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Extracellular polymeric substances extraction induced the increased purification performance of percoll density gradient centrifugation for anammox bacteria
Chemical Engineering Journal 287 (2016) 529–536
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Extracellular polymeric substances extraction induced the increased purification performance of percoll density gradient centrifugation for anammox bacteria Zuotao Zhang a,b, Zheng Gong c, Sitong Liu a,b,⇑, Jinren Ni a,b a b c
Department of Environmental Engineering, Peking University, Beijing 100871, China Key Laboratory of Water and Sediment Sciences, Ministry of Education of China, Beijing 100871, China School of Life Science, Liaoning Normal University, South Liushu Street 1, Dalian 116081, China
h i g h l i g h t s PDGC pretreatments by EPS extraction and ultrasonication were evaluated. EPS extraction significantly increased purification performance of PDGC. 95% purification rate achieved without first centrifugation to remove big flocs. Two orders of magnitude higher purified biomass was obtained compared to previous. Involved mechanism was clarified by bacteria aggregating behaviors.
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Article history: Received 11 August 2015 Received in revised form 21 November 2015 Accepted 25 November 2015 Available online 28 November 2015 Keywords: Anammox Nitrogen removal Percoll density gradient centrifugation Extracellular polymeric substances Bacteria isolation
a b s t r a c t Anaerobic ammonium oxidation (anammox) is a novel wastewater treatment process. The intrinsic nature of anammox bacteria makes their traditional isolation from a consortium impossible. This study presents a detailed assessment of different pretreatments for percoll density gradient centrifugation (PDGC) to purify anammox bacteria and proposes that the extraction of extracellular polymeric substances (EPS) could significantly increase the purification performance. The results demonstrate that both EPS extraction using cation exchange resin for 2 h and mild ultrasound at an optimal power of 7.2 kJ were suitable pretreatments to increase the purification efficiency in comparison to PDGC performed directly from an original consortium (optimal conditions identified as 8000 rpm, 30 min). Importantly, the implementation of EPS extraction before ultrasound was verified to have the capacity to further increase the purification efficiency up to 95%, which demonstrates the prominent effects of EPS extraction on the PDGC performance. Compared to the previous report, EPS extraction was applied instead of the first centrifugation prior to PDGC to remove large flocs, which correspondingly increased the purified amounts. To clarify the involved mechanism, the bacteria aggregating behaviors before and after EPS extraction over time were visually tracked using a particle size analyzer. The results of this study help to obtain large amounts of anammox bacteria and elucidate the anammox characteristics and communities in both natural and engineered systems. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Anaerobic ammonium oxidation (anammox) bacteria, which oxidize ammonium to dinitrogen gas using nitrite as the electron ⇑ Corresponding author at: College of Environmental Science and Engineering, Peking University, Yiheyuan Road, No. 5, Haidian District, Beijing 100871, China. Tel./fax: +86 10 62754290. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.cej.2015.11.084 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.
acceptor under anaerobic conditions, have been recognized as important microorganisms for the global nitrogen cycle [1–3]. The anammox process for wastewater treatment (WWT) has also received widespread attention due to its cost effectiveness in the treatment of wastewater with a low C/N ratio and its environmental friendliness [4,5]. Although many research groups around the world have cultivated and enriched anammox bacteria since their discovery, the investigation of these bacteria continues to be hindered by bottlenecks [6–8]. The slow growth rate and strict metabolism
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conditions not only directly limit their wide application in WWT but also result in the difficult isolation of anammox bacteria from the consortium via the traditional spread plate method [1,9]. Moreover, anammox bacteria are reportedly inclined to aggregate into flocculates [10]. These intrinsic characteristics further make the conventional isolation of anammox bacteria very difficult. The isolation of anammox bacteria is very significant, not only to understand their physical or biochemical properties but also to shorten their cultivation period. The selection of a proper seeding sludge has been considered very critical for the start-up of anammox reactors [11–13]. Given the low growth rate of anammox bacteria and the complexities involved in the competition with other bacteria, such as heterotrophic and inert microorganisms, as well as the inhibition by organic compounds or oxidants that emerge during bacteria death, the gradual domestication of seeding sludge to stimulate anammox activity is very timeconsuming [12]. Therefore, the direct isolation of anammox bacteria from seeding sludge has considerable potential to accelerate reactor start-up. The performance of anammox reactor deteriorated under some conditions. Some heterotrophic bacteria reportedly grow and ultimately significantly decrease the anammox percentage in the consortium after a long-term deterioration of anammox reactors [14,15]. In this case, studying the isolation of anammox bacteria helps to avoid unnecessary competition with other bacteria, which are hopefully advances the recovery process. As reported in Nature, Strous et al. [1] applied percoll density gradient centrifugation (PDGC) for the first time to isolate anammox bacteria, which has been confirmed to be a suitable method to purify anammox bacteria. This procedure is mainly based on the difference in the densities formed in different layers for cell deposition [16]. The anammox consortium was initially scattered by mild ultrasound and centrifuged, during which large aggregates were removed and dispersed cells were obtained. The anammox bacteria are sensitive to sonication and single, intact cells were almost absent in the aggregates [17]. Thus, after the first centrifugation, most of the consortium had been removed and only small amounts of dispersed cells remained. Subsequently, PDGC was performed to obtain purified anammox consortium. Although some previous studies mainly focused on the application of PDGC [1,18], little attention had been paid to the different procedures of PDGC and further systemically investigated this separation process for anammox bacteria. The purification efficiencies of PDGC mainly rely on the dispersion degree of cells. The number of dispersed cells positively correlates with the number of pure bacteria and purification efficiencies [16]. However, dispersed cells are not easily obtained in an anammox consortium because these bacteria inherently form flocs [1,10]. Extracellular polymeric substances (EPS) serve as the bond between bacteria and have been considered to play an important role in the formation of flocs [19]. Recent research also demonstrated that the decreased EPS content directly lead to the dispersal of anammox granules [20]. This study attempted to indentify whether EPS extraction from anammox consortium could be used as a part of pretreatment procedure of PDGC. In this study, experiments were carried out to identify the purification performance of PDGC for an anammox consortium. The objectives of this study were as follows: (1) to determine the optimal parameters for different pretreatment procedures; (2) to analyze the effects of pretreatments on the purification efficiency; (3) to clarify the mechanism of EPS extraction to increase the PDGC performance according to the associated conglobating performance of anammox bacteria. Remarks were thereby focused on the contribution to widen the knowledge and application of PDGC for anammox bacteria purification, which would help to further accelerate the investigation of anammox bacteria and the anammox process for WWT.
