摘要
好氧颗粒污泥是一类物理结构致密、沆降性能优异的生物聚集体,胞外多聚物(Extracellular Polymeric Substances, EPS)对其形成与结构稳定化具有重要作用。然而,目前污泥EPS提取与分析方法存在局限性,影响颗粒污泥主要成分EPS的组成含量分析,限制颗粒污泥形成机理的探析。论文比较研究了不同提取方法对生物聚集体EPS主要组分提取效率的影响;在此基础上,应用三维荧光光谱(3D-EEM)、傅里叶红外光谱(FTIR)等先进技术研究好氧污泥颗粒化过程EPS组分变化,并结合不同形态结构的颗粒污泥EPS组分分析,初步揭示影响好氧颗粒污泥形成及结构稳定化的EPS关键组分。主要获得以下研究结论:
1、鉴于目前污泥EPS提取方法存在不同组分提取效率差异大等问题,以普通絮体污泥、好氧颗粒污泥以及结构失稳的好氧颗粒污泥为研究对象,应用3D-EEM技术分析阳离子交换树脂法(CER)、硫化物法(Sulfide)、CER+Sulfide法、甲酰胺+NaOH法、加热法(Heat)五种常见提取方法对污泥EPS提取效率的影响。结果表明,实验选取的五种污泥EPS提取方法的破细胞程度均较小,其中甲酰胺+NaOH法、Heat法处理后的污泥EPS提取量远高于CER法、Sulfide法及CER+Sulfide法,表明甲酰胺+NaOH法、Heat法具有较高的EPS提取效率。3D-EEM分析进一步揭示,CER法、Sulfide法及CER+Sulfide法提取的污泥EPS组分较单一、不全面;甲酰胺+NaOH法虽具有最高的EPS提取效率,但不能有效提取污泥EPS中的芳香族类蛋白,不适于污泥EPS后续功能组分分析。相比而言,Heat法可全面提取污泥EPS中的三大类具有荧光特性的芳香族类蛋白、类富里酸、类胡敏酸,可作为较优的污泥EPS提取方法。
2、应用3D-EEM技术研究处理模拟城镇污水好氧颗粒污泥反应器启动过程污泥EPS组分变化发现,颗粒化过程代表芳香族类蛋白的荧光特征峰PeakB、D和F (Ex/Em=220-230/358.5-364、280-290/360-368、230/308.5)的响应值逐渐增强,其中酪氨酸类蛋白(Peak F)在颗粒污泥中的高度富集揭示其在颗粒结构维持中的重要作用;代表类胡敏酸的荧光特征峰Peak C (Ex/Em=350-360/445.5-452.5)的响应值在污泥颗粒化过程中呈先增后减再稳定的趋势,结合有关文献分析其在污泥颗粒化过程中利于微生物聚集。结合FTIR分析发现,p-折叠蛋白(1650-1640cm-1)、天冬氨酸(1400cm-1)在污泥颗粒化过程大量存在,且其响应值变化趋势与PN含量变化基本一致;属芳香族类蛋白的酪氨酸(1517cm-1)亦在成熟颗粒内富集,印证了3D-EEM的分析结果;此外,吸收波段为1200-1000cm-1的多糖组分在污泥颗粒化过程发生显著的结构转变,结合有关文献认为不同结构多糖物质的转化影响污泥颗粒化,有关机理待进一步探析。
3、研究不同形态结构的好氧颗粒污泥发现,相比稳态好氧颗粒,失稳颗粒污泥的PN, PS含量大幅增加,但芳香族类蛋白(Peak B、D)明显减少,证实芳香族类蛋白在颗粒污泥结构中具有重要作用;类胡敏酸(Peak C)响应值随颗粒污泥的失稳而增加,表明该组分的过量富集影响颗粒污泥稳定性。FTIR分析亦证实酪氨酸类蛋白(1517cm-1)的存在以及颗粒化过程多糖物质(1200-1000cm’)结构的转变对颗粒污泥形成与稳定化影响较大,揭示这两类物质是颗粒污泥EPS的关键组分。
The aerobic granular sludge is a class of microbial aggregates with compact physical structure and excellent settleability. Extracellular polymeric substances(EPS), as major compents of aerobic granular sludge, plays an important role in the formation and stability of aerobic granule. As the limitation of EPS extract and analytical methods, it's difficult to analysis the main EPS components and reveal the mechanism of aerobic sludge granulation. Based on the comparison of different EPS extraction methods for microbial aggregates, three-dimensional fluorescence spectroscopy (3D-EEM) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the key components of EPS during the formation and stablility of aerobic granular sludge. Main conclusions were as follows:
1、In view of the different effectiveness of various methods for EPS extraction from flocs sludge, unstable and stable aerobic granular sludge,3D-EEM was applied to investigate EPS extract efficiencies of cation exchange resin (CER), Sulfide, CER combined Sulfide, Formamide combined NaOH and Heat methods. It was found that the cells was negligibly broken to all of the extract methods. EPS concents extracted by Formamide+NaOH and Heat methods were higher than those by CER, Sulfide and CER+Sulfide methods, which suggested that Formamide+NaOH and Heat method had a higher EPS extraction efficiency. Analysis of 3D-EEM revealed that the EPS components extracted by CER, Sulfide and CER+Sulfide methods were incomplete, and Formamide+NaOH method, which could not effectively extract aromatic protein-like, was not suitable for the following analysis of sludge EPS composition. In contrast, the Heat method, which could fully extract EPS components including aromatic protein-like, fulvic acid-like and visible humic acid-like, was the best EPS extraction method for microbial aggregates.
