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Comparison of extraction methods for quantifying extracellular polymers in biofilms
~
Pergamon
War Sci T«h Vol. 39. No.7, pp. 211-218.1999 ro 1999/AWQ
Published by Elsevier SCIence LId Pnnted in Ore.r Britain. All righrs reserved
PH: 50273-1223(99)00170-5
0273-1223/99 $2000 + 0.00
COMPARISON OF EXTRACTION METHODS FOR QUANTIFYING EXTRACELLULAR POLYMERS IN BIOFILMS Xiaoqi Zhang"', Paul L. Bishop'" and Brian K. Kinkle"'''' • Department o/Civil and Environmental Engineering, University o/Cincinnati, Cincinnati, OH 4522/·0071. USA .. Department 0/ Biological Sciences, University 0/ Cincinnati. Cincinnati. OH 4522/-0006. USA
ABSTRACT Five commonly used extraction methods - regular ceninfugation. EDTA extraction. ultracentrifugation, steaming extraction and regular centrifugation With formaldehyde (ReF) - were selected to study their effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfale reducing and mtrifyingldenitrifying biofilm samples. Biofilm EPS extraction yields were represenled by carbohydrate and protein concentrations; the amount of cell lysIS during the extractions was indicated by DNA concentratlOn. The results showed !hat analyzing wash walers is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield. dependIng on the extracllOn method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the steaming extraction gave the greatest protem yield, and the other three extraction methods gave approximately equivalent amounts of carbohydrale and proteins for both types of biofilm. DNA in the EPS was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions. ~ I999lA WQ Published by Elsevier Science Ud. All rights reserved
KEYWORDS Aerobic/sulfate reducing; biofilms; carbohydrate; DNA; extracellular polymeric substances (EPS); extraction methods; nitrifying/denitrifying; proteins. INTRODUCTION Microbial extracellular polymeric substances (EPS) are high molecular-weight mucous secretions of bacteria and micraalgae. They range from tight capsules, which closely bind cells, to the loosely attached slime material. Biofilm EPS possesses many important functions in water and wastewater treatment. including anchoring the microorganisms near food sources, protecting them from dehydration and toxic substances, and providing ion exchange properties due to negatively-charged surface functional groups which allow them to bind cationic species such as heavy metals (Sutherland. 1977). The EPS composition determines many important properties ofbiofilm such as density. porosity, difTusivity, strength, elasticity, frictional resistance. thermal conductivity, and metabolic activity. More information about their compositions will contribute to a better understanding of the physical and physiological behavior ofbiofilms in environmental systems. 21 I
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X. ZHANG et al.
Many ~ethods have been used to extract the EPS from different bacterial cultures and activated sludges for analysIs. These extraction methods have included use of ammonium hydroxide, sodium hydroxide, ethylene-diaminetetraacetic aCId (EDTA), sulfuric acid, trichloroacetic acid, boiling benzene, ultrasonication. blending, high-speed centrifugation, and extraction by boiling or autoclaving (Brown and Lester. 1980). However, most of these extraction studies were perfonned on activated sludge (Brown and Lester, 1980, Gehr and Henry, 1983, Morgan et al., 1990, Urbain et al., 1993, Jia et al., 1996, Fr01und et al., 1996); very little research has been conducted on biofilm samples. The chemical compositions of the EPS are usually reported in the literature to be very heterogeneous. Carbohydrate predominates and represents up to 65% of extracellular materials (Horan and Eccles, 1986); other substances are also present, such as proteins. nucleic acids and lipids (Goodwin and Forster, 1985). Their exopolymer component ratios vary, depending on the sample source and extraction technique (Morgan et al., 1990). For example, Horan and Eccles (1986), Forster and Clarke (1983) and Morgan et al. (1990) found more carbohydrate than proteins in activated sludges, with a ratio range of 0.16-0.70. Karapanagiotis et al., (1989), Forster (1982) and Morgan et al. (1990) found more proteins than carbohydrate in digested sludges, with a ratio range of 1.1-5.1. In this study, five extraction methods, which are currently used for activated sludge, were evaluated for the extraction of EPS from biofilm samples grown under aerobic/sulfate reduction and nitrification/denitrification conditions. The five extraction methods are: (I) regular centrifugation, (2) EDTA extraction, (3) ultracentrifugation, (4) steaming extraction, and (5) regular centrifugation with fonnaldehyde. The five extraction methods were compared on the same samples to examine their effectiveness and repeatability and to detennine to what degree they may cause cell lysis. Minimum cell lysis is desired during the extractions to avoid contamination by intracellular material. To evaluate the degree of cell disruption that may be caused during the extractions, pellets in the biofilm were intentionally lysed and the amounts of DNA from the EPS and the pellets were compared. Researchers dealing with the EPS of activated sludge nonnally wash their activated sludge samples first and then discard the wash solution. Gehr and Henry (1983) proposed, though, that an initial washing step will remove exopolymer material from activated sludge. Presumably, the washing step will wash out some loosely attached extracellular polymers. The contribution of the washing step to the EPS collected from biofilm samples was verified and the amount of its contribution was further examined in this research. METHODS Bio.,films Biofilrns are typically heterogeneous structures. Many biofilms may essentially be considered to be layered. with an aerobic layer overlying anoxic or anaerobic layers. This is true ofbiofilms in which both nitrification and denitrification occur, and in ones where sulfate reduction occurs in lower, anoxic layers.
