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Novel aerobic granular sludge culture strategy: Using granular sludge Anammox process effluent as a biocatalyst
Bioresource Technology 294 (2019) 122156
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Novel aerobic granular sludge culture strategy: Using granular sludge Anammox process effluent as a biocatalyst
T
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Peng Tanga, Deshuang Yua, Guanghui Chena,b, , Peiyu Zhanga, Xiaoxia Wanga, Chengcheng Liua, Shuo Huanga a b
School of Environmental Science and Engineering, Qingdao University, Qingdao 266071, PR China National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Beijing University of Technology, Beijing 100124, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Aerobic granular sludge Anammox Quorum sensing Signal molecules Community structure
The effluent from granular sludge Anammox process was used as a biocatalyst to improve the culture rate of aerobic granular sludge, and the internal causes of this effect were studied. In this study, in sequencing batch reactor, the formation and changes of AGS was monitored with granular sludge Anammox process effluent added before and after. The community structure of AGS was analyzed by molecular biology methods. The results showed ammonia utilizing rate increased from 3.41 to 5.96 mgNH4+-N/ (g VSS·h), NO2–-N maintained a high accumulation rate, and the denitrification performance remained stable. On 40th day, the diameter of the AGS reached 3.5 mm, and the concentrations of PN and PS reached 330.5 and 62.9 mg/gVSS, respectively. The community structure has changed. Nitrosomonas (31.7%) became the new dominant bacteria. Signal molecules contained in the effluent as inter-species signal molecules could enhance the formation of AGS.
1. Introduction At present, traditional nitrification and denitrification are still recognized as the most economical, effective and promising nitrogen removal processes. aerobic granular sludge (AGS) is a granular microbial aggregate formed by the aggregation and growth of a large number of
⁎
bacteria. Compared with flocculated activated sludge, AGS has advantages in appearance, settlement performance, impact resistance, biomass and structure. However, the formation of aerobic granular sludge is a relatively complex process, subject to many conditions, such as inoculation sludge, substrate composition, organic load, reactor type and mode of operation (Adav and Lee, 2008). Therefore, research on
Corresponding author. E-mail address: [email protected] (G. Chen).
https://doi.org/10.1016/j.biortech.2019.122156 Received 18 August 2019; Received in revised form 12 September 2019; Accepted 14 September 2019 Available online 16 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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provided by CH3COONa, and concentrations were prepared as needed for the study. The trace element comprised the following components (mg/L): 1 FeSO4·7H2O, 0.2 H3BO3, 0.2 MnCl2·4H2O, 0.2 ZnSO4·7H2O, 0.1 CuSO4·5H2O, 0.2 NiCl2·6H2O, 0.3 CoCl2·6H2O.
the formation mechanism of aerobic granular sludge and how to accelerate the cultivation of aerobic granular sludge has become a hot topic in recent years. The mechanism for rapid granulation mainly included the crystal nucleus hypothesis and the selection pressure hypothesis, which have been accepted by many scholars (Lettinga et al., 1980; Zhang et al., 2016). The crystal nucleus hypothesis states that formation of AGS is similar to the crystallization process, and inoculation sludge, inorganic salt precipitation or inert organic material can be used as crystal nucleus for particle growth. Based on crystal nuclei, AGS is finally formed by microbial growth and secretion of EPS. The selection pressure hypothesis can be divided into hydraulic selection pressure and biological selection pressure. The sludge is selected by the hydraulic selection pressure determined by the reactor structure and hydraulic conditions while simultaneously changing the nutrient load to form biological selection pressure on the surviving microorganisms. At present, much research has been done on accelerating the cultivation of aerobic granular sludge. According to the theory of crystal nucleus hypothesis, Long (2014a) inoculated aerobic granules as the nuclei and carriers of the new granules, and cultured stable aerobic particles within 18 days in a pilot scale sequencing batch reactor. AGS was successfully cultivated within 18 days by inoculating with 30% (w/w) mature aerobic granules (Long et al., 2015b). Liu (2018) seeded xanthan gum to enhance the aerobic sludge granulation and increased the cohesion within the sludge. Liu and Tay (2002) observed that a higher H/D ratio could provide a longer circular flowing trajectory, which created an effective hydraulic attrition for microbial aggregation. Lei (2004) studied the relationship between granule formation and settling times (5, 10, 15 and 20 min), and Wang et al. (2013) shortened the settling time and cultured aerobic granular sludge with good activity in 30 days. For AGS coupled with Anammox processes, the effluent from granular sludge Anammox process was accidentally found to affect the granulation process of aerobic sludge. To further investigate this phenomenon, in this study, synthetic wastewater was prepared similar to the effluent, and the traditional culture method for shortening the settling time was compared. By measuring EPS, community structure and other characteristics, the effects of granular sludge Anammox process effluent on the granulation of aerobic sludge were quantitatively clarified, and the mechanism was revealed, providing a simple and reliable way for the cultivation of AGS.
2.1.3. Chemical analysis of water quality All water samples obtained from the SBR reactor were filtered with a 0.45 μm filter membrane and used for water quality measurement. NH4+-N, NO2–-N, NO3–-N and TN were measured according to The National Environmental Protection Standard of the People's Republic (2002) with a UV spectrophotometer (UV-5200). The ammonia utilizing rate (AUR) calculated by the methods of Sánchez (2005) and Yu (2018) was used to characterize the rate of the ammoxidation reaction. A sludge volume index (SVI) and measuring of mixed liquor suspended solids (MLSS) were carried out according to standard methods. pH and dissolved oxygen (DO) were measured with the Multi 3620 IDS meter. COD was measured using the Lianhua Technology COD Rapid Tester.
2.2. Characterization of nitrifying granules 2.2.1. Evaluation of aerobic granular sludge size AGS was observed with an Olympus SZ61 stereomicroscope. When the AGS was small, the diameter was calculated with the eyepiece ruler; when the AGS was larger, the diameters were determined by the wet sieving separation method (Zhang and Jiang, 2019). In a phosphate buffer, the granular sludge mixture was passed through the standard sample sieves of 7, 10, 18, and 35 mesh, and then, the particles trapped on each sieve were collected. The ratio of AGS of the different particle diameter ranges was obtained by measuring the VSS of the AGS on the standard sieve.
2.2.2. Extracellular polymeric substances extraction and High-throughput sequencing analysis EPS in the reactors were extracted according to the procedure of Yang and Li (2009) every three days. In brief, 50 mL of a sludge mixture was dewatered by centrifugation at 4000r/min for 5 min. The supernatant was discarded and the volume was replenished by phosphate buffer. The mixture was extracted by water bath at 80 °C for 30 min, and then centrifuged for 30 min at 5000 r/min. The supernatant was filtered with a 0.22 μm membrane filter, and the filtrate was an aqueous solution containing the EPS. The content was characterized by protein (PN) and polysaccharide (PS). The extracellular proteins (PN) were measured by the Coomassie brilliant blue method (Bradford, 1976) with Albumin form bovine serum (BSA) as the standard; polysaccharides (PS) were measured by the anthrone method (Lever, 1972) with glucose as the standard. DNA extraction quality was determined by 0.8% agarose gel electrophoresis, and DNA was quantified by ultraviolet spectrophotometer. PCR amplification of variable regions (single or continuous multiple) of rRNA genes or specific gene fragments was performed by targeting sequences, such as microbial ribosomal RNA that can reflect the composition and diversity of the bacterial community. PCR amplification products were detected by 2% agarose gel electrophoresis, and the target fragments were cut and recovered. According to the preliminary quantitative results from electrophoresis, the recovered products of PCR amplification were quantified by fluorescence. Using the quantitative results, the samples were mixed according to the corresponding proportion according to the sequencing requirements of each sample. After the sequencing library was prepared, the samples were sequenced by 2 × 300 bp double-terminal sequencing on the MiSeq sequencing machine.