2. Material and methods 2.1. Percoll density gradient centrifugation Ten percent of the anammox consortium used in this study consisted of the bacterium Ca. Brocadia fulgida [21]. The restored consortium existed as flocs form. This consortium was first washed three times with phosphate buffer solution (PBS). The PBS was composed of 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4 with pH of 7.2. After centrifugation for 5 min at 5000 rpm to remove the supernatant, a mixture of PBS buffer and percoll solution was added at a volume ratio of 3.1: 6.9. The dispersed cells were suspended in the mixture by vortexing, and PDGC was performed subsequently. The optimal centrifugation time and speed were determined with different tests. The anammox cells then appeared as a red band at the lower part of the gradient. This red band was carefully extracted with a syringe needle to yield the purified anammox consortium after being washed in PBS. The purification efficiency and bacterial death percentage were measured via Fluorescence in situ hybridization (FISH) and a Flow cytometer (FCM), respectively. The separation efficiency was analyzed according to the dry weight ratio of the purified consortium to that of the original mixture. The dry weight was determined according to standard methods [22]. 2.2. Mild ultrasound The cells were treated with ultrasound using an ultrasound instrument with a tip diameter of 9.5 nm (Schentz Biotechnology Ltd., China). Twenty milliliters of anammox consortium at a concentration of 0.72 g VSS L 1 was used for ultrasound at ambient temperatures (20–30 °C). The optimized ultrasound time and power were determined based on many trials. 2.3. EPS extraction EPS was extracted from the consortium using the cation exchange resin (CER) method [23–25] en milliliters of anammox consortium was transferred to an extraction beaker with baffles, and the CER was added. The suspension was stirred at 200 rpm and 4 °C. Various extraction times were tested to determine the optimum value. The extracted EPS were then harvested by centrifugation of the suspension, which consisted of CER and consortium, for 1 min at 10,000 rpm. The protein concentration of the EPS solution was determined using the Lowry method. The polysaccharide content was determined by Lowry method [26]. The Zeta electric potential was measured using a Nano-ZS90. 2.4. Size distribution of the anammox consortium The size distributions of the anammox consortium were determined using a Laser Particle Size Analyzer (LPSA, Malvern Instruments Ltd., UK), which can identify particle sizes at a size resolution of 0.02 lm and create a frequency distribution to accurately describe the floc sizes. 2.5. Sample fixation and FISH The consortium to be analyzed was fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and then stored in a mixture of PBS and ethanol at a final ethanol concentration of 50% and 20 °C. Ten microliters of bacteria were withdrawn and placed in the hybridization wells, which were then dehydrated in 50%, 80% and 100% ethanol. Hybridization was performed at 46 °C for 90 min by using 1 lL 16S rRNA-targeted oligonucleotide probe and 9 lL
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hybridization buffer [21]. The probe PLA46 GACTTGCATGCCTAATCC labeled with Cy3 at the 5’ end was applied in this investigation to target Planctomycetes [27]. The formamide concentrations used in the hybridization buffer and the washing buffer were described previously [27,28]. After the hybridization, the samples were stained with DAPI (5 lg mL 1, 10 lL) for 15 min in the dark at 4 °C. The images were obtained with an acquisition system LSM 50 META laser scanning microscope (Zeiss, LSM510 META) controlled by confocal software, v3.2 (Zeiss, Germany). The percentages of anammox bacteria accounting for the total population were obtained with a standard software package delivered with the instrument (version 4.0). For each sample, 10 different fields were selected, and the average value was calculated. 2.6. Bacterial death and live analysis The consortium was first filtered with filtration membranes with pore size of 200 lm especially designed for FCM analysis (Saitaike Ltd., China). The filtrate was harvested and washed three times in 4 °C PBS. The bacteria suspension was subsequently diluted with PBS to 200 lL. Five microliters of Propidium Iodide (PI) (Kaiji Biotechnology Co. Ltd, China) was used as a counter stain to identify the dead bacteria and added to each 100 lL of cell suspension. After incubating at room temperature (20 °C) for 15 min, 400 lL of PBS was added, and the solution was gently mixed. The samples were collected with a FACS Calibur flow cytometer for detection, which was equipped with laser excitation capabilities at 535 nm for PI fluorescence and used at a low flow rate. The percentages of fluorescence-positive cells were then determined using the SUMMIT V4.0 software to reflect the percentages of the dead bacteria. 3. Results and discussion 3.1. Performance of direct PDGC for anammox consortium purification Ten percent of the original consortium consisted of the anammox bacterium Ca. B. fulgida, as identified by FISH analysis (figure was not shown). A preliminary experiment was performed to compare the FISH results by PLA46 and BFU613, which probe was especially designed for B. fulgida [28]. The data show that the detected amounts are the same. Considering avoiding the potential effects produced by the autofluorescent of B. fulgida, we applied PLA46 labeled with Cy3 to detect the target bacteria. B. fulgida could exhibit autofluorescence characteristics when being excitated at light wavelength of 352 nm and 442 nm [28]. In this study, the probe PLA46 was labeled with Cy3, which could appear fluorescence at exciting light wavelength of 515 nm, different from the case of autofluorescence. In order to track the Cy3 dyed PLA46, we only set the excitation wavelength at 515 nm for CLSM analysis. In this way, the autofluorescence of B. fulgida could not affect the results. To isolate anammox bacteria from the consortium, we first explored PDGC without any pretreatment. The purification efficiencies of PDGC, namely the percentages of anammox bacteria in the purified consortium, varied with the centrifugation time and speed (Fig. 1). When the centrifugation speed was set to 1000 rpm, the purification efficiencies remained low (<20%), even when the centrifugation time was extended to 60 min, indicating that the anammox percentages in the purified consortium remained low. These findings suggest that the PDGC performance depends on the formation of different layers with different densities [16]. The low centrifugation speed was not sufficient for the percoll deposition and thus led to poor purification efficiencies. The situation was markedly improved at a centrifugation speed
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of 5000 rpm. Extending the centrifugation time was verified to significantly benefit the purification efficiency, which reached a value of 45% at 60 min. The efficiency was maximized at centrifugation speed of 8000 rpm; the purification efficiency increased to nearly 50% at this speed when the centrifugation time was 30 min. Further extending the time to 60 min resulted in comparatively lower purification efficiencies. Moreover, the purification performance evidently worsened when centrifugation speed was further increased to 10,000 rpm, as shown in Fig. 1. In our case, the optimal PDGC parameters were identified as a centrifugation speed of 8000 rpm and centrifugation time of 30 min. These conditions yielded the highest purification efficiency of 50%, indicating that nearly 50% of the purified consortium consisted of anammox bacteria. The purity of the anammox consortium was visibly increased compared to the original mixture, which consisted of only 10% anammox bacteria. Nevertheless, the purity obtained here was still not satisfactory to meet the requirements to further investigate the anammox characteristics [1]. The second performance of PDGC using the purified consortium and optimal parameters failed to further increase the purification efficiency. Thus, advanced pretreatment and optimization of PDGC still needed to be explored. 3.2. Evaluation of EPS extraction as the potential pretreatment of PDGC The consortium generally tends to aggregate and form flocs, which was the main reason for the low purification efficiencies. The major challenge to increase the PDGC performance lies in obtaining dispersed single cells. EPS refers to the compounds released by and enveloping the bacterial cells, which can adhere to each other and finally result in the aggregation of bacteria [29]. In this experiment, the potential of EPS extraction as a pretreatment of PDGC was investigated. The EPS can be extracted via several methods, among which CER is prominent because it ensures the integrity of cells; thus it was selected in this study [23]. Although 600 rpm was previously applied for EPS extraction by CER, the low velocities of 400 rpm and 200 rpm were also chosen for EPS extraction from activated sludge [24,25,30]. For different kinds of organism, the optimal extraction conditions are slightly discrepant. We have tried the different velocities for EPS extraction. The results showed if 300 rpm was applied, DNA concentration in the extracted EPS solution evidently increased, meaning the obvious bacteria damage occurred. With the aim to obtain anammox bacteria as much as possible after PDGC, we select 200 rpm for EPS extraction by CER. Fig. 2 clearly shows that prolonging the extraction time from 1 to 9 h could significantly increase the amounts of extracted EPS, as indicated by the increased protein and polysaccharide concentrations in the EPS solution. In addition, prolonging the extraction time also varied the Zeta electric potential. Besides Zeta electric potential could reflect the cell aggregation performance, bacteria hydrophobicity may also have relationship with aggregation, based on the assumption of double layer compression in DLVO theory. However, according to the report by Tsuneda et al. [31], the absolute values of Zeta electric potential, rather than hydrophobicity, correlated with the cell aggregation. Thus, in this study, Zeta electric potential was only selected to indicate the change of bacteria aggregation. The increasing absolute value of this electric potential implied that the aggregation performance of the flocs weakened [31], which was regarded as good sign for the dispersion of the consortium to obtain the single cells. Accordingly, the purification efficiencies were verified to be evidently increased as the extraction time was prolonged, which was expected. However, the gradual increase in the bacteria death percentage in the consortium as the extraction time was prolonged was also
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Fig. 1. Performance of direct PDGC for anammox purification. (a–d) show the varied purification efficiencies with the centrifugation time at centrifugation speeds of 1000 rpm, 5000 rpm, 8000 rpm and 10,000 rpm.