2, The results of sludge EPS analysis during aerobic sludge granulation using 3D-EEM showed that the response values of fluorescence peak B, D and F (Ex/Em=220-230/358.5-364,280-290/360-368,230/308.5) which represent aromatic protein-like gradually increased along with sludge granulation, and it's found that tyrosine protein-like(peak F, one of the aromatic protein-like) was enhanced granular sludge. The response value of fluorescence peak C (Ex/Em=350-360/445.5-452.5) which represent humic acid-like firstly increased and then decreased to a stable value, it's speculated that humic acid-like also favor microbial aggregates. Further analysis with FTIR indicated thatβ-sheet protein (1650-1640cm-1) and aspartic acid (1400cm-1) existed in the whole sludge granulation, and their variation tendency was consistent with PN content. The enrichment of tyrosine protein-like (1517cm-1) was also found in mature granule, and it's further confirmed its importance in the granular stability. In addition, the variation of absorption band between 1200-1000cm"1 which represent polysaccharide demonstrated that the structural shift of polysaccharide favors the aerobic sludge granulation and the mechanism about it need further research.
3. The analysis of stable-and unstable aerobic granule EPS showed that the PN and PS contents of unstable aerobic granule significantly increased compared to the stable granule, but it's worth noting that aromatic protein-like (Ex/Em=220-230/342-368.5,230/308.5) was greatly decreased, which comfirmed the importance of aromatic protein-like in maintaining structural stability of aerobic granular sludge. Futhermore excessive humic acid-like (Ex/Em=360/447.5-452) would result in the loss of granular stability. FTIR results demonstrated that the existence of tyrosine protein-like(1517cm-1) and the structural shift of polysaccharide(1200-1000cm-1) played a critical role in the formation and stability of aerobic granule, and they are the key EPS components of aerobic granular sludge.
引文
[1]Liu Y, Tay JH. The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge. Water Research,2002,36:1653-1665.
[2]Forster CF, Quarmby J. The physical characteristics of anaerobic granular sludges in relation to their internal architecture. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology,1995,67:103-110.
[3]Tay JH, Liu QS, Liu Y. The effects of shear force on the formation, structure and metabolism of aerobic granules. Applied Microbiology and Biotechnology,2001,57: 227-233.
[4]Liu Y, Tay JH. State of the art of bio granulation technology for wastewater treatment. Biotechnology Advances,2004,22:533-563.
[5]Ni BJ, Xie WM, Liu SG, et al. Granulation of activated sludge in a pilot-scale sequencing batch reactor for the treatment of low-strength municipal wastewater. Water Research,2009,43:751-761.
[6]Jiang HL, Tay JH, Tay STL. Aggregation of immobilized activated sludge cells into aerobically grown microbial granules for the aerobic biodegradation of phenol. Letters in Applied Microbiology,2002,35:439-445.
[7]McSwain BS, Irvine RL, Hausner M, et al. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Applied and Environmental Microbiology,2005,71:1051-1057.
[8]Wang ZP, Liu LL, Yao H, et al. Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors. Chemosphere,2006,63:1728-1735.
[9]Chen MY, Lee DJ, Tay JH. Distribution of extracellular polymeric substances in aerobic granules. Applied Microbiology and Biotechnology,2007,73:1463-1469.