2. Material and methods 2.1. Cultivation of aerobic granular sludge 2.1.1. Reactor configuration and operating conditions The main experiment was completed in the sequencing batch reactor (SBR), as shown in Fig. 1B. The operating cycle of the SBR reactor was 6 h, including influent, aeration, settling, and effluent periods. The seed sludge was obtained from the return sludge of a municipal wastewater facility in Qingdao, China. The experiment was divided into two main stages. In the first stage, the synthetic wastewater was prepared with tap water. The settling time was adjusted every five days, and the flocculated sludge with poor settling performance was washed out of the reactor. In the second stage, the synthetic wastewater was completely prepared with granular sludge Anammox process effluent that stably operated in an UASB reactor (Fig. 1A) in the laboratory (Tang et al., 2019). The influent was synthetic wastewater that prepared with tap water, and the effluent mainly contains trace amounts of NO2–-N and a small amount of NO3–-N. More detailed operation process and operation conditions of the experiment are shown in Table 1 and Fig. 2. 2.1.2. Wastewater composition The components and concentrations for the synthetic wastewater are shown in Table 2. NH4+-N was provided by NH4Cl, COD was 2
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Fig. 1. Configuration of the sequencing batch reactor (SBR) and operating procedures. Table 1 Operation conditions for the SBR over the 40-day operational period.
Table 2 Chemical composition of the synthetic wastewater.
Working volume
pH
Temperature
Airflow rate
Drainage ratio
Component
Concentration (g/L)
Component
Concentration (g/L)
8L
7.5–8.0
32 °C
2 L/min
3/8
CaCl2·2H2O KH2PHO4 MgSO4
0.01 0.02 0.04
NaHCO3 CH3COONa NH4Cl
1.2 – –
3. Results and discussion 3.1. Reactor performances 3.1.1. Ammonia nitrogen removal In the first stage, the influent NH4+-N concentration was maintained at 60 mg/L. With environmental changing, large numbers of cells autolyzed in the initial experiment, causing fluctuations in indicators such as NH4+-N. On the 5th day, the aerobic sludge in the reactor had complete nitrification performance. The influent concentrations of NH4+-N, NO2–-N and NO3–-N (inf NH4+-N, inf NO2–-N, and inf NO3–-N) were 59.3, 6.1 and 13.3 mg/L, respectively, and the effluent concentrations of NH4+-N, NO2–-N and NO3–-N(eff NH4+-N, eff NO2–-N, and eff NO3–-N) were 0.54, 8.38 and 65.9 mg/L, respectively. During the process of shortening the settling time, a large amount of sludge with poor settlement performance was removed from the reactor every five days. On the 7th to 20th days, NH4+-N was completely oxidized, but the eff NO2–-N began to increase, and the eff NO3–-N gradually decreased. On the 20th day, the inf NH4+-N, inf NO2–-N, and inf NO3–N in the reactor were 60.8, 19.8 and 2.2 mg/L, respectively, and the effluent concentrations were 0, 72.3 and 9.4 mg/L, respectively. From the 21st day to the end of the experiment, with the settling time kept at 3 min, synthetic wastewater was prepared granular sludge Anammox process effluent. Fig. 3 shown that the accumulation rate of NO2–-N continued to increase, and the eff NO3–-N was still low. On the 40th
Fig. 3. Variation of NH4+-N, NO2–-N, and NO3–-N during operation.
day, the inf NH4+-N, inf NO2–-N, and inf NO3–-N were 79.3, 19.3 and 4.8 mg/L, respectively, and the effluent concentrations were 0, 87.5 and 8.9 mg/L, respectively. From the 20th to 40th days, the sludge was washed regularly with clean water every seven days to avoid excessive accumulation of NO2–-N.
Fig. 2. Operation process for the SBR over the 40-day operational period. 3
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performance from the reactor. On the 15th day, a small amount of tiny granular sludge with a diameter of 0.3 mm appeared in the reactor. The shape was close to a sphere or an ellipsoid, but the mechanical strength was poor and AGS was easily broken. On the 20th day, a large amount of loosely structured, irregularly shaped AGS appeared in the reactor. The diameter of a small amount of AGS increased to 0.5 mm and had a yellow outer layer and a dark core. From the 21st day, the average diameter of the AGS and granulation rate increased rapidly. On the 25th day, the diameter of the AGS had increased to 0.8–1.0 mm, with a smooth and round appearance. The color of the loose AGS was a uniform pale yellow; the tightly structured AGS had a yellowish outer layer and a brown or greyish white core. during the next 15 days, AGS was further cultivated and matured. On the 30th day, 1.2–1.5 mm granules had obviously increased and eventually became the advantageous range. on the 35th day, the diameter of the AGS was 1.8–2.5 mm. On the 40th days, the largest AGS particle size has reached 3.5 mm and tended to be stable during the following time. It can be intuitively observed that the diameter of the AGS increased rapidly after the addition of the effluent from UASB reactor, and the mature AGS had good elasticity and mechanical strength.
Fig. 4. Variation of COD, MLSS and SVI during operation.
To further compare the nitrification performance of AGS in the first and second stages, water samples on the 20th and 40th day were taken every half hour, and the concentrations of COD, NH4+-N, NO2–-N, NO3–-N and TN were determined. On the 20th day, the inf NH4+-N was 60.7 mg/L. The ammoxidation reaction was completed at 6th hour and AUR was 3.41 mgNH4+-N/(g VSS·h). On the 40th day, the inf NH4+-N was 80.7 mg/L. the ammoxidation reaction was completed at the 3rd hour, and AUR was 5.96 mgNH4+-N/(g VSS·h). In the typical cycle, the overall profile of TN slowly decreased, from 70.2 and 98.5 mg/L to 61.1and 88.90 mg/L, respectively. In comparison, the nitrification performance of AGS on the 40th day was significantly better than on 20th day, but the AGS at the end of the two stages that maintained short-cut nitrification and denitrification performances.
3.2.2. EPS and PN/PS EPS is a macromolecular substance secreted by microorganisms and can change the physicochemical properties of cell surfaces. The main components include proteins, polysaccharides, humic acids and oil. These substances are beneficial for the aggregation of microbial cells and play an important role in the formation and stabilization of granular sludge (Rusanowska et al., 2019). The composition and properties of EPS changed during the granulation process of aerobic sludge (Fig. 5). In the early stage of domestication, the death of a large number of bacteria reduced the content of PS and PN. On the 5th day, PS and PN were 154.9 and 41.6 mg/gVSS, respectively, and PS/PN was 3.72. To adapt to changes in COD and settling time, microorganisms in activated sludge changed corresponding to their physiological and biochemical characteristics (Wang et al., 2019). On the 19th day, PS and PN were 187.7 and 45.7 mg/ gVSS, respectively, and PS/PN was 4.05. On the 21st to 40th days, PS, PN and PS/PN increased rapidly and reached maximum values of 330.5 mg/gVSS, 62.9 mg/gVSS, and 5.25, respectively. At the end of the experiment, the growth rate of PS and PN slowed and stabilized. The growth rate of PS and PN was analyzed in the same way as that of MLSS. During the 5th to 20th days, the growth slopes of PS and PN were 2.86 and 0.39, respectively; during the 21st to 40th days, the growth slopes of PS and PN were 8.25 and 0.98, respectively. By comparing the growth slopes, it was determined that the signal molecules had an appreciable effect on the growth rates of PS and PN. The results indicated that protein played a key role in the granulation of aerobic sludge, and many researchers have determined the same conclusion (Rusanowska et al., 2019; Yan et al., 2015). This trend was consistent with the diameter change of the AGS, and both the PN
3.1.2. COD, MLSS and SVI In the first 5 days, the SBR reactor was in the domestication stage without added CH3COONa. Large amounts of bacteria died with aeration, and sludge with poor settlement performance was washed out of the reactor. During this time, as shown in Fig. 4, MLSS rapidly decreased from 4813 to 2409 mg/L, SVI decreased from 238 to 183 mL/g, and the settlement performance was still poor. From the 6th to 20th days, the COD was controlled at 500 mg/L, and the settling time was continuously adjusted three times. Fine sand-like granular sludge appeared in the reaction on the 20th day, and the sludge was better retained in the reactor. MLSS slowly rose, and on the 20th day, the MLSS was 2965 mg/L. SVI decreased rapidly, from 167 mL/g on 6th day to 101 mL/g on 20th day, and the settlement performance improved significantly. From the 21st day, COD and settling time were maintained, and synthetic wastewater was prepared with the effluent from granular sludge Anammox process. MLSS increased to 4510 mg/L, and the SVI fell to the minimum of 68 mL/g on the 40th day and remained stable. To compare the speed of MLSS growth in the two stages, the data for MLSS during the 5th to 20th and 21st to 40th days were linearly fitted by Origin Pro 2017, and the regression equation was obtained. The slope of the regression line was used to indicate the growth speed of MLSS at different stages. During the 5th to 20th days, the growth slope of MLSS was 36.12; during the 16th to 40th days, the growth slope of MLSS was 85.09. By comparing the growth slopes, it can be determined that the growth speed of MLSS in second stage was faster than in first stage. The results showed that the granular sludge Anammox process effluent effectively enhanced the ability of sludge to coagulate, effectively reducing bacterial loss and accelerating the granulation of AGS. 3.2. Formation of AGS and EPS 3.2.1. Morphology changes during the aerobic sludge granulation process The appearance and diameter of AGS was clearly observed using a stereo microscope. On the 10th day, using the microscope, it was found that the sludge consisted of a large number of fine micelle particles that were connected by filamentous bacteria. The appearance was brown, the structure was loose, and the sedimentation was poor. The shorter settling time washed out flocculated sludge without good settling
Fig. 5. Variations of EPS during sludge granulation. 4
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Fig. 6. Microbial population dynamics of AGS at different levels. A, phylum; B, genus. The range of breakpoint: 15–25.