Fig. 2. EPS extraction as a potential pretreatment for PDGC. (a) Shows the protein and polysaccharides concentration in the extracted EPS for different extraction times. (b) Shows the Zeta potential of the consortium after EPS extraction. (c) and (d) present the purification efficiency and dead bacteria percentage for different extraction times, respectively.
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noteworthy. Although CER is a mild method of EPS extraction and has been verified to exert little damage on the cells, the destruction of cells could not be completely avoided. The bacteria death percentage especially increased for an extraction time of 3 h. Considering the increased purification efficiency and bacteria death percentage, the optimal extraction time was determined to be 2 h. At these conditions, PDGC yielded an anammox consortium purity of 70% when EPS extraction was applied as a pretreatment, which is much higher than the purity directly obtained by PDGC without any pretreatment. 3.3. Evaluation of mild ultrasound as the potential pretreatment for PDGC As proposed previously [1], the application of mild ultrasound as a pretreatment for PDGC is a good strategy to disperse the flocs into single cells. The ultrasound performance has been acknowledged to be determined by the ultrasound energy, which could be calculated by multiplying the ultrasound time and power. In this part, different ultrasound times in range of 4–28 s were tested at a power of 300 W to determine the optimal value for achieving high purification efficiencies and low cell destruction levels (Fig. 3). The purification efficiencies positively correlated with the ultrasound energy from 1.2 to 7.2 kJ. The highest purification efficiency of 65% was obtained for an ultrasound energy of 7.2 kJ, suggesting that the optimal time was 24 s when the ultrasound power was set to 300 W. The purification efficiency did not markedly increase when the ultrasound energy was increased further, but an abrupt increase in the bacterial death percentage was noticed. The strong ultrasound inevitably harmed the cells and finally led to the death of bacteria in the consortium. Based on the above results, the optimal ultrasound conditions were 24 s and 300 W for an ultrasound energy of 7.2 kJ. This ultrasound energy was higher than that reported in a previous study [1], in which 30 s and 150 W were applied. The main difference between these two procedures (present and previous) is that the first round of centrifugation before PDGC in the previous study, which aimed to remove the main body of the consortium, was not applied in our case. This difference could directly lead to the remarked increase in the amounts of purified bacteria. Irrespective of the first centrifugation, the purity of the anammox consortium also decreased because not all of the flocs were dispersed and some flocs remained in the suspension. 3.4. Evaluation of a combination of EPS extraction and mild ultrasound as pretreatment for PDGC To increase the purification performance of PDGC, EPS extraction and mild ultrasound were applied sequentially as a
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pretreatment to examine their effects on the purification efficiency and bacterial death percentage. Specifically, EPS extraction was first performed to severely weaken the coagulation force of different flocs, followed by the application of mild ultrasound to further disperse the aggregates in an effort to yield as many single, dispersed cells as possible. All of the parameters for PDGC centrifugation, EPS extraction and ultrasound obtained in Sections 3.1–3.3 were adopted in this case. Fig. 4(a–c) visually compare the purification efficiencies, bacterial death percentages and separation efficiencies of PDGC for different pretreatments. Direct PDGC without any pretreatment resulted in a purified consortium that contained only 50% more anammox bacteria than the original. The results further illustrated that the application of EPS extraction and mild ultrasound as a pretreatment significantly enhanced the purification efficiency compared to that of PDGC performed alone. When the EPS extraction was performed for 2 h, the purification efficiency was 70%. This efficiency was 65% when using mild ultrasound at 7.2 kJ. The most attractive result here was a purification efficiency of up to 95% when EPS extraction and mild ultrasound were sequentially applied as a pretreatment, suggesting the higher purification performance of PDGC. These treatments inevitably damaged the bacteria. A comparison of sequential EPS extraction and mild ultrasound indicated a bacterial death percentage of 38%, in contrast to the 5% rate in the original consortium. Both the EPS extraction and ultrasound dispersed aggregates and simultaneously destroyed bacteria [32]. Sole pretreatment EPS extraction (CER, 2 h) or mild ultrasound (7.2 kJ) also resulted in bacteria death, with average rates of 20% and 34%, respectively, which significantly hinders the application of pretreatments for PDGC. However, an examination of the dead bacteria in the purified consortium indicated that these problems were negligible, because PDGC also performed well in separating cell fragments from the whole cells according to their different densities. Fig. 4(d) clearly indicates that the percentage of bacterial death remained low (<10%) in the purified consortium after PDGC. In addition, the separation efficiencies, namely the amounts of the purified consortium that account for the original mixture, decreased when EPS extraction was combined with ultrasound. The percentages were 5% for the direct PDGC, 4% for the sole pretreatment and 3.4% for the combined application of EPS extraction and ultrasound. In current study, the final step of EPS extraction was to harvest EPS after centrifugation at 9000g and 4 °C for 20 min. Ultrasonication was applied after EPS extraction with aim to disperse the flocs formed during centrifugation process. Because EPS extraction induced the changed Zeta potential and aggregation performance, the dispersed cells were not easy to aggregate again. It is highly beneficial to increase PDGC efficiency, which mainly relies on the
Fig. 3. Mild ultrasound as a potential pretreatment for PDGC. (a) and (b) represent the purification efficiency and dead bacteria percentage for different ultrasound powers, respectively.
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Fig. 4. Comparison of PDGC performance by different pretreatments. (a) and (b) show the purification efficiency and dead bacteria percentage after the pretreatment. (c) Shows the dead bacteria percentage in the purified consortium. (d) Shows the separation efficiency.
dispersion degree of cells. We also tried the reverse sequence (ultrasonication applied before EPS extraction). However, because the formed aggregation during the centrifugation process could not be dispersed very well, the PDGC efficiency was much lower. The sequential combination of EPS extraction and ultrasound was identified as a suitable and reliable pretreatment for PDGC in order to achieve high purification efficiency. Ultrasound is well known to help disperse flocs; thus, the potential mechanism by which EPS extraction increases the purification efficiency needs to be further underlined. 3.5. Mechanism of the effects of EPS extraction on centrifugation performance EPS is well known to play important roles in the formation of bacteria aggregates [29]. LPSA was used to analyze the features of aggregated bacteria after the application of ultrasound. Fig. 5 shows the change in the frequency distribution of particle diameters over time when EPS extraction was not applied. The frequency was as high as 15% with a particle diameter of 832 nm soon after ultrasound. This frequency varied significantly over time, being observed at 26% with a particle diameter of 1664 nm after 10 min, 29% with a particle diameter of 3646 nm after 20 min and 35% with a diameter of 7692 nm after 30 min. These data vividly display the change in bacteria aggregation after ultrasound. The mutual adhesion of cells allowed cells to rapidly aggregate again after being dispersed. However, the situation changed dramatically when the EPS extraction was performed prior to ultrasound. The corresponding descriptions of the change in the particle diameters over time are shown in Fig. 6. According to the calculated results, the highest
frequency distribution was identified in a range of 800–1100 nm. Although the bacteria also aggregated after some time, the particle diameters remained low. The frequency distribution after 10 min was highest at 912 nm with a value of 29%. This value increased to 34% after 20 min, and then the highest frequency of 30% appeared at 1062 nm after 30 min. Collectively, Figs. 5 and 6 show that EPS extraction helps to avoid bacteria aggregation and ensures the long-term dispersion of cells, which can reliably and considerably increase the PDGC efficiency. PDGC procedure was first put forward by Strous et al. [1], which contains the approaches of mild ultrasonication, centrifugation to remove big biofilm fragments and PDGC. Other previous works just employed this method to isolate anammox bacteria from the consortium [18,33]. In current study, EPS extraction was applied instead of the first centrifugation to remove big flocs prior to PDGC, which corresponding increased the purified amounts. In the case of anammox consortium applied this study, approximately 1.4% consortium was harvested after PDGC, two orders of magnitude higher in contrast to previous method. Our present results verify that EPS extraction could improve PDGC efficiency. For the next step, the work should be focused on the following two aspects: (1) because different sludge has different properties, PDGC performance under different sludge conditions should be further investigated. (2) To quicken the start-up of anammox reactor, the select of suitable sludge will be very important. The exploration of PDGC procedure in this study with the prominent advantage of large purification amounts could be used as the preliminary treatment before inoculating bacteria to the reactor. In this aspect, we could efficiently develop a new strategy to fast enrich anammox bacteria and effectively overcome the obstacle of the rare anammox bacteria source for its process application.