Aerobic/sulfate reducing biofilms and nitrifying/denitrifying biofilms were grown in laboratory-scale rotating drum biofilm reactors (RDBRs). Biofilm growth occurs on the surface of the RDBR rotating drum and removable sampling slats which arc coated with shrink tubing. A more detailed description of the reactor system can be found in Hanner (1991) and Harmer and Bishop (1992). The aerobic/sulfate reducing biofilm reactor was initially seeded with wastewater and digested sludge from a municipal/industrial wastewater treatment plant in Cincinnati, Ohio. The reactor was continuously fed with: yeast extract, 50 mg/I; sodium lactate (C 3Hs0 3Na), 156 mg/I; KH2P04, 120 mg/l; NaHC03, 180 mg/l; NH4CI, 100 mg/I; CaCI 2 -2H 20, 15 mg/I; MgS04 -7H 20, 60 mg/I; FeCh- 4H 20, 1.5 mg/l; Na2S04, 200 mg/l. The pH was maintained at 7.2-7.3 and dissolved oxygen (DO) was kept at 1.5-3.5 mg/1. The nitrifying/denitrifying biofilm reactor was seeded with nitrifiers and secondary effluent from a trickling filter wastewater treatment plant in Dayton, Ohio. It was continuously fed with: bacto peptone, 25 mg/l; yeast extract, 25 mg/I; NH4C1, 95 mg/I; K2C0 3, 320 mg/I; K2HP04. 25 mg/I; KH 2P04, 10 mg/I; MgS04 -7H 20, 11.25 mg/l; CaCh-2H20, 13.75 mg/I; FeCh -6H20, 0.125 mg/I; MnS04-H20, 0.0112 mg/1; CUS04. 0.0007 mg/I; Na2Mo04-2H20, 0.0004 mg/I; 2n504-71-1 20, 0.0120 mg/1. The pl-l and DO were kept at 7.5 and 1.5-3.5mg/I, respectively. Biofilm samples were collected from the sampling slats and total solids (TS) were measured to represent biofilm mass.
Quantifying extracellular polymers in biofilms
213
BPS e~tractiQn meth~s All biofilm samples were analyzed in triplicate. Four steps were involved in extractmg and collectmg the EPS from the biofilm samples (Figure I). Biofilm samples Washing, recover slime material (A)
Figure J. Procedure for the extrachon method compansons.
Step J: Washing (recover slime material). Biofilm samples (about 1 g) were put into centrifuge tubes along with 25 ml MilIiQ water (Millipore). The tubes were shaken gently, and then centrifuged at 3,500 rpm (6,ooOg) for 10 minutes. The liquid was decanted from the centrifuge tubes, and collected as the slime material. Step 2: Stripping (recover capsule-bound material). After the washing step, the tubes containing the biofilm pellets were filled with another 25 ml MilIiQ water. The contents were blended in a vortex blender (Oster, 867-28L) at high speed for 1 minute to recover the capsule-bound material.