degrade COD and organic acids (Kim et al., 2013; Li et al., 2018). With the special structural characteristics of granular sludge (aerobic, anoxic, anaerobic zones), anaerobic denitrifying bacteria can exist in the aerobic system. The relative abundance of Thauera (d) decreased from 12.5% to 7.5%, that of Denitratesoma (c) increased from 0.4% to 1.0%, and that of an unidentified_f_Comamonadaceae (b), which can denitrify, increased from 0.1% to 1.2% (Kang et al., 2018). The relative abundance of genus (unidentified_f_PHOS-HE51(g), uncultured_f_Anaerolineaceae (h)) that belong to Polyphosphate accumulating organisms (PAOs) (Baldwin et al., 2015; McIlroy et al., 2017) and other genera (uncultured_f_Cytophagaceae (j), uncultured_f_ODP1230B8 (k), Phaselicystis (m), Sorangium(n)) also changed appreciably. The results of the Alpha diversity analysis are shown in Table 3. The Chao1 and the ACE indices in the Alpha diversity analysis were key indicators for characterizing the richness of the flora: the higher the index value, the higher the richness of the community. Compared with the PN2 sample, the Chao1 index and the ACE index of the PN sample increased (He et al., 2018, Wang et al., 2019). This indicated that the microbial community richness showed an upward trend after 20 days of synthetic wastewater treatment with the high concentration of signal molecules. The Simpson and the Shannon indices were influenced by species richness and uniformity in the sample community and could be used to characterize the diversity of the bacteria: the higher the Simpson index, the larger the diversity of the community; the higher the Shannon index, the greater the complexity of the community. In Table 3, it can be seen that the Shannon index decreased and that the Simpson index decreased synchronously in the PN sample compared with the PN2 sample. This indicated that the diversity of the sludge community structure reduced, and the proportion of dominant bacteria increased after signal molecules were added. When comparing the two samples, the relative abundance of Nitrospirae in the aerobic system was at a low level. In the first stage, with shorter settling times, a large amount of sludge was washed out of the reactor. Since the proliferation rate of AOB is faster than NOB, NOB were gradually eliminated from the system, and AOB became the dominant bacteria . However, in the second stage, only a small amount
content and the PN/PS increased with increasing particle size (Nancharaiah and Kiran Kumar Reddy, 2018). 3.3. High-throughput sequencing analysis For investigating the effects of the granular sludge Anammox process effluent on the AGS community structure, sludge samples were taken on the 20th day (named PN2) and on the 40th day (named PN) and stored at −37 °C. After the sludge was pre-treated, the samples were sequenced on the MiSeq sequencing machine. Significant differences between the two samples in both diversity and relative abundance at the phylum level were observed from the comparison results (Fig. 6A). The main community of the PN2 sample consisted of five phylums: Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Planctomycetes. However, the dominant populations of the PN sample has only three phylums: Proteobacteria, Bacteroidetes, and Chloroflexi. Proteobacteria and Bacteroidetes were one of the largest phylum in the bacterial domain. The species and genetic diversity were very abundant, which determined that the groups covered a wide range of physiological metabolic types (Kang et al., 2018). The relative abundance of Proteobacteria decreased from 65.7% to 55.8% and the relative abundance of Bacteroidetes increased from 6.2% to 31.9% in sample PN2 and PN, respectively, with the increasing or decreasing of other bacteria in relative abundance. To further investigate the changes in community structure at the genus level, the bacteria with relatively large abundance changes or the functional bacteria were plotted as a histogram (Fig. 6B). The relative abundance of Nitrosomonas (a) in the PN sample was 31.7%, which was 28% more than in the PN2 sample. This sequencing result directly explained the different rates of ammonia oxidation in the typical cycles of the 21st and 40th days. In the PN2 and PN samples, the content of NOB (Nitrospira and Nitrobacter) was very low, and the relative abundance of Nitrospira was 0 and 0.1%, respectively; the relative abundance of Nitrobacter was 0.3% and 0, respectively. In modern taxonomic systems, some of the bacteria in Rhodocyclaceae and Rhodospirillaceae belong to purple non-sulfur bacteria. The purple non-sulfur bacteria can use organic matter to carry out the heterotrophic treatment under the conditions of dark aerobic or micro-aerobic conditions. The relative abundances of uncultured_f_Rhodocyclaceae (e) and uncultured_f_Rhodospirillaceae (f) varied greatly from 26.8% and 6.65% to 0.2% and 0, respectively. Conversely, uncultured_f_Saprospiraceae (l) increased from 0.1% to 13.3%, and Chryseolinea (i) increased from 0.1% to 5.4%. They can replace the functions of purple non-sulfur bacteria and
Table 3 Alpha diversity analysis for samples collected at different times.
5
Sample
Chao 1
ACE
Shannon
Simpson
PN2 PN
497.41 521.68
510.57 526.65
5.41 5.04
0.91 0.89
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Table 4 Different cultivation methods of AGS. Major cultivation method
Results of experiment
Refs.
Externally add xanthan gum
Xanthan gum enhanced the aerobic sludge granulation; granulation finished on the 20th day GAC accelerated the granulation, but had no obvious effect on community structure. Completed AGS cultivation on the 30th day Cultivation of AGS was successful with 15 rpm AGS was cultured successfully within 20 days; Nitrosomonas (31.7%) became dominant bacteria.
Liu et al. (2018)
Externally add granular activated carbon (GAC) Continuously reduce settling time Set different rotating speed Use granular sludge Anammox process effluent as a biocatalyst
Tao et al. (2017) Wang et al. (2013) Devlin and Oleszkiewicz (2018) This study
the community structure of AGS changed significantly before and after the addition of signal molecules. The aerobic, anoxic and anaerobic layers affected the community structure of AGS. However, considering the large changes in the community structure, it was speculated that the signal molecules had a major impact on the structural development of diverse groups of bacterial. The high-throughput results indicated that the microbial community richness showed an upward trend, the diversity of the sludge community structure reduced, and the proportion of dominant bacteria increased, after 20 days of synthetic wastewater prepared with the high concentration of signal molecules. Added signal molecules activated the target gene expression and the gene encoding enzymes after binding to a receptor in the cell membrane or cytoplasm (Fuqua and Winans, 1994; Schikora et al., 2016). AI-2, DSF and AHLs could control the specific genes, which sped up the reproduction of bacteria and make them dominant (the relative abundance of Nitrosomonas from 3.7% to 31.7%) but also inhibited the activity of bacteria and eliminated them eliminated (the relative abundance of uncultured_f_Rhodocyclaceae(k) from 26.8% to 6.65%). The methods and results of the latest researchers for AGS culture were summarized (Table 4). They were more based on the theory of the crystal nucleus hypothesis and the selection pressure hypothesis. But substances being added externally to provide crystal nucleus or selection pressure being changed would increase the operating costs or more complex operations to engineering applications. In this study, signal molecules contained in effluent were directly applied to bacteria to promote the secretion of EPS and accelerate the formation of AGS. At the same time, signal molecules can optimize the structure of the flora, make the ammonia oxidizing bacteria become dominant bacteria, and increase the rate of ammonia oxidation. As a self-congregating strain, Anammox bacteria (AnAOB) can produce signal molecules that play a positive role in the granulation of aerobic sludge. Signal molecules are expensive and it is difficult to add signal molecules directly to the aerobic system with considering the costs. The effluent from granular sludge Anammox process contains a high concentration of signal molecules and is easily obtained and used in sewage treatment plants, which has high application value. These findings will have a positive impact on the redesign of the simultaneous partial nitrification/Anammox (SPN/A) process and the coupling of other processes and will have an important effect on the application of Anammox and AGS processes in engineering.