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Fig. 5. Change in the frequency distribution of consortium diameters over time after ultrasound without EPS extraction.
Fig. 6. Change in the frequency distribution of consortium diameters over time when EPS extraction was performed before ultrasound.
4. Conclusions In summary, the optimum parameters for PDGC were determined, and the different pretreatments for PDGC were compared
to obtain high purification efficiency. When PDGC was performed directly on the original consortium, the optimal centrifugation conditions were determined to be 8000 rpm and 30 min, which yielded a purification efficiency of 50% from a consortium that
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contained only 10% anammox bacteria. A pretreatment of either EPS extraction or mild ultrasound could considerably increase the purification efficiency of PDGC. The optimal conditions for EPS extraction were 2 h duration for the CER method, which yielded a purification efficiency of 70%. The optimal ultrasound power was determined to be 7.2 kJ, which yielded 65% purification efficiency. Importantly, EPS extraction followed by ultrasound was verified to dramatically increase the purification efficiency to approximately 95%, which demonstrated the prominent effects of EPS extraction on the potential increase in the PDGC performance. Finally, the bacteria aggregating behavior was visually described by selectively applying EPS extraction to underline the EPS effects on the increase in the purification efficiency. The results presented here were beneficial to obtain high amounts of purified anammox bacteria and thus provided deep insight in the anammox characteristics and community in both natural and engineered systems. Acknowledgment The authors are grateful to the National Natural Science Foundation of China (Nos. 51308007 and 51478006) for the financial support. References [1] M. Strous, J.A. Fuerst, E.H.M. Kramer, S. Logemann, G. Muyzer, K.T. van de Pas-Schoonen, R. Webb, J.G. Kuenen, M.S.M. Jetten, Missing lithotroph identified as new planctomycete, Nature 400 (1999) 446–449. [2] B.L. Hu, L.D. Shen, X.Y. Xu, P. Zheng, Anaerobic ammonium oxidation (anammox) in different natural ecosystems, Biochem. Soc. Trans. 39 (6) (2011) 1811–1816. [3] M.G. Prokopenko, M.B. Hirst, L. De Brabandere, D.J.P. Lawrence, W.M. Berelson, J. Granger, B.X. Chang, S. Dawson, E.J. Crane III, L. Chong, B. Thamdrup, A. Townsend-small, D.M. Sigman, Nitrogen losses in anoxic marine sediments driven by thioploca–anammox bacterial consortia, Nature 500 (2013) 194– 198. [4] J.G. Kuenen, Anammox bacteria: from discovery to application, Nat. Rev. Microbiol. 6 (2008) 320–326. [5] W.R. Abma, W. Driessen, R. Haarhuis, M.C. van Loosdrecht, Upgrading of sewage treatment plant by sustainable and cost-effective separate treatment of industrial wastewater, Water Sci. Technol. 61 (7) (2010) 1715–1722. [6] M.S.M. Jetten, M. Schmid, K. van de Pas-Schoonen, J. Sinninghe Damste, M. Strous, Anammox organisms: enrichment, cultivation and environmental analysis, Methods Enzymol. 397 (2005) 34–57. [7] Q. Banihani, N. Hadadin, A. Jamrah, Start-up of anaerobic ammonium oxidation (anammox) from conventional return activated sludge in up-flow anaerobic sludge blanket reactor for autotrophic nitrogen removal from wastewater, Jor. J. Civ. Eng. 6 (1) (2012) 17–27. [8] S. Uyanik, O.K. Bekmezci, A. Yurtsever, Strategies for successful anammox enrichment at laboratory scale, Clean – Soil, Air, Water 39 (7) (2011) 653–657. [9] B. Kartal, L. van Niftrik, J.T. Keltjens, H.J. Op den Camp, M.S. Jetten, Anammoxgrowth physiology cell biology and metabolism, Adv. Microb. Physiol. 60 (2012) 211–262. [10] H.F. Lu, P. Zheng, Q.X. Ji, H.T. Zhang, J.Y. Ji, L. Wang, S. Ding, T.T. Chen, J.Q. Zhang, C.J. Tang, J.W. Chen, The structure, density and settlability of anammox granular sludge in high rate reactors, Bioresour. Technol. 123 (2012) 312–317. [11] A. Dapena-Mora, S.W.H. van Hulle, J.L. Campos, R. Mendez, P.A. Vanrolleghem, M.S.M. Jetten, Enrichment of anammox biomass from municipal activated sludge: experimental and modeling results, J. Chem. Technol. Biotechnol. 79 (2004) 1421–1428. [12] N. Chamchoi, S. Nitisoravut, Anammox enrichment from different conventional sludges, Chemosphere 66 (11) (2007) 2225–2232.
