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Spectroscopic characterization of dissolved organic matter from sludge solubilization treatment by micro-bubble technology
Ecological Engineering 106 (2017) 94–100
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Spectroscopic characterization of dissolved organic matter from sludge solubilization treatment by micro-bubble technology Lei Wang a,b , Ying-Jun Li c , Ying Xiong d , Xu-Hui Mao a , Lie-Yu Zhang b , Jian-Feng Xu b,e , Wen-Bing Tan b , Jin-Sheng Wang b , Tong-Tong Li b , Bei-Dou Xi a,b,∗ , Di-Hua Wang a,∗∗ a
School of Resource and Environmental Sciences, Wuhan University, Hubei, Wuhan 430079, PR China Groundwater and Environmental System Engineering Innovation Base, State Environmental Protection Key Laboratory of Simulation and Control of Groundwater Pollution, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China c Beijing Vocational College of Agriculture, Beijing 100012, PR China d Key Laboratory of Urban Stormwater System and Water Environment, Beijing University of Civil Engineering and Architecture, Beijing 100044, PR China e School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China b
a r t i c l e
i n f o
Article history: Received 10 November 2016 Received in revised form 13 April 2017 Accepted 20 May 2017 Available online 3 June 2017 Keywords: Micro-bubble Sludge solubilization SEM PCR-DGGE Excitation-emission matrix (EEM) spectra
a b s t r a c t In this study, the micro-bubble were used for the sludge solubilization process. In order to investigate the enhancement of sludge solubilization by micro-bubbles, the performance of the micro-bubble system and a conventional bubble system (the control one) was compared in terms of sludge solubilization. The performance in the micro-bubble reactor was much better than the control one, as a result total suspended solid (TSS) was rapidly decreased to 2325.19 TSS L−1 . To investigate the mechanism of the sludge solubilization, the constituents of dissolved organic matter (DOM) from the sludge solubilization was investigated using fluorescence spectroscopy. Compared to the DOM from the reactor using conventional bubbles, the DOM from the micro-bubbles reactor contained higher protein- and humic-like substances. Moreover, the total integrated fluorescence intensity (TOT) were constantly decreased by 18.37% in the control one and increased by 34.39% in the micro-bubble reactor. The results mean that the sludge were effectively solubilized, and organic molecules were released. Polymerase chain reactionsdenaturing gradient gel electrophoresis (PCR-DGGE) was used to analyse the microbial communities after a micro-bubble process. The dominant bacteria in the micro-bubble reactor were identified as Delftia sp., Sphenophorus levis and Stenotrophomonas sp., which were benefit to the cleavage of refractory organics. Scanning Electron Microscopy (SEM) inspection showed that the sludge after the micro-bubble process presented more pores and channels on the sludge surface; the micro-bubbles was good at forming zoogloea, which was helpful for oxygen and mass transfer. Overall, the results indicate that the micro-bubbles aeration could improve the solubilization of sludge. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction In recent decades, tougher effluent stipulations and the growth of population promote the sewage networks, thus the amount of sewage sludge has rapidly increased for the development of wastewater treatment plants(WWTPs). The treatment and disposal
∗ Corresponding author at: Groundwater and Environmental System Engineering Innovation Base, State Environmental Protection Key Laboratory of Simulation and Control of Groundwater Pollution, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China. ∗∗ Corresponding author. E-mail addresses: [email protected] (B.-D. Xi), [email protected] (D.-H. Wang).
of sewage sludge increase the operational costs of WWTPs in an industrial scale by approximately 50–65% (Wei et al., 2003; Liu et al., 2011), which causes a great challenge for the ecological environment governance (Lloret et al., 2013). For reducing these drawbacks, sludge digestion can be considered as one of the traditional biological techniques for the stabilization of sewage sludge discharged from wastewater treatment plants (Chu et al., 2008; Borowski and Szopa, 2007). Digestion can be performed under either aerobic or anaerobic conditions. The stabilization mechanism for digestion is involved with the degradation of organic components in sludge by metabolism of micro-organisms (Wang et al., 2009; Bernard and Gray, 2000). However, several problems and disadvantages still exist for the typical sludge digestion technologies, such as the large water content, the low biodegradability and a high energy cost. These disadvantages can be overcome on an
http://dx.doi.org/10.1016/j.ecoleng.2017.05.032 0925-8574/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
L. Wang et al. / Ecological Engineering 106 (2017) 94–100
industrial scale by the solubilization treatment of the excess sludge (Wang et al., 2008; Chu et al., 2008); therefore, the micro-bubble technology was introduced to sludge digestion pretreatment by some scholars (Von Gunten, 2003; Kim et al., 2010). Due to high inner pressure, micro-bubbles will shrink and collapse beneath the water surface (Poh et al., 2014; Li et al., 2006). Previous work has shown that free radicals can be generated by the collapse of micro-bubbles without dynamic stimulus (Takahashi et al., 2007). Moreover, hydroxyl radicals with higher standard redox potential not only have shown immediate reactivity with the majority of organic compounds, but also the application of micro-bubble technology to sewage sludge could improve the biodegradability and cell disintegration (Von Gunten, 2003; Kim et al., 2010). Sewage sludge often contains many types of particulate and refractory organic compounds, such as suspended solids, soluble organics with more recalcitrant. Customarily, they are poorly biodegradable and inherently toxic, which makes it difficult to apply traditional biological treatments (Saktaywin et al., 2005; Ge et al., 2011). Some researchers have shown that the application of a micro-bubble collapse technique leads to the decomposition of refractory organic compounds (Li et al., 2009). Although micro-bubble technology has shown the capability of hydrolyzing refractory organic compounds, systematic studies regarding the transformation of organic matter from sewdge sludge during treatment with micro-bubble technology are still needed. The solubilization treatment with micro-bubble technology may lead to fluctuations on the organic constituents and relative biodegradability of the sewage sludge. The total view about fluorescent properties of DOM can be indicated by three-dimensional fluorescence excitation-emission matrix (EEM), and it can be used in structural identiflcation and stability assessment about organic matters (He et al., 2011a). Therefore, to elucidate the mechanism of sludge solubilization using micro-bubbles, the DOM component can be characterized using EEM spectra. Furthermore, identifying the predominant bacteria in the granules and correlating their population dynamics with sludge solubilization need to be further investigated. We used PCR-DGGE amplified 16S ribosomal RNA (rRNA) gene fragments on a full-scale to investigate the distinctive bacterial community in the solubilization treatment with micro-bubble technology. DNA-based molecular biology technique is particularly effective for the characterization of microbial diversity and community structure about sludge samples. Furthermore, microbial response during sludge solubilization treatment were compared and discussed (Li et al., 2008; Zhang et al., 2011). At the same time, we also used SEM to inspect the microbial organisms grown in the sludge and sludge structures to verify the influence of micro-bubbles on granule morphology during sludge granulation (Li et al., 2008). In this study, the performances of the micro-bubble generator and a conventional gas diffuser were compared to investigate the mechanism of sludge solubilization. The objectives of this paper were to observe the characteristic and transformation of the DOM of sewage sludge with micro-bubbles. In addition, the evolution of microbial was also discussed.