of sludge was washed out of the reactor, but NOB did not recover. It was speculated that some substances contained in the effluent of the granular sludge Anammox process had a greater impact on NOB. 3.4. Effects of signal molecules During growth, the bacteria produce signal molecules, called Autoinducers (AIs) and secrete them into the surrounding environment. Bacteria monitor the number of surrounding bacteria by sensing the concentration of signal molecules. When the population density reaches a certain threshold, the bacteria regulate the expression of specific genes, and this communication between bacteria is called Quorum sensing (QS). The study published by Davies (1998) on the effects of QS on bacterial biofilm formation provided a new perspective on promoting bacterial aggregate growth and reducing loss. Studies have shown that signal molecules can regulate the physiological behavior of bacteria, affect the synthesis of extracellular polymer substances (EPS) and biofilm formation by bacteria (Ren et al., 2010), and affect the micro-ecological composition and function of sludge and the proliferation rate of bacteria, thus affecting the structure and dominance of bacteria (Li et al., 2014). The types of signal molecules found in the current research could be divided into intra- and inter-species signal molecules and universal signal molecules, including Auto-inducer-2 (AI-2), diffusible signal molecules (DSF), and N-acyl-homoserine lactones (AHLs) (Deng et al., 2011). Adding vanillin and porcine kidney acylase I to block the AHL-based QS reduced AHLs, weakened the mechanical strength of the granular sludge and reduced the production of EPS (Lv et al., 2014; Zhao et al., 2016). Tang (2015) confirmed that C6-HSL could increase Anammox bacteria activity and growth rate by conducting a series of batch tests adding three AHLs (C6-HSL, C8-HSL, C12-HSL); Zhang (2019) showed that the settling performance of the granules was improved significantly due to addition of C8-HSL, which contributed to the high TN removal efficiency of the reactor. At present, in water pollution control engineering, the theory of quorum sensing and signal molecules regulating bacterial physiological behavior is widely used in the study of accelerating the granulation of Anammox sludge. However, little research has been done on aerobic nitrifying sludge. It is unclear whether the signal molecules have an influent on the culture of AGS and whether they would affect the community structure of AGS. Numbers studies have confirmed that the supernatant of granular sludge Anammox process effluent contains a variety of signal molecules (Zhao et al., 2018b). Li et al. (2014) used A. tumefaciens NTL4 to prove the existence of AHLs in activated sludge. Based on experimental results and data analysis of MSLL and EPS, it was speculated that the high concentration of signal molecules could induce quorum sensing between aerobic bacteria, promoting the secretion of EPS by microorganisms and microbial attachment in AGS. Amorim (2018) and Wang et al. (2019) also studied the community structure of AGS. Compared with their conclusions, it was found that there was a significant difference in the dominant population and the relative abundance of the same bacteria. This indicated that different culture methods and substrate concentrations had a great impact on the community structure of AGS. Based on the above experimental results,
4. Conclusions The results showed that the effluent of granular sludge Anammox process used as a biocatalyst could accelerate the granulation process of aerobic sludge effectively. After synthetic wastewater prepared with granular sludge Anammox process effluent, the average diameter of the AGS increased rapidly from 0.5 to 3.5 mm within 20 days, and the content of PN and PS increased from 187.7 and 45.7 mg/gVSS to 330.5 and 62.9 mg/gVSS, respectively. Nitrosomonas (31.7%), uncultured_f_Saprospiraceae (13.3%), and unidentified_f_PHOS-HE51 (9.5%) became the new dominant bacteria. The results demonstrated that inter-species signal molecules in effluent could be transmitted and 6
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function between bacteria of different species.
Long, B., Yang, C.Z., Pu, W.H., Yang, J.K., Jiang, G.S., Dan, J.F., Li, C.Y., Liu, F.B., 2014. Rapid cultivation of aerobic granular sludge in a pilot scale sequencing batch reactor. Bioresource Technol. 166 (8), 57–63. Long, B., Yang, C., Pu, W., Yang, J., Liu, F., Zhang, L., Cheng, K., 2015. Rapid cultivation of aerobic granular sludge in a continuous flow reactor. J. Environ. Chem. Eng. 3 (4), 2966–2973. Lv, J., Wang, Y., Zhong, C., Li, Y., Hao, W., Zhu, J., 2014. The effect of quorum sensing and extracellular proteins on the microbial attachment of aerobic granular activated sludge. Bioresource Technol. 152, 53–58. McIlroy, S.J., Kirkegaard, R.H., Dueholm, M.S., Fernando, E., Karst, S.M., Albertsen, M., Nielsen, P.H., 2017. Culture-independent analyses reveal novel anaerolineaceae as abundant primary fermenters in anaerobic digesters treating waste activated sludge. Front. Microbiol.(8). Nancharaiah, Y.V., Kiran Kumar Reddy, G., 2018. Aerobic granular sludge technology: mechanisms of granulation and biotechnological applications. Bioresource Technol. 247, 1128–1143. Ren, T., Yu, H., Li, X., 2010. The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation. Appl. Microbiol. Biot. 88 (3), 789–797. Rusanowska, P., Cydzik-Kwiatkowska, A., Świątczak, P., Wojnowska-Baryła, I., 2019. Changes in extracellular polymeric substances (EPS) content and composition in aerobic granule size-fractions during reactor cycles at different organic loads. Bioresource Technol. 272, 188–193. Sánchez, O., Aspé, E., Martí, M.C., Roeckel, M., 2005. Rate of ammonia oxidation in a synthetic saline wastewater by a nitrifying mixed-culture. J. Chem. Technol. Biotechnol. 80 (11), 1261–1267. Schikora, A., Schenk, S.T., Hartmann, A., 2016. Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group. Plant Mol. Biol. 90 (6), 605–612. Tang, X., Liu, S., Zhang, Z., Zhuang, G., 2015. Identification of the release and effects of AHLs in Anammox culture for bacteria communication. Chem. Eng. J. 273, 184–191. Tang, P., Yu, D., Chen, G., Zhang, P., Wang, X., Lv, T., Liu, C., Huang, S., 2019. Rapid cultivation of anaerobic ammonium oxidation granular sludge and inhibition kinetics of granular sludge (in Chinese). Environ. Sci. 40 (9). Tao, J., Qin, L., Liu, X., Li, B., Chen, J., You, J., Shen, Y., Chen, X., 2017. Effect of granular activated carbon on the aerobic granulation of sludge and its mechanism. Bioresour. Technol. 236, 60–67. The State Environmental Protection Administration. Water and wastewater monitoring and analysis method. 2002. Fourth Ed. Beijing: China Environmental Science Press. Wang, X., Chen, Z., Shen, J., Zhao, X., Kang, J., 2019. Impact of carbon to nitrogen ratio on the performance of aerobic granular reactor and microbial population dynamics during aerobic sludge granulation. Bioresource Technol. 271, 258–265. Wang, C., Li, J., Zhao, B., Wang, Y., Liu, G., 2013. Rapid cultivation of aerobic granular sludge and analysis of sludge characteristics (in Chinese). J. Central South Univers. (Science and Technology) 06, 2623–2628. Yan, L., Liu, Y., Wen, Y., Ren, Y., Hao, G., Zhang, Y., 2015. Role and significance of extracellular polymeric substances from granular sludge for simultaneous removal of organic matter and ammonia nitrogen. Bioresource Technol. 179, 460–466. Yang, S., Li, X., 2009. Influences of extracellular polymeric substances (EPS) on the characteristics of activated sludge under non-steady-state conditions. Process Biochem. 44 (1), 91–96. Yu, L., Li, R., Delatolla, R., Zhang, R., Yang, X., Peng, D., 2018. Natural continuous influent nitrifier immigration effects on nitrification and the microbial community of activated sludge systems. J. Environ. Sci.-China 7, 159–167. Zhang, Q., Hu, J., Lee, D., 2016a. Aerobic granular processes: Current research trends. Bioresource Technol. 210, 74–80. Zhang, W., Jiang, F., 2019. Membrane fouling in aerobic granular sludge (AGS)-membrane bioreactor (MBR): Effect of AGS size. Water Res. 157, 445–453. Zhang, J., Li, J., Zhao, B., Zhang, Y., Wang, X., Chen, G., 2019. Long-term effects of Nacyl-homoserine lactone-based quorum sensing on the characteristics of ANAMMOX granules in high-loaded gnomic for reactors. Chemosphere 218, 632–642. Zhao, R., Zhang, H., Zou, X., Yang, F., 2016. Effects of inhibiting acylated homoserine lactones (AHLs) on anammox activity and stability of granules'. Curr. Microbiol. 73 (1), 108–114. Zhao, R., Zhang, H., Zhang, F., Yang, F., 2018. Fast start-up Anammox process using Acylhomoserine lactones (AHLs) containing supernatant. J. Environ. Sci.-China 65, 127–132.