[13] B.L. Hu, L.D. Shen, S. Liu, C. Cai, T.T. Chen, B. Kartal, H.R. Harhangi, H.J. Op den Camp, L.P. Lou, X.Y. Xu, P. Zheng, M.S. Jetten, Enrichment of an anammox bacterial community from a flooded paddy soil, Environ. Microbiol. Rep. 6 (3) (2013) 483–489. [14] B. Ni, M. Ruscalleda, B.F. Smets, Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in anammox biofilm, Water Res. 46 (15) (2012) 4645–4652. [15] M.S.I. Mozumder, C. Picioreanu, Effects of heterotrophic growth on autotrophic nitrogen removal in a granular sludge reactor, Environ. Technol. 35 (5–8) (2014) 1027–1037. [16] B.L. Kreamer, J.L. Staecker, N. Sawada, G.L. Sattler, M.T. Hsia, H.C. Pitot, Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations, Vitro Cell. Dev. Biol. 22 (4) (1986) 201– 211. [17] M. Strous, J.G. Kuenen, J.A. Fuerst, M. Wagner, M.S.M. Jetten, The anammox case – A new experimental manifesto for microbiological eco-physiology, Antonie Van Leeuwenhoek 81 (1–4) (2002) 693–702. [18] S. Liu, Z. Gong, F. Yang, H. Zhang, L. Shi, K. Furukawa, Combined process of urea nitrogen removal in anaerobic anammox co-culture reactor, Bioresour. Technol. 99 (6) (2008) 1722–1728. [19] S. Balasubramanian, S. Yan, R.D. Tyagi, R.Y. Surampalli, Extracellular polymeric substances (EPS) producing bacterial strains of municipal wastewater sludge: isolation, molecular identification, EPS characterization and performance for sludge settling and dewatering, Water Res. 44 (7) (2010) 2253–2266. [20] T. Chen, P. Zheng, L.D. Shen, C.J. Tang, S. Ding, Dispersal and control of anammox granular sludge at high substrate concentrations, Biotechnol. Bioprocess Eng. 17 (5) (2012) 1093–1102. [21] S. Liu, Z. Zhang, J. Ni, Effects of Ca2+ on activity restoration of the damaged anammox consortium, Bioresour. Technol. 143 (2013) 315–321. [22] APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Baltimore, MD, 1998. [23] B. Frolund, R. Palmgren, K. Keiding, P.H. Nielsen, Extraction of extracellular polymers from activated sludge using a cation exchange resin, Water Res. 30 (1996) 1749–1758. [24] J. Wawrzynczyk, E. Szewczyk, O. Norrlow, E. Szwajcer Dey, Application of enzymes, sodium, tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge, J. Biotechnol. 130 (2007) 274–281. [25] X. Hou, S. Liu, Z. Zhang, Role of extracellular polymeric substance in determining the high aggregation ability of anammox sludge, Water Res. 75 (2015) 51–62. [26] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [27] A. Neef, R. Amann, H. Schlesner, K.H. Schleifer, Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes, Microbiology 144 (1998) 3257–3266. [28] B. Kartal, L. van Niftrik, J. Rattray, J.L. van de Vossenberg, M.C. Schmid, J. Sinninghe Damsté, M.S.M. Jetten, M. Strous, Candidatus ‘‘Brocadia fulgida”: an autofluorescent anaerobic ammoniumoxidizing bacterium, FEMS Microbiol. Ecol. 63 (1) (2008) 46–55. [29] X.M. Liu, G.P. Sheng, H.W. Luo, F. Zhang, S.J. Yuan, J. Xu, R.J. Zeng, J.G. Wu, H.Q. Yu, Contribution of extracellular polymeric substances (EPS) to the sludge aggregation, Environ. Sci. Technol. 44 (11) (2010) 4355–4360. [30] Y. Zhang, F. Wang, X. Zhu, J. Zeng, Q. Zhao, X. Jiang, Extracellular polymeric substances govern the development of biofilm and mass transfer of polycyclic aromatic hydrocarbons for improved biodegradation, Bioresour. Technol. 193 (2015) 274–280. [31] S. Tsuneda, H. Aikawa, H. Hayashi, A. Yuasa, A. Hirata, Extracellular polymeric substances responsible for bacterial adhesion onto solid surface, FEMS Microbiol. Lett. 223 (2) (2003) 287–292. [32] Z. Liang, W. Li, S. Yang, P. Du, Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge, Chemosphere 81 (5) (2010) 626–632. [33] B. Kartal, J. Rattray, L.A.V. Niftrik, J.V.D. Vossenberg, M.C. Schmid, R.I. Webb, S. Schouten, J.A. Fuerst, J.S. Damsté, M.S.M. Jetten, M. Strous, Candidatus ‘‘Anammoxoglobus propionicus” a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria, Syst. Appl. Microbiol. 30 (1) (2007) 39–49.
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Extracellular polymeric substances extraction induced the increased purification performance of percoll density gradient centrifugation for anammox bacteria Zuotao Zhang a,b, Zheng Gong c, Sitong Liu a,b,⇑, Jinren Ni a,b a b c
Department of Environmental Engineering, Peking University, Beijing 100871, China Key Laboratory of Water and Sediment Sciences, Ministry of Education of China, Beijing 100871, China School of Life Science, Liaoning Normal University, South Liushu Street 1, Dalian 116081, China
h i g h l i g h t s PDGC pretreatments by EPS extraction and ultrasonication were evaluated. EPS extraction significantly increased purification performance of PDGC. 95% purification rate achieved without first centrifugation to remove big flocs. Two orders of magnitude higher purified biomass was obtained compared to previous. Involved mechanism was clarified by bacteria aggregating behaviors.
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Article history: Received 11 August 2015 Received in revised form 21 November 2015 Accepted 25 November 2015 Available online 28 November 2015 Keywords: Anammox Nitrogen removal Percoll density gradient centrifugation Extracellular polymeric substances Bacteria isolation
a b s t r a c t Anaerobic ammonium oxidation (anammox) is a novel wastewater treatment process. The intrinsic nature of anammox bacteria makes their traditional isolation from a consortium impossible. This study presents a detailed assessment of different pretreatments for percoll density gradient centrifugation (PDGC) to purify anammox bacteria and proposes that the extraction of extracellular polymeric substances (EPS) could significantly increase the purification performance. The results demonstrate that both EPS extraction using cation exchange resin for 2 h and mild ultrasound at an optimal power of 7.2 kJ were suitable pretreatments to increase the purification efficiency in comparison to PDGC performed directly from an original consortium (optimal conditions identified as 8000 rpm, 30 min). Importantly, the implementation of EPS extraction before ultrasound was verified to have the capacity to further increase the purification efficiency up to 95%, which demonstrates the prominent effects of EPS extraction on the PDGC performance. Compared to the previous report, EPS extraction was applied instead of the first centrifugation prior to PDGC to remove large flocs, which correspondingly increased the purified amounts. To clarify the involved mechanism, the bacteria aggregating behaviors before and after EPS extraction over time were visually tracked using a particle size analyzer. The results of this study help to obtain large amounts of anammox bacteria and elucidate the anammox characteristics and communities in both natural and engineered systems. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Anaerobic ammonium oxidation (anammox) bacteria, which oxidize ammonium to dinitrogen gas using nitrite as the electron ⇑ Corresponding author at: College of Environmental Science and Engineering, Peking University, Yiheyuan Road, No. 5, Haidian District, Beijing 100871, China. Tel./fax: +86 10 62754290. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.cej.2015.11.084 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.
acceptor under anaerobic conditions, have been recognized as important microorganisms for the global nitrogen cycle [1–3]. The anammox process for wastewater treatment (WWT) has also received widespread attention due to its cost effectiveness in the treatment of wastewater with a low C/N ratio and its environmental friendliness [4,5]. Although many research groups around the world have cultivated and enriched anammox bacteria since their discovery, the investigation of these bacteria continues to be hindered by bottlenecks [6–8]. The slow growth rate and strict metabolism
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conditions not only directly limit their wide application in WWT but also result in the difficult isolation of anammox bacteria from the consortium via the traditional spread plate method [1,9]. Moreover, anammox bacteria are reportedly inclined to aggregate into flocculates [10]. These intrinsic characteristics further make the conventional isolation of anammox bacteria very difficult. The isolation of anammox bacteria is very significant, not only to understand their physical or biochemical properties but also to shorten their cultivation period. The selection of a proper seeding sludge has been considered very critical for the start-up of anammox reactors [11–13]. Given the low growth rate of anammox bacteria and the complexities involved in the competition with other bacteria, such as heterotrophic and inert microorganisms, as well as the inhibition by organic compounds or oxidants that emerge during bacteria death, the gradual domestication of seeding sludge to stimulate anammox activity is very timeconsuming [12]. Therefore, the direct isolation of anammox bacteria from seeding sludge has considerable potential to accelerate reactor start-up. The performance of anammox reactor deteriorated under some conditions. Some heterotrophic bacteria reportedly grow and ultimately significantly decrease the anammox percentage in the consortium after a long-term deterioration of anammox reactors [14,15]. In this case, studying the isolation of anammox bacteria helps to avoid unnecessary competition with other bacteria, which are hopefully advances the recovery process. As reported in Nature, Strous et al. [1] applied percoll density gradient centrifugation (PDGC) for the first time to isolate anammox bacteria, which has been confirmed to be a suitable method to purify anammox bacteria. This procedure is mainly based on the difference in the densities formed in different layers for cell deposition [16]. The anammox consortium was initially scattered by mild ultrasound and centrifuged, during which large aggregates were removed and dispersed cells were obtained. The anammox bacteria are sensitive to sonication and single, intact cells were almost absent in the aggregates [17]. Thus, after the first centrifugation, most of the consortium had been removed and only small amounts of dispersed cells remained. Subsequently, PDGC was performed to obtain purified anammox consortium. Although some previous studies mainly focused on the application of PDGC [1,18], little attention had been paid to the different procedures of PDGC and further systemically investigated this separation process for anammox bacteria. The purification efficiencies of PDGC mainly rely on the dispersion degree of cells. The number of dispersed cells positively correlates with the number of pure bacteria and purification efficiencies [16]. However, dispersed cells are not easily obtained in an anammox consortium because these bacteria inherently form flocs [1,10]. Extracellular polymeric substances (EPS) serve as the bond between bacteria and have been considered to play an important role in the formation of flocs [19]. Recent research also demonstrated that the decreased EPS content directly lead to the dispersal of anammox granules [20]. This study attempted to indentify whether EPS extraction from anammox consortium could be used as a part of pretreatment procedure of PDGC. In this study, experiments were carried out to identify the purification performance of PDGC for an anammox consortium. The objectives of this study were as follows: (1) to determine the optimal parameters for different pretreatment procedures; (2) to analyze the effects of pretreatments on the purification efficiency; (3) to clarify the mechanism of EPS extraction to increase the PDGC performance according to the associated conglobating performance of anammox bacteria. Remarks were thereby focused on the contribution to widen the knowledge and application of PDGC for anammox bacteria purification, which would help to further accelerate the investigation of anammox bacteria and the anammox process for WWT.