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Fig. 1. A schematic diagram of reactor for sludge stabilization process.
250 mm and a working volume of 30 L. In order to investigate the enhancement of sludge solubilization by micro-bubbles, the performance of the micro-bubble system and a conventional bubble system (the control one) was compared in terms of sludge solubilization. Air was fed into the reactor at a flow rate of 0.5 L/min by the micro-bubble generator compared with a conventional gas diffuser. Micro-bubble generator (MG) was made by Benzhou New Technology (Nano Bubbles) Promotion Co. (Chaoyang district, Beijing city, China) which created micro-bubbles using a gyratory accelerator and an ejector. The air was supplied through a titanium porous diffuser positioned at the bottom of the reactor. The MG titanium porous diffuser was cylindrical with a pore size of 45 m. The mean diameter of the bubbles from the micro-bubble system was below 0.42 m and their numerical density exceeded 2.9 × 104 counts/mL. Conventional bubble generator manufactured by Nanjing Jing Lan Environmental protection equipment manufacturing Co., Ltd. (Nanjing city, China). Activated sludge from an anaerobic tank in a continuous WWTP (Shunyi district, Beijing city, China) was used in this experiment. The sludge concentration used in these experiments was about 3000 mg TSS L−1 . 2.2. Sample collection and storage Water samples (50 mL) were collected from the reactors every hour and filtered using 0.45 m Whatman glass fiber filters. Water samples were stored at −20 ◦ C in fridge, and they were used for DOC and Fluorescence spectroscopy. Sludge samples (20 g) were collected from the reactors every hour and maintained in a dark room at −80 ◦ C in fridge. Sludge samples were used for PCR-DGGE and SEM within 72 h. Total DNA was directly extracted from sludge samples and was maintained in a dark room at −80 ◦ C in fridge which was used within 36 h.
2. Materials and methods
2.3. Analytical methods
2.1. Experimental equipment
The sludge samples, which were centrifuged at 4 ◦ C and the rotate speed of 10000 rpm for 10 min, were heated at 105 ◦ C to measure TSS. The dissolved organic carbon (DOC) concentrations of all samples were measured using an Analytik Jena Multi N/C 2100 DOC analyzer (Analytik Jena, Jena, Germany) and were set at approximately 15 mg L−1 for the fluorescence analyses. Fluorescence spectroscopy was measured using a luminescence spectrometer (Hitachi F-7000, Japan) with a sampling interval of 5 nm for both
A schematic diagram of the experimental equipment is provided in Fig. 1. The experimental system includes three parts: the water-bath system, the reactor and the diffuser. The temperature was maintained at 35 ◦ C ± 2.5 ◦ C throughout a thermostated jacket using the water-bath system. A laboratory-scale reactor, which was fabricated by Plexiglas glass, contained an internal diameter of
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Fig. 2. Changes in the TSS and DOC concentrations during the reaction processes.
the excitation and emission monochromators, and the scan speed was set to 2400 nm min−1 during the analysis process Excitationemission matrix (EEM) spectra were obtained by subsequently scanning the emission spectra from 280 to 550 nm with a 5 nm increment as the excitation wavelength increased from 200 nm to 450 nm (He et al., 2011b). SEM was subsequently performed to examine the structure of sludge. The sample was freeze-dried, sputter coated with gold and examined via SEM (JEOL JSM-6390LV, Japan) at an accelerating voltage of 10 kV. The total DNA was extracted using a DNA gel extraction kit (BioTeke Corporation) for 16S rDNA PCR amplification. The total DNA from the sludge samples was used as a template to amplify the V3 variable region in the bacterial 16S rDNA gene via PCR with universal primers 341F-GC (5 -GC-clamp-CCTACGGGAGGCAGCAG-3 ), (5 -ATTACCGCGGCTGCTGG-3 ), and GC-clamp (5 534R CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG-3 ) (Florez and Mayo, 2006). The reclaimed DNA was then used as a template to reamplify the bands with the same pair of primers (341f-534r) under the previously described PCR conditions. These sequences were then compared with similar sequences from the Gen Bank DNA database via BLAST analysis (basic logical alignment search tool, BLAST at NCBI) (Li et al., 2006).
3. Results and discussion 3.1. TSS and DOC The TSS and DOC concentrations of all samples obtained from the two reactors are presented in Fig. 2. The TSS and DOC contents gradually decreased in the control one and reached final values of 2860.1 mg TSS L−1 and 17.4 mg L−1 with increasing reaction time. As reported by He et al., microorganisms only use organic matters that are dissolved in water. As the DOC content of the DOM increased, the biodegradable compounds increased and microorganisms become more active (He et al., 2011a). Therefore, the decreasing of DOC in the control one is probably due to the endogenous metabolism of microorganisms, and some organic compounds were completely oxidized to carbon dioxide. The decline of TSS was relied on better performance of sedimentation in aerobic condition. When sludge was exposured to micro-bubble, the TSS in the micro-bubble reactor rapidly decreased to 2325.19 TSS L−1 , the rate of sludge solubilization was obviously faster than the control one. Whereas the DOC content in the micro-bubble reactor decreased during the first 2 h and then increased from 76.15 mg L−1 to 93.83 mg L−1 . The DOC content at 12 h was much greater, and the only difference between the two reactors depended on the microbubbles. Thus, the increase in DOC concentration was caused by difference in bubble characteristics between the two reactors. As Chu et al. reported, sludge disintegration process can be described as sequential decomposition reactions of the cell destruction, sol-
ubilization and subsequent oxidation of the released organics into carbon dioxide. Micro-bubble could dissolve sludge and the organic substances released from the cells (Chu et al., 2008; Saktaywin et al., 2005). In addition, Jaffé et al. (2004) reported that the change of DOC concentration reflected the increased amount of DOM. Therefore, the uncommon change of the DOC is thought to be connected with the rupture of sludge and the delivery of DOM from supernatan of sludge. The fluorescence spectroscopy was consequently examined to give an insight into the DOM during a micro-bubble process.