Acknowledgements The work was supported by the Shandong key research and development program (2019GSF110014), the postdoctoral Science Foundation Project of China (2018M630053), the National Natural Science Foundation of China (51778304) Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122156. References Adav, S.S., Lee, D.J., 2008. Extraction of extracellular polymeric substances from aerobic granule with compact interior structure. J. Hazard. Mater. 154 (1–3), 1120–1126. Amorim, C.L., Alves, M., Castro, P.M.L., Henriques, I., 2018. Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticals. Ecotox. Environ. Safe. 147, 905–912. Baldwin, S.A., Khoshnoodi, M., Rezadehbashi, M., Taupp, M., Hallam, S., Mattes, A., Sanei, H., 2015. The microbial community of a passive biochemical reactor treating arsenic, zinc, and sulfate-rich seepage. Frontiers in Bioengineering and Biotechnology 3 (27). Bradford, M.M., 1976. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (s 1–2), 248–254. Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., Greenberg, E.P., 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280 (5361), 295–298. Deng, Y., Wu, J., Tao, F., Zhang, L., 2011. Listening to a new language: DSF-based quorum sensing in gram-negative bacteria. Chem. Rev. 111 (1), 160–173. Devlin, T.R., Oleszkiewicz, J.A., 2018. Cultivation of aerobic granular sludge in continuous flow under various selective pressure. Bioresource Technol. 253, 281–287. Fuqua, W.C., Winans, S.C., 1994. A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J. Bacteriol. 176 (10), 2796–2806. He, S., Chen, Y., Qin, M., Mao, Z., Yuan, L., Niu, Q., Tan, X., 2018. Effects of temperature on anammox performance and community structure. Bioresource Technol. 260, 186–195. Kang, A.J., Brown, A.K., Wong, C.S., Huang, Z., Yuan, Q., 2018. Variation in bacterial community structure of aerobic granular and suspended activated sludge in the presence of the antibiotic sulfamethoxazole. Bioresource Technol. 261, 322–328. Kim, J., Alkawally, M., Brady, A.L., Rijpstra, W.I.C., Sinninghe Damsté, J.S., Dunfield, P.F., 2013. Chryseolinea serpens gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from soil. Int. J. Syst. Evol. Micr. 63 (Pt 2), 654. Lei, Q., Yu, L., Tay, J.H., 2004. Effect of settling time on aerobic granulation in sequencing batch reactor. Biochem. Eng. J. 21 (1), 47–52. Lettinga, G., Velsen, A.F.M.V., Hobma, S.W., Zeeuw, W.D., Klapwijk, A., 1980. Use of the upflow sludge blanket (usb) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnol. Bioeng. 22 (4), 699–734. Lever, M., 1972. A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47 (1), 273–279. Li, Y., Lv, J., Zhong, C., Hao, W., Wang, Y., Zhu, J., 2014. Performance and role of N-acylhomoserine lactone (AHL)-based quorum sensing (QS) in aerobic granules. J. Environ. Sci. (China) 26 (8), 1615–1621. Li, J., Ye, W., Wei, Q., Wei, D., Ngo, H.H., Guo, W., Qiao, Y., Xu, W., Du, B., 2018. System performance and microbial community succession in a partial nitrification biofilm reactor in response to salinity stress. Bioresource Technol. 270, 512–518. Liu, Y., Tay, J.H., 2002. The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge. Water Res. 36 (7), 1653–1665. Liu, S., Zhan, H., Xie, Y., Shi, W., Wang, S., 2018. Rapid cultivation of aerobic granular sludge by xanthan gum in SBR reactors. Water Sci. Technol. 2017 (2), 360–369.
7
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Novel aerobic granular sludge culture strategy: Using granular sludge Anammox process effluent as a biocatalyst
T
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Peng Tanga, Deshuang Yua, Guanghui Chena,b, , Peiyu Zhanga, Xiaoxia Wanga, Chengcheng Liua, Shuo Huanga a b
School of Environmental Science and Engineering, Qingdao University, Qingdao 266071, PR China National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Beijing University of Technology, Beijing 100124, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Aerobic granular sludge Anammox Quorum sensing Signal molecules Community structure
The effluent from granular sludge Anammox process was used as a biocatalyst to improve the culture rate of aerobic granular sludge, and the internal causes of this effect were studied. In this study, in sequencing batch reactor, the formation and changes of AGS was monitored with granular sludge Anammox process effluent added before and after. The community structure of AGS was analyzed by molecular biology methods. The results showed ammonia utilizing rate increased from 3.41 to 5.96 mgNH4+-N/ (g VSS·h), NO2–-N maintained a high accumulation rate, and the denitrification performance remained stable. On 40th day, the diameter of the AGS reached 3.5 mm, and the concentrations of PN and PS reached 330.5 and 62.9 mg/gVSS, respectively. The community structure has changed. Nitrosomonas (31.7%) became the new dominant bacteria. Signal molecules contained in the effluent as inter-species signal molecules could enhance the formation of AGS.
1. Introduction At present, traditional nitrification and denitrification are still recognized as the most economical, effective and promising nitrogen removal processes. aerobic granular sludge (AGS) is a granular microbial aggregate formed by the aggregation and growth of a large number of
⁎
bacteria. Compared with flocculated activated sludge, AGS has advantages in appearance, settlement performance, impact resistance, biomass and structure. However, the formation of aerobic granular sludge is a relatively complex process, subject to many conditions, such as inoculation sludge, substrate composition, organic load, reactor type and mode of operation (Adav and Lee, 2008). Therefore, research on
Corresponding author. E-mail address: [email protected] (G. Chen).
https://doi.org/10.1016/j.biortech.2019.122156 Received 18 August 2019; Received in revised form 12 September 2019; Accepted 14 September 2019 Available online 16 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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provided by CH3COONa, and concentrations were prepared as needed for the study. The trace element comprised the following components (mg/L): 1 FeSO4·7H2O, 0.2 H3BO3, 0.2 MnCl2·4H2O, 0.2 ZnSO4·7H2O, 0.1 CuSO4·5H2O, 0.2 NiCl2·6H2O, 0.3 CoCl2·6H2O.
the formation mechanism of aerobic granular sludge and how to accelerate the cultivation of aerobic granular sludge has become a hot topic in recent years. The mechanism for rapid granulation mainly included the crystal nucleus hypothesis and the selection pressure hypothesis, which have been accepted by many scholars (Lettinga et al., 1980; Zhang et al., 2016). The crystal nucleus hypothesis states that formation of AGS is similar to the crystallization process, and inoculation sludge, inorganic salt precipitation or inert organic material can be used as crystal nucleus for particle growth. Based on crystal nuclei, AGS is finally formed by microbial growth and secretion of EPS. The selection pressure hypothesis can be divided into hydraulic selection pressure and biological selection pressure. The sludge is selected by the hydraulic selection pressure determined by the reactor structure and hydraulic conditions while simultaneously changing the nutrient load to form biological selection pressure on the surviving microorganisms. At present, much research has been done on accelerating the cultivation of aerobic granular sludge. According to the theory of crystal nucleus hypothesis, Long (2014a) inoculated aerobic granules as the nuclei and carriers of the new granules, and cultured stable aerobic particles within 18 days in a pilot scale sequencing batch reactor. AGS was successfully cultivated within 18 days by inoculating with 30% (w/w) mature aerobic granules (Long et al., 2015b). Liu (2018) seeded xanthan gum to enhance the aerobic sludge granulation and increased the cohesion within the sludge. Liu and Tay (2002) observed that a higher H/D ratio could provide a longer circular flowing trajectory, which created an effective hydraulic attrition for microbial aggregation. Lei (2004) studied the relationship between granule formation and settling times (5, 10, 15 and 20 min), and Wang et al. (2013) shortened the settling time and cultured aerobic granular sludge with good activity in 30 days. For AGS coupled with Anammox processes, the effluent from granular sludge Anammox process was accidentally found to affect the granulation process of aerobic sludge. To further investigate this phenomenon, in this study, synthetic wastewater was prepared similar to the effluent, and the traditional culture method for shortening the settling time was compared. By measuring EPS, community structure and other characteristics, the effects of granular sludge Anammox process effluent on the granulation of aerobic sludge were quantitatively clarified, and the mechanism was revealed, providing a simple and reliable way for the cultivation of AGS.