2. Material and methods 2.1. Percoll density gradient centrifugation Ten percent of the anammox consortium used in this study consisted of the bacterium Ca. Brocadia fulgida [21]. The restored consortium existed as flocs form. This consortium was first washed three times with phosphate buffer solution (PBS). The PBS was composed of 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4 with pH of 7.2. After centrifugation for 5 min at 5000 rpm to remove the supernatant, a mixture of PBS buffer and percoll solution was added at a volume ratio of 3.1: 6.9. The dispersed cells were suspended in the mixture by vortexing, and PDGC was performed subsequently. The optimal centrifugation time and speed were determined with different tests. The anammox cells then appeared as a red band at the lower part of the gradient. This red band was carefully extracted with a syringe needle to yield the purified anammox consortium after being washed in PBS. The purification efficiency and bacterial death percentage were measured via Fluorescence in situ hybridization (FISH) and a Flow cytometer (FCM), respectively. The separation efficiency was analyzed according to the dry weight ratio of the purified consortium to that of the original mixture. The dry weight was determined according to standard methods [22]. 2.2. Mild ultrasound The cells were treated with ultrasound using an ultrasound instrument with a tip diameter of 9.5 nm (Schentz Biotechnology Ltd., China). Twenty milliliters of anammox consortium at a concentration of 0.72 g VSS L 1 was used for ultrasound at ambient temperatures (20–30 °C). The optimized ultrasound time and power were determined based on many trials. 2.3. EPS extraction EPS was extracted from the consortium using the cation exchange resin (CER) method [23–25] en milliliters of anammox consortium was transferred to an extraction beaker with baffles, and the CER was added. The suspension was stirred at 200 rpm and 4 °C. Various extraction times were tested to determine the optimum value. The extracted EPS were then harvested by centrifugation of the suspension, which consisted of CER and consortium, for 1 min at 10,000 rpm. The protein concentration of the EPS solution was determined using the Lowry method. The polysaccharide content was determined by Lowry method [26]. The Zeta electric potential was measured using a Nano-ZS90. 2.4. Size distribution of the anammox consortium The size distributions of the anammox consortium were determined using a Laser Particle Size Analyzer (LPSA, Malvern Instruments Ltd., UK), which can identify particle sizes at a size resolution of 0.02 lm and create a frequency distribution to accurately describe the floc sizes. 2.5. Sample fixation and FISH The consortium to be analyzed was fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and then stored in a mixture of PBS and ethanol at a final ethanol concentration of 50% and 20 °C. Ten microliters of bacteria were withdrawn and placed in the hybridization wells, which were then dehydrated in 50%, 80% and 100% ethanol. Hybridization was performed at 46 °C for 90 min by using 1 lL 16S rRNA-targeted oligonucleotide probe and 9 lL
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hybridization buffer [21]. The probe PLA46 GACTTGCATGCCTAATCC labeled with Cy3 at the 5’ end was applied in this investigation to target Planctomycetes [27]. The formamide concentrations used in the hybridization buffer and the washing buffer were described previously [27,28]. After the hybridization, the samples were stained with DAPI (5 lg mL 1, 10 lL) for 15 min in the dark at 4 °C. The images were obtained with an acquisition system LSM 50 META laser scanning microscope (Zeiss, LSM510 META) controlled by confocal software, v3.2 (Zeiss, Germany). The percentages of anammox bacteria accounting for the total population were obtained with a standard software package delivered with the instrument (version 4.0). For each sample, 10 different fields were selected, and the average value was calculated. 2.6. Bacterial death and live analysis The consortium was first filtered with filtration membranes with pore size of 200 lm especially designed for FCM analysis (Saitaike Ltd., China). The filtrate was harvested and washed three times in 4 °C PBS. The bacteria suspension was subsequently diluted with PBS to 200 lL. Five microliters of Propidium Iodide (PI) (Kaiji Biotechnology Co. Ltd, China) was used as a counter stain to identify the dead bacteria and added to each 100 lL of cell suspension. After incubating at room temperature (20 °C) for 15 min, 400 lL of PBS was added, and the solution was gently mixed. The samples were collected with a FACS Calibur flow cytometer for detection, which was equipped with laser excitation capabilities at 535 nm for PI fluorescence and used at a low flow rate. The percentages of fluorescence-positive cells were then determined using the SUMMIT V4.0 software to reflect the percentages of the dead bacteria. 3. Results and discussion 3.1. Performance of direct PDGC for anammox consortium purification Ten percent of the original consortium consisted of the anammox bacterium Ca. B. fulgida, as identified by FISH analysis (figure was not shown). A preliminary experiment was performed to compare the FISH results by PLA46 and BFU613, which probe was especially designed for B. fulgida [28]. The data show that the detected amounts are the same. Considering avoiding the potential effects produced by the autofluorescent of B. fulgida, we applied PLA46 labeled with Cy3 to detect the target bacteria. B. fulgida could exhibit autofluorescence characteristics when being excitated at light wavelength of 352 nm and 442 nm [28]. In this study, the probe PLA46 was labeled with Cy3, which could appear fluorescence at exciting light wavelength of 515 nm, different from the case of autofluorescence. In order to track the Cy3 dyed PLA46, we only set the excitation wavelength at 515 nm for CLSM analysis. In this way, the autofluorescence of B. fulgida could not affect the results. To isolate anammox bacteria from the consortium, we first explored PDGC without any pretreatment. The purification efficiencies of PDGC, namely the percentages of anammox bacteria in the purified consortium, varied with the centrifugation time and speed (Fig. 1). When the centrifugation speed was set to 1000 rpm, the purification efficiencies remained low (<20%), even when the centrifugation time was extended to 60 min, indicating that the anammox percentages in the purified consortium remained low. These findings suggest that the PDGC performance depends on the formation of different layers with different densities [16]. The low centrifugation speed was not sufficient for the percoll deposition and thus led to poor purification efficiencies. The situation was markedly improved at a centrifugation speed
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of 5000 rpm. Extending the centrifugation time was verified to significantly benefit the purification efficiency, which reached a value of 45% at 60 min. The efficiency was maximized at centrifugation speed of 8000 rpm; the purification efficiency increased to nearly 50% at this speed when the centrifugation time was 30 min. Further extending the time to 60 min resulted in comparatively lower purification efficiencies. Moreover, the purification performance evidently worsened when centrifugation speed was further increased to 10,000 rpm, as shown in Fig. 1. In our case, the optimal PDGC parameters were identified as a centrifugation speed of 8000 rpm and centrifugation time of 30 min. These conditions yielded the highest purification efficiency of 50%, indicating that nearly 50% of the purified consortium consisted of anammox bacteria. The purity of the anammox consortium was visibly increased compared to the original mixture, which consisted of only 10% anammox bacteria. Nevertheless, the purity obtained here was still not satisfactory to meet the requirements to further investigate the anammox characteristics [1]. The second performance of PDGC using the purified consortium and optimal parameters failed to further increase the purification efficiency. Thus, advanced pretreatment and optimization of PDGC still needed to be explored. 