3.2. Excitation-emission matrix (EEM) spectra The EEM fluorescence spectra of the sludge from both the control one and the micro-bubble reactor are shown in Fig. 3. Three main peaks, i.e., peak 1 (Ex/Em: 225/340 nm; tyrosine protein-like substances), peak 2 (Ex/Em: 275/340 nm; tryptophan protein-like substances) and peak 3 (Ex/Em: 325/400 nm; humic acid-like substances), were detected in all samples from both the control one and the micro-bubble reactor (He et al., 2011b). It was interesting to note that peak 4 (Ex/Em: 280/400 nm; fulvic acid-like substances) appeared in the micro-bubble reactor at the time of 8 h and its intensity became more and more stronger thereafter. As the reaction process proceeded, the intensities of peaks 1 and 2 in the control one were dramatically reduced (Fig. 3). According to Hudson et al. (2008), peaks 1 and 2 are related to proteinderived and soluble microbial byproduct-like materials. Therefore, the increase in the intensities of peaks 1 and 2 is attributed to the degradation of organic matter as a result of microbial activity, suggesting that a part of the pollutants were removed in the control one. Compared with the peaks 1 and 2, the intensity of peak 3 changed little due to its high degree of humification or maturation. These results suggest that the biological bio-oxidation of humiclike organic matter proceeded slowly, and the humic substances were difficult for the microorganism to be utilized (He et al., 2011b). The above results suggest that some easily biodegradable compounds, such as proteins, were continuously consumed and finally disappeared, leaving only the humic substances in the control one with common bubbles. Compared with protein-like materials, the humic substances were difficult for the microorganisms to use. The changes of fluorescent matter in the micro-bubble reactor were different from that in the control one. Peaks 1 and 2 were remarkably increased in the micro-bubble reactor, and peak 3 increased as the reaction progressed as well. Furthermore, peak 4 suddenly emerged at the ninth hour and continuously arose during the later period, which indicated the formation of fulvic acid-like substances. These results demonstrated that, compared to the control one, more DOM presented in the micro-bubble reactor, which was related to the unique function of the micro-bubbles. It appears that the effective transformation of DOM in sludge was affected by micro-bubble. Similar results were also observed by other schol-
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Fig. 3. Fluorescence spectra of the DOM from the control one (a) and the micro-bubble reactor (b).
ars that organic matters were released with the solubilization of the sludge in the micro-bubble system (Chu et al., 2007). This is in a good agreement with the previous report that the changes
were related to decomposition of dead cells and macromolecular organics (Qu et al., 2012). The results convincingly show that the
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Fig. 6. DGGE lanes of samples from the control one (left) and the micro-bubble reactor (right). The lanes for the two samples were cutted from one profile.
micro-bubble distinctly enhanced the solubilization of sludge, and increased the DOM contents in aqueous phase. As shown in Fig. 4, the total integrated fluorescence intensity (TOT) were constantly decreased by 18.37% in the control one and increased by 34.39% in the micro-bubble reactor. It means that the DOM was degraded in the control one, and new dissolved organic components were supplied in the micro-bubble reactor due to the micro-bubble effect. This phenomenon was consistent with the previous study, which indicated that micro-bubbles could accelerate the formation of hydroxyl radicals and the higher hydroxyl radicals produced in micro-bubble system may contribute to rapid sludge solubilization (Chu et al., 2008). Therefore, it can be concluded that the sludge were effectively solubilized. The Pi,n values of the five EEM regions for the DOM and its corresponding fractions during the sludge solubilization process are shown in Fig. 5. The Pi,n values for Regions I, II and IV in the DOM decreased in the control one as time progressed, whereas those for Regions III and V increased significantly during the process, indicating that a decrease in tyrosine-like and tryptophan-like materials, as well as an increase in humic- and fulvic-like substances in the reactor using conventional bubbles. Hudson et al. (2008) reported that tyrosine-like and tryptophan-like materials are easily biodegraded with regard to humic- and fulvic-like substances. Therefore, with the disappearance of some easily degradable compounds such as aromatic proteins, the remaining refractory compounds gradually dominated the DOM in the control one; these compounds can consequently be used to assess sludge stability, and this quantitative result is consistent with the location of the EEM peaks.
The micro-bubble causes the disarray in Pi,n during the process. The Pi,n of the Regions I, II and IV exhibited a decreasing trend, whereas Pi,n of the Regions III and V were increased with time. Similar with the TSS disintegration, the Regions I, II and IV decreased apparently at 2-h. Protein-like compounds, especially those that are present as free molecules or bound in polypeptide, are unstable and easily utilized by microorganisms. It can be inferred that the simple aromatic proteins and soluble microbial byproduct-like materials in sludge were utilized by microorganisms. Similar results were also observed in the study reported by Jouraiphy et al. (2008). However, the Regions I, II and IV increased at the 4th and the 6th hour. According to Takahashi et al. (2007), free radicals can be generated during the natural micro-bubble shrinkage process. The sludge solubilization process has been suggested as the sequential processes of suspended solids disintegration, solubilization of the solids (cells) and mineralization of the soluble organic matter released from the microbial cells (Ahn et al., 2002). Micro-bubble can destroy the cell walls of sludge and solubilize or oxidize them to organic substances.The sludge was broken down, and the bio-macromolecules was released and transformed into small molecules. As reported by Yan, the Pi,n variation of the microbubble reactor may be caused by a combination of biological and chemical effects, which resulted in the disorders of DOM in different periods (Yan et al., 2009). The Regions III and V were dominated in the micro-bubble reactor until the 8th hour, the conversion of DOM changed as the above trend again.
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Fig. 5. The corresponding fractions of Pi,n values for the DOM from the control one (a) and the micro-bubble reactor (b).
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Fig. 7. SEM images of sludge from the control one (a) and the micro-bubble reactor (b).