For purposes of extraction method comparisons, liquid from the washing step and the stripping step were combined and brought to a volume of 50 mt. Step 3: Extraction (separate the stripped materialfrom the cells). Five extraction methods were applied and compared. These included:
(1) Rel:U1ar centriful:atjon (lia et al., 1996, with modification). 10 ml of the combined sample were centrifuged at 12,000 rpm (lI,227g) for 30 minutes at room temperature. (2) forA extraction (Brown and Lester, 1980). 10 ml of 2% EDTA was added to 10 ml of the combined sample and left quiescently for 3 hours at 4°C. The centrifuge tubes were then centrifuged at 14,000 g for 20 minutes at 4°C. (3) U1tracentriful:atjon (Brown and Lester, 1980). 10 ml of the combined sample were centrifuged at 33,000 g for 10 minutes at 4°C. The pellets obtained were resuspended with another 10 ml MilliQ water, and centrifugation was repealed for another 10 minutes to enhance the extraction.
(4) Slearnjnl: extraction (Brown and Lesler, 1980). 10 ml of the combined samples were sleamed in an autoclave at 80 aC under I bar pressure for 10 minutes and then centrifuged while slill hot at 8,000 g for 10 minutes; during centrifugation, the temperature was reduced to 15°C. The steaming treatment was used in an
X. ZHANG et al.
214
autoclaving under normal autoclave attempt to reduce the disruptive effects on the cells of boiling or conditions (120°C, 16 bars). The same extraction procedure (5) ~e~ular centriful:atjon with foona1dehyde (RCF) (Jia et al., 1996). g 0.22% formaldehyde was containin NaCI 8.5% ml 25 descn~ed above was used on I g samples, except that was centrifuged at 12,000 sample d combine the of ml 10 step. stripping the in water added Instead ofMilliQ ge with an SS-34 rotor Centrifu ed Superspe ted Refrigera rpm (11,227 g) for 30 minutes in a Sorvall RC-5B (Du Pont). centrifugation after each extraction Step 4: Filtering and collectmg the EPS. The supernatant obtained on were free of cells. The pellets samples that was filtered through 0.22 j.!m cellulose acetate filters to ensure study. lysis cell the for used ifnot were discarded yields. the same procedure described In order to evaluate the contribution of the washing step to biofilm EPS material were treated separately to capsule the above was replicated. except that the slime material and obtain separate EPS yields. were represented by carbohydrate and ChemIcal composition qna[ysiy The EPS yields in the extracts ation. A HP 8453 UV-Vis diode-array concentr DNA by protein concentrations, and cell lysis was indicated (Gerhardt et al., 1994) method was acid ulfuric phenol-s A used. was r hotomete (190 - 1100 nm) spectrop tion of the Bradford (1976) method, modifica A . used to quantitY Carbohydrate, with glucose as the standard proteins, with Bovine Serum quantify to used was l), Chemica (Pierce e procedur ie called the Coomass was used to quantitY DNA, 1994) al., et t (Gerhard method Albumin (BSA) as the standard. A fluorometric . standard the as sperm with salmon s with protein analysis. Theref~re, a~1 Protein dialysis. Formaldehyde used in the RCF extraction interfere or I Molecularporous DialySIS Spectra/p a through put were n the samples treated after the RCF extractio hyde. The dialysis membranes were formalde the remove to m) (Spectru 8,000 6 MWCO with e Membran An optimum dialysis time of 5-7 placed in a beaker containing 2 liter of continuously stirred MilliQ water. hour time period. 88 an over ations concentr protein ng monitori by ed determin was hours Biofilm samples Washing , recover (A)
Collect pellets Grind in liquid N" freeze-th aw three times Add extracllo n buffer and proteinas e K
Carbohy drate DNA
Figure 2. Procedure for lysing the pellets from biotilms.