2.1.3. Chemical analysis of water quality All water samples obtained from the SBR reactor were filtered with a 0.45 μm filter membrane and used for water quality measurement. NH4+-N, NO2–-N, NO3–-N and TN were measured according to The National Environmental Protection Standard of the People's Republic (2002) with a UV spectrophotometer (UV-5200). The ammonia utilizing rate (AUR) calculated by the methods of Sánchez (2005) and Yu (2018) was used to characterize the rate of the ammoxidation reaction. A sludge volume index (SVI) and measuring of mixed liquor suspended solids (MLSS) were carried out according to standard methods. pH and dissolved oxygen (DO) were measured with the Multi 3620 IDS meter. COD was measured using the Lianhua Technology COD Rapid Tester.
2.2. Characterization of nitrifying granules 2.2.1. Evaluation of aerobic granular sludge size AGS was observed with an Olympus SZ61 stereomicroscope. When the AGS was small, the diameter was calculated with the eyepiece ruler; when the AGS was larger, the diameters were determined by the wet sieving separation method (Zhang and Jiang, 2019). In a phosphate buffer, the granular sludge mixture was passed through the standard sample sieves of 7, 10, 18, and 35 mesh, and then, the particles trapped on each sieve were collected. The ratio of AGS of the different particle diameter ranges was obtained by measuring the VSS of the AGS on the standard sieve.
2.2.2. Extracellular polymeric substances extraction and High-throughput sequencing analysis EPS in the reactors were extracted according to the procedure of Yang and Li (2009) every three days. In brief, 50 mL of a sludge mixture was dewatered by centrifugation at 4000r/min for 5 min. The supernatant was discarded and the volume was replenished by phosphate buffer. The mixture was extracted by water bath at 80 °C for 30 min, and then centrifuged for 30 min at 5000 r/min. The supernatant was filtered with a 0.22 μm membrane filter, and the filtrate was an aqueous solution containing the EPS. The content was characterized by protein (PN) and polysaccharide (PS). The extracellular proteins (PN) were measured by the Coomassie brilliant blue method (Bradford, 1976) with Albumin form bovine serum (BSA) as the standard; polysaccharides (PS) were measured by the anthrone method (Lever, 1972) with glucose as the standard. DNA extraction quality was determined by 0.8% agarose gel electrophoresis, and DNA was quantified by ultraviolet spectrophotometer. PCR amplification of variable regions (single or continuous multiple) of rRNA genes or specific gene fragments was performed by targeting sequences, such as microbial ribosomal RNA that can reflect the composition and diversity of the bacterial community. PCR amplification products were detected by 2% agarose gel electrophoresis, and the target fragments were cut and recovered. According to the preliminary quantitative results from electrophoresis, the recovered products of PCR amplification were quantified by fluorescence. Using the quantitative results, the samples were mixed according to the corresponding proportion according to the sequencing requirements of each sample. After the sequencing library was prepared, the samples were sequenced by 2 × 300 bp double-terminal sequencing on the MiSeq sequencing machine.
2. Material and methods 2.1. Cultivation of aerobic granular sludge 2.1.1. Reactor configuration and operating conditions The main experiment was completed in the sequencing batch reactor (SBR), as shown in Fig. 1B. The operating cycle of the SBR reactor was 6 h, including influent, aeration, settling, and effluent periods. The seed sludge was obtained from the return sludge of a municipal wastewater facility in Qingdao, China. The experiment was divided into two main stages. In the first stage, the synthetic wastewater was prepared with tap water. The settling time was adjusted every five days, and the flocculated sludge with poor settling performance was washed out of the reactor. In the second stage, the synthetic wastewater was completely prepared with granular sludge Anammox process effluent that stably operated in an UASB reactor (Fig. 1A) in the laboratory (Tang et al., 2019). The influent was synthetic wastewater that prepared with tap water, and the effluent mainly contains trace amounts of NO2–-N and a small amount of NO3–-N. More detailed operation process and operation conditions of the experiment are shown in Table 1 and Fig. 2. 2.1.2. Wastewater composition The components and concentrations for the synthetic wastewater are shown in Table 2. NH4+-N was provided by NH4Cl, COD was 2
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Fig. 1. Configuration of the sequencing batch reactor (SBR) and operating procedures. Table 1 Operation conditions for the SBR over the 40-day operational period.
Table 2 Chemical composition of the synthetic wastewater.
Working volume
pH
Temperature
Airflow rate
Drainage ratio
Component
Concentration (g/L)
Component
Concentration (g/L)
8L
7.5–8.0
32 °C
2 L/min
3/8
CaCl2·2H2O KH2PHO4 MgSO4
0.01 0.02 0.04
NaHCO3 CH3COONa NH4Cl
1.2 – –
3. Results and discussion 3.1. Reactor performances 3.1.1. Ammonia nitrogen removal In the first stage, the influent NH4+-N concentration was maintained at 60 mg/L. With environmental changing, large numbers of cells autolyzed in the initial experiment, causing fluctuations in indicators such as NH4+-N. On the 5th day, the aerobic sludge in the reactor had complete nitrification performance. The influent concentrations of NH4+-N, NO2–-N and NO3–-N (inf NH4+-N, inf NO2–-N, and inf NO3–-N) were 59.3, 6.1 and 13.3 mg/L, respectively, and the effluent concentrations of NH4+-N, NO2–-N and NO3–-N(eff NH4+-N, eff NO2–-N, and eff NO3–-N) were 0.54, 8.38 and 65.9 mg/L, respectively. During the process of shortening the settling time, a large amount of sludge with poor settlement performance was removed from the reactor every five days. On the 7th to 20th days, NH4+-N was completely oxidized, but the eff NO2–-N began to increase, and the eff NO3–-N gradually decreased. On the 20th day, the inf NH4+-N, inf NO2–-N, and inf NO3–N in the reactor were 60.8, 19.8 and 2.2 mg/L, respectively, and the effluent concentrations were 0, 72.3 and 9.4 mg/L, respectively. From the 21st day to the end of the experiment, with the settling time kept at 3 min, synthetic wastewater was prepared granular sludge Anammox process effluent. Fig. 3 shown that the accumulation rate of NO2–-N continued to increase, and the eff NO3–-N was still low. On the 40th
Fig. 3. Variation of NH4+-N, NO2–-N, and NO3–-N during operation.
day, the inf NH4+-N, inf NO2–-N, and inf NO3–-N were 79.3, 19.3 and 4.8 mg/L, respectively, and the effluent concentrations were 0, 87.5 and 8.9 mg/L, respectively. From the 20th to 40th days, the sludge was washed regularly with clean water every seven days to avoid excessive accumulation of NO2–-N.
Fig. 2. Operation process for the SBR over the 40-day operational period. 3
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performance from the reactor. On the 15th day, a small amount of tiny granular sludge with a diameter of 0.3 mm appeared in the reactor. The shape was close to a sphere or an ellipsoid, but the mechanical strength was poor and AGS was easily broken. On the 20th day, a large amount of loosely structured, irregularly shaped AGS appeared in the reactor. The diameter of a small amount of AGS increased to 0.5 mm and had a yellow outer layer and a dark core. From the 21st day, the average diameter of the AGS and granulation rate increased rapidly. On the 25th day, the diameter of the AGS had increased to 0.8–1.0 mm, with a smooth and round appearance. The color of the loose AGS was a uniform pale yellow; the tightly structured AGS had a yellowish outer layer and a brown or greyish white core. during the next 15 days, AGS was further cultivated and matured. On the 30th day, 1.2–1.5 mm granules had obviously increased and eventually became the advantageous range. on the 35th day, the diameter of the AGS was 1.8–2.5 mm. On the 40th days, the largest AGS particle size has reached 3.5 mm and tended to be stable during the following time. It can be intuitively observed that the diameter of the AGS increased rapidly after the addition of the effluent from UASB reactor, and the mature AGS had good elasticity and mechanical strength.