3.3. PCR-DGGE To investigate the influence of microorganism on the solubilization of sludge, and to provide a better understanding of the difference between a common bubble and a micro-bubble for the potential use of sludge solubilization, the microbial community of the two samples was analyzed. The PCR-DGGE (Fig. 6) obtained in different reactors could reflect the total bacterial DNA diversification of the sludge during the solubilization process. The number and intensity of migrating bands showed a distinct difference between the control one and the micro-bubble reactor. The biodiversity indexes were 3.36 and 3.91, respectively. To find the specific microbial species caused by the micro-bubble effect, the bands from the two samples were also selected for sequence analysis. In the control one, the specific bands showed maximum homology to Acinetobacter junii, Enterobacter sp. and Bacillus sp. All of these bacteria were suitable for a reactor with aerobic environment. For example, Acinetobacter junii was used as an inoculum to trigger the release of phosphate from activated sludge systems when the cells reached the stationary growth phase (Momba and Cloete, 1996). The present study suggested that Enterobacter sp. and Bacillus sp. were commonly isolated from different sources including activated sludge or sediment (Thirumala et al., 2010; Fang et al., 2010). The DGGE analysis also showed that more complicated genetic diversity were greatly affected by the micro-bubbles. Three strains of Gramnegative bacteria in the micro-bubble reactor were identified as Delftia sp. Sphenophorus levis and Stenotrophomonas sp. were considered as the dominant strains in the samples extracted from the micro-bubble reactor. It was reported that Delftia sp. was responsible for the biodegradation of organic matter Organic matter that was difficult to degrade was first degraded by Delftia sp. and then converted to another smaller compound, which was then further solubilized (Xiao et al., 2009). Zhou et al. (2011) have reported that Stenotrophomonas sp. is capable of degrading toxic organic matter to nontoxic compounds, and the presence of Sphenophorus levis is related to the release of small organic compounds and benefits from the fermentation process. The microbial community structure is the cornerstone of the function and characteristics of a sludge. It was found that bacteria in the micro-bubble reactor could enhance the solubilization of hardto-cleave organic matter compounds. Because microorganisms are the main factor for sludge solubilization, the easily biodegradable compounds in the DOM lead to more microbial diversity and activity, which may be an important mechanism of sludge reduction achieved by micro-bubble technology. We also inferred that the specific functional microbial species selected by the micro-bubbles could enhance the biodegradability and the DOM content of sludge. Thus, the results mentioned above indicated that micro-bubbles could change the microbial community, and the specific strains survived in the micro-bubble reactor were effectively cleaving
refractory organics, which is beneficial for the sludge solubilization process. 3.4. Scanning electron microscopy Cell clusters, pores and extracellular polymeric substances were found in sludge (Zhan et al., 2006). As indicated in the SEM image in (Fig. 7), a large quantity of cocci and rods are observed within in the EPS framework. After continuous aeration for 12 h, the sludge from the control one (Fig. 7a) had a deformed structure. Its surface contained fewer pores and channels, and the zoogloea structure from the sludge was misshapen. In addition, no orbicular cells were observed; all the zoogloea were gathered into a mass. However, in the micro-bubble system, the smaller bubbles, which exist in great amounts, might rupture much faster and produce a higher speed jet, thus the associated mechanical stresses are likely to be higher. As a result, the structure of sludge from the micro-bubble reactor (Fig. 7b) had more pores and channels on the sludge surface. The zoogloea were widely dispersed with complete bacterial structures, demonstrating that the micro-bubbles are form zoogloea well and immobilize microorganisms, which is helpful for oxygen and mass transfer. 4. Conclusions In this study, the micro-bubble were used for the sludge solubilization process and the sludge were effectively solubilized by microbubbles. The spectra characterization analysis showed the total DOM of sewage sludge significantly increased by microbubble, and the component were progressively more available to microorganisms. Moreover, the PCR-DGGE results indicated that micro-bubbles could change the special strains, which were benefit for the cleavage of refractory organics. In addition, detected by SEM, micro-bubbles could form zoogloea well, which is helpful for oxygen and mass transfer. Therefore, the application of microbubble technology was effective at the release of DOM and the growth of microbial, by which the micro-bubble could enhance the treatment of sewage sludge. Currently, micro-bubble technology for sludge reduction mainly includes the enhanced ozonation of the biomass. The use of micro-bubbles can decrease the operating costs and improve the efficiency for sludge solubilization. This artical may provide a suitable way to utilize micro-bubble for sludge solubilization. Acknowledgements This work was kindly supported by the National Sciencetechnology Support Plan Projects of China (2015BAL04B01) and the Fundamental Research Funds for the Central Universities (Wuhan University-Duke initiative project 201604398).