during the extractions, the pellets Celll.l'Sis stuc[y In order to evaluate the degree of cell lysis that occurred (Zhou et aJ., 1996). Figure 2 method aw freeze-th a were collected after the RCF extraction and lysed using
Quantifying eXllacellular polymers in biofiIms
215
shows the procedure for lysing the pellets from biofilms. DNA, carbohydrate and proteins from the pellets were quantified. In order to verifY the accuracy of results, the EPS and the lysed pellets were combined and analyzed for DNA, carbohydrate and proteins. RESULTS AND DISCUSSION Contribution of the wasbinK step to the BPS yields It was found that the carbohydrate extraction yield contributed by the washing step varied from 8-50% among the different extraction methods (see Figure 3), which verifies that collecting the wash water from the washing step is essential in biofilm EPS analysis (Gehr and Henry, t 983). Ultracentrifugation (33,000 g) contributed the greatest amount of carbohydrate to the EPS yields from the washing step. Depending on the method used, the more the carbohydrate collected from the washing step, the less the carbohydrate recovered from the stripping step. M
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Extraction method cowpansons Figures 4 and 5 show the extracted carbohydrate, protein and DNA concentrations measured by each extraction method. Among the methods, for both types of biofilms, the RCF extraction provided the greatest amount of carbohydrate, but only relatively small amounts of proteins because formaldehyde cross-links proteins and make them hard to detect. The steammg extraction procedure helps to release the stripped material from the pellets; it gave the second greatest amount of carbohydrate and the greatest amount of proteins. The other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins. In both types of biofilms, the protein yield was lower than the carbohydrate yield In the EPS. The RCF method gave the highest yield ratio of carbohydrate to proteins: 13 7 for the aerobic/sulfate reducing biofilms, and t t.O for the nitrifying!denitrifying biofilms; the rest of the methods gave a range of ratios: 1.54-2.23 for the aerobic/sulfate reducing biofilms, and 2.99-4.64 for the nitrifyingldemtrifying biotilms. A perequlslte for a good extraction method is to remove exopolymer material effectively, with minimal cell lysis (Gehr and Henry, 1983). DNA was used as the cell lysis indicator. The amounts of DNA after extraction may be due either to its release from the exopolymer matrix or from cell disruption. DNA concentrations were very low in both aerobic/sulfate reducing and nitrifymgldenitrifying biofilms, the highestbeing(4.17± 1.53) x 1O"llglllgTS. Cell lySIS study. Although the amounts of DNA in the extracted EPS were very small, it is still difficult to conclude that there was no cell lysis during any of the extractions without knowing the DNA amounts in the pellets. Therefore, an experiment was designed to study the DNA amounts in the pellets, together with a comparison of DNA amounts in the EPS and the blOfilms. Carbohydrate and protein concentrations were measured as well (FIgure 6). A great amount of DNA 18.52 ± 1.55) x 10'3 IJgllJg TS) was detected in the pellets. There was only (0,68 ± 0.15) x 10'3 1Jg!1!8 TS in the EPS. DNA amounts in the pellets were 27 hmes higher than in the EPS on a total solids basis. Generally, greater than 90 % of the cells will be lysed using the freezc-thaw method (Zhou et al., 1996). Apparently, no significant cell lysis occurred during any extractions. The sum of the DNA from the EPS and the pellets almost exactly equaled the DNA amounts in the blOfilms, which verified the accuracy of the measurement.
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Quantifymg extracellular polymers in biotilms
217
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Figure 6. Blofibn composition (sulfate/reducmg biofilms, RCF extraction method),
CONCLUSIONS Collecting the wash solutions is essential in analyzing the EPS from biofilm; its contribution varied from 8-50% of total carbohydrate yields, depending on the extraction method. The five extraction methods presented a general trend in their ability to extract the EPS from aerobic/sulfate reducing and nitrit)'ing/denitrit)'ing biofilms. The RCF extraction gave the greatest carbohydrate yield and steaming extraction gave the greatest protein yield for extracted EPS from both types of biofilms. The other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins. DNA concentrations in the EPS were 27 times smaller than in the pellets, indicating no obvious cell lysis occurred during the five different extractions. ACKNOWLEDGMENT We gratefully acknowledge Tong Yu, Jin Li, Qingzhong Wu and Mark Kramer of the University of Cincinnati for their assistance with reactor operation. This study was funded by the Superfund Basic Research Programs of the National Institutes of Environmental Health Sciences (NIEHS), USA. REFERENCES Bradford, M M. (1976). A rapId and sensitive method for the quantification of microgram quantities of protein utilizing the pnnclple of protein-dye binding. Allalyr Bwchem., n, 248-54. Brown. M. J. and Lester, J. N. (1980). Companson of bacterial exlracellular polymer extraction methods. Appl. Ellviroll. M,crobiol.,40(2). 179-185
Forster, C. F. (1982). Sludge surfaces and theIr relation to the rheology of sewage sludge suspensions. J Chem. Techllol. BlOrechllol.• 32, 799-807.
