction coefficient of 23.97 mM cm-1 for the difference in absorption between the peak at 550 nm and the trough at 535 nm. The heme c content values are expressed as µmol g-1 VSS. 2.7 X-ray powder diffraction (XRD) Anammox granules for XRD analysis were previously dried and calcined in an oven at 500 °C for 2 h to remove the organic fraction. XRD analysis was performed using a Multiflex diffractometer (Bruker D8, Germany) with monochromatic Cu Kα1 radiation in the 2θ region between 5° and 90°. X-ray profile fitting was carried out using the Jade 6.5 program. 2.8 Strength of granules The microbial granule strength is described as an abrasion as a result of exposure to different shear rates, which can be evaluated by an ultrasonic method (Wan et al., 2013). In this process, 10 mL of randomly selected anammox granules were placed into a 50-mL conical flask that contained with 40 mL of deionized water, and then the tube was placed in an ultrasonic bath at 28 kHz, 70 W. The ultrasonic was intermittently applied at 2.5 s (on) - 3 s (off) cycles. The suspension turbidity data under ultrasound were measured spectrophotometrically at 600 nm. In this abrasion experiment at a constant shear rate, the production of fines (XF) per unit of volume and time was a first-order process in the concentration of larger granules (XNF). ୢూ ௧
= Kܺ
(1)
where K is a constant for the strength of the granules under the conditions applied. 2.9 Analytical procedures
The ammonium, nitrite, nitrate, total phosphate, VSS and suspended solid concentrations, as well as the pH and settling velocity (VS), were determined using standard methods (APHA et al., 2005). Anammox granules were taken regularly from the lab reactors to measure the SAA in ex situ batch experiments (Zhang et al., 2016). The LDH activity was detected using the UV spectrometer according to the protocols of the LDH assay kit (Jiancheng, China).The mean granule diameter (MGD) was measured using an image analysis system (QCOLite) with a Leica DM2LB microscope and a digital camera (Canon S30). Scanning electron microscopy (SEM) was used to observe the granule morphology using the methods reported by Tang et al. 2011. The Visual MINTEQ (VMINTEQ.v3.1) model was used to calculate the chemical equilibrium in the reactor. The total genomic DNA of each sample was extracted using the Power Soil DNA Kit (Mo Bio Laboratories, Carlsbad, CA). PCR amplification of the 16S rRNA gene was carried out using a primer set: Pla46F (5’-GAG TTT GAT CMT GGC TCA G-3’) and 1545R (5’-ACG GYT ACC TTG TTA CGA CTT-3’). For the detection of total bacteria and anammox bacteria, the 16S rRNA gene and hydrazine synthase structural genes (hzsA) were quantified with primer sets 338F -518R and hzsA_1597F-hzsA_1857R, respectively (Harhangi et al., 2012). The copy numbers were determined in triplicate using a CFX96 Touch™ thermocycler and a real-time detection system (Bio-Rad, CA, USA). Details are presented in the Supplementary Materials. An analysis of variance (ANOVA) was used to test the significance of the results, and p < 0.05 was considered statistically significant.