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Liu, S.G., Zhu, N.W., Loretta, Y., 2011. The one-stage autothermal thermophilic aerobic digestion for sewage sludge treatment. Chem. Eng. J. 174, 564–570. Lloret, E., Pastor, L., Pradas, P., Pascual, J.A., 2013. Semi full-scale thermophilic anaerobic digestion (TAnD) for advanced treatment of sewage sludge: stabilization process and pathogen reduction. Chem. Eng. J. 232, 42–50. Momba, M.N.B., Cloete, T.E., 1996. The relationship of biomass to phosphate uptake by Acinetobacter junii in activated sludge mixed liquor. Water Res. 30, 364–370. Poh, P.E., Ong, W.Y.J., Lau, E.V., Chong, M.N., 2014. Investigation on micro-bubble flotation and coagulation for the treatment of anaerobically treated palm oil mill effluent (POME). J. Environ. Chem. Eng. 2, 1174–1181. Qu, F.S., Liang, H., He, J.G., Ma, J., Wang, Z.Z., Yu, H.R., Li, G.B., 2012. Characterization of dissolved extracellular organic matter (dEOM) and bound extracellular organic matter (bEOM) of Microcystis aeruginosa and their impacts on UF membrane fouling. Water Res. 46, 2881–2890. Saktaywin, W., Tsuno, H., Nagare, H., Soyama, T., Weerapakkaroon, J., 2005. Advanced sewage treatment process with excess sludge reduction and phosphorus recovery. Water Res. 39, 902–910. Takahashi, M., Chiba, K., Li, P., 2007. Free-radical generation from collapsing microbubble in the absence of a dynamic stimulus. J. Phys. Chem. B 111, 1343–1347. Thirumala, M., Reddy, S.V., Mahmood, S.K., 2010. Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge. J. Ind. Microbiol. Biotechnol. 37, 271–278. Von Gunten, U., 2003. Ozonation of drinking water: part I. Oxidation kinetics and product formation. Water Res. 37, 1443–1467. Wang, Z.W., Wu, Z.C., Hua, J., Wang, X.H., Du, X.Z., Hua, H., 2008. Application of flatsheet membrane to thickening and digestion of waste activated sludge (WAS). Hazard. Mater. J. 154, 535–542. Wang, X.H., Wu, Z.C., Wang, Z.W., Yin, X., Du, X.Z., 2009. Floc destruction and its impact on dewatering properties in the process of using flat-sheet membrane for simultaneous thickening and digestion of waste activated sludge. Bioresour. Technol. 100, 1937–1942. Wei, Y.S., Van Houten, R.T., Borger, A.R., Eikelboom, D.H., Fan, Y.B., 2003. Minimization of excess sludge production for biological wastewater treatment. Water Res. 37, 4453–4467. Xiao, C., Ning, J., Yan, H., Sun, X., Hu, J., 2009. Biodegradation of aniline by a newly isolated Delftia sp. XYJ6. Chin. J. Chem. Eng. 17, 500–505. Yan, S.T., Chu, L.B., Xing, X.H., Yu, A.F., Sun, X.L., Jurcik, B., 2009. Analysis of the mechanism of sludge ozonation by a combination of biological and chemical approaches. Water Res. 43, 195–203. Zhan, X.M., Rodgers, M., O’Reilly, E., 2006. Biofilm growth and characteristics in an alternating pumpe d sequencing batch biofilm reactor (APSBBR). Water Res. 40, 817–825. Zhang, B., Ji, M., Qiu, Z.G., Liu, H.N., Wang, J.F., Li, J.W., 2011. Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system. Bioresour. Technol. 102, 2474–2480. Zhou, B., Yuan, R., Shi, C., Yu, L., Gu, J., Zhang, C., 2011. Biodegradation of geosmin in drinking water by novel bacteria isolated from biologically active carbon. J. Environ. Sci. (China) 23, 816–823.
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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Spectroscopic characterization of dissolved organic matter from sludge solubilization treatment by micro-bubble technology Lei Wang a,b , Ying-Jun Li c , Ying Xiong d , Xu-Hui Mao a , Lie-Yu Zhang b , Jian-Feng Xu b,e , Wen-Bing Tan b , Jin-Sheng Wang b , Tong-Tong Li b , Bei-Dou Xi a,b,∗ , Di-Hua Wang a,∗∗ a
School of Resource and Environmental Sciences, Wuhan University, Hubei, Wuhan 430079, PR China Groundwater and Environmental System Engineering Innovation Base, State Environmental Protection Key Laboratory of Simulation and Control of Groundwater Pollution, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China c Beijing Vocational College of Agriculture, Beijing 100012, PR China d Key Laboratory of Urban Stormwater System and Water Environment, Beijing University of Civil Engineering and Architecture, Beijing 100044, PR China e School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China b
a r t i c l e
i n f o
Article history: Received 10 November 2016 Received in revised form 13 April 2017 Accepted 20 May 2017 Available online 3 June 2017 Keywords: Micro-bubble Sludge solubilization SEM PCR-DGGE Excitation-emission matrix (EEM) spectra
a b s t r a c t In this study, the micro-bubble were used for the sludge solubilization process. In order to investigate the enhancement of sludge solubilization by micro-bubbles, the performance of the micro-bubble system and a conventional bubble system (the control one) was compared in terms of sludge solubilization. The performance in the micro-bubble reactor was much better than the control one, as a result total suspended solid (TSS) was rapidly decreased to 2325.19 TSS L−1 . To investigate the mechanism of the sludge solubilization, the constituents of dissolved organic matter (DOM) from the sludge solubilization was investigated using fluorescence spectroscopy. Compared to the DOM from the reactor using conventional bubbles, the DOM from the micro-bubbles reactor contained higher protein- and humic-like substances. Moreover, the total integrated fluorescence intensity (TOT) were constantly decreased by 18.37% in the control one and increased by 34.39% in the micro-bubble reactor. The results mean that the sludge were effectively solubilized, and organic molecules were released. Polymerase chain reactionsdenaturing gradient gel electrophoresis (PCR-DGGE) was used to analyse the microbial communities after a micro-bubble process. The dominant bacteria in the micro-bubble reactor were identified as Delftia sp., Sphenophorus levis and Stenotrophomonas sp., which were benefit to the cleavage of refractory organics. Scanning Electron Microscopy (SEM) inspection showed that the sludge after the micro-bubble process presented more pores and channels on the sludge surface; the micro-bubbles was good at forming zoogloea, which was helpful for oxygen and mass transfer. Overall, the results indicate that the micro-bubbles aeration could improve the solubilization of sludge. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction In recent decades, tougher effluent stipulations and the growth of population promote the sewage networks, thus the amount of sewage sludge has rapidly increased for the development of wastewater treatment plants(WWTPs). The treatment and disposal
∗ Corresponding author at: Groundwater and Environmental System Engineering Innovation Base, State Environmental Protection Key Laboratory of Simulation and Control of Groundwater Pollution, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China. ∗∗ Corresponding author. E-mail addresses: [email protected] (B.-D. Xi), [email protected] (D.-H. Wang).