Forster. C. F. and Clarke, A. R. (1983). Theproducbon of polymer from activated sludge byethanohc extraction and its relation to treatment plant operation. Waf. POIIUf. Control. 82, 43()-433. Fr+lund, 8., Palmgren. R.. Keiding, K., and Nielsen, P. H. (1996). Exlraction of eXlracellular polymers from acllvated sludge usmg a calion exchange resm. Waf. Re,·., 30(8). 1749-1758. Gehr, R and Henry. J. G. (1983). Removal of extracellular material- techniques and pitfalls. Waf. Res .• 17(12). 1743-1748. Gerhardt. P.• Murray. R. G. E., Wood. W. A. and Krieg, N. R. (1994) Mefhods for General and Molecular Bacteriology. Amencan Society for MIcrobiology, Washington, D.C. GoodWin. J.A.S. and Forster, C. F. (1985). A further examinatIon inlo the composition of activated sludge surfaces in relation to their selliement characteristics. Waf. Res., 19, 527-533. Harmer. C. (1991). Factors affecling secondary ulllization kinetics of 8%0 dye in wastewater biofilms. M.S. Thesis, UDlversity of Cmcmnatl, Cincinnati, OhIO. U.S.A. Harmer. C. and BIshop. P. L. (1992). Transformation of azo dye AO-7 by waslewater blofibns. Wat. Sci. Tech., 26(3/4). 627-636. Horan, N. J. and Eccles. C. R. (1986). Purification and characterization of extracellular polysaccharide from activaled slUdge. Wat. Res. 20. 1427-1432.
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Jia. X. S., Furumai, H. and Fang, Herbert H. P. (1996). Yields of biomass and extracellular polymers m four anaerobic sludges. Environ. Tech .. 17.283-291. Karapanagiotls, N. K., Rudd, T., Sterritt, R. M. and Lester, 1. N. (1989). Extraction and characterization of extracellular polymers In digested sewage sludge. J. Chem. Technol. Biotechnol., 44,107-120. Morgan, J. W., Forster, C. F. and Evison, L. (1990). A comparative study of the nature of biopolymer extracted from anaerobic and acnvated sludges. Wat. Res., 24(6), 743-750. Sutherland, I. W. (1977). Bacterial exopolysaccharides - their nature and production. In: Surface Carbohydrates of the Procaryotic Cells, I. W. Sutherland (Ed.), Academic Press, Loodon, England, pp. 27-96. Urbam. V.. Block, J. C. and Manem, J. (1993). Bioflocculation m activated sludge: an analytical approach. Wat. Res., 27(5), 829-838. Zhou, J. Bruns, M. A. and Tiejie, J. M. (1996). DNA recovery from soils of diverse composition. Appl. Environ. Microbiol., 62(3),316-322.
Pergamon
War Sci T«h Vol. 39. No.7, pp. 211-218.1999 ro 1999/AWQ
Published by Elsevier SCIence LId Pnnted in Ore.r Britain. All righrs reserved
PH: 50273-1223(99)00170-5
0273-1223/99 $2000 + 0.00
COMPARISON OF EXTRACTION METHODS FOR QUANTIFYING EXTRACELLULAR POLYMERS IN BIOFILMS Xiaoqi Zhang"', Paul L. Bishop'" and Brian K. Kinkle"'''' • Department o/Civil and Environmental Engineering, University o/Cincinnati, Cincinnati, OH 4522/·0071. USA .. Department 0/ Biological Sciences, University 0/ Cincinnati. Cincinnati. OH 4522/-0006. USA
ABSTRACT Five commonly used extraction methods - regular ceninfugation. EDTA extraction. ultracentrifugation, steaming extraction and regular centrifugation With formaldehyde (ReF) - were selected to study their effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfale reducing and mtrifyingldenitrifying biofilm samples. Biofilm EPS extraction yields were represenled by carbohydrate and protein concentrations; the amount of cell lysIS during the extractions was indicated by DNA concentratlOn. The results showed !hat analyzing wash walers is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield. dependIng on the extracllOn method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the steaming extraction gave the greatest protem yield, and the other three extraction methods gave approximately equivalent amounts of carbohydrale and proteins for both types of biofilm. DNA in the EPS was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions. ~ I999lA WQ Published by Elsevier Science Ud. All rights reserved
KEYWORDS Aerobic/sulfate reducing; biofilms; carbohydrate; DNA; extracellular polymeric substances (EPS); extraction methods; nitrifying/denitrifying; proteins. INTRODUCTION Microbial extracellular polymeric substances (EPS) are high molecular-weight mucous secretions of bacteria and micraalgae. They range from tight capsules, which closely bind cells, to the loosely attached slime material. Biofilm EPS possesses many important functions in water and wastewater treatment. including anchoring the microorganisms near food sources, protecting them from dehydration and toxic substances, and providing ion exchange properties due to negatively-charged surface functional groups which allow them to bind cationic species such as heavy metals (Sutherland. 1977). The EPS composition determines many important properties ofbiofilm such as density. porosity, difTusivity, strength, elasticity, frictional resistance. thermal conductivity, and metabolic activity. More information about their compositions will contribute to a better understanding of the physical and physiological behavior ofbiofilms in environmental systems. 21 I