3 Results and discussion 3.1 Short-term effects of phosphate on anammox granules Previous batch experiments showed that phosphate at low levels (≤ 5 mM), acting as a buffer medium, slightly stimulated the specific anammox activity of anammox granules. Conversely, 10 mM-phosphate inhibited the SAA by 10%, and the inhibitory effects were exacerbated as the phosphate concentrations increased further. The IC50 value was calculated as 49.6 ± 6.4 mM by model simulation. Moreover, based on the kinetic experiments with various substrate levels, the inhibitory effect of phosphate at 20 mM on anammox granules was considered as uncompetitive inhibition pattern that decreased the maximum substrate uptake rate but enhanced the affinity of biomass to substrates. Specifically, phosphate does not directly bond with the enzymes of anammox metabolism but bond with complexes of enzymes and substrates, thereby forming triple-complexes. These triple-complexes could not be converted to products, resulting in decreased enzyme activity. Accordingly, the inhibitory effect of phosphate on anammox granules can be defined as a reversible inhibition process. However, when the anammox granules were exposed to high level of phosphate (≥ 50 mM) for 10 hours, the liquid phase acquired an orange coloration, which could indicate the cell lysis. In order to confirm this fact, UV–vis absorption spectra of the liquid phase from the batch tests were analyzed (Fig. 1a). A maximum peak between 400 and 410 nm was observed, similar to the previous study in which anammox biomass was shocked by high temperature of 45 °C (Dosta et al., 2008). Therefore, the orange color of the liquid phase could be attributed to the segregation of cytochrome c (Fig. S1). Indeed,
abnormally higher heme c content was detected in the liquid phase (Fig. 1b). Heme is an indispensable part of key anammox enzymes, including hydrazine synthase (HZS) and hydrazine dehydrogenase (HDH) (Jetten et al., 2009). Furthermore, the significantly higher LDH release (Fig. 1c) indicated that high levels of phosphate increased the permeability or induced the rupture of the cell membrane, thereby releasing intracellular polymeric substances to extracellular locations. This can be used for explaining the accumulation of protein and polysaccharide in the liquid phase (Fig. 1d). In terms of these results, taken as a whole, we updated the understanding of the phosphate inhibition mode: phosphate at range of 10 mM to 20 mM (may be higher but in this study 20 mM was determined) caused uncompetitive inhibition pattern, while phosphate higher than 50 mM induced an irreversible loss of the activity. However, cytoplasm leakage usually occurs when cells are exposed to hypotonic media, thus how to explain the mechanism of high level phosphate interacting with anammox cells needs further research. The results will trigger a radical reevaluation of the application of phosphate buffer in the bio-system containing anammox cells. -----------------------------------------------Fig.1 ----------------------------------------------3.2 Evolution of physiological characteristics 3.2.1 Heme content and specific anammox activity To further assess the long-term effects of phosphates, two continuous-flow UASB reactors were operated as described above. The specific anammox activity slightly increased by 8.3% at 10.8 mgP L-1 (Fig. 2a), consistent with the acute exposure results.
When influent phosphate concentration increased to 52.9 mgP L-1, the specific anammox activity was decreased by 19.3 %, incompatible with the acute exposure results that low level (≤ 5 mM) of phosphate stimulated specific anammox activity. It seemed that the anammox granules were susceptible to phosphate as high as 52.9 mgP L-1. However, as the influent phosphate concentration of R1 increased stepwise to 500 mg L-1 afterwards, the anammox granules recovered and showed equal activity with that of R0, indicating the gradually increased tolerance of anammox granules during long-term acclimatization. Furthermore, heme plays an important role in AnAOB energy metabolism and cell synthesis and producing the characteristic carmine color of anammox sludge (Tang et al., 2011). In this study, no significant differences in heme c contents were observed between two anammox granules over the course of a 195-day operation (Fig. 2b, independent-samples t-test, p > 0.05). -----------------------------------------------Fig.2 ----------------------------------------------3.2.2 Dehydrogenase activity The vast majority of redox reactions in the organisms were catalyzed by dehydrogenase and oxidase; dehydrogenase, an intracellular enzyme, is intimately involved in the oxidative phosphorylation process inside the cell and is an important indicator of microbial activity (Zhang et al., 2015a). As shown in Fig.2c, the presence of phosphates at 52.9 mgP L-1 remarkably stimulated TTC-DHA levels. Further, the TTC-DHA of anammox granules in R1 was 3.8-fold higher than that in R0 on Day 175. Interestingly, the electron transport rate as indicated by TTC-DHA was stimulated rather than inhibited, which appears to conflict with the observation of acute exposure.
