Aerobic Granulation in a for the Treatment of Piggery Wastewater

Dalei Zhang1*, Yanan Wang1, Hongwei Li1, Shaoran Wang2, Yumei Jing3

ABSTRACT: This study investigated the formation of aerobic granules strategy on the treatment of swine wastewater and found that fed with digested piggery wastewater. After 42 days of cultivation, small feeding ratio had a more significant effect on the removal of yellow granules with mean diameter of 0.4 mm were first observed in the phosphorus and nitrogen than on the removal of chemical reactor. Scanning electron microscope pictures showed the granules oxygen demand (COD). Huang et al. (2011) found that high were compact, round structures with clear outer shapes and mainly 2þ 2þ composed of filamentous bacteria. Maximum concentrations of K and Ca would be helpful for removing and ammonia removal ratios were 90.1 and 91.7%, respectively. The nutrients in piggery wastewater. Previous studies have shown Monod equation, which was used to describe ammonium utilization, great improvements in treating piggery wastewater, but there are yielded a maximum rate of 6.25 mg (g volatile suspended solids)1 h1. still some problems that hinder treatment (Latif et al., 2011). The measurement of extracellular polymeric substances (EPS) content Rich nitrogenous compounds in the wastewater make active and three-dimensional excitation and emission matrix results showed biomass densification, granular processes, and, consequently, that the EPS concentration increased during the granulation process. microorganism retention in the reactor difficult, limiting the use Fluorescence in situ hybridization analysis showed significant amounts of upflow anaerobic blanket (UASB) reactors. of nitrifying bacteria in the aerobic granules. Results in this study provide insights to the treatment of piggery wastewater using aerobic granular Because of its promise for biological wastewater treatment, sludge. Water Environ. Res., 85, 239 (2013). immobilized-bacteria technology has been extensively investi- gated (Adav et al., 2008a; Choi et al., 2011; Kim et al., 2011). KEYWORDS: aerobic granules, piggery wastewater, sequencing batch Compared with conventional , aerobic granules reactors, nitrogen removal, fluorescence in situ hybridization. have good settling properties, dense microbial structure, high doi:10.2175/106143012X13560205145136 biomass concentration, and good capacity to deal with toxic media (Adav, Lee, Show, and Tay, 2008; Su and Yu, 2005). Taking advantage of individual granule properties, aerobic granules were used to treat high-strength wastewaters containing Introduction organics, nitrogen, phosphorus, and toxic substances (Moussavi Livestock and poultry breeding have developed rapidly in et al., 2010; Shi et al., 2010; Winkler et al., 2011). Considerable China in recent years. However, this rapid development has research has been carried out on nitrogen removal by aerobic caused difficult environmental pollution issues. Large quantities granules, and because of the good results obtained, aerobic of wastewater containing ammonium, phosphorus, and organic granulation seems to be a suitable process for treating piggery matter have been discharged to the environment, causing wastewater. However, aerobic granules have not yet been applied serious environmental problems such as eutrophication in lakes in the treatment of piggery wastewater, and there is still a lack of and rivers. As is typical of livestock and poultry breeding knowledge about aerobic granulation processes fed with piggery wastewater, piggery wastewater is considered to have a drastic wastewater. effect on the environment and human health, as it contains high The main purpose of the present study, therefore, was to concentrations of nitrogen, phosphorous, and organic matter investigate the feasibility of cultivating aerobic granules using (Han et al., 2011). piggery wastewater in a sequencing batch reactor (SBR). The Many researchers have sought solutions for treating piggery formation process, physicochemical parameters, activities, and wastewater (Han et al., 2011; Xu & Shen, 2011). Zhang et al. microbial community of aerobic granules were investigated in (2011) studied anaerobic codigestion of piggery wastewater and food waste and identified the key factors governing codigestion this work. The information provided is useful for understanding performance. Han et al. (2011) investigated the effect of feeding the mechanism of aerobic granulation, as well as its future application in the treatment of piggery wastewater.

1 School of Environmental and Municipal Engineering, Qingdao Materials and Methods Technological University, Qingdao 266033, China. Inoculated Sludge and Wastewater Content. The reactor 2 Qingdao Research Academy of Environment Science, Qingdao, 266000, China. was inoculated with 2.0 L of sludge from the Qingdao Municipal Wastewater Treatment Plant located in Shandong, China. The 3 Sewage Wastewater Treatment Plant of Haibohe, Qingdao, 266000, China. sludge contained mixed liquor suspended solids (MLSS) of 2.8 g/ L, mixed liquor volatile suspended solids (MLVSS) of 1.82 g/L, a * School of Environmental and Municipal Engineering, Qingdao Technological University, Qingdao 266033, China; e-mail: MLVSS/MLSS ratio of 0.65, and a sludge volume index (SVI) of [email protected]. 73.46 mL/g.