[10]Adav SS, Lee DJ, Tay JH. Extracellular polymeric substances and structural stability of aerobic granule. Water Research,2008,42:1644-1650.
[11]Morgan JW, Forster CF, Evison L. A comparative-study of the nature of biopolymers extracted from anaerobic and activated sludges. Water Research,1990, 24:743-750.
[12]Liu YQ, Liu Y, Tay JH. The effects of extracellular polymeric substances on the formation and stability of biogranules. Applied Microbiology and Biotechnology, 2004,65:143-148.
[13]Pevere A, Guibaud G, van Hullebusch ED, et al. Effect of Na+ and Ca2+ on the aggregation properties of sieved anaerobic granular sludge. Colloids and Surfaces a-Physicochemical and Engineering Aspects,2007,306:142-149.
[14]Tsuneda S, Aikawa H, Hayashi H, et al. Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. Ferns Microbiology Letters, 2003,223:287-292.
[15]Laspidou CS, Rittmann BE. A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water Research, 2002,36:2711-2720.
[16]Li XY, Yang SF. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Research,2007,41:1022-1030.
[17]Liu XM, Sheng GP, Luo HW, et al. Contribution of Extracellular Polymeric Substances (EPS) to the Sludge Aggregation. Environmental Science & Technology, 2010,44:4355-4360.
[18]Ryder C, Byrd M, Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr. Opin. Microbiol.,2007,10:644-648.
[19]Byrd MS, Sadovskaya I, Vinogradov E, et al. Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol. Microbiol.,2009, 73:622-638.
[20]Ma LM, Conover M, Lu HP, et al. Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix. Plos Pathogens,2009,5:1-11.
[21]Watnick PI, Kolter R. Steps in the development of a Vibrio cholerae El Tor biofilm. Mol. Microbiol.,1999,34:586-595.
[22]Danese PN, Pratt LA, Kolter R. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. Journal of Bacteriology, 2000,182:3593-3596.
[23]Sutherland IW. The biofilm matrix-an immobilized but dynamic microbial environment. Trends Microbiol.,2001,9:222-227.
[24]Neu TR, Kuhlicke U, Lawrence JR. Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms. Applied and Environmental Microbiology, 2002,68:901-909.
[25]Conrad A, Kontro M, Keinanen MM, et al. Fatty acids of lipid fractions in extracellular polymeric substances of activated sludge flocs. Lipids,2003,38: 1093-1105.
[26]Jahn A, Nielsen PH. Cell biomass and exopolymer composition in sewer biofilms. Water Science and Technology,1998,37:17-24.
[27]Kaplan JB, Velliyagounder K, Ragunath C, et al. Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms. Journal of Bacteriology,2004,186: 8213-8220.
[28]Lynch DJ, Fountain TL, Mazurkiewicz JE, et al. Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture. FEMS Microbiol. Lett.,2007,268:158-165.
[29]Higgins MJ, Novak JT. Characterization of exocellular protein and its role in bioflocculation. Journal of Environmental Engineering-Asce,1997,123:479-485.
[30]Mora P, Rosconi F, Fraguas LF, et al. Azospirillum brasilense Sp7 produces an outer-membrane lectin that specifically binds to surface-exposed extracellular polysaccharide produced by the bacterium. Archives of Microbiology,2008,189: 519-524.
[31]Tielker D, Hacker S, Loris R, et al. Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation. Microbiology-Sgm,2005,151:1313-1323.
[32]Diggle SP, Stacey RE, Dodd C, el al. The galactophilic lectin, Lec A, contributes to biofilm development in Pseudomonas aeruginosa. Environ. Microbiol.,2006,8: 1095-1104.
[33]Johansson EMV, Crusz SA, Kolomiets E, et al. Inhibition and Dispersion of Pseudomonas aeruginosa Biofilms by Glycopeptide Dendrimers Targeting the Fucose-Specific Lectin LecB. Chemistry & Biology,2008,15:1249-1257.
[34]Branda SS, Chu F, Kearns DB, et al. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol.,2006,59:1229-1238.
[35]Lasa I, Penades JR. Bap:A family of surface proteins involved in biofilm formation. Res. Microbiol.,2006,157:99-107.
[36]Rice KC, Bayles KW. Molecular control of bacterial death and lysis. Microbiology and Molecular Biology Reviews,2008,72:85-109.