of sewage sludge increase the operational costs of WWTPs in an industrial scale by approximately 50–65% (Wei et al., 2003; Liu et al., 2011), which causes a great challenge for the ecological environment governance (Lloret et al., 2013). For reducing these drawbacks, sludge digestion can be considered as one of the traditional biological techniques for the stabilization of sewage sludge discharged from wastewater treatment plants (Chu et al., 2008; Borowski and Szopa, 2007). Digestion can be performed under either aerobic or anaerobic conditions. The stabilization mechanism for digestion is involved with the degradation of organic components in sludge by metabolism of micro-organisms (Wang et al., 2009; Bernard and Gray, 2000). However, several problems and disadvantages still exist for the typical sludge digestion technologies, such as the large water content, the low biodegradability and a high energy cost. These disadvantages can be overcome on an
http://dx.doi.org/10.1016/j.ecoleng.2017.05.032 0925-8574/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
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industrial scale by the solubilization treatment of the excess sludge (Wang et al., 2008; Chu et al., 2008); therefore, the micro-bubble technology was introduced to sludge digestion pretreatment by some scholars (Von Gunten, 2003; Kim et al., 2010). Due to high inner pressure, micro-bubbles will shrink and collapse beneath the water surface (Poh et al., 2014; Li et al., 2006). Previous work has shown that free radicals can be generated by the collapse of micro-bubbles without dynamic stimulus (Takahashi et al., 2007). Moreover, hydroxyl radicals with higher standard redox potential not only have shown immediate reactivity with the majority of organic compounds, but also the application of micro-bubble technology to sewage sludge could improve the biodegradability and cell disintegration (Von Gunten, 2003; Kim et al., 2010). Sewage sludge often contains many types of particulate and refractory organic compounds, such as suspended solids, soluble organics with more recalcitrant. Customarily, they are poorly biodegradable and inherently toxic, which makes it difficult to apply traditional biological treatments (Saktaywin et al., 2005; Ge et al., 2011). Some researchers have shown that the application of a micro-bubble collapse technique leads to the decomposition of refractory organic compounds (Li et al., 2009). Although micro-bubble technology has shown the capability of hydrolyzing refractory organic compounds, systematic studies regarding the transformation of organic matter from sewdge sludge during treatment with micro-bubble technology are still needed. The solubilization treatment with micro-bubble technology may lead to fluctuations on the organic constituents and relative biodegradability of the sewage sludge. The total view about fluorescent properties of DOM can be indicated by three-dimensional fluorescence excitation-emission matrix (EEM), and it can be used in structural identiflcation and stability assessment about organic matters (He et al., 2011a). Therefore, to elucidate the mechanism of sludge solubilization using micro-bubbles, the DOM component can be characterized using EEM spectra. Furthermore, identifying the predominant bacteria in the granules and correlating their population dynamics with sludge solubilization need to be further investigated. We used PCR-DGGE amplified 16S ribosomal RNA (rRNA) gene fragments on a full-scale to investigate the distinctive bacterial community in the solubilization treatment with micro-bubble technology. DNA-based molecular biology technique is particularly effective for the characterization of microbial diversity and community structure about sludge samples. Furthermore, microbial response during sludge solubilization treatment were compared and discussed (Li et al., 2008; Zhang et al., 2011). At the same time, we also used SEM to inspect the microbial organisms grown in the sludge and sludge structures to verify the influence of micro-bubbles on granule morphology during sludge granulation (Li et al., 2008). In this study, the performances of the micro-bubble generator and a conventional gas diffuser were compared to investigate the mechanism of sludge solubilization. The objectives of this paper were to observe the characteristic and transformation of the DOM of sewage sludge with micro-bubbles. In addition, the evolution of microbial was also discussed.
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Fig. 1. A schematic diagram of reactor for sludge stabilization process.
250 mm and a working volume of 30 L. In order to investigate the enhancement of sludge solubilization by micro-bubbles, the performance of the micro-bubble system and a conventional bubble system (the control one) was compared in terms of sludge solubilization. Air was fed into the reactor at a flow rate of 0.5 L/min by the micro-bubble generator compared with a conventional gas diffuser. Micro-bubble generator (MG) was made by Benzhou New Technology (Nano Bubbles) Promotion Co. (Chaoyang district, Beijing city, China) which created micro-bubbles using a gyratory accelerator and an ejector. The air was supplied through a titanium porous diffuser positioned at the bottom of the reactor. The MG titanium porous diffuser was cylindrical with a pore size of 45 m. The mean diameter of the bubbles from the micro-bubble system was below 0.42 m and their numerical density exceeded 2.9 × 104 counts/mL. Conventional bubble generator manufactured by Nanjing Jing Lan Environmental protection equipment manufacturing Co., Ltd. (Nanjing city, China). Activated sludge from an anaerobic tank in a continuous WWTP (Shunyi district, Beijing city, China) was used in this experiment. The sludge concentration used in these experiments was about 3000 mg TSS L−1 . 2.2. Sample collection and storage Water samples (50 mL) were collected from the reactors every hour and filtered using 0.45 m Whatman glass fiber filters. Water samples were stored at −20 ◦ C in fridge, and they were used for DOC and Fluorescence spectroscopy. Sludge samples (20 g) were collected from the reactors every hour and maintained in a dark room at −80 ◦ C in fridge. Sludge samples were used for PCR-DGGE and SEM within 72 h. Total DNA was directly extracted from sludge samples and was maintained in a dark room at −80 ◦ C in fridge which was used within 36 h.
2. Materials and methods
2.3. Analytical methods
2.1. Experimental equipment
The sludge samples, which were centrifuged at 4 ◦ C and the rotate speed of 10000 rpm for 10 min, were heated at 105 ◦ C to measure TSS. The dissolved organic carbon (DOC) concentrations of all samples were measured using an Analytik Jena Multi N/C 2100 DOC analyzer (Analytik Jena, Jena, Germany) and were set at approximately 15 mg L−1 for the fluorescence analyses. Fluorescence spectroscopy was measured using a luminescence spectrometer (Hitachi F-7000, Japan) with a sampling interval of 5 nm for both
A schematic diagram of the experimental equipment is provided in Fig. 1. The experimental system includes three parts: the water-bath system, the reactor and the diffuser. The temperature was maintained at 35 ◦ C ± 2.5 ◦ C throughout a thermostated jacket using the water-bath system. A laboratory-scale reactor, which was fabricated by Plexiglas glass, contained an internal diameter of
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the excitation and emission monochromators, and the scan speed was set to 2400 nm min−1 during the analysis process Excitationemission matrix (EEM) spectra were obtained by subsequently scanning the emission spectra from 280 to 550 nm with a 5 nm increment as the excitation wavelength increased from 200 nm to 450 nm (He et al., 2011b). SEM was subsequently performed to examine the structure of sludge. The sample was freeze-dried, sputter coated with gold and examined via SEM (JEOL JSM-6390LV, Japan) at an accelerating voltage of 10 kV. The total DNA was extracted using a DNA gel extraction kit (BioTeke Corporation) for 16S rDNA PCR amplification. The total DNA from the sludge samples was used as a template to amplify the V3 variable region in the bacterial 16S rDNA gene via PCR with universal primers 341F-GC (5 -GC-clamp-CCTACGGGAGGCAGCAG-3 ), (5 -ATTACCGCGGCTGCTGG-3 ), and GC-clamp (5 534R CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG-3 ) (Florez and Mayo, 2006). The reclaimed DNA was then used as a template to reamplify the bands with the same pair of primers (341f-534r) under the previously described PCR conditions. These sequences were then compared with similar sequences from the Gen Bank DNA database via BLAST analysis (basic logical alignment search tool, BLAST at NCBI) (Li et al., 2006).