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Table 1—Characteristics of the digested wastewater. HM 550, Thermo Fisher Scientific, Kalamazoo, Michigan) and mounted on gelatin-coated glass slides. After air drying at room Parametersa Digested wastewater temperature, hybridization was conducted following the method CODt (g/L) 53.5 6 2.3 described by Sekiguchi (1999). CODs (g/L) 6.9 6 1.4 A carboxy-X-rhodamine (ROX) labeled Nso190 probe þ NH4 -N (g/L) 4.3 6 1.0 (50CGATCCCCTGCTTTTCTCC30) targeting ammonia-oxi- SS (g/kg) 38.7 6 2.4 dizing bacteria (AOB) and a fluorescein isothiocyanate (FITC) pH 8.0 6 0.3 labeled NIT3 probe (50CCTGTGCTCCATGCTCCG30) target- a þ CODt ¼ total COD, CODs ¼ soluble COD, NH4 -N ¼ ammonium nitrogen, ing nitrite-oxidizing bacteria (NOB) were used. Hybridized and SS ¼ suspended solids. samples were viewed with a fluorescence microscope (DM6000 B, Leica Microsystems, Wetzlar, Germany). Digital images were Undiluted, digested piggery wastewater was collected from a analyzed using Image Pro Plus (version 6.0, Media Cybernetics, methane tank of a pig farm in Qingdao, China. The main Inc., Rockville, Maryland). Three samples were measured to characteristics of the digested wastewater are presented in Table determine average values, and at least 10 different fields were 1. The digested wastewater was centrifuged at 8000 rpm for 1 examined for each sample. hour to eliminate suspended matter and then was diluted and Analysis. The COD, MLSS, MLVSS, SVI, ammonium, nitrate, used as influent to the SBR. The organic loading rate (OLR) was and nitrite concentrations were measured according to Standard 6.9 to 7.2 kg COD m3 d1, and the influent nitrogen Methods (APHA et al., 1998). The pH was measured with a concentration was 120 to 160 mg/L. Some other necessary digital, portable pH meter. Extracellular polymeric substances trace elements were added to ensure the growth of bacteria (Shi (EPS) were extracted by cation exchange resin according to the et al., 2010). The pH of the diluted wastewater was 7.5 6 0.1. method developed by Frolund et al. (1996). Protein (PN) was Reactor Set-Up and Operation. The experiments were measured by the Lowry method (modified) with bovine serum performed in an SBR with a working volume of 4.0 L, internal albumin as standard, and polysaccharide (PS) content was diameter of 70 mm, and height of 1200 mm. Air was introduced analyzed by the anthrone method with glucose as standard through a diffuser by pump; superficial upflow air velocity was (Frolund et al., 1996; Lowry, 1956). The granule structure and 14 mm/s. The dissolved oxygen concentration was 4.8 mg/L. surface morphology were viewed via an SEM (Quanta 200 FEG, The reactor was operated at room temperature (25 6 28C). FEI Company, Hillsboro, Oregon). The reactor was operated sequentially in a 4-hour cycle, including a filling time of 8 minutes, aeration of 224 minutes, Results and Discussion settling of 6 minutes, and effluent withdrawal of 2 minutes. The Formation and Physical Characteristics of Aerobic Gran- settling time of the reactor was decreased from 6 minutes to 2 ules. The initial seeding sludge had a fluffy and irregular minutes on day 30, as a shorter settling time was beneficial to structure as is typical of flocculent activated sludge. As shown in granule formation (Adav et al., 2009). The volumetric exchange Figure 1, sludge properties changed during the granulation ratio was 50%, and the hydraulic retention time (HRT) was 8 process: Settling properties gradually improved, and MLSS hours. increased with cultivated time, consistent with previous studies Three-Dimensional Excitation and Emission Matrix Fluo- (Ma et al., 2011; Su and Yu, 2005). There was not much sludge rescence Spectroscopy. Three-dimensional excitation and washed out from the reactor as reported by others, probably emission matrix (EEM) spectra are a series of emission spectra because a settling time of 6 minutes was used in the initial 30 over a range of excitation wavelengths, and they can be used to days. Settling time was decreased from 6 minutes to 2 minutes identify fluorescent compounds present in complex mixtures. on the 30th day, because a shorter settling time was found to be The spectra, which were measured with a fluorescence spectrophotometer (Cary Eclipse, Varian Inc., Palo Alto, California), were collected with subsequent scanning emission spectra from 200 to 550 nm at 5.0-nm increments by varying the excitation wavelength from 200 to 450 nm at 5.0-nm increments. The spectra were recorded at a scan rate of 1200 nm/min, using excitation and emission slit bandwidths of 5 nm. The voltage of the photomultiplier tube was set to 800 V for high level light detection. The blank scans were performed at an interval of 10 using deionized water. Identification of Nitrifying Bacteria Composition. Gran- ules collected from the reactor on the 60th day were used for scanning electron microscopy (SEM) observation and fluores- cence in situ hybridization (FISH) analysis. Granules were fixed in 4% freshly prepared paraformaldehyde solution for 4 to 6 hours at 48C and then washed with phosphate-buffered saline. The fixed granules were dehydrated by successive passages through 50, 80, and 100% ethanol (three times). The granules were then embedded in melted paraffin wax compound. Sections Figure 1—Profiles of SVI and MLSS of aerobic granules in the of 20-lm thickness were cut with a rotary microtome (Microm process.