3.2. Evaluation of EPS extraction as the potential pretreatment of PDGC The consortium generally tends to aggregate and form flocs, which was the main reason for the low purification efficiencies. The major challenge to increase the PDGC performance lies in obtaining dispersed single cells. EPS refers to the compounds released by and enveloping the bacterial cells, which can adhere to each other and finally result in the aggregation of bacteria [29]. In this experiment, the potential of EPS extraction as a pretreatment of PDGC was investigated. The EPS can be extracted via several methods, among which CER is prominent because it ensures the integrity of cells; thus it was selected in this study [23]. Although 600 rpm was previously applied for EPS extraction by CER, the low velocities of 400 rpm and 200 rpm were also chosen for EPS extraction from activated sludge [24,25,30]. For different kinds of organism, the optimal extraction conditions are slightly discrepant. We have tried the different velocities for EPS extraction. The results showed if 300 rpm was applied, DNA concentration in the extracted EPS solution evidently increased, meaning the obvious bacteria damage occurred. With the aim to obtain anammox bacteria as much as possible after PDGC, we select 200 rpm for EPS extraction by CER. Fig. 2 clearly shows that prolonging the extraction time from 1 to 9 h could significantly increase the amounts of extracted EPS, as indicated by the increased protein and polysaccharide concentrations in the EPS solution. In addition, prolonging the extraction time also varied the Zeta electric potential. Besides Zeta electric potential could reflect the cell aggregation performance, bacteria hydrophobicity may also have relationship with aggregation, based on the assumption of double layer compression in DLVO theory. However, according to the report by Tsuneda et al. [31], the absolute values of Zeta electric potential, rather than hydrophobicity, correlated with the cell aggregation. Thus, in this study, Zeta electric potential was only selected to indicate the change of bacteria aggregation. The increasing absolute value of this electric potential implied that the aggregation performance of the flocs weakened [31], which was regarded as good sign for the dispersion of the consortium to obtain the single cells. Accordingly, the purification efficiencies were verified to be evidently increased as the extraction time was prolonged, which was expected. However, the gradual increase in the bacteria death percentage in the consortium as the extraction time was prolonged was also
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Fig. 1. Performance of direct PDGC for anammox purification. (a–d) show the varied purification efficiencies with the centrifugation time at centrifugation speeds of 1000 rpm, 5000 rpm, 8000 rpm and 10,000 rpm.
Fig. 2. EPS extraction as a potential pretreatment for PDGC. (a) Shows the protein and polysaccharides concentration in the extracted EPS for different extraction times. (b) Shows the Zeta potential of the consortium after EPS extraction. (c) and (d) present the purification efficiency and dead bacteria percentage for different extraction times, respectively.
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noteworthy. Although CER is a mild method of EPS extraction and has been verified to exert little damage on the cells, the destruction of cells could not be completely avoided. The bacteria death percentage especially increased for an extraction time of 3 h. Considering the increased purification efficiency and bacteria death percentage, the optimal extraction time was determined to be 2 h. At these conditions, PDGC yielded an anammox consortium purity of 70% when EPS extraction was applied as a pretreatment, which is much higher than the purity directly obtained by PDGC without any pretreatment. 3.3. Evaluation of mild ultrasound as the potential pretreatment for PDGC As proposed previously [1], the application of mild ultrasound as a pretreatment for PDGC is a good strategy to disperse the flocs into single cells. The ultrasound performance has been acknowledged to be determined by the ultrasound energy, which could be calculated by multiplying the ultrasound time and power. In this part, different ultrasound times in range of 4–28 s were tested at a power of 300 W to determine the optimal value for achieving high purification efficiencies and low cell destruction levels (Fig. 3). The purification efficiencies positively correlated with the ultrasound energy from 1.2 to 7.2 kJ. The highest purification efficiency of 65% was obtained for an ultrasound energy of 7.2 kJ, suggesting that the optimal time was 24 s when the ultrasound power was set to 300 W. The purification efficiency did not markedly increase when the ultrasound energy was increased further, but an abrupt increase in the bacterial death percentage was noticed. The strong ultrasound inevitably harmed the cells and finally led to the death of bacteria in the consortium. Based on the above results, the optimal ultrasound conditions were 24 s and 300 W for an ultrasound energy of 7.2 kJ. This ultrasound energy was higher than that reported in a previous study [1], in which 30 s and 150 W were applied. The main difference between these two procedures (present and previous) is that the first round of centrifugation before PDGC in the previous study, which aimed to remove the main body of the consortium, was not applied in our case. This difference could directly lead to the remarked increase in the amounts of purified bacteria. Irrespective of the first centrifugation, the purity of the anammox consortium also decreased because not all of the flocs were dispersed and some flocs remained in the suspension. 3.4. Evaluation of a combination of EPS extraction and mild ultrasound as pretreatment for PDGC To increase the purification performance of PDGC, EPS extraction and mild ultrasound were applied sequentially as a
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pretreatment to examine their effects on the purification efficiency and bacterial death percentage. Specifically, EPS extraction was first performed to severely weaken the coagulation force of different flocs, followed by the application of mild ultrasound to further disperse the aggregates in an effort to yield as many single, dispersed cells as possible. All of the parameters for PDGC centrifugation, EPS extraction and ultrasound obtained in Sections 3.1–3.3 were adopted in this case. Fig. 4(a–c) visually compare the purification efficiencies, bacterial death percentages and separation efficiencies of PDGC for different pretreatments. Direct PDGC without any pretreatment resulted in a purified consortium that contained only 50% more anammox bacteria than the original. The results further illustrated that the application of EPS extraction and mild ultrasound as a pretreatment significantly enhanced the purification efficiency compared to that of PDGC performed alone. When the EPS extraction was performed for 2 h, the purification efficiency was 70%. This efficiency was 65% when using mild ultrasound at 7.2 kJ. The most attractive result here was a purification efficiency of up to 95% when EPS extraction and mild ultrasound were sequentially applied as a pretreatment, suggesting the higher purification performance of PDGC. These treatments inevitably damaged the bacteria. A comparison of sequential EPS extraction and mild ultrasound indicated a bacterial death percentage of 38%, in contrast to the 5% rate in the original consortium. Both the EPS extraction and ultrasound dispersed aggregates and simultaneously destroyed bacteria [32]. Sole pretreatment EPS extraction (CER, 2 h) or mild ultrasound (7.2 kJ) also resulted in bacteria death, with average rates of 20% and 34%, respectively, which significantly hinders the application of pretreatments for PDGC. However, an examination of the dead bacteria in the purified consortium indicated that these problems were negligible, because PDGC also performed well in separating cell fragments from the whole cells according to their different densities. Fig. 4(d) clearly indicates that the percentage of bacterial death remained low (<10%) in the purified consortium after PDGC. In addition, the separation efficiencies, namely the amounts of the purified consortium that account for the original mixture, decreased when EPS extraction was combined with ultrasound. The percentages were 5% for the direct PDGC, 4% for the sole pretreatment and 3.4% for the combined application of EPS extraction and ultrasound. In current study, the final step of EPS extraction was to harvest EPS after centrifugation at 9000g and 4 °C for 20 min. Ultrasonication was applied after EPS extraction with aim to disperse the flocs formed during centrifugation process. Because EPS extraction induced the changed Zeta potential and aggregation performance, the dispersed cells were not easy to aggregate again. It is highly beneficial to increase PDGC efficiency, which mainly relies on the
Fig. 3. Mild ultrasound as a potential pretreatment for PDGC. (a) and (b) represent the purification efficiency and dead bacteria percentage for different ultrasound powers, respectively.