3. Results and discussion 3.1. TSS and DOC The TSS and DOC concentrations of all samples obtained from the two reactors are presented in Fig. 2. The TSS and DOC contents gradually decreased in the control one and reached final values of 2860.1 mg TSS L−1 and 17.4 mg L−1 with increasing reaction time. As reported by He et al., microorganisms only use organic matters that are dissolved in water. As the DOC content of the DOM increased, the biodegradable compounds increased and microorganisms become more active (He et al., 2011a). Therefore, the decreasing of DOC in the control one is probably due to the endogenous metabolism of microorganisms, and some organic compounds were completely oxidized to carbon dioxide. The decline of TSS was relied on better performance of sedimentation in aerobic condition. When sludge was exposured to micro-bubble, the TSS in the micro-bubble reactor rapidly decreased to 2325.19 TSS L−1 , the rate of sludge solubilization was obviously faster than the control one. Whereas the DOC content in the micro-bubble reactor decreased during the first 2 h and then increased from 76.15 mg L−1 to 93.83 mg L−1 . The DOC content at 12 h was much greater, and the only difference between the two reactors depended on the microbubbles. Thus, the increase in DOC concentration was caused by difference in bubble characteristics between the two reactors. As Chu et al. reported, sludge disintegration process can be described as sequential decomposition reactions of the cell destruction, sol-
ubilization and subsequent oxidation of the released organics into carbon dioxide. Micro-bubble could dissolve sludge and the organic substances released from the cells (Chu et al., 2008; Saktaywin et al., 2005). In addition, Jaffé et al. (2004) reported that the change of DOC concentration reflected the increased amount of DOM. Therefore, the uncommon change of the DOC is thought to be connected with the rupture of sludge and the delivery of DOM from supernatan of sludge. The fluorescence spectroscopy was consequently examined to give an insight into the DOM during a micro-bubble process.
3.2. Excitation-emission matrix (EEM) spectra The EEM fluorescence spectra of the sludge from both the control one and the micro-bubble reactor are shown in Fig. 3. Three main peaks, i.e., peak 1 (Ex/Em: 225/340 nm; tyrosine protein-like substances), peak 2 (Ex/Em: 275/340 nm; tryptophan protein-like substances) and peak 3 (Ex/Em: 325/400 nm; humic acid-like substances), were detected in all samples from both the control one and the micro-bubble reactor (He et al., 2011b). It was interesting to note that peak 4 (Ex/Em: 280/400 nm; fulvic acid-like substances) appeared in the micro-bubble reactor at the time of 8 h and its intensity became more and more stronger thereafter. As the reaction process proceeded, the intensities of peaks 1 and 2 in the control one were dramatically reduced (Fig. 3). According to Hudson et al. (2008), peaks 1 and 2 are related to proteinderived and soluble microbial byproduct-like materials. Therefore, the increase in the intensities of peaks 1 and 2 is attributed to the degradation of organic matter as a result of microbial activity, suggesting that a part of the pollutants were removed in the control one. Compared with the peaks 1 and 2, the intensity of peak 3 changed little due to its high degree of humification or maturation. These results suggest that the biological bio-oxidation of humiclike organic matter proceeded slowly, and the humic substances were difficult for the microorganism to be utilized (He et al., 2011b). The above results suggest that some easily biodegradable compounds, such as proteins, were continuously consumed and finally disappeared, leaving only the humic substances in the control one with common bubbles. Compared with protein-like materials, the humic substances were difficult for the microorganisms to use. The changes of fluorescent matter in the micro-bubble reactor were different from that in the control one. Peaks 1 and 2 were remarkably increased in the micro-bubble reactor, and peak 3 increased as the reaction progressed as well. Furthermore, peak 4 suddenly emerged at the ninth hour and continuously arose during the later period, which indicated the formation of fulvic acid-like substances. These results demonstrated that, compared to the control one, more DOM presented in the micro-bubble reactor, which was related to the unique function of the micro-bubbles. It appears that the effective transformation of DOM in sludge was affected by micro-bubble. Similar results were also observed by other schol-
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Fig. 3. Fluorescence spectra of the DOM from the control one (a) and the micro-bubble reactor (b).
ars that organic matters were released with the solubilization of the sludge in the micro-bubble system (Chu et al., 2007). This is in a good agreement with the previous report that the changes
were related to decomposition of dead cells and macromolecular organics (Qu et al., 2012). The results convincingly show that the
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Fig. 6. DGGE lanes of samples from the control one (left) and the micro-bubble reactor (right). The lanes for the two samples were cutted from one profile.
micro-bubble distinctly enhanced the solubilization of sludge, and increased the DOM contents in aqueous phase. As shown in Fig. 4, the total integrated fluorescence intensity (TOT) were constantly decreased by 18.37% in the control one and increased by 34.39% in the micro-bubble reactor. It means that the DOM was degraded in the control one, and new dissolved organic components were supplied in the micro-bubble reactor due to the micro-bubble effect. This phenomenon was consistent with the previous study, which indicated that micro-bubbles could accelerate the formation of hydroxyl radicals and the higher hydroxyl radicals produced in micro-bubble system may contribute to rapid sludge solubilization (Chu et al., 2008). Therefore, it can be concluded that the sludge were effectively solubilized. The Pi,n values of the five EEM regions for the DOM and its corresponding fractions during the sludge solubilization process are shown in Fig. 5. The Pi,n values for Regions I, II and IV in the DOM decreased in the control one as time progressed, whereas those for Regions III and V increased significantly during the process, indicating that a decrease in tyrosine-like and tryptophan-like materials, as well as an increase in humic- and fulvic-like substances in the reactor using conventional bubbles. Hudson et al. (2008) reported that tyrosine-like and tryptophan-like materials are easily biodegraded with regard to humic- and fulvic-like substances. Therefore, with the disappearance of some easily degradable compounds such as aromatic proteins, the remaining refractory compounds gradually dominated the DOM in the control one; these compounds can consequently be used to assess sludge stability, and this quantitative result is consistent with the location of the EEM peaks.