This may be attributed to the appropriate pH environment: the long-term presence of phosphates acts as a buffer providing favorable culture conditions for microbial metabolism. 3.2.3 EPS amounts and composition EPS, a complex high-molecular-weight mixture of polymers excreted by microorganisms, is a major component in microbial aggregates and maintains these aggregates together in a three-dimensional matrix (Sheng et al., 2010). The total EPS production of R0 increased from 162.5 to 192.2 mg g-1VSS in the initial phase and then maintained stable. The EPS production of R1 initially increased up to 256.4 mg g-1 VSS with the application of phosphate at 52.9 mgP L-1, and reversed as the phosphate concentrations increased (Fig.2c). Finally, the EPS production of R1 was decreased to and maintained at approximately 85% of that of R0 over a long period of time (Days 105-175). Three peaks were easily identified in the 3D-EEM fluorescence spectra (Fig. 3). The first main peak was located at the excitation/emission wavelengths (Ex/Em) of 275/340-350 nm (Peak A); the second was observed at the Ex/Em of 225/340-350 nm (Peak B); and the third was identified at the Ex/Em of 225/305 nm (Peak C). These three peaks have been reported as protein-like peaks, in which the fluorescence is associated with soluble microbial by-product-like materials or tryptophan-containing proteins(Peak A), aromatic proteins-like substances (Peak B) and tyrosine-containing proteins (Peak C) (Zhang et al., 2015b). These protein-like components excreted by microorganisms, were indicative of different intracellular metabolic regulation
mechanisms. Obviously, three peak intensities (on Day 195) were reduced due to the decreased content of EPS, indicating that the presence of phosphate at high level impacted the anammox metabolism or modified the co-metabolism microflora in granules. The locations of Peak A and Peak B were red-shifted to longer wavelengths (Table 2), which may be attributed to the presence of carbonyl-containing substituents, carboxyl constituents, hydroxyl, alkoxyl, and amino groups (Ren et al., 2015). Therefore, the presence of phosphate at high level resulted in the structural differences in these protein-like compounds. -----------------------------------------------Fig. 3 --------------------------------------------------------------------------------------------Table 2----------------------------------------------3.3 Variations of physic-chemical properties 3.3.1 Microbially induced HAP precipitation in anammox granules XRD, an efficient tool for distinguishing crystalline minerals from those of amorphous structures, was used to identify the species of P minerals in the granule samples. As illustrated in Fig. S2, a number of distinct peaks in the diffractogram reflect the presence of crystalline forms. By comparison with a portable document format standard card (2004) using Jade 6.5, most of the stronger peaks coincide well with those of the hydroxyapatite (HAP, Ca5(PO4)3(OH)) pattern (FOM = 3.1). This result agrees with a previous report that HAP is a major phosphate mineral in aerobic granules (Mañas et al., 2011). Although several different P minerals have been previously reported in granules, such as whitlockite (Ca3(PO4)2, Ca18Mg2H2(PO4)14) and struvite (NH4Mg PO4·6H2O) (Huang et al., 2015; Lin et al., 2013), they were not
detected in the anammox granules in this study. Moreover, the low saturation index of whitlockite implied that whitlockite was poorly or very temporarily formed. Among the calcium phosphate family, HAP is considered the most stable and insoluble (Huang et al., 2015). Still, whether other intermediates are present or not remains unknown due to the limitation of XRD for micro-amounts of minerals. The influent pH and composition play important roles in phosphate precipitation due to their influence on the saturation index of different minerals. The Visual MINTEQ (VMINTEQ.v3.1) model was used to estimate the possible minerals precipitated in the reactor. The struvite saturation index was close to zero throughout all the phase due to ammonia consumption by anammox, indicating that the ammonium and magnesium concentrations were too low to cause struvite precipitation under these conditions. The production of vivianite was negligible because of the trace Fe element in the influent. Concerning calcium phosphates, hydroxyapatite was the most stable phase among the calcium phosphates that showed oversaturation conditions throughout the experiment (Table S1). Although the turbidity slightly increased with increased phosphates concentrations, no visible-shaped precipitation was observed in the influent or effluent. The evidence indicates that HAP only precipitated within the anammox biomass matrix and not in other places in the reactor, meaning that confined conditions were more favorable for HAP formation or accumulation than the conditions in the bulk. Two explanations can be proposed: (i) higher local pH and (ii) a special combination pattern. First, the internal pH can be higher than the bulk pH because of the acidity reduction in the