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Fig. 4. Comparison of PDGC performance by different pretreatments. (a) and (b) show the purification efficiency and dead bacteria percentage after the pretreatment. (c) Shows the dead bacteria percentage in the purified consortium. (d) Shows the separation efficiency.
dispersion degree of cells. We also tried the reverse sequence (ultrasonication applied before EPS extraction). However, because the formed aggregation during the centrifugation process could not be dispersed very well, the PDGC efficiency was much lower. The sequential combination of EPS extraction and ultrasound was identified as a suitable and reliable pretreatment for PDGC in order to achieve high purification efficiency. Ultrasound is well known to help disperse flocs; thus, the potential mechanism by which EPS extraction increases the purification efficiency needs to be further underlined. 3.5. Mechanism of the effects of EPS extraction on centrifugation performance EPS is well known to play important roles in the formation of bacteria aggregates [29]. LPSA was used to analyze the features of aggregated bacteria after the application of ultrasound. Fig. 5 shows the change in the frequency distribution of particle diameters over time when EPS extraction was not applied. The frequency was as high as 15% with a particle diameter of 832 nm soon after ultrasound. This frequency varied significantly over time, being observed at 26% with a particle diameter of 1664 nm after 10 min, 29% with a particle diameter of 3646 nm after 20 min and 35% with a diameter of 7692 nm after 30 min. These data vividly display the change in bacteria aggregation after ultrasound. The mutual adhesion of cells allowed cells to rapidly aggregate again after being dispersed. However, the situation changed dramatically when the EPS extraction was performed prior to ultrasound. The corresponding descriptions of the change in the particle diameters over time are shown in Fig. 6. According to the calculated results, the highest
frequency distribution was identified in a range of 800–1100 nm. Although the bacteria also aggregated after some time, the particle diameters remained low. The frequency distribution after 10 min was highest at 912 nm with a value of 29%. This value increased to 34% after 20 min, and then the highest frequency of 30% appeared at 1062 nm after 30 min. Collectively, Figs. 5 and 6 show that EPS extraction helps to avoid bacteria aggregation and ensures the long-term dispersion of cells, which can reliably and considerably increase the PDGC efficiency. PDGC procedure was first put forward by Strous et al. [1], which contains the approaches of mild ultrasonication, centrifugation to remove big biofilm fragments and PDGC. Other previous works just employed this method to isolate anammox bacteria from the consortium [18,33]. In current study, EPS extraction was applied instead of the first centrifugation to remove big flocs prior to PDGC, which corresponding increased the purified amounts. In the case of anammox consortium applied this study, approximately 1.4% consortium was harvested after PDGC, two orders of magnitude higher in contrast to previous method. Our present results verify that EPS extraction could improve PDGC efficiency. For the next step, the work should be focused on the following two aspects: (1) because different sludge has different properties, PDGC performance under different sludge conditions should be further investigated. (2) To quicken the start-up of anammox reactor, the select of suitable sludge will be very important. The exploration of PDGC procedure in this study with the prominent advantage of large purification amounts could be used as the preliminary treatment before inoculating bacteria to the reactor. In this aspect, we could efficiently develop a new strategy to fast enrich anammox bacteria and effectively overcome the obstacle of the rare anammox bacteria source for its process application.
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Fig. 5. Change in the frequency distribution of consortium diameters over time after ultrasound without EPS extraction.
Fig. 6. Change in the frequency distribution of consortium diameters over time when EPS extraction was performed before ultrasound.
4. Conclusions In summary, the optimum parameters for PDGC were determined, and the different pretreatments for PDGC were compared
to obtain high purification efficiency. When PDGC was performed directly on the original consortium, the optimal centrifugation conditions were determined to be 8000 rpm and 30 min, which yielded a purification efficiency of 50% from a consortium that
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contained only 10% anammox bacteria. A pretreatment of either EPS extraction or mild ultrasound could considerably increase the purification efficiency of PDGC. The optimal conditions for EPS extraction were 2 h duration for the CER method, which yielded a purification efficiency of 70%. The optimal ultrasound power was determined to be 7.2 kJ, which yielded 65% purification efficiency. Importantly, EPS extraction followed by ultrasound was verified to dramatically increase the purification efficiency to approximately 95%, which demonstrated the prominent effects of EPS extraction on the potential increase in the PDGC performance. Finally, the bacteria aggregating behavior was visually described by selectively applying EPS extraction to underline the EPS effects on the increase in the purification efficiency. The results presented here were beneficial to obtain high amounts of purified anammox bacteria and thus provided deep insight in the anammox characteristics and community in both natural and engineered systems. Acknowledgment The authors are grateful to the National Natural Science Foundation of China (Nos. 51308007 and 51478006) for the financial support. References [1] M. Strous, J.A. Fuerst, E.H.M. Kramer, S. Logemann, G. Muyzer, K.T. van de Pas-Schoonen, R. Webb, J.G. Kuenen, M.S.M. Jetten, Missing lithotroph identified as new planctomycete, Nature 400 (1999) 446–449. [2] B.L. Hu, L.D. Shen, X.Y. Xu, P. Zheng, Anaerobic ammonium oxidation (anammox) in different natural ecosystems, Biochem. Soc. Trans. 39 (6) (2011) 1811–1816. [3] M.G. Prokopenko, M.B. Hirst, L. De Brabandere, D.J.P. Lawrence, W.M. Berelson, J. Granger, B.X. Chang, S. Dawson, E.J. Crane III, L. Chong, B. Thamdrup, A. Townsend-small, D.M. Sigman, Nitrogen losses in anoxic marine sediments driven by thioploca–anammox bacterial consortia, Nature 500 (2013) 194– 198. [4] J.G. Kuenen, Anammox bacteria: from discovery to application, Nat. Rev. Microbiol. 6 (2008) 320–326. [5] W.R. Abma, W. Driessen, R. Haarhuis, M.C. van Loosdrecht, Upgrading of sewage treatment plant by sustainable and cost-effective separate treatment of industrial wastewater, Water Sci. Technol. 61 (7) (2010) 1715–1722. [6] M.S.M. Jetten, M. Schmid, K. van de Pas-Schoonen, J. Sinninghe Damste, M. Strous, Anammox organisms: enrichment, cultivation and environmental analysis, Methods Enzymol. 397 (2005) 34–57. [7] Q. Banihani, N. Hadadin, A. Jamrah, Start-up of anaerobic ammonium oxidation (anammox) from conventional return activated sludge in up-flow anaerobic sludge blanket reactor for autotrophic nitrogen removal from wastewater, Jor. J. Civ. Eng. 6 (1) (2012) 17–27. [8] S. Uyanik, O.K. Bekmezci, A. Yurtsever, Strategies for successful anammox enrichment at laboratory scale, Clean – Soil, Air, Water 39 (7) (2011) 653–657. [9] B. Kartal, L. van Niftrik, J.T. Keltjens, H.J. Op den Camp, M.S. Jetten, Anammoxgrowth physiology cell biology and metabolism, Adv. Microb. Physiol. 60 (2012) 211–262. [10] H.F. Lu, P. Zheng, Q.X. Ji, H.T. Zhang, J.Y. Ji, L. Wang, S. Ding, T.T. Chen, J.Q. Zhang, C.J. Tang, J.W. Chen, The structure, density and settlability of anammox granular sludge in high rate reactors, Bioresour. Technol. 123 (2012) 312–317. [11] A. Dapena-Mora, S.W.H. van Hulle, J.L. Campos, R. Mendez, P.A. Vanrolleghem, M.S.M. Jetten, Enrichment of anammox biomass from municipal activated sludge: experimental and modeling results, J. Chem. Technol. Biotechnol. 79 (2004) 1421–1428. [12] N. Chamchoi, S. Nitisoravut, Anammox enrichment from different conventional sludges, Chemosphere 66 (11) (2007) 2225–2232.
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