Fig. 4. Variation of COD, MLSS and SVI during operation.
To further compare the nitrification performance of AGS in the first and second stages, water samples on the 20th and 40th day were taken every half hour, and the concentrations of COD, NH4+-N, NO2–-N, NO3–-N and TN were determined. On the 20th day, the inf NH4+-N was 60.7 mg/L. The ammoxidation reaction was completed at 6th hour and AUR was 3.41 mgNH4+-N/(g VSS·h). On the 40th day, the inf NH4+-N was 80.7 mg/L. the ammoxidation reaction was completed at the 3rd hour, and AUR was 5.96 mgNH4+-N/(g VSS·h). In the typical cycle, the overall profile of TN slowly decreased, from 70.2 and 98.5 mg/L to 61.1and 88.90 mg/L, respectively. In comparison, the nitrification performance of AGS on the 40th day was significantly better than on 20th day, but the AGS at the end of the two stages that maintained short-cut nitrification and denitrification performances.
3.2.2. EPS and PN/PS EPS is a macromolecular substance secreted by microorganisms and can change the physicochemical properties of cell surfaces. The main components include proteins, polysaccharides, humic acids and oil. These substances are beneficial for the aggregation of microbial cells and play an important role in the formation and stabilization of granular sludge (Rusanowska et al., 2019). The composition and properties of EPS changed during the granulation process of aerobic sludge (Fig. 5). In the early stage of domestication, the death of a large number of bacteria reduced the content of PS and PN. On the 5th day, PS and PN were 154.9 and 41.6 mg/gVSS, respectively, and PS/PN was 3.72. To adapt to changes in COD and settling time, microorganisms in activated sludge changed corresponding to their physiological and biochemical characteristics (Wang et al., 2019). On the 19th day, PS and PN were 187.7 and 45.7 mg/ gVSS, respectively, and PS/PN was 4.05. On the 21st to 40th days, PS, PN and PS/PN increased rapidly and reached maximum values of 330.5 mg/gVSS, 62.9 mg/gVSS, and 5.25, respectively. At the end of the experiment, the growth rate of PS and PN slowed and stabilized. The growth rate of PS and PN was analyzed in the same way as that of MLSS. During the 5th to 20th days, the growth slopes of PS and PN were 2.86 and 0.39, respectively; during the 21st to 40th days, the growth slopes of PS and PN were 8.25 and 0.98, respectively. By comparing the growth slopes, it was determined that the signal molecules had an appreciable effect on the growth rates of PS and PN. The results indicated that protein played a key role in the granulation of aerobic sludge, and many researchers have determined the same conclusion (Rusanowska et al., 2019; Yan et al., 2015). This trend was consistent with the diameter change of the AGS, and both the PN
3.1.2. COD, MLSS and SVI In the first 5 days, the SBR reactor was in the domestication stage without added CH3COONa. Large amounts of bacteria died with aeration, and sludge with poor settlement performance was washed out of the reactor. During this time, as shown in Fig. 4, MLSS rapidly decreased from 4813 to 2409 mg/L, SVI decreased from 238 to 183 mL/g, and the settlement performance was still poor. From the 6th to 20th days, the COD was controlled at 500 mg/L, and the settling time was continuously adjusted three times. Fine sand-like granular sludge appeared in the reaction on the 20th day, and the sludge was better retained in the reactor. MLSS slowly rose, and on the 20th day, the MLSS was 2965 mg/L. SVI decreased rapidly, from 167 mL/g on 6th day to 101 mL/g on 20th day, and the settlement performance improved significantly. From the 21st day, COD and settling time were maintained, and synthetic wastewater was prepared with the effluent from granular sludge Anammox process. MLSS increased to 4510 mg/L, and the SVI fell to the minimum of 68 mL/g on the 40th day and remained stable. To compare the speed of MLSS growth in the two stages, the data for MLSS during the 5th to 20th and 21st to 40th days were linearly fitted by Origin Pro 2017, and the regression equation was obtained. The slope of the regression line was used to indicate the growth speed of MLSS at different stages. During the 5th to 20th days, the growth slope of MLSS was 36.12; during the 16th to 40th days, the growth slope of MLSS was 85.09. By comparing the growth slopes, it can be determined that the growth speed of MLSS in second stage was faster than in first stage. The results showed that the granular sludge Anammox process effluent effectively enhanced the ability of sludge to coagulate, effectively reducing bacterial loss and accelerating the granulation of AGS. 3.2. Formation of AGS and EPS 3.2.1. Morphology changes during the aerobic sludge granulation process The appearance and diameter of AGS was clearly observed using a stereo microscope. On the 10th day, using the microscope, it was found that the sludge consisted of a large number of fine micelle particles that were connected by filamentous bacteria. The appearance was brown, the structure was loose, and the sedimentation was poor. The shorter settling time washed out flocculated sludge without good settling
Fig. 5. Variations of EPS during sludge granulation. 4
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Fig. 6. Microbial population dynamics of AGS at different levels. A, phylum; B, genus. The range of breakpoint: 15–25.
degrade COD and organic acids (Kim et al., 2013; Li et al., 2018). With the special structural characteristics of granular sludge (aerobic, anoxic, anaerobic zones), anaerobic denitrifying bacteria can exist in the aerobic system. The relative abundance of Thauera (d) decreased from 12.5% to 7.5%, that of Denitratesoma (c) increased from 0.4% to 1.0%, and that of an unidentified_f_Comamonadaceae (b), which can denitrify, increased from 0.1% to 1.2% (Kang et al., 2018). The relative abundance of genus (unidentified_f_PHOS-HE51(g), uncultured_f_Anaerolineaceae (h)) that belong to Polyphosphate accumulating organisms (PAOs) (Baldwin et al., 2015; McIlroy et al., 2017) and other genera (uncultured_f_Cytophagaceae (j), uncultured_f_ODP1230B8 (k), Phaselicystis (m), Sorangium(n)) also changed appreciably. The results of the Alpha diversity analysis are shown in Table 3. The Chao1 and the ACE indices in the Alpha diversity analysis were key indicators for characterizing the richness of the flora: the higher the index value, the higher the richness of the community. Compared with the PN2 sample, the Chao1 index and the ACE index of the PN sample increased (He et al., 2018, Wang et al., 2019). This indicated that the microbial community richness showed an upward trend after 20 days of synthetic wastewater treatment with the high concentration of signal molecules. The Simpson and the Shannon indices were influenced by species richness and uniformity in the sample community and could be used to characterize the diversity of the bacteria: the higher the Simpson index, the larger the diversity of the community; the higher the Shannon index, the greater the complexity of the community. In Table 3, it can be seen that the Shannon index decreased and that the Simpson index decreased synchronously in the PN sample compared with the PN2 sample. This indicated that the diversity of the sludge community structure reduced, and the proportion of dominant bacteria increased after signal molecules were added. When comparing the two samples, the relative abundance of Nitrospirae in the aerobic system was at a low level. In the first stage, with shorter settling times, a large amount of sludge was washed out of the reactor. Since the proliferation rate of AOB is faster than NOB, NOB were gradually eliminated from the system, and AOB became the dominant bacteria . However, in the second stage, only a small amount
content and the PN/PS increased with increasing particle size (Nancharaiah and Kiran Kumar Reddy, 2018). 3.3. High-throughput sequencing analysis For investigating the effects of the granular sludge Anammox process effluent on the AGS community structure, sludge samples were taken on the 20th day (named PN2) and on the 40th day (named PN) and stored at −37 °C. After the sludge was pre-treated, the samples were sequenced on the MiSeq sequencing machine. Significant differences between the two samples in both diversity and relative abundance at the phylum level were observed from the comparison results (Fig. 6A). The main community of the PN2 sample consisted of five phylums: Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Planctomycetes. However, the dominant populations of the PN sample has only three phylums: Proteobacteria, Bacteroidetes, and Chloroflexi. Proteobacteria and Bacteroidetes were one of the largest phylum in the bacterial domain. The species and genetic diversity were very abundant, which determined that the groups covered a wide range of physiological metabolic types (Kang et al., 2018). The relative abundance of Proteobacteria decreased from 65.7% to 55.8% and the relative abundance of Bacteroidetes increased from 6.2% to 31.9% in sample PN2 and PN, respectively, with the increasing or decreasing of other bacteria in relative abundance. To further investigate the changes in community structure at the genus level, the bacteria with relatively large abundance changes or the functional bacteria were plotted as a histogram (Fig. 6B). The relative abundance of Nitrosomonas (a) in the PN sample was 31.7%, which was 28% more than in the PN2 sample. This sequencing result directly explained the different rates of ammonia oxidation in the typical cycles of the 21st and 40th days. In the PN2 and PN samples, the content of NOB (Nitrospira and Nitrobacter) was very low, and the relative abundance of Nitrospira was 0 and 0.1%, respectively; the relative abundance of Nitrobacter was 0.3% and 0, respectively. In modern taxonomic systems, some of the bacteria in Rhodocyclaceae and Rhodospirillaceae belong to purple non-sulfur bacteria. The purple non-sulfur bacteria can use organic matter to carry out the heterotrophic treatment under the conditions of dark aerobic or micro-aerobic conditions. The relative abundances of uncultured_f_Rhodocyclaceae (e) and uncultured_f_Rhodospirillaceae (f) varied greatly from 26.8% and 6.65% to 0.2% and 0, respectively. Conversely, uncultured_f_Saprospiraceae (l) increased from 0.1% to 13.3%, and Chryseolinea (i) increased from 0.1% to 5.4%. They can replace the functions of purple non-sulfur bacteria and
Table 3 Alpha diversity analysis for samples collected at different times.