anammox reaction. The higher pH increased HAP supersaturation, thereby resulting in formation or accumulation in the inner region of anammox granules. Moreover, the dense structure of the granules encouraged the accumulation of HAP in the granule core and repressed the solubilization of the crystals (Huang et al., 2015). Second, recent studies have also revealed the considerable accumulation of P in the EPS of sludge in the enhanced biological phosphorus removal process (Zhang et al., 2013), implying a non-negligible role for EPS. AnAOB cells are enclosed by a matrix of EPS, which contains abundant functional groups such as carboxyl, phosphoric, amine, and hydroxyl groups that are negatively charged at neutral pH and are able to form organometallic complexes with multivalent cations via electrostatic attraction or other interactions (Liu and Fang, 2002). Ca2+ is the main multivalent cation binding to extracellular PS molecules (Jiang et al., 2003), thereby acting as a bridge interconnecting these components. Experimental observations suggest a scenario involving correlation with core formation, as depicted in Fig. 4. Once the granules surface is covered with precipitates, fresh bacterial cells tended to attach to the formed core to develop a large aggregate, which may be a survival strategy to avoid being washed out. -----------------------------------------------Fig. 4----------------------------------------------3.3.2 Granule diameter, settling properties and mechanical properties The granulation of anammox microorganisms resulted in increased granule diameters from 1.64 to 2.35 mm in R0 (Table 3). Due to stable protein to polysaccharide ratio (PN/PS) and VSS/SS ratios, no visible variations in settling
velocity or mechanical strength were observed during the 175-day operation. However, the floating ratio (volume ratio of floating sludge and total sludge) increased up to 20.6% on Day 175, as induced by a high shear force during long-term high-loaded operation with high hydraulic loading. The floating granules were large particles with hollow inside. These hollows were likely formed by bacterial decay deep inside the granules due to starvation or to the accumulation of dinitrogen gas under the limitations of mass transfer, which can be trapped inside the granules and contribute to the formation of gas pockets (Chen et al., 2014). Regarding R1, the long-term addition of phosphate caused a lower VSS/SS ratio that was triggered by the formation of an HAP core. Generally, a low VSS/SS ratio resulted in an inadequate biomass of bioreactors; however, the VSS concentration of R1 was greater than R0. In addition, the gradual decline in the PN/PS indicated a higher strength and better settleability. Indeed, these novel anammox granules possessed excellent settleability (settling velocity of approximately 285 m h-1), superior granule diameter (7.08 mm in average, Fig. S2b) and remarkable mechanical strength (4.4-fold higher than R0). Moreover, there were almost no floating granules. -----------------------------------------------Table 3----------------------------------------------3.4 Bacterial community dynamics The phylogenetic analysis revealed that the dominant anammox strain present in the two reactors on Day 195 belonged to the same species (99% related to Ca. Kuenenia stuttgartiensis), indicating no shift in the dominant anammox strain throughout the experimental period (Fig. 5). However, Q-PCR results showed that the
AnAOB population in R1 on Day 195 was significantly higher than that of R0. Indeed, the SEM images showed that the cell distribution in the granules of R1 was much denser than that of R0 (Fig. S3a). However, the relative abundance of AnAOB was declined due to the explosion of total bacteria. 