212
X. ZHANG et al.
Many ~ethods have been used to extract the EPS from different bacterial cultures and activated sludges for analysIs. These extraction methods have included use of ammonium hydroxide, sodium hydroxide, ethylene-diaminetetraacetic aCId (EDTA), sulfuric acid, trichloroacetic acid, boiling benzene, ultrasonication. blending, high-speed centrifugation, and extraction by boiling or autoclaving (Brown and Lester. 1980). However, most of these extraction studies were perfonned on activated sludge (Brown and Lester, 1980, Gehr and Henry, 1983, Morgan et al., 1990, Urbain et al., 1993, Jia et al., 1996, Fr01und et al., 1996); very little research has been conducted on biofilm samples. The chemical compositions of the EPS are usually reported in the literature to be very heterogeneous. Carbohydrate predominates and represents up to 65% of extracellular materials (Horan and Eccles, 1986); other substances are also present, such as proteins. nucleic acids and lipids (Goodwin and Forster, 1985). Their exopolymer component ratios vary, depending on the sample source and extraction technique (Morgan et al., 1990). For example, Horan and Eccles (1986), Forster and Clarke (1983) and Morgan et al. (1990) found more carbohydrate than proteins in activated sludges, with a ratio range of 0.16-0.70. Karapanagiotis et al., (1989), Forster (1982) and Morgan et al. (1990) found more proteins than carbohydrate in digested sludges, with a ratio range of 1.1-5.1. In this study, five extraction methods, which are currently used for activated sludge, were evaluated for the extraction of EPS from biofilm samples grown under aerobic/sulfate reduction and nitrification/denitrification conditions. The five extraction methods are: (I) regular centrifugation, (2) EDTA extraction, (3) ultracentrifugation, (4) steaming extraction, and (5) regular centrifugation with fonnaldehyde. The five extraction methods were compared on the same samples to examine their effectiveness and repeatability and to detennine to what degree they may cause cell lysis. Minimum cell lysis is desired during the extractions to avoid contamination by intracellular material. To evaluate the degree of cell disruption that may be caused during the extractions, pellets in the biofilm were intentionally lysed and the amounts of DNA from the EPS and the pellets were compared. Researchers dealing with the EPS of activated sludge nonnally wash their activated sludge samples first and then discard the wash solution. Gehr and Henry (1983) proposed, though, that an initial washing step will remove exopolymer material from activated sludge. Presumably, the washing step will wash out some loosely attached extracellular polymers. The contribution of the washing step to the EPS collected from biofilm samples was verified and the amount of its contribution was further examined in this research. METHODS Bio.,films Biofilrns are typically heterogeneous structures. Many biofilms may essentially be considered to be layered. with an aerobic layer overlying anoxic or anaerobic layers. This is true ofbiofilms in which both nitrification and denitrification occur, and in ones where sulfate reduction occurs in lower, anoxic layers.