5
Sample
Chao 1
ACE
Shannon
Simpson
PN2 PN
497.41 521.68
510.57 526.65
5.41 5.04
0.91 0.89
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Table 4 Different cultivation methods of AGS. Major cultivation method
Results of experiment
Refs.
Externally add xanthan gum
Xanthan gum enhanced the aerobic sludge granulation; granulation finished on the 20th day GAC accelerated the granulation, but had no obvious effect on community structure. Completed AGS cultivation on the 30th day Cultivation of AGS was successful with 15 rpm AGS was cultured successfully within 20 days; Nitrosomonas (31.7%) became dominant bacteria.
Liu et al. (2018)
Externally add granular activated carbon (GAC) Continuously reduce settling time Set different rotating speed Use granular sludge Anammox process effluent as a biocatalyst
Tao et al. (2017) Wang et al. (2013) Devlin and Oleszkiewicz (2018) This study
the community structure of AGS changed significantly before and after the addition of signal molecules. The aerobic, anoxic and anaerobic layers affected the community structure of AGS. However, considering the large changes in the community structure, it was speculated that the signal molecules had a major impact on the structural development of diverse groups of bacterial. The high-throughput results indicated that the microbial community richness showed an upward trend, the diversity of the sludge community structure reduced, and the proportion of dominant bacteria increased, after 20 days of synthetic wastewater prepared with the high concentration of signal molecules. Added signal molecules activated the target gene expression and the gene encoding enzymes after binding to a receptor in the cell membrane or cytoplasm (Fuqua and Winans, 1994; Schikora et al., 2016). AI-2, DSF and AHLs could control the specific genes, which sped up the reproduction of bacteria and make them dominant (the relative abundance of Nitrosomonas from 3.7% to 31.7%) but also inhibited the activity of bacteria and eliminated them eliminated (the relative abundance of uncultured_f_Rhodocyclaceae(k) from 26.8% to 6.65%). The methods and results of the latest researchers for AGS culture were summarized (Table 4). They were more based on the theory of the crystal nucleus hypothesis and the selection pressure hypothesis. But substances being added externally to provide crystal nucleus or selection pressure being changed would increase the operating costs or more complex operations to engineering applications. In this study, signal molecules contained in effluent were directly applied to bacteria to promote the secretion of EPS and accelerate the formation of AGS. At the same time, signal molecules can optimize the structure of the flora, make the ammonia oxidizing bacteria become dominant bacteria, and increase the rate of ammonia oxidation. As a self-congregating strain, Anammox bacteria (AnAOB) can produce signal molecules that play a positive role in the granulation of aerobic sludge. Signal molecules are expensive and it is difficult to add signal molecules directly to the aerobic system with considering the costs. The effluent from granular sludge Anammox process contains a high concentration of signal molecules and is easily obtained and used in sewage treatment plants, which has high application value. These findings will have a positive impact on the redesign of the simultaneous partial nitrification/Anammox (SPN/A) process and the coupling of other processes and will have an important effect on the application of Anammox and AGS processes in engineering.
of sludge was washed out of the reactor, but NOB did not recover. It was speculated that some substances contained in the effluent of the granular sludge Anammox process had a greater impact on NOB. 3.4. Effects of signal molecules During growth, the bacteria produce signal molecules, called Autoinducers (AIs) and secrete them into the surrounding environment. Bacteria monitor the number of surrounding bacteria by sensing the concentration of signal molecules. When the population density reaches a certain threshold, the bacteria regulate the expression of specific genes, and this communication between bacteria is called Quorum sensing (QS). The study published by Davies (1998) on the effects of QS on bacterial biofilm formation provided a new perspective on promoting bacterial aggregate growth and reducing loss. Studies have shown that signal molecules can regulate the physiological behavior of bacteria, affect the synthesis of extracellular polymer substances (EPS) and biofilm formation by bacteria (Ren et al., 2010), and affect the micro-ecological composition and function of sludge and the proliferation rate of bacteria, thus affecting the structure and dominance of bacteria (Li et al., 2014). The types of signal molecules found in the current research could be divided into intra- and inter-species signal molecules and universal signal molecules, including Auto-inducer-2 (AI-2), diffusible signal molecules (DSF), and N-acyl-homoserine lactones (AHLs) (Deng et al., 2011). Adding vanillin and porcine kidney acylase I to block the AHL-based QS reduced AHLs, weakened the mechanical strength of the granular sludge and reduced the production of EPS (Lv et al., 2014; Zhao et al., 2016). Tang (2015) confirmed that C6-HSL could increase Anammox bacteria activity and growth rate by conducting a series of batch tests adding three AHLs (C6-HSL, C8-HSL, C12-HSL); Zhang (2019) showed that the settling performance of the granules was improved significantly due to addition of C8-HSL, which contributed to the high TN removal efficiency of the reactor. At present, in water pollution control engineering, the theory of quorum sensing and signal molecules regulating bacterial physiological behavior is widely used in the study of accelerating the granulation of Anammox sludge. However, little research has been done on aerobic nitrifying sludge. It is unclear whether the signal molecules have an influent on the culture of AGS and whether they would affect the community structure of AGS. Numbers studies have confirmed that the supernatant of granular sludge Anammox process effluent contains a variety of signal molecules (Zhao et al., 2018b). Li et al. (2014) used A. tumefaciens NTL4 to prove the existence of AHLs in activated sludge. Based on experimental results and data analysis of MSLL and EPS, it was speculated that the high concentration of signal molecules could induce quorum sensing between aerobic bacteria, promoting the secretion of EPS by microorganisms and microbial attachment in AGS. Amorim (2018) and Wang et al. (2019) also studied the community structure of AGS. Compared with their conclusions, it was found that there was a significant difference in the dominant population and the relative abundance of the same bacteria. This indicated that different culture methods and substrate concentrations had a great impact on the community structure of AGS. Based on the above experimental results,
4. Conclusions The results showed that the effluent of granular sludge Anammox process used as a biocatalyst could accelerate the granulation process of aerobic sludge effectively. After synthetic wastewater prepared with granular sludge Anammox process effluent, the average diameter of the AGS increased rapidly from 0.5 to 3.5 mm within 20 days, and the content of PN and PS increased from 187.7 and 45.7 mg/gVSS to 330.5 and 62.9 mg/gVSS, respectively. Nitrosomonas (31.7%), uncultured_f_Saprospiraceae (13.3%), and unidentified_f_PHOS-HE51 (9.5%) became the new dominant bacteria. The results demonstrated that inter-species signal molecules in effluent could be transmitted and 6
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function between bacteria of different species.
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