The micro-bubble causes the disarray in Pi,n during the process. The Pi,n of the Regions I, II and IV exhibited a decreasing trend, whereas Pi,n of the Regions III and V were increased with time. Similar with the TSS disintegration, the Regions I, II and IV decreased apparently at 2-h. Protein-like compounds, especially those that are present as free molecules or bound in polypeptide, are unstable and easily utilized by microorganisms. It can be inferred that the simple aromatic proteins and soluble microbial byproduct-like materials in sludge were utilized by microorganisms. Similar results were also observed in the study reported by Jouraiphy et al. (2008). However, the Regions I, II and IV increased at the 4th and the 6th hour. According to Takahashi et al. (2007), free radicals can be generated during the natural micro-bubble shrinkage process. The sludge solubilization process has been suggested as the sequential processes of suspended solids disintegration, solubilization of the solids (cells) and mineralization of the soluble organic matter released from the microbial cells (Ahn et al., 2002). Micro-bubble can destroy the cell walls of sludge and solubilize or oxidize them to organic substances.The sludge was broken down, and the bio-macromolecules was released and transformed into small molecules. As reported by Yan, the Pi,n variation of the microbubble reactor may be caused by a combination of biological and chemical effects, which resulted in the disorders of DOM in different periods (Yan et al., 2009). The Regions III and V were dominated in the micro-bubble reactor until the 8th hour, the conversion of DOM changed as the above trend again.
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Fig. 7. SEM images of sludge from the control one (a) and the micro-bubble reactor (b).
3.3. PCR-DGGE To investigate the influence of microorganism on the solubilization of sludge, and to provide a better understanding of the difference between a common bubble and a micro-bubble for the potential use of sludge solubilization, the microbial community of the two samples was analyzed. The PCR-DGGE (Fig. 6) obtained in different reactors could reflect the total bacterial DNA diversification of the sludge during the solubilization process. The number and intensity of migrating bands showed a distinct difference between the control one and the micro-bubble reactor. The biodiversity indexes were 3.36 and 3.91, respectively. To find the specific microbial species caused by the micro-bubble effect, the bands from the two samples were also selected for sequence analysis. In the control one, the specific bands showed maximum homology to Acinetobacter junii, Enterobacter sp. and Bacillus sp. All of these bacteria were suitable for a reactor with aerobic environment. For example, Acinetobacter junii was used as an inoculum to trigger the release of phosphate from activated sludge systems when the cells reached the stationary growth phase (Momba and Cloete, 1996). The present study suggested that Enterobacter sp. and Bacillus sp. were commonly isolated from different sources including activated sludge or sediment (Thirumala et al., 2010; Fang et al., 2010). The DGGE analysis also showed that more complicated genetic diversity were greatly affected by the micro-bubbles. Three strains of Gramnegative bacteria in the micro-bubble reactor were identified as Delftia sp. Sphenophorus levis and Stenotrophomonas sp. were considered as the dominant strains in the samples extracted from the micro-bubble reactor. It was reported that Delftia sp. was responsible for the biodegradation of organic matter Organic matter that was difficult to degrade was first degraded by Delftia sp. and then converted to another smaller compound, which was then further solubilized (Xiao et al., 2009). Zhou et al. (2011) have reported that Stenotrophomonas sp. is capable of degrading toxic organic matter to nontoxic compounds, and the presence of Sphenophorus levis is related to the release of small organic compounds and benefits from the fermentation process. The microbial community structure is the cornerstone of the function and characteristics of a sludge. It was found that bacteria in the micro-bubble reactor could enhance the solubilization of hardto-cleave organic matter compounds. Because microorganisms are the main factor for sludge solubilization, the easily biodegradable compounds in the DOM lead to more microbial diversity and activity, which may be an important mechanism of sludge reduction achieved by micro-bubble technology. We also inferred that the specific functional microbial species selected by the micro-bubbles could enhance the biodegradability and the DOM content of sludge. Thus, the results mentioned above indicated that micro-bubbles could change the microbial community, and the specific strains survived in the micro-bubble reactor were effectively cleaving
refractory organics, which is beneficial for the sludge solubilization process. 3.4. Scanning electron microscopy Cell clusters, pores and extracellular polymeric substances were found in sludge (Zhan et al., 2006). As indicated in the SEM image in (Fig. 7), a large quantity of cocci and rods are observed within in the EPS framework. After continuous aeration for 12 h, the sludge from the control one (Fig. 7a) had a deformed structure. Its surface contained fewer pores and channels, and the zoogloea structure from the sludge was misshapen. In addition, no orbicular cells were observed; all the zoogloea were gathered into a mass. However, in the micro-bubble system, the smaller bubbles, which exist in great amounts, might rupture much faster and produce a higher speed jet, thus the associated mechanical stresses are likely to be higher. As a result, the structure of sludge from the micro-bubble reactor (Fig. 7b) had more pores and channels on the sludge surface. The zoogloea were widely dispersed with complete bacterial structures, demonstrating that the micro-bubbles are form zoogloea well and immobilize microorganisms, which is helpful for oxygen and mass transfer. 4. Conclusions In this study, the micro-bubble were used for the sludge solubilization process and the sludge were effectively solubilized by microbubbles. The spectra characterization analysis showed the total DOM of sewage sludge significantly increased by microbubble, and the component were progressively more available to microorganisms. Moreover, the PCR-DGGE results indicated that micro-bubbles could change the special strains, which were benefit for the cleavage of refractory organics. In addition, detected by SEM, micro-bubbles could form zoogloea well, which is helpful for oxygen and mass transfer. Therefore, the application of microbubble technology was effective at the release of DOM and the growth of microbial, by which the micro-bubble could enhance the treatment of sewage sludge. Currently, micro-bubble technology for sludge reduction mainly includes the enhanced ozonation of the biomass. The use of micro-bubbles can decrease the operating costs and improve the efficiency for sludge solubilization. This artical may provide a suitable way to utilize micro-bubble for sludge solubilization. Acknowledgements This work was kindly supported by the National Sciencetechnology Support Plan Projects of China (2015BAL04B01) and the Fundamental Research Funds for the Central Universities (Wuhan University-Duke initiative project 201604398).
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