Aerobic/sulfate reducing biofilms and nitrifying/denitrifying biofilms were grown in laboratory-scale rotating drum biofilm reactors (RDBRs). Biofilm growth occurs on the surface of the RDBR rotating drum and removable sampling slats which arc coated with shrink tubing. A more detailed description of the reactor system can be found in Hanner (1991) and Harmer and Bishop (1992). The aerobic/sulfate reducing biofilm reactor was initially seeded with wastewater and digested sludge from a municipal/industrial wastewater treatment plant in Cincinnati, Ohio. The reactor was continuously fed with: yeast extract, 50 mg/I; sodium lactate (C 3Hs0 3Na), 156 mg/I; KH2P04, 120 mg/l; NaHC03, 180 mg/l; NH4CI, 100 mg/I; CaCI 2 -2H 20, 15 mg/I; MgS04 -7H 20, 60 mg/I; FeCh- 4H 20, 1.5 mg/l; Na2S04, 200 mg/l. The pH was maintained at 7.2-7.3 and dissolved oxygen (DO) was kept at 1.5-3.5 mg/1. The nitrifying/denitrifying biofilm reactor was seeded with nitrifiers and secondary effluent from a trickling filter wastewater treatment plant in Dayton, Ohio. It was continuously fed with: bacto peptone, 25 mg/l; yeast extract, 25 mg/I; NH4C1, 95 mg/I; K2C0 3, 320 mg/I; K2HP04. 25 mg/I; KH 2P04, 10 mg/I; MgS04 -7H 20, 11.25 mg/l; CaCh-2H20, 13.75 mg/I; FeCh -6H20, 0.125 mg/I; MnS04-H20, 0.0112 mg/1; CUS04. 0.0007 mg/I; Na2Mo04-2H20, 0.0004 mg/I; 2n504-71-1 20, 0.0120 mg/1. The pl-l and DO were kept at 7.5 and 1.5-3.5mg/I, respectively. Biofilm samples were collected from the sampling slats and total solids (TS) were measured to represent biofilm mass.
Quantifying extracellular polymers in biofilms
213
BPS e~tractiQn meth~s All biofilm samples were analyzed in triplicate. Four steps were involved in extractmg and collectmg the EPS from the biofilm samples (Figure I). Biofilm samples Washing, recover slime material (A)
Figure J. Procedure for the extrachon method compansons.
Step J: Washing (recover slime material). Biofilm samples (about 1 g) were put into centrifuge tubes along with 25 ml MilIiQ water (Millipore). The tubes were shaken gently, and then centrifuged at 3,500 rpm (6,ooOg) for 10 minutes. The liquid was decanted from the centrifuge tubes, and collected as the slime material. Step 2: Stripping (recover capsule-bound material). After the washing step, the tubes containing the biofilm pellets were filled with another 25 ml MilIiQ water. The contents were blended in a vortex blender (Oster, 867-28L) at high speed for 1 minute to recover the capsule-bound material.
For purposes of extraction method comparisons, liquid from the washing step and the stripping step were combined and brought to a volume of 50 mt. Step 3: Extraction (separate the stripped materialfrom the cells). Five extraction methods were applied and compared. These included:
(1) Rel:U1ar centriful:atjon (lia et al., 1996, with modification). 10 ml of the combined sample were centrifuged at 12,000 rpm (lI,227g) for 30 minutes at room temperature. (2) forA extraction (Brown and Lester, 1980). 10 ml of 2% EDTA was added to 10 ml of the combined sample and left quiescently for 3 hours at 4°C. The centrifuge tubes were then centrifuged at 14,000 g for 20 minutes at 4°C. (3) U1tracentriful:atjon (Brown and Lester, 1980). 10 ml of the combined sample were centrifuged at 33,000 g for 10 minutes at 4°C. The pellets obtained were resuspended with another 10 ml MilliQ water, and centrifugation was repealed for another 10 minutes to enhance the extraction.
(4) Slearnjnl: extraction (Brown and Lesler, 1980). 10 ml of the combined samples were sleamed in an autoclave at 80 aC under I bar pressure for 10 minutes and then centrifuged while slill hot at 8,000 g for 10 minutes; during centrifugation, the temperature was reduced to 15°C. The steaming treatment was used in an
X. ZHANG et al.
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autoclaving under normal autoclave attempt to reduce the disruptive effects on the cells of boiling or conditions (120°C, 16 bars). The same extraction procedure (5) ~e~ular centriful:atjon with foona1dehyde (RCF) (Jia et al., 1996). g 0.22% formaldehyde was containin NaCI 8.5% ml 25 descn~ed above was used on I g samples, except that was centrifuged at 12,000 sample d combine the of ml 10 step. stripping the in water added Instead ofMilliQ ge with an SS-34 rotor Centrifu ed Superspe ted Refrigera rpm (11,227 g) for 30 minutes in a Sorvall RC-5B (Du Pont). centrifugation after each extraction Step 4: Filtering and collectmg the EPS. The supernatant obtained on were free of cells. The pellets samples that was filtered through 0.22 j.!m cellulose acetate filters to ensure study. lysis cell the for used ifnot were discarded yields. the same procedure described In order to evaluate the contribution of the washing step to biofilm EPS material were treated separately to capsule the above was replicated. except that the slime material and obtain separate EPS yields. were represented by carbohydrate and ChemIcal