[37]Stinson MW, Bergey EJ. Isolation of a heart-binding and kidney-binding protein from guoup-A Streptococci. Infection and Immunity,1982,35:335-342.
[38]Goodman SD, Obergfell KP, Jurcisek JA, et al. Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunology,2011,4:625-637.
[39]Azeredo J, Oliveira R. The role of exopolymers produced by Sphingomonas paucimobilis in biofilm formation and composition. Biofouling,2000,16:17-27.
[40]Vilain S, Pretorius JM, Theron J, et al. DNA as an Adhesin:Bacillus cereus Requires Extracellular DNA To Form Biofilms. Applied and Environmental Microbiology,2009,75:2861-2868.
[41]Whitchurch CB, Tolker-Nielsen T, Ragas PC, et al. Extracellular DNA required for bacterial biofilm formation. Science,2002,295:1487-1487.
[42]Wu JF, Xi CW. Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix. Applied and Environmental Microbiology,2009,75: 5390-5395.
[43]Thomas VC, Thurlow LR, Boyle D, et al. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. Journal of Bacteriology,2008,190:5690-5698.
[44]Watanabe M, Sasaki K, Nakashimada Y, et al. Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp. Applied microbiology and biotechnology,1998,50:682-691.
[45]Qin ZQ, Ou YZ, Yang LA, et al. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology-Sgm,2007,153: 2083-2092.
[46]Petersen FC, Tao L, Scheie AA. DNA binding-uptake system:a link between cell-to-cell communication and biofilm formation. Journal of Bacteriology,2005,187: 4392-4400.
[47]Zhang XQ, Bishop PL. Biodegradability of biofilm extracellular polymeric substances. Chemosphere,2003,50:63-69.
[48]Tay JH, Liu QS, Liu Y. The role of cellular polysaccharides in the formation and stability of aerobic granules. Letters in Applied Microbiology,2001,33:222-226.
[49]Adav SS, Lee DJ. Extraction of extracellular polymeric substances from aerobic granule with compact interior structure. Journal of Hazardous Materials,2008,154: 1120-1126.
[50]Qin L, Tay JH, Liu Y. Selection pressure is a driving force of aerobic granulation in sequencing batch reactors. Process Biochemistry,2004,39:579-584.
[51]Wang F, Yang FL, Zhang XW, et al. Effects of cycle time on properties of aerobic granules in sequencing batch airlift reactors. World Journal of Microbiology & Biotechnology,2005,21:1379-1384.
[52]Chen MY, Lee DJ, Tay JH, et al. Staining of extracellular polymeric substances and cells in bioaggregates. Applied Microbiology and Biotechnology,2007,75:-467-474.
[53]Seviour T, Pijuan M, Nicholson T, et al. Understanding the Properties of Aerobic Sludge Granules as Hydrogels. Biotechnology and Bioengineering,2009,102: 1483-1493.
[54]Seviour TW, Lambert LK, Pijuan M, et al. Selectively inducing the synthesis of a key structural exopolysaccharide in aerobic granules by enriching for Candidatus "Competibacter phosphatis". Applied Microbiology and Biotechnology,2011,92: 1297-1305.
[55]Zhu L, Xu X, Luo W, et al. Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor. Journal of Applied Microbiology,2008,104:152-160.
[56]Adav SS, Lee DJ, Lai JY. Proteolytic activity in stored aerobic granular sludge and structural integrity. Bioresource Technology,2009,100:68-73.
[57]Zhang LL, Feng XX, Zhu NW, et al. Role of extracellular protein in the formation and stability of aerobic granules. Enzyme and Microbial Technology,2007, 41:551-557.
[58]Sheng GP, Yu HQ, Yu Z. Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila. Applied microbiology and biotechnology,2005,67:125-130.
[59]Nielsen PH, Keiding K. Disintegration of activated sludge flocs in presence of sulfide. Water Research,1998,32:313-320.
[60]Sheng GP, Yu HQ. Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy. Water Research,2006,40:1233-1239.
[61]Wuertz S, Spaeth R, Hinderberger A, et al. A new method for extraction of extracellular polymeric substances from biofilms and activated sludge suitable for direct quantification of sorbed metals. Water Science and Technology,2001,43: 25-31.
[62]Wu J, Xi C. Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix. Applied and Environmental Microbiology,2009,75: 5390-5395.
[63]Ras M, Lefebvre D, Derlon N, et al. Extracellular polymeric substances diversity of biofilms grown under contrasted environmental conditions. Water Research,2011, 45:1529-1538.