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It has been demonstrated that EPS play a crucial role in the formation of granules, such as affecting hydrophobicity of cells, enhancing polymeric interaction, and physically bridging neighboring cells to each other by altering the negative charges on the bacterial surface (Adav, Lee, and Tay, 2008; Wang et al., 2006). Thus, large EPS content would be helpful for aerobic granulation. It could be supposed, therefore, that the increase of EPS content in aerobic granules observed in the present study would help the formation and support the mechanical stability of granules. Three-dimensional EEM spectroscopy was applied in this experiment to identify the fluorescent compounds and charac- terize the organic material that was present in low concentra- tions. As can be seen in Figure 3, four fluorescent peaks were found. Peak A was located at the excitation/emission wavelength (Ex/Em) of 265 nm/365 nm and was considered an aromatic protein. Peak B was located at an Ex/Em of 270 nm/295 nm, which was reported as a tyrosine protein-like material. Peak C Figure 2—Extracellular polymeric substances of granules during was observed at an Ex/Em of 235 nm/295 nm, while Peak D was the operation. observed at an Ex/Em of 240 nm/355 nm. These peaks were attributed to soluble microbial by-product-like material, such as more beneficial to granule formation (Adav et al., 2009; Shi et al., protein- and humic-like material (Chen et al., 2003; Sheng and 2010). After 42 days, small yellow granules with a mean diameter Yu, 2006). Figure 3 clearly shows that the fluorescence intensities of 0.4 mm were first observed in reactor. These granules of the four peaks increased with cultivation time, an indication gradually became the dominant form of biomass in the reactor. that there was a trend that EPS concentration increased during On the 60th day, samples of the granules were taken from the the granulation process. This result was consistent with the reactor, and their microstructure was further examined by SEM determination of EPS in Figure 2. (not shown). The granules had a compact and round structure Performance of Reactor and Removal Efficiencies. Ammo- with a clear outer shape. Amplification showed the granules to nia and COD profiles within the SBR throughout the granulation be dense, compact bacterial structures, with filamentous bacteria process are presented in Figure 4. It can be observed that surrounding the granules. ammonium concentration in the effluent was higher than 30 Character of Extracellular Polymeric Substances in Aer- mg/L at the beginning of the operation, an indication that obic Granules. Extracellular polymeric substances are sticky activated sludge could not remove all of the ammonium. Effluent metabolic products secreted by bacteria and mainly composed of ammonium concentration then gradually decreased, and the PS, PN nucleic acids, lipids, and other polymeric compounds ammonium removal ratio reached as high as 86%. There was a (Adav, Lee, Show, and Tay, 2008; Lee et al., 2010). According to similar trend for COD; the COD removal ratio increased from previous studies, EPS are very important for aerobic granules, 82% to 90% after 20 days and remained at a high level until the such as for providing nutrition to microorganisms and end of the experiment. A resulting benefit was that more sludge protecting microorganisms from toxic shocks. The EPS content stayed in the reactor. The settling properties of the aerobic determined at different stages in the present study is presented granules were much better than that of the activated sludge, thus in Figure 2. In the initial 30 days, PN and PS increased sharply, there were enough bacteria left to keep the reactor with high and then the EPS concentration remained steady within a performance. certain range. A significant trend in which PN increased faster Previous studies have also demonstrated that aerobic granules than PS was observed. This phenomenon was in accordance with have superior ability to remove pollutants such as ammonium, a previous study (Adav, Lee, and Tay, 2008). COD, and phosphorus (Abdullah et al., 2011). Aerobic granules

Figure 3—Excitation and emission matrix fluorescence spectra of EPS: (a) on day 10 and (b) on day 60.