3.5 Correlation between granule properties and reactor performance As the influent phosphates concentration of R1 increased stepwise from 2 mg L-1 to 500 mg L-1 during the 195-day operation, no significant differences were observed in the nitrogen removal performance of the two reactors in terms of NRR. Chronic response of anammox granules to phosphate seem to be incompatible with the acute response. The phosphates in the bulk reactor were close to the influent concentration; thus, this difference cannot be explained by the decreased phosphates due to the accumulation of HAP. The variations in SAA level and heme c content confirmed the adaptability of anammox granules with the aid of gradual acclimatization. Afterwards, the nitrogen loading rate (NLR) was gradually improved to 21 kgN m-3 d-1 in parallel with a shortening HRT. R1 showed higher potential of nitrogen removal than R0 (Table 4). Surprisingly, high level of phosphate appears to be completely compatible with anammox granules. Finally, novel anammox granules with HAP cores were generated that possessed excellent settleability, huge granule diameter and superior mechanical strength. A previous report suggested the accumulation of inorganic precipitates in aerobic granules, which enhanced granule strength but reduced bioactivity (Ren et al., 2008). In this study, the HAP core not only increased the mechanical properties of anammox granules without reducing bioactivity but also
provided a stable microbial carrier for anammox organisms to grow on, resulting in the accumulation of high concentrations of biomass in a reactor despite working at extremely high hydraulic up-flow velocity; this was the crucial factor contributing to the very high NRR. -----------------------------------------------Table 4----------------------------------------------3.6 Implications of this work With the recent advances in anammox-based processes in mainstream treatment, a net energy-producing sewage treatment plant providing effective nutrient removal is nearing feasibility (Gilbert et al., 2014; Lotti et al., 2014). The results of this study indicated that the presence of phosphate may be beneficial and facilitate the application of anammox in the pre-processing treatment of sewage. Additionally, in terms of the application of anammox-based processes in the mainstream, how to obtain sufficient biomass retention in the high flow rate is one of the three major challenges (Gao et al., 2014). These anammox granules that were immobilized by HAP cores with excellent settleability and superior biomass attachment may provide a useful reference. Future work will be necessary to determine the practical conditions to take advantage of these anammox granules during the treatment of real wastewater. 4. Conclusions Acute exposure to high-level phosphate (≥50 mM) induced the cytoplasm leakage. The presence of phosphate as high as 500 mgP L-1 showed no obvious adverse effects on the physiological characteristics of anammox granules during continuous-flow operation. Moreover, no shift in the dominant anammox strain was observed, as well
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Figure Captions Fig. 1 Short-term effects of phosphate on anammox granules. (a) UV–vis absorption spectra; (b) Heme c concentrations; (c) LDH release; (d) Dissolved organic matter compositions (DOM) and contents of the liquid phase after 10-h exposure. PN: protein; PS: polysaccharide.
Fig. 2 Long-term effects of phosphate on the physiological characteristics of anammox granules. (a) Specific anammox activity (SAA); (b) Heme c content; (c) TTC-Dehydrogenase activity (TTC-DHA); (d) Extracellular polymeric substance (EPS) amounts and compositions of the anammox granules during different phases. PN: protein; PS: polysaccharide.
Fig. 3 Three-dimensional EEM fluorescence spectra of EPS. Anammox granules sampled from R0 (a) and R1 (b) on Day 175. Peak A: tryptophan-containing proteins; Peak B: aromatic proteins-like substances; Peak C: tyrosine-containing proteins.
Fig. 4 Mechanism for the formation of anammox granules with a microbially induced hydroxyapatite (HAP) core.
Fig. 5 Phylogenetic tree of anammox bacteria showing the positions of the clones (a: CK-Pla: R0 and P-Pla: R1) and (b) 16S rRNA gene copy numbers of total bacteria and hzsA obtained from the anammox granules in the two reactors after operation for 195 days.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Table 1 Total phosphate concentrations in the influent and effluent of R1 during different phases. Time (d)
Influent (mgP L-1)
Effluent (mgP L-1)
1-15
2.07 ± 0.23
1.95 ± 0.31
16-30
5.14 ± 0.23
4.93 ± 0.19
30-45
10.8 ± 0.6
10.4 ± 0.6
46-60
20.5 ± 0.8
19.8 ± 0.7
61-75
52.9 ± 5.0
50.2 ± 4.6
76-90
79.7 ± 6.9
74.0 ± 7.2
91-105
124.0 ± 4.7
116.5 ± 6.0
106-120
162.5 ± 3.8
152.2 ± 4.4
121-135
208.5 ± 6.4
197.5 ± 7.3
136-150
301.7 ± 19.0
291.5 ± 22.0
151-165
406.7 ± 6.8
383.2 ± 21.2
166-195
498.9 ± 22.8
481. 9 ± 34.2
Table 2 EEM spectra parameters of the EPS extracted from the anammox granules sampled from two reactors on Day 175.