[64]Read RR, Costerton JW. Purification and characterization of adhesive exopolysaccharides from Pseudomonas-putida and Pseudomonas-fluorescens. Canadian Journal of Microbiology,1987,33:1080-1090.
[65]D'Abzac P, Bordas F, Van Hullebusch E, et al. Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges:comparison of chemical and physical extraction protocols. Applied microbiology and biotechnology,2010,85: 1589-1599.
[66]Dignac MF, Urbain V, Rybacki D, et al. Chemical description of extracellular polymers:Implication on activated sludge floe structure. Water Science and Technology,1998,38:45-53.
[67]Liu H, Fang HHP. Extraction of extracellular polymeric substances (EPS) of sludges. Journal of Biotechnology,2002,95:249-256.
[68]Sheng GP, Yu HQ, Yue ZB. Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances. Applied microbiology and biotechnology,2005,69:216-222.
[69]Liang Z, Li W, Yang S, et al. Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge. Chemosphere,2010,81:626-632.
[70]Zita A, Hermansson M. Effects of bacterial cell surface structures and hydrophobicity on attachment to activated sludge floes. Applied and Environmental Microbiology,1997,63:1168-1170.
[71]Novak JT, Sadler ME, Murthy SN. Mechanisms of floe destruction during anaerobic and aerobic digestion and the effect on conditioning and dewatering of biosolids. Water Research,2003,37:3136-3144.
[72]Park C, Novak JT. Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water Research,2007,41: 1679-1688.
[73]Park C, Novak JT, Helm RF, et al. Evaluation of the extracellular proteins in full-scale activated sludges. Water Research,2008,42:3879-3889.
[74]Frolund B, Palmgren R, Keiding K, et al. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Research,1996,30: 1749-1758.
[75]Brown MJ, Lester JN. Comparison of bacterial extracellular polymer extraction methods. Applied and Environmental Microbiology,1980,40:179-185.
[76]Platt RM, Geesey GG, Davis JD, et al. Isolation and partial chemical-analysis of firmly bound exopolysaccharide from adherent cells of a fresh-water sediment bacterium. Canadian Journal of Microbiology,1985,31:675-680.
[77]Vieira EGJ, Henriques SB. Comparison of the Lowry and coomassie blue methods for the determination of protein-concentration. Brazilian Journal of Medical and Biological Research,1992,25:583-591.
[78]Hatate H, Ohgai M, Murase N, et al. Estimation of protein content in marine microalgae using the Lowry method. Fisheries Science,1998,64:168-169.
[79]Wnuk B. Trials on the application of the Lowry method for detemination of protein content in sugar beet juices. Listy Cukrovarnicke a Reparske,2011,127: 105-109.
[80]Frolund B, Griebe T, Nielsen PH. Enzymatic activity in the activated sludge floc matrix. Applied Microbiology and Biotechnology,1995,43:755-761.
[81]Wu J, Xi C. Enzymatic method for extracting extracellular DNA in biofilm matrix. Cold Spring Harbor protocols,2010:1-3.
[82]Steinberger RE, Holden PA. Extracellular DNA in single-and multiple-species unsaturated biofilms. Applied and Environmental Microbiology,2005,71:5404-5410.
[83]Pietramellara G, Ascher J, Borgogni F, et al. Extracellular DNA in soil and sediment:fate and ecological relevance. Biol. Fertil. Soils,2009,45:219-235.
[84]Beech IB, Cheung CWS, Johnson DB, et al. Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy. Biofouling,1996,10:65-77.
[85]Li X, Logan BE. Analysis of bacterial adhesion using a gradient force analysis method and colloid probe atomic force microscopy. Langmuir,2004,20:8817-8822.
[86]Zhang T, Fang HHP. Quantification of extracellular polymeric substances in biofilms by confocal laser scanning microscopy. Biotechnology Letters,2001,23: 405-409.
[87]Li DH, Ganczarczyk JJ. Structure of activated sludge flocs. Biotechnology and Bioengineering,1990,35:57-65.
[88]Macleod FA, Guiot SR, Costerton JW. Electron microscopic examination of the extracellular polymeric substances in anaerobic granular biofilms. World Journal of Microbiology & Biotechnology,1995,11:481-485.
[89]Decho AW, Kawaguchi T. Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast (R) resin. Biotechniques,1999,27:1246-1252.