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Figure 4—Profile of removal performances of the reactor. were cultivated to treat high-strength, agro-based wastewater, and the COD and ammonium removal ratios were all as high as 90% at the end of the experiment. It was also reported that aerobic granules could completely remove 4-chloroaniline at concentrations as high as 8.18 g/L (Zhu et al., 2011). It was assumed that the excellent settling properties and abundant bacterial community would make aerobic granules to be a better choice for hard-to-treat wastewater. To study the nitrification process of granules, cycle experi- ments were carried out on the 60th day. It can be seen clearly in Figure 5 that ammonium was almost converted to nitrite as time progressed. Nitrite concentration increased during the initial 2.5 hours and then was converted to nitrate by NOB in the final 1.5 hours, resulting in large increases in nitrate concentration. Further, the Monod equation was used to model ammonium utilization as follows:

Figure 6—Fluorescence in situ hybridization images of aerobic granules collected on the 60th day: (a) ROX-labeled probe Nso190 and FITC-labeled probe NIT3 (green), (b) ROX-labeled probe Nso190, and (c) FITC-labeled probe NIT3.

q S q ¼ max ð1Þ K þ S where q is the ammonium utilization rate, mg (g VSS)1 h1; 1 qmax is the maximum ammonium utilization rate, mg (g VSS) Figure 5—Nitrification profiles observed in one cycle on day 60. h1; K is the apparent half-rate constant, mg/L; and S is the