Peak A
Peak B
Peak C
Sample EX/EM (nm) Intensity EX/EM (nm) Intensity EX/EM (nm) Intensity R0
275/340
598.8
225/345
585.8
225/305
575.8
R1
275/350
503.5
225/350
515.2
225/305
440.2
Table 3 Physic-chemical properties of anammox granules during different phases. R eactor
R 0
R 1
Tim e (d)
VS (m h )
Floatin g ratiob (%)
10
56.3±21.7
0
45
50.7±14.2
5.3
75
55.4±19.2
10.4
105
56.9±14.2
15.6
135
58.6±12.1
17.6
175
58.5±23.0
20.6
10
55.4±22.1
0
45
58.8±25.6
5.1
75
67.9±24.3
9.7
105
77.0±36.5
7.6
135 175 a
a
-1
168.6±69. 1 285.5±68. 7
3.1 ≈0
MGDc (mm) 1.64±1.0 5 1.69±0.9 6 1.73±1.3 2 1.96±1.2 5 2.21±0.7 8 2.35±1.2 3 1.61±1.1 1 1.67±1.2 3 2.13±0.8 5 3.10±1.3 2 5.06±1.3 8 7.08±2.3 1
VS S (g L-1) 22.4 25.4 26.5 26.4 28.6 29.5 21.2 24.5 26.9 28.7 32.0 35.8
SS (g L-1) 29. 9 32. 2 36. 8 34. 3 32. 9 34. 3 28. 3 33. 1 39. 0 43. 4 76. 2 99. 4
VSS/S S
Mechanica l strength (K)
0.75
0.1414
0.76
-
0.72
0.1394
0.77
-
0.87
-
0.86
0.1349
0.75
0.1421
0.74
-
0.69
0.1002
0.66
-
0.42
-
0.36
0.0305
: Settling velocity (VS); b: Volume ratio of floating sludge and total sludge; c:Mean
granule diameter (MGD)
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Table 4 Operation parameters of anammox reactors during different phases. N LR a
ime (d)
T (k gN m3
R0 Q b
(L d1 )
H R T (h )
Vu p
c
( m h1 )
d-1 ) 1 11. 1. 0. 20 -15 2 2 29 1 11. 1. 0. 20 6-60 2 2 29 6 11. 1. 0. 1-15 20 2 2 29 0 1 11. 1. 0. 51-1 20 2 2 29 95 1 14. 0. 0. 96-2 25 1 96 36 02 2 16. 0. 0. 03-2 30 9 80 44 11 2 21. 37 0. 0. 12-2 0 .5 64 54 20 a
c
NR Rd
NRE e
pHinf
pHeff
(%)
NR Rd
NRE e
pHinf
pHeff
(%)
10.2 ±0.2 10.1 ±0.2
90.6 ±0.6 89.8 ±1.5
7.83± 8.32± 0.14 0.13 7.77± 8.34± 0.08 0.14
10.1 ±0.1 10.0 ±0.2
90.1 ±0.6 89.1 ±1.5
7.80± 8.38± 0.11 0.05 7.76± 8.37± 0.09 0.20
9.9± 0.2
85.5 ±1.3
7.91± 8.48± 0.10 0.09
9.9± 0.3
88.4 ±2.3
7.80± 8.20± 0.03 0.05
10.0 ±0.3
88.7 ±2.5
7.95± 8.42± 0.09 0.08
10.3 ±0.3
91.5 ±2.2
7.89± 7.95± 0.05 0.07
12.1 ±0.5
85.6 ±3.5
7.90± 8.53± 0.13 0.05
12.7 ±0.2
90.9 ±1.2
7.80± 7.81± 0.03 0.12
12.4 ±0.8
73.1 ±4.6
7.89± 8.66± 0.15 0.72
14.5 ±0.2
86.7 ±1.4
7.82± 7.82± 0.05 0.10
11.1 ±0.7
52.3 ±3.4
7.88± 8.76± 0.11 0.11
17.3 ±0.6
82.2 ±2.8
7.80± 7.81± 0.03 0.09
: Nitrogen loading rate (NLR)
b
R1
: Quantity of flow (Q)
: Hydraulic up-flow velocity (Vup)
d: Nitrogen removal rate (NRR) e: Nitrogen removal efficiency (NRE)
34
Highlights Acute exposure to high-level phosphate (≥ 50 mM) induced the cytoplasm leakage. Phosphate up to 500 mgP L-1 showed no obvious adverse effects on physiological characteristics. HAP core improved the physic-chemical properties of anammox granules.
35
Graphical abstract
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