[90]Klausen MM, Thomsen TR, Nielsen JL, et al. Variations in microcolony strength of probe-defined bacteria in activated sludge flocs. Fems Microbiology Ecology,2004, 50:123-132.
[91]Webb HK, Vi Khanh T, Hasan J, et al. Physico-mechanical characterisation of cells using atomic force microscopy-Current research and methodologies. Journal of Microbiological Methods,2011,86:131-139.
[92]van der Aa BC, Dufrene YF. In situ characterization of bacterial extracellular polymeric substances by AFM. Colloids and Surfaces B-Biointerfaces,2002,23: 173-182.
[93]Metzger U, Lankes U, Fischpera K, et al. The concentration of polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum pullulans as revealed by (13)C CPMAS NMR spectroscopy. Applied Microbiology and Biotechnology, 2009,85:197-206.
[94]Michel C, Beny C, Delorme F, et al. New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria. Applied microbiology and biotechnology,2009,82:371-378.
[95]Sheng GP, Yu HQ, Wang CM. FTIR-spectral analysis of two photosynthetic H-2-producing strains and their extracellular polymeric substances. Applied Microbiology and Biotechnology,2006,73:204-210.
[96]Ramesh A, Lee DJ, Hong SG. Soluble microbial products (SMP) and soluble extracellular polymeric substances (EPS) from wastewater sludge. Applied Microbiology and Biotechnology,2006,73:219-225.
[97]Badireddy AR, Chellam S, Gassman PL, et al. Role of extracellular polymeric substances in bioflocculation of activated sludge microorganisms under glucose-controlled conditions. Water Research,2010,44:4505-4516.
[98]Pan XL, Liu J, Zhang DY, et al. A comparison of five extraction methods for extracellular polymeric substances (EPS) from biofilm by using three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy. Water Sa,2010,36: 111-116.
[99]Omoike A, Chorover J. Spectroscopic study of extracellular polymeric substances from Bacillus subtilis:Aqueous chemistry and adsorption effects. Biomacromolecules,2004,5:1219-1230.
[100]Lattner D, Flemming HC, Mayer C. (13)C-NMR study of the interaction of bacterial alginate with bivalent cations. International Journal of Biological Macromolecules,2003,33:81-88.
[101]Comte S, Guibaud G, Baudu M. Effect of extraction method on EPS from activated sludge:An HPSEC investigation. Journal of Hazardous Materials,2007,140: 129-137.
[102]Park C, Novak JT, Helm RF, et al. Evaluation of the extracellular proteins in full-scale activated sludges. Water Research,2008,42:3879-3889.
[103]Gerdes G, Dunajtschikpiewak K, Riege H, et al. Structural diversity of biogenic carbonate particles in microbial mats. Sedimentology,1994,41:1273-1294.
[104]Flemming HC, Wingender J. Relevance of microbial extracellular polymeric substances (EPSs)-Part I:Structural and ecological aspects. Water Science and Technology,2001,43:1-8.
[105]Flemming HC, Wingender J. Relevance of microbial extracellular polymeric substances (EPSs)-Part II:Technical aspects. Water Science and Technology,2001, 43:9-16.
[106]Mayer C, Moritz R, Kirschner C, et al. The role of intermolecular interactions: studies on model systems for bacterial biofilms. International Journal of Biological Macromolecules,1999,26:3-16.
[107]Raszka A, Chorvatova M, Wanner J. The role and significance of extracellular polymers in activated sludge. Part Ⅰ:Literature review. Acta Hydrochimica Et Hydrobiologica,2006,34:411-424.
[108]Ge LY, Deng HH, Gao HW, et al. Optimized Extraction Protocol for Extracellular Polymeric Substances (EPS) From Two Activated Sludges. Research Journal of Chemistry and Environment,2010,14:78-82.
[109]Ge LY, Deng HH, Wang HW, et al. Comparison of extraction methods for quantifying extracellular polymers in activated sludges. Fresenius Environmental Bulletin,2007,16:299-303.
[110]Dominguez L, Rodriguez M, Prats D. Effect of different extraction methods on bound EPS from MBR sludges. Part Ⅰ:Influence of extraction methods over three-dimensional EEM fluorescence spectroscopy fingerprint. Desalination,2010, 261:19-26.
[111]Adav SS, Lee DJ. Characterization of extracellular polymeric substances (EPS) from phenol degrading aerobic granules. Journal of the Taiwan Institute of Chemical Engineers,2011,42:645-651.