242 Water Environment Research, Volume 85, Number 3 Zhang et al. ammonium concentration, mg/L (Liu et al., 2010; Shi et al., References 2010). Therefore, the kinetic equation for ammonium utilization Abdullah, N.; Ujang, Z.; Yahya, A. (2011) Aerobic Granular Sludge in the reactor was Formation for High Strength Agro-Based Wastewater Treatment. Bioresour. Technol., 102, 6778–6781. 6:25S Adav, S. S.; Lee, D.-J.; Lai, J.-Y. (2009) Aerobic Granulation in Sequencing q ¼ ð2Þ 18:16 þ S Batch Reactors at Different Settling Times. Bioresour. Technol., 100, 1 1 5359–5361. and qmax was 6.25 mg (g VSS) h . Compared with previous Adav, S. S.; Lee, D.-J.; Show, K.-Y.; Tay, J.-H. (2008) Aerobic Granular studies, qmax was significantly lower than a value of 18.00 mg (g Sludge: Recent Advances. Biotechnol. Adv., 26, 411–423. 1 1 1 1 VSS) h but much higher than 3.29 mg (g VSS) h Adav, S. S.; Lee, D.-J.; Tay, J.-H. (2008) Extracellular Polymeric reported for an airlift reactor (Carvallo et al., 2002; Shi et al., Substances and Structural Stability of Aerobic Granules. Water 2010). Most importantly, the aerobic granules were cultivated Res., 42, 1644–1650. with real wastewater in this study, whereas synthetic wastewater American Public Health Association; American Water Works Associa- was used in the other studies. It is well known that real tion; Water Environment Federation (1998) Standard Methods for wastewater is more difficult to treat, as its contents are complex. the Examination of Water and Wastewater, 20th ed.; American Public Health Association: Washington, D.C. Thus, the maximum ammonium utilization rate achieved in this Carvallo, L.; Carrera, J.; Chamy, R. (2002) Nitrifying Activity Monitoring study was respectable and proved that treating real wastewater and Kinetic Parameters Determination in a Biofilm Airlift Reactor by aerobic granules is feasible. by Respirometry. Biotechnol. Lett., 24, 2063–2066. Fluorescence In Situ Hybridization of Nitrifying Bacteria Chen, W.; Westerhoff, P.; Leenheer, J. A.; Booksh, K. (2003). Fluorescence in Aerobic Granules. The FISH images of the aerobic granules Excitation–Mission Matrix Regional Integration to Quantify collected from the reactor on the 60th day are presented in Spectra for Dissolved Organic Matter. Environ. Sci. Technol., 37, Figure 6. The granules were hybridized with a ROX-labeled 5701–5710. Nso190 probe as the AOB domain specific probe (Figure 6b) and Choi, H.-J.; Yu, S.-W.; Lee, S.-M.; Yu, S.-Y. (2011) Effects of Potassium a FITC-labeled NIT3 probe as the Nitrobacter domain specific and Magnesium in the Enhanced Biological Phosphorus Removal Process Using a Membrane Bioreactor. Water Environ. Res., 83, probe (Figure 6c). The FISH images (Figure 6a) illustrate that the 613–621. AOB were close to the granule surface, while the NOB were in Frolund, B.; Palmgren, R.; Keiding, K.; Nielsen, P. H. (1996) Extraction of the inner layer of the granules. This distribution is similar to that Extracellular Polymers from Activated Sludge Using a Cation previously reported (Shi et al., 2010). Quantitative FISH image Exchange Resin. Water Res., 30, 1749–1758. analyses of the samples showed that the ratios of AOB and NOB Han, Z.; Zhu, J.; Ding, Y.; Wu, W.; Chen, Y.; Zhang, R.; Wang, L. (2011) to total active bacteria were 33 6 2.5 and 256 1.8%, respectively. Effect of Feeding Strategy on the Performance of Sequencing Batch It can also be seen from Figure 6 that a great deal of nitrifying Reactor with Dual Anoxic Feedings for Swine Wastewater bacteria, including AOB and NOB, surrounded the granules, Treatment. Water Environ. Res., 83, 643–649. ensuring that the nitrifying process progressed smoothly. Huang, H. M.; Xu, C. L.; Zhang, W. (2011) Removal of Nutrients from Piggery Wastewater Using Struvite Precipitation and Pyrogenation Conclusions Technology. Bioresour. Technol., 102, 2523–2528. Kim, H.-S.; Schuler, A. J.; Gunsch, C. K.; Pei, R.; Gellner, J.; Boltz, J. P.; Aerobic granules were successfully formed with piggery Freudenberg, R. G.; Dodson, R. (2011) Comparison of Conventional wastewater. The physicochemical parameters, activities, and and Integrated Fixed-Film Activated Sludge Systems: Attached- and microbial community of the granules were also investigated in Suspended-Growth Functions and Quantitative Polymerase Chain this study. Settling properties of sludge were improved Reaction Measurements. Water Environ. Res., 83, 627–635. significantly as SVI decreased from 73.46 mL/g to 36 mL/g. Latif, M. A.; Ghufran, R.; Wahid, Z. A.; Ahmad, A. (2011) Integrated Granules with compact structures were observed after 42 days. Application of Upflow Anaerobic Sludge Blanket Reactor for the Performance of the aerobic granules in removing COD and Treatment of Wastewaters. Water Res., 45, 4683–4699. ammonium in the piggery wastewater was excellent, and the Lee, D.-J.; Chen, Y.-Y.; Show, K.-Y.; Whiteley, C. G.; Tay, J.-H. (2010) maximum ammonium utilization rate could reach as high as Advances in Aerobic Granule Formation and Granule Stability in 28, 6.25 mg (g VSS)1 h1. The FISH images of the granules showed the Course of Storage and Reactor Operation. Biotechnol. Adv., 919–934. that AOB and NOB were in rich abundance and surrounded the Liu, L.; Gao, D.-W.; Zhang, M.; Fu, Y. (2010) Comparison of Ca2þ and granules. According to the results obtained in this study, the Mg2þ Enhancing Aerobic Granulation in SBR. J. Hazard. Mater., aerobic granules showed superior abilities in removing COD and 181, 382–387. ammonium in piggery wastewater, providing insights to the Lowry, O. H.; Rosebrough, N.; Farr, A. L.; Randall, R. J. (1956) Protein development of aerobic granular sludge in piggery wastewater Measurements Using Folin Phenol Reagent. J. Biol. Chem., 193, treatment. 265–275. Ma, D.-Y.;Wang, X.-H.; Song, C.; Wang, S.-G.; Fan, M.-H.; Li, X.-M. Acknowledgments (2011) Aerobic Granulation for Methylene Blue Biodegradation in a The author gratefully acknowledges the financial support Sequencing Batch Reactor. Desalination, 276, 233–238. provided by the National Natural Science Foundation of China Moussavi, G.; Barikbin, B.; Mahmoudi, M. (2010) The Removal of High (NSFC) (51008164), Shandong Youth Scientists Award Fund Concentrations of Phenol from Saline Wastewater Using Aerobic Granular SBR. Chem. Eng. J., 158, 498–504. (2010BSE09015), Natural Science Foundation of Qingdao (11-2- Sekiguchi, Y.; Kamagata, Y.; Nakamura, K.; Ohashi, A.; Harada, H. (1999) 4-4-(5)-gch), and Chinese National Key Projects of Water Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Pollution Control and Reclamation (2009ZX07424-003). 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