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Insight into the influence of magnesium on the properties of heterotrophic denitrifying granules
Ecological Engineering 92 (2016) 62–66
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Insight into the influence of magnesium on the properties of heterotrophic denitrifying granules Hui Chen a,b , Qian-Qian Chen a,b , Zhi-Jian Shi a,b , Jia-Jia Xu a,b , Miao-Miao He a,b , Man-Ling Shi a,b,∗ , Ren-Cun Jin a,b,∗ a b
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310036, China
a r t i c l e
i n f o
Article history: Received 25 November 2015 Received in revised form 22 February 2016 Accepted 21 March 2016 Keywords: UASB Denitrification Granule properties Magnesium
a b s t r a c t This paper examined the influence of magnesium on denitrifying sludge. Two up-flow anaerobic sludge bed (UASB) reactors were used in the study. R1 was continuously supplied with 50 mg L−1 of divalent magnesium in the wastewater and R0 was used as a control without magnesium. On the 258th day, the sludge properties in R0 and R1 were analyzed when the nitrogen loading rates were both 36.0 kgN m−3 d−1 and the nitrogen removal rates of R0 and R1 were 33.8 and 34.6 kgN m−3 d−1 , respectively. The diameters of R0 and R1 were 0.95and1.35 mm, settling velocity (74.5 ± 20.8 and102.0 ± 22.6 m h−1 ), extracellular polymeric substance (60.8 ± 2.1 and76.0 ± 2.7 mg g−1 VSS)and specific denitrifying activity (794.4 ± 9.6 and825.6 ± 4.9 mgN d−1 ) were also compared. These results addressed that the addition of magnesium to denitrifying reactor is an effective alternative to enhance sludge properties. © 2016 Published by Elsevier B.V.
1. Introduction Denitrifying process is a promising technology for nitrogen removal from wastewater, which is a key step in nitrogen cycle(Li et al., 2013; Monballiu et al., 2013). Many studies have been undertaken to establish stable denitrifying reactors and cultivate denitrifying granules (Chen et al., 2014; Li et al., 2013). Anaerobic granulation is a novel environmental technology that has been extensively studied in the area of denitrification (Chen et al., 2014; Li et al., 2013; Xing et al., 2015). Compared with flocculent sludge, anaerobic denitrifying granules have a denser structure, better settling ability, greater biomass retention and greater tolerance to shock loadings (De Kreuk and van Loosdrecht, 2004; Etterer and Wilderer, 2001). Aggregated granules can increase cell quantity and enhance bacterial information exchange and cooperative actions (Kartal et al., 2011; Strous et al., 1999). Moreover, it has been suggested that the aggregated granules and macromolecular crowding could improve the robustness of gene expression (Tan et al., 2013). Anaerobic granulation typically requires 2–4 months of careful operation. Many factors play key roles during the formation of granules, e.g., the nature of influent carbon source,
∗ Corresponding authors. E-mail addresses: [email protected] (M.-L. Shi), [email protected], [email protected] (R.-C. Jin). http://dx.doi.org/10.1016/j.ecoleng.2016.03.038 0925-8574/© 2016 Published by Elsevier B.V.
food-to-microorganism ratio and pH (Sajjad and Kim, 2015; Yang et al., 2008).Divalent cations, such as calcium and magnesium, play important roles during granulation by reducing charges on cell surfaces or bridging extracellular polymeric substances (EPS) (Flemming and Wingender, 2010). However, previous studies focused on the cultivation of denitrifying sludge under different conditions, and few studies have compared the differences in granular properties of the sludge cultivated with various strategies. Therefore, the purpose of this study was to describe the differences between the granules cultivated with and without magnesium and to evaluate the influence of magnesium on granule properties during the operation.
2. Materials and methods 2.1. Experimental set-up Two identical up-flow anaerobic sludge bed (UASB) reactors, R0 and R1 , were used to cultivate denitrifying granules with working volumes of 1.25 L (Fig. 1). The UASB reactors were designed with an internal diameter of 60 mm. According to previous studies, 40–50 mg L−1 Mg2+ or Ca2+ were effective for accelerating sludge granulation (Liu et al., 2010; Liu and Sun, 2011). Therefore, R1 was supplied with 50 mg L−1 of Mg2+ in the influent, and R0 was used as a control without Mg2+ . Both reactors were placed in a thermostatic
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rheological behavior was conducted in a measuring glass according to Su and Yu (2005). 2.4. Statistical analysis Variance analyses (ANOVA) were conducted to compare the data between the two granules. The statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) V 13.0. Correlations were considered significant at the 95% confidence interval (p < 0.05). 3. Results and discussion Sludge samples were collected from the reactors and their properties were analyzed on the 258th day when the reactors reached a maximum nitrogen loading rate(NLR)of 36.0 kg N m−3 d−1 , and the corresponding organic loading rate (OLR) was 216.0 kg COD m−3 d−1 . Fig. 1. Schematic diagram of the denitrifying UASB reactor. Table 1 Synthetic wastewater composition. Composition Concentration (mg L−1 ) Composition
Concentration (mg L−1 )
Na2 HPO4 NaH2 PO4 NaHCO3 CaCl2 Peptone
100 1.25c 1.25c add as required add as required
2655 2375 400 0.5 240
Yeast extract Trace element Ia Trace element IIb Nitrate Glucose
The ingredient of the trace element I (g × L−1 ): 5.000 EDTA, 9.14 FeSO4 ·7H2 O. The ingredient of the trace element II (g × L−1 ): 15.000 EDTA, 0.430 ZnSO4 ·7H2 O, 0.240CoCl2 ·6H2 O, 0.990 MnCl2 ·4H2 O, 0.250CuSO4 ·5H2 O, 0.220 NaMoO4 ·2H2 O, 0.210 NiCl2 ·6H2 O, 0.014 H3 BO4 . c Milliliter per liter wastewater. a
b
room at 35 ± 1 ◦ C without light to avoid the growth of phototrophic organisms. 2.2. Synthetic wastewater Synthetic wastewater was prepared. Glucose and sodium nitrate were selected as the C and N sources, respectively. The chemical oxygen demand (COD) to nitrate-nitrogen (COD/NO3 − -N) ratio was 6.0 which was higher than the theoretical stoichiometric ratio (4.9) for complete denitrification (including bacterial growth) (Franco et al., 2006), guaranteeing the stability of denitrifying reaction resulted from organic matter as limiting substrate. The synthetic wastewater composition is listed in Table 1. The initial COD and inorganic NO3 − -N concentrations were set at 300 and 50 mg L−1 , respectively. The wastewater for R0 and R1 were identical except for Mg2+ . The pH of the synthetic wastewater was maintained within 6.9–7.3 and was adjusted by the addition of 1 mol L−1 of sodium hydroxide and hydrochloric acid solutions.
3.1. Size distribution, density and settling ability of the granules The sludge in the reactors gradually aggregated and several pictures of the sludge morphology were taken (e.g., flocs and granules; Fig. 2). It was evident from the graphs that the size of the sludge continuously became larger, and the color changed from black/grey to cream-colored. Moreover, the granules from R1 had smoother surfaces and larger sizes than the ones from R0 . The aggregated microbial granules have been found to be highly amorphous (Jiang and Logan, 1991). The diameter, density and settling ability are important indices that describe the sludge property. These details are summarized in Table 2. The sludge density was higher in R1 than the one in R0 . Furthermore, the differences in diameters and settling velocities were extremely significant (p < 0.01). The percentage of particles with diameter below 0.5 mm was 52.2% in R0 , while the corresponding value in R1 was 40.4%. In R0 , the granules with diameters exceeding 5.0 mm accounted for 2.9%, and the value in R1 was 4.9%. The average settling velocity of the granules was lower than 126.36 ± 13.32 m h−1 as obtained by Li et al. (2013). This velocity was fast enough to keep the biomass inside the reactors compared with the results achieved by Zhang et al. (2013). The obtained results demonstrated that the magnesium supplementation greatly increased the settling velocity (from 74.5 ± 20.8 to 102.0 ± 22.6 m h−1 ). Commonly, granules are aggregates of microbial origin without the requirement fora carrier and the settling velocity was higher than 10 m h−1 , a level faster than that of the sludge flocs (Tay et al., 2006). In summary, these results demonstrated that the addition of magnesium in wastewater enhanced the properties of the denitrifying granules, including the diameter, settling velocity and density. These are key factors in maintaining the biomass and the stability of the reactor. 3.2. Rheological characteristics of the granules
2.3. Analytical methods The determination of suspended solids (SS) and volatile suspended solids (VSS) levels was performed using standard methods (APHA, 2005). The granule diameter was determined according to the method described by Jin et al. (2013). EPS were extracted using the ‘heating’ method, and the extracellular protein (PN) and polysaccharide (PS) concentrations were determined as described by Ma et al. (2012). The granule morphology was observed using a stereoscope (EZ4HD, LEICA, Germany). The settling velocity (VS ) and specific denitrification activity (SDA) were determined using the methods described by Chen et al. (2015). The determination of
Rheology describes the formation of a body under the influence of mechanical stress. Thus, rheology is a valuable tool for characterizing the non-Newtonian properties of sludge suspensions since it can scientifically quantify the flow behaviors of real processes (Su and Yu, 2005). As previously mentioned, the SS content influences the apparent viscosity (a ) of the microbial granule-containing mixed liquor (Liu et al., 2009). Fig. 3 shows the relationship between the a and the SS. For the granules from R0 , the a increased from 260.2 ± 10.0 to 1341.3 ± 50.4 mPa s when the SS in the measuring glass increased from 4.2 to 24.1 g L−1 . For R1 , the corresponding values were 290.5 ± 10.6 and 1973.3 ± 340.2 mPa s when the SS in the
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Table 2 Comparison of the characteristics of the sludge from R0 and R1. Mean diameter (mm) R0 R1 Statistical analysis (p)
0.95 1.35 0
Density (g mL−1 ) 0.9989 ± 0.0795 1.0084 ± 0.0909 0.693
Settling velocity (m h−1 ) 74.5 ± 20.8 102.0 ± 22.6 0
Fig. 2. Aggregation progress of the denitrifying granules. (A) Seeding sludge. (B) Early granulation time. (C) Mature denitrifying granules in R0 . (D) Mature denitrifying granules in R1 .
measuring glass increased from 4.4 to 24.0 g L−1 . In summary, the magnesium supplementation increased the viscosity. It is evident from Fig. 3 that the a increased exponentially with the SS concentration. This was in good agreement with the results achieved by other researchers (Mu and Yu, 2006; Su and Yu, 2005). 3.3. EPS content and SDA Microbial EPS consists of a rich matrix of polymers of primarily polysaccharides and proteins (Liu et al., 2009). The microbial cells are naturally embedded in the aggregates, together with the EPS, making up the aggregates. Thus, the physical properties of the microorganisms depend on both the EPS and the functional bacteria (Chen et al., 2013). The EPS content is important for the granulation process due to the influence of the EPS on the surface charge and energy, which affects the adsorption and adherence properties (Su and Yu, 2005). Moreover, the EPS effectively hydrates the granule surfaces and protects granules against shear forces and gas bubbles, maintaining the stability of the granules (Hulshoff Pol et al., 2004). The EPS contents in R0 and R1 were 60.8 ± 2.1 and 76.0 ± 2.7 mg EPS g−1 VSS (extremely significant difference,
p < 0.01), respectively, with a PN/PS ratio of 9.6 ± 0.5 and 6.2 ± 0.6. The PN/PS ratio for granules is typically expressed to evaluate the settling ability of the granules. Ahigher PN/PS ratio indicates a worse settling ability (Chen et al., 2015). Due to a better settling ability, the sludge was easily maintained in the reactor, leading to the higher biomass concentration in R1 than in R0 (19.9 and 17.8 g L−1 for SS while 15.0 and 13.1 g L−1 for VSS in the reactors). In this study, polysaccharides in the R0 sludge was 5.76 ± 0.09 mg g−1 VSS, while the value in R1 was 10.58 ± 1.28 mg g−1 VSS due to the enhancement with magnesium. Polysaccharides play an important role in maintaining the structural integrity of anaerobic granules (Liu et al., 2004). High polysaccharide levels have been reported in the presence of excess divalent ions, such as Fe2+ , Mg2+ and Ca2+ (Jiang et al., 2003; Shen et al., 1993; Veiga et al., 1997). Polysaccharides establish a strong and sticky framework and contribute to the stable structure of the granule. Moreover, the polysaccharide functional groups, such as hydroxyls, interact with Mg2+ to form a rigid, non-deformable polymeric gel-like matrix and further enhance the structural stability of the sludge (Costerton et al., 1987; Sutherland, 2001).
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Table 3 Comparison of the maximum achieved SDAs with other studies. Sludge form
Reactor
SDA (mgN h−1 g−1 VSS)
Reference
Suspended sludge Suspended sludge Granules Granules Granules Granules
Batch Continuous SBR SBR USB UASB
648 170 729.6 28.8 319.2 794.4 (without Mg2+ ) 825.6 (with Mg2+ )
Akunna et al., 1993 Carrera et al., 2003 Fernandez-Nava et al., 2008 Adav et al., 2010 Pagacova et al., 2010 This study
where P is the reactor potential (kg N m−3 d−1 ). According to Eq. (1), the P values of R0 and R1 were 10.4 and 12.4 kg N m−3 d−1 , respectively. However, during the experiment, the highest achieved NRRs of R0 and R1 were 33.9 and 34.6 kg N m−3 d−1 , respectively. This difference between the calculated and experimented values was probably due to the effects attributed to the continuous feeding. Better mass transfer/mixing was achieved and adverse conditions were avoided, e.g., substrate inhibition. There were significant differences between the biomass sampled from R0 and R1 . The magnesium supplementation promoted the EPS content, SDA and other properties, resulting in the superior capacity of the R1 reactor. Sludge granulation was enhanced in two ways: (1) Mg2+ played an important activator role on enzymes and stimulated enzyme reactions associated with the syntheses of cell materials (Brdjanovic et al., 1996), and (2) Mg2+ neutralized the negative charges on the cell surfacesto promote sludge granulation (Morgan et al., 1990). 4. Conclusions After the continuous operation of the UASB reactors, significant differences in the sludge properties were observed (p < 0.05)Due to the addition of magnesium in the influent in R1 , the sludge properties, including granule sizes, EPS, SDA and settling velocity, were effectively improved. It was an alternative to cultivate denitrifying granules with enhanced properties by adding magnesium in the influent. In practical applications, denitrifying biomass with enhanced physical and chemical properties can be achieved by providing divalent magnesium. Acknowledgements The authors wish to thank the National Key Technologies R&D Program of China (No. 2012BAC13B02) and the Natural Science Foundation of China (No. 51278162 and No. 51578204) for their partial support of this study. References Fig. 3. Relation between the apparent viscosity (a ) and SS. (A) The R0 profile. (B) The R1 profile.
The SDA could be utilized as another index to evaluate the performance of the denitrifying reactor. In this study, the SDAs of the sludge from R0 and R1 were 794.4 ± 9.6 and 825.6 ± 4.9 mgN h−1 g−1 VSS, respectively, and the difference between the SDAs of the reactors was significant (p < 0.05). In fact, the SDAs obtained in this study were higher than those reported previously (Table 3). Due to a higher biomass SDA in R1 , R1 exhibited greater nitrogen and organic removal rates (NRR and ORR) than those in R0 (34.6 versus 33.9 kg N m−3 d−1 and 128.5 versus 116.5 kg COD m−3 d−1 ) on the 258th day when the NLR and OLR were 36 kg N m−3 d−1 and 216 kg COD m−3 d−1 , respectively. The potential of the reactor was calculated according to Eq. (1): P = VSS × SDA
(1)
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Chen, H., He, L.L., Liu, A.N., Guo, Q., Zhang, Z.Z., Jin, R.C., 2015. Start-up of granule-based denitrifying reactors with multiple magnesium supplementation strategies. J. Environ. Manage. 155, 204–211. Costerton, J.W., Cheng, K.J., Geesey, G.G., Ladd, T.I., Nickel, J.C., Dasgupta, M., Marrie, T.J., 1987. Bacterial biofilm in nature and disease. Ann. Rev. Microbiol. 41, 435–464. De Kreuk, M.K., van Loosdrecht, M.C.M., 2004. Selection of slow growing organisms as means for improving aerobic granular sludge stability. Water Sci. Technol. 49 (11-12), 9–17. Etterer, T., Wilderer, P.A., 2001. Generation and properties of aerobic granular sludge. Water Sci. Technol. 43 (3), 19–26. Fernandez-Nava, Y., Maranon, E., Soons, J., Castrillon, L., 2008. Denitrification of wastewater containing high nitrate and calcium concentrations. Bioresour. Technol. 99, 7976–7981. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633. Franco, A., Roca, E., Lema, J.M., 2006. Granulation in high-load denitrifying upflow sludge bed (USB) pulsed reactors. Water Res. 40, 871–880. Hulshoff Pol, L.W., Lopes, S.I.D., Lettinga, G., Lens, P.N.L., 2004. Anaerobic sludge granulation. Water Res. 38, 1376–1389. Jiang, Q., Logan, B.E., 1991. Fractal dimensions of aggregates determined from steady state size distributions. Environ. Sci. Technol. 25, 2031–2038. Jiang, H.L., Tay, J.H., Liu, Y., Tay, S.T.L., 2003. Ca2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors. Biotechnol. Lett. 25, 95–99. Jin, R.C., Yang, G.F., Zhang, Q.Q., Ma, C., Yu, J.J., Xing, B.S., 2013. The effect of sulfide inhibition on the ANAMMOX process. Water Res. 47, 1459–1469. Kartal, B., Maalacke, W.J., de Almeida, N.M., 2011. Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130. Li, W., Zheng, P., Wang, L., Zhang, M., Lu, H.F., Xing, Y.J., Zhang, J.Q., Wang, R., Song, J., Ghulam, A., 2013. Physical characteristics and formation mechanism of denitrifying granular sludge in high-load reactor. Bioresour. Technol. 142, 683–687. Liu, Y.J., Sun, D.D., 2011. Calcium augmentation for enhanced denitrifying granulation in sequencing batch reactors. Process Biochem. 46, 987–992. Liu, Y., Yang, S.F., Tay, J.H., 2004. Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria. J. Biotechnol. 108, 161–169. Liu, X.W., Sheng, G.P., Yu, H.Q., 2009. Physicochemical characteristics of microbial granules. Biotechnol. Adv. 27, 1061–1070. Liu, L., Gao, D.W., Zhang, M., Fu, Y., 2010. Comparison of Ca2+ and Mg2+ enhancing aerobic granulation in SBR. J. Hazard. Mater. 181, 382–387. Ma, C., Jin, R.C., Yang, G.F., Yu, J.J., Xing, B.S., Zhang, Q.Q., 2012. Impacts of transient salinity shock loads on Anammox process performance. Bioresour. Technol. 112, 124–130.
Monballiu, A., Desmidt, E., Ghyselbrecht, K., De Clippeleir, H., Van Hulle, S.W.H., Verstraete, W., Meesschaert, B., 2013. Enrichment of anaerobic ammonium oxidizing (Anammox) bacteria from OLAND and conventional sludge: features and limitations. Sep. Purif. Technol. 104, 130–137. Morgan, J.M., Forster, C.F., Evison, L., 1990. A comparative study of the nature ofbiopolymers extracted from anaerobic and activated sludges. Water Res. 24, 743–750. Mu, Y., Yu, H.Q., 2006. Rheological and fractal characteristics of granular sludge in an upflow anaerobic reactor. Water Res. 40, 3596–3602. Pagacova, P., Galbova, K., Drtil, M., Jonatova, I., 2010. Denitrification in USB reactor with granulated biomass. Bioresour. Technol. 101, 150–156. Sajjad, M., Kim, K.S., 2015. Studies on the interactions of Ca2+ and Mg2+ with EPS and their role in determining the physicochemical characteristics of granular sludges in SBR system. Process Biochem. 55, 966–972. Shen, C.F., Kosaric, N., Blaszczyk, R., 1993. The effect of selected heavy metals (Ni: coand Fe) on anaerobic granules and their extracellular polymeric substance (EPS). Water Res. 27, 25–33. Strous, M., Fuerst, J.A., Kramer, E.H., 1999. Missing lithograph identified as new planctomycete. Nature 400, 446–449. Sutherland, I.W., 2001. Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147, 3–9. Su, K.Z., Yu, H.Q., 2005. Formation and characterization of aerobic granules in a sequencing batch reactor treating soybean-processing wastewater. Environ. Sci. Technol. 39, 2818–2827. Tan, C., Saurabh, S., Bruchez, M.P., 2013. Molecular crowding shapes gene expression in synthetic cellular nanosystems. Nat. Nanotechnol. 8, 602–608. Tay, J.H., Tay, S.T.L., Liu, Y., Show, K.Y., Ivanov, V., 2006. Biogranulation technologies for wastewater treatment. Waste Manag. Ser. 6, 1–277. Veiga, M.C., Jain, M.K., Wu, W.M., Hollingsworth, R.I., Zeikus, J.G., 1997. Composition and role of extracellular polymers in methanogenic granules. Appl. Environ. Microbiol. 63, 403–407. Xing, B.S., Guo, Q., Yang, G.F., Zhang, Z.Z., Li, P., Guo, L.X., Ren, R.C., 2015. The properties of anaerobic ammonium oxidation (anammox) granules: roles of ambient temperature salinity and calcium concentration. Sep. Purif. Technol. 147, 311–318. Yang, S.F., Li, X.Y., Yu, H.Q., 2008. Formation and characterization of fungal and bacterial granules under different feeding alkalinity and pH conditions. Process Biochem. 43, 8–14. Zhang, Q.Q., Yang, G.F., Wang, H., Wu, K., Jin, R.C., Zheng, P., 2013. Estimating the recovery of ANAMMOX performance from inhibition by copper (II) and oxytetracycline (OTC). Sep. Purif. Technol. 113, 90–103.
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Insight into the influence of magnesium on the properties of heterotrophic denitrifying granules Hui Chen a,b , Qian-Qian Chen a,b , Zhi-Jian Shi a,b , Jia-Jia Xu a,b , Miao-Miao He a,b , Man-Ling Shi a,b,∗ , Ren-Cun Jin a,b,∗ a b
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310036, China
a r t i c l e
i n f o
Article history: Received 25 November 2015 Received in revised form 22 February 2016 Accepted 21 March 2016 Keywords: UASB Denitrification Granule properties Magnesium
a b s t r a c t This paper examined the influence of magnesium on denitrifying sludge. Two up-flow anaerobic sludge bed (UASB) reactors were used in the study. R1 was continuously supplied with 50 mg L−1 of divalent magnesium in the wastewater and R0 was used as a control without magnesium. On the 258th day, the sludge properties in R0 and R1 were analyzed when the nitrogen loading rates were both 36.0 kgN m−3 d−1 and the nitrogen removal rates of R0 and R1 were 33.8 and 34.6 kgN m−3 d−1 , respectively. The diameters of R0 and R1 were 0.95and1.35 mm, settling velocity (74.5 ± 20.8 and102.0 ± 22.6 m h−1 ), extracellular polymeric substance (60.8 ± 2.1 and76.0 ± 2.7 mg g−1 VSS)and specific denitrifying activity (794.4 ± 9.6 and825.6 ± 4.9 mgN d−1 ) were also compared. These results addressed that the addition of magnesium to denitrifying reactor is an effective alternative to enhance sludge properties. © 2016 Published by Elsevier B.V.
1. Introduction Denitrifying process is a promising technology for nitrogen removal from wastewater, which is a key step in nitrogen cycle(Li et al., 2013; Monballiu et al., 2013). Many studies have been undertaken to establish stable denitrifying reactors and cultivate denitrifying granules (Chen et al., 2014; Li et al., 2013). Anaerobic granulation is a novel environmental technology that has been extensively studied in the area of denitrification (Chen et al., 2014; Li et al., 2013; Xing et al., 2015). Compared with flocculent sludge, anaerobic denitrifying granules have a denser structure, better settling ability, greater biomass retention and greater tolerance to shock loadings (De Kreuk and van Loosdrecht, 2004; Etterer and Wilderer, 2001). Aggregated granules can increase cell quantity and enhance bacterial information exchange and cooperative actions (Kartal et al., 2011; Strous et al., 1999). Moreover, it has been suggested that the aggregated granules and macromolecular crowding could improve the robustness of gene expression (Tan et al., 2013). Anaerobic granulation typically requires 2–4 months of careful operation. Many factors play key roles during the formation of granules, e.g., the nature of influent carbon source,
∗ Corresponding authors. E-mail addresses: [email protected] (M.-L. Shi), [email protected], [email protected] (R.-C. Jin). http://dx.doi.org/10.1016/j.ecoleng.2016.03.038 0925-8574/© 2016 Published by Elsevier B.V.
food-to-microorganism ratio and pH (Sajjad and Kim, 2015; Yang et al., 2008).Divalent cations, such as calcium and magnesium, play important roles during granulation by reducing charges on cell surfaces or bridging extracellular polymeric substances (EPS) (Flemming and Wingender, 2010). However, previous studies focused on the cultivation of denitrifying sludge under different conditions, and few studies have compared the differences in granular properties of the sludge cultivated with various strategies. Therefore, the purpose of this study was to describe the differences between the granules cultivated with and without magnesium and to evaluate the influence of magnesium on granule properties during the operation.
2. Materials and methods 2.1. Experimental set-up Two identical up-flow anaerobic sludge bed (UASB) reactors, R0 and R1 , were used to cultivate denitrifying granules with working volumes of 1.25 L (Fig. 1). The UASB reactors were designed with an internal diameter of 60 mm. According to previous studies, 40–50 mg L−1 Mg2+ or Ca2+ were effective for accelerating sludge granulation (Liu et al., 2010; Liu and Sun, 2011). Therefore, R1 was supplied with 50 mg L−1 of Mg2+ in the influent, and R0 was used as a control without Mg2+ . Both reactors were placed in a thermostatic
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rheological behavior was conducted in a measuring glass according to Su and Yu (2005). 2.4. Statistical analysis Variance analyses (ANOVA) were conducted to compare the data between the two granules. The statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) V 13.0. Correlations were considered significant at the 95% confidence interval (p < 0.05). 3. Results and discussion Sludge samples were collected from the reactors and their properties were analyzed on the 258th day when the reactors reached a maximum nitrogen loading rate(NLR)of 36.0 kg N m−3 d−1 , and the corresponding organic loading rate (OLR) was 216.0 kg COD m−3 d−1 . Fig. 1. Schematic diagram of the denitrifying UASB reactor. Table 1 Synthetic wastewater composition. Composition Concentration (mg L−1 ) Composition
Concentration (mg L−1 )
Na2 HPO4 NaH2 PO4 NaHCO3 CaCl2 Peptone
100 1.25c 1.25c add as required add as required
2655 2375 400 0.5 240
Yeast extract Trace element Ia Trace element IIb Nitrate Glucose
The ingredient of the trace element I (g × L−1 ): 5.000 EDTA, 9.14 FeSO4 ·7H2 O. The ingredient of the trace element II (g × L−1 ): 15.000 EDTA, 0.430 ZnSO4 ·7H2 O, 0.240CoCl2 ·6H2 O, 0.990 MnCl2 ·4H2 O, 0.250CuSO4 ·5H2 O, 0.220 NaMoO4 ·2H2 O, 0.210 NiCl2 ·6H2 O, 0.014 H3 BO4 . c Milliliter per liter wastewater. a
b
room at 35 ± 1 ◦ C without light to avoid the growth of phototrophic organisms. 2.2. Synthetic wastewater Synthetic wastewater was prepared. Glucose and sodium nitrate were selected as the C and N sources, respectively. The chemical oxygen demand (COD) to nitrate-nitrogen (COD/NO3 − -N) ratio was 6.0 which was higher than the theoretical stoichiometric ratio (4.9) for complete denitrification (including bacterial growth) (Franco et al., 2006), guaranteeing the stability of denitrifying reaction resulted from organic matter as limiting substrate. The synthetic wastewater composition is listed in Table 1. The initial COD and inorganic NO3 − -N concentrations were set at 300 and 50 mg L−1 , respectively. The wastewater for R0 and R1 were identical except for Mg2+ . The pH of the synthetic wastewater was maintained within 6.9–7.3 and was adjusted by the addition of 1 mol L−1 of sodium hydroxide and hydrochloric acid solutions.
3.1. Size distribution, density and settling ability of the granules The sludge in the reactors gradually aggregated and several pictures of the sludge morphology were taken (e.g., flocs and granules; Fig. 2). It was evident from the graphs that the size of the sludge continuously became larger, and the color changed from black/grey to cream-colored. Moreover, the granules from R1 had smoother surfaces and larger sizes than the ones from R0 . The aggregated microbial granules have been found to be highly amorphous (Jiang and Logan, 1991). The diameter, density and settling ability are important indices that describe the sludge property. These details are summarized in Table 2. The sludge density was higher in R1 than the one in R0 . Furthermore, the differences in diameters and settling velocities were extremely significant (p < 0.01). The percentage of particles with diameter below 0.5 mm was 52.2% in R0 , while the corresponding value in R1 was 40.4%. In R0 , the granules with diameters exceeding 5.0 mm accounted for 2.9%, and the value in R1 was 4.9%. The average settling velocity of the granules was lower than 126.36 ± 13.32 m h−1 as obtained by Li et al. (2013). This velocity was fast enough to keep the biomass inside the reactors compared with the results achieved by Zhang et al. (2013). The obtained results demonstrated that the magnesium supplementation greatly increased the settling velocity (from 74.5 ± 20.8 to 102.0 ± 22.6 m h−1 ). Commonly, granules are aggregates of microbial origin without the requirement fora carrier and the settling velocity was higher than 10 m h−1 , a level faster than that of the sludge flocs (Tay et al., 2006). In summary, these results demonstrated that the addition of magnesium in wastewater enhanced the properties of the denitrifying granules, including the diameter, settling velocity and density. These are key factors in maintaining the biomass and the stability of the reactor. 3.2. Rheological characteristics of the granules
2.3. Analytical methods The determination of suspended solids (SS) and volatile suspended solids (VSS) levels was performed using standard methods (APHA, 2005). The granule diameter was determined according to the method described by Jin et al. (2013). EPS were extracted using the ‘heating’ method, and the extracellular protein (PN) and polysaccharide (PS) concentrations were determined as described by Ma et al. (2012). The granule morphology was observed using a stereoscope (EZ4HD, LEICA, Germany). The settling velocity (VS ) and specific denitrification activity (SDA) were determined using the methods described by Chen et al. (2015). The determination of
Rheology describes the formation of a body under the influence of mechanical stress. Thus, rheology is a valuable tool for characterizing the non-Newtonian properties of sludge suspensions since it can scientifically quantify the flow behaviors of real processes (Su and Yu, 2005). As previously mentioned, the SS content influences the apparent viscosity (a ) of the microbial granule-containing mixed liquor (Liu et al., 2009). Fig. 3 shows the relationship between the a and the SS. For the granules from R0 , the a increased from 260.2 ± 10.0 to 1341.3 ± 50.4 mPa s when the SS in the measuring glass increased from 4.2 to 24.1 g L−1 . For R1 , the corresponding values were 290.5 ± 10.6 and 1973.3 ± 340.2 mPa s when the SS in the
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Table 2 Comparison of the characteristics of the sludge from R0 and R1. Mean diameter (mm) R0 R1 Statistical analysis (p)
0.95 1.35 0
Density (g mL−1 ) 0.9989 ± 0.0795 1.0084 ± 0.0909 0.693
Settling velocity (m h−1 ) 74.5 ± 20.8 102.0 ± 22.6 0
Fig. 2. Aggregation progress of the denitrifying granules. (A) Seeding sludge. (B) Early granulation time. (C) Mature denitrifying granules in R0 . (D) Mature denitrifying granules in R1 .
measuring glass increased from 4.4 to 24.0 g L−1 . In summary, the magnesium supplementation increased the viscosity. It is evident from Fig. 3 that the a increased exponentially with the SS concentration. This was in good agreement with the results achieved by other researchers (Mu and Yu, 2006; Su and Yu, 2005). 3.3. EPS content and SDA Microbial EPS consists of a rich matrix of polymers of primarily polysaccharides and proteins (Liu et al., 2009). The microbial cells are naturally embedded in the aggregates, together with the EPS, making up the aggregates. Thus, the physical properties of the microorganisms depend on both the EPS and the functional bacteria (Chen et al., 2013). The EPS content is important for the granulation process due to the influence of the EPS on the surface charge and energy, which affects the adsorption and adherence properties (Su and Yu, 2005). Moreover, the EPS effectively hydrates the granule surfaces and protects granules against shear forces and gas bubbles, maintaining the stability of the granules (Hulshoff Pol et al., 2004). The EPS contents in R0 and R1 were 60.8 ± 2.1 and 76.0 ± 2.7 mg EPS g−1 VSS (extremely significant difference,
p < 0.01), respectively, with a PN/PS ratio of 9.6 ± 0.5 and 6.2 ± 0.6. The PN/PS ratio for granules is typically expressed to evaluate the settling ability of the granules. Ahigher PN/PS ratio indicates a worse settling ability (Chen et al., 2015). Due to a better settling ability, the sludge was easily maintained in the reactor, leading to the higher biomass concentration in R1 than in R0 (19.9 and 17.8 g L−1 for SS while 15.0 and 13.1 g L−1 for VSS in the reactors). In this study, polysaccharides in the R0 sludge was 5.76 ± 0.09 mg g−1 VSS, while the value in R1 was 10.58 ± 1.28 mg g−1 VSS due to the enhancement with magnesium. Polysaccharides play an important role in maintaining the structural integrity of anaerobic granules (Liu et al., 2004). High polysaccharide levels have been reported in the presence of excess divalent ions, such as Fe2+ , Mg2+ and Ca2+ (Jiang et al., 2003; Shen et al., 1993; Veiga et al., 1997). Polysaccharides establish a strong and sticky framework and contribute to the stable structure of the granule. Moreover, the polysaccharide functional groups, such as hydroxyls, interact with Mg2+ to form a rigid, non-deformable polymeric gel-like matrix and further enhance the structural stability of the sludge (Costerton et al., 1987; Sutherland, 2001).
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Table 3 Comparison of the maximum achieved SDAs with other studies. Sludge form
Reactor
SDA (mgN h−1 g−1 VSS)
Reference
Suspended sludge Suspended sludge Granules Granules Granules Granules
Batch Continuous SBR SBR USB UASB
648 170 729.6 28.8 319.2 794.4 (without Mg2+ ) 825.6 (with Mg2+ )
Akunna et al., 1993 Carrera et al., 2003 Fernandez-Nava et al., 2008 Adav et al., 2010 Pagacova et al., 2010 This study
where P is the reactor potential (kg N m−3 d−1 ). According to Eq. (1), the P values of R0 and R1 were 10.4 and 12.4 kg N m−3 d−1 , respectively. However, during the experiment, the highest achieved NRRs of R0 and R1 were 33.9 and 34.6 kg N m−3 d−1 , respectively. This difference between the calculated and experimented values was probably due to the effects attributed to the continuous feeding. Better mass transfer/mixing was achieved and adverse conditions were avoided, e.g., substrate inhibition. There were significant differences between the biomass sampled from R0 and R1 . The magnesium supplementation promoted the EPS content, SDA and other properties, resulting in the superior capacity of the R1 reactor. Sludge granulation was enhanced in two ways: (1) Mg2+ played an important activator role on enzymes and stimulated enzyme reactions associated with the syntheses of cell materials (Brdjanovic et al., 1996), and (2) Mg2+ neutralized the negative charges on the cell surfacesto promote sludge granulation (Morgan et al., 1990). 4. Conclusions After the continuous operation of the UASB reactors, significant differences in the sludge properties were observed (p < 0.05)Due to the addition of magnesium in the influent in R1 , the sludge properties, including granule sizes, EPS, SDA and settling velocity, were effectively improved. It was an alternative to cultivate denitrifying granules with enhanced properties by adding magnesium in the influent. In practical applications, denitrifying biomass with enhanced physical and chemical properties can be achieved by providing divalent magnesium. Acknowledgements The authors wish to thank the National Key Technologies R&D Program of China (No. 2012BAC13B02) and the Natural Science Foundation of China (No. 51278162 and No. 51578204) for their partial support of this study. References Fig. 3. Relation between the apparent viscosity (a ) and SS. (A) The R0 profile. (B) The R1 profile.
The SDA could be utilized as another index to evaluate the performance of the denitrifying reactor. In this study, the SDAs of the sludge from R0 and R1 were 794.4 ± 9.6 and 825.6 ± 4.9 mgN h−1 g−1 VSS, respectively, and the difference between the SDAs of the reactors was significant (p < 0.05). In fact, the SDAs obtained in this study were higher than those reported previously (Table 3). Due to a higher biomass SDA in R1 , R1 exhibited greater nitrogen and organic removal rates (NRR and ORR) than those in R0 (34.6 versus 33.9 kg N m−3 d−1 and 128.5 versus 116.5 kg COD m−3 d−1 ) on the 258th day when the NLR and OLR were 36 kg N m−3 d−1 and 216 kg COD m−3 d−1 , respectively. The potential of the reactor was calculated according to Eq. (1): P = VSS × SDA
(1)
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