[112]Her N, Amy G, McKnight D, et al. Characterization of DOM as a function of MW by fluorescence EEM and HPLC-SEC using UVA, DOC, and fluorescence detection. Water Research,2003,37:4295-4303.
[113]Coble PG. Characterization of marine and terrestrial DOM in seawater using excitation emission matrix spectroscopy. Marine Chemistry,1996,51:325-346.
[114]Coble PG, Green SA, Blough NV, et al. Characterisation of dissolved organic matter in the black-sea by fluorescence spectroscopy. Nature,1990,348:432-435.
[115]DuBois M, Gilles KA, Hamilton JK, et al. Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry,1956,28: 350-356.
[116]Li ZH, Wang XC, Kuba T, et al. Porous structure and spatial characteristics of aerobic granules. Int. J. Environ. Pollut.,2011,45:25-35.
[117]Wu FC, Tanoue E. Isolation and partial characterization of dissolved copper-complexing ligands in streamwaters. Environmental Science & Technology, 2001,35:3646-3652.
[118]Wang Q, Du GC, Chen J. Aerobic granular sludge cultivated under the selective pressure as a driving force. Process Biochem.,2004,39:557-563.
[119]Du S, Zhang ZH, Zhang YF, et al., Characteristics of Aerobic Granular Sludge Cultivated by Promoting and Inhibiting Filamentous Bacteria, Environmental Biotechnology and Materials Engineering,2011,183-185:1075-1079.
[120]Beun JJ, Hendriks A, Van Loosdrecht MCM, et al. Aerobic granulation in a sequencing batch reactor. Water Research,1999,33:2283-2290.
[121]Quarmby J, Forster CF. An examination of the structure of USAB granules. Water Research,1995,29:2449-2454.
[122]Wang ZW, Liu Y, Tay JH. Distribution of EPS and cell surface hydrophobicity in aerobic granules. Applied Microbiology and Biotechnology,2005,69:469-473.
[123]Wingender J, Neu TR, Flemming HC. Microbial Extracellular Polymeric Substances:Characterization, Structures and Function. Springer-Verlag.:Berlin Heidelberg,1999.
[124]Hong K, Sun S, Tian W, et al. A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Applied microbiology and biotechnology,1999,51:523-526.
[125]Wang ZW, Wu ZC, Yin X, et al. Membrane fouling in a submerged membrane bioreactor (MBR) under sub-critical flux operation:Membrane foulant and gel layer characterization. Journal of Membrane Science,2008,325:238-244.
[126]Mascarenhas M, Dighton J, Arbuckle GA. Characterization of plant carbohydrates and changes in leaf carbohydrate chemistry due to chemical and enzymatic degradation measured by microscopic ATR FT-IR spectroscopy. Applied Spectroscopy,2000,54:681-686.
[127]Barth A, Zscherp C. What vibrations tell us about proteins. Quarterly Reviews of Biophysics,2002,35:369-430.
[128]Barth A. The infrared absorption of amino acid side chains. Progress in Biophysics & Molecular Biology,2000,74:141-173.
[129]Chen Y, Jiang W, Liang DT, et al. Aerobic granulation under the combined hydraulic and loading selection pressures. Bioresource Technology,2008,99: 7444.7449.
[130]Sturm BSM, Irvine RL. Dissolved oxygen as a key parameter to aerobic granule formation. Water Science and Technology,2008,58:781-787.
[131]Pan S, Tay JH, He YX, et al. The effect of hydraulic retention time on the stability of aerobically grown microbial granules. Letters in Applied Microbiology, 2004,38:158-163.
[132]Adav SS, Lee DJ, Lai JY. Aerobic granulation in sequencing batch reactors at different settling times. Bioresource Technology,2009,100:5359-5361.
[133]Seviour T, Pijuan M, Nicholson T, et al. Gel-forming exopolysaccharides explain basic differences between structures of aerobic sludge granules and floccular sludges. Water Research,2009,43:4469-4478.
[134]Punal A, Brauchi S, Reyes JG, et al. Dynamics of extracellular polymeric substances in UASB and EGSB reactors treating medium and low concentrated wastewaters. Water Science and Technology,2003,48:41-49.
[135]Chen W, Westerhoff P, Leenheer JA, et al. Fluorescence excitation-Emission matrix regional integration to quantify spectra for dissolved organic matter. Environmental Science & Technology,2003,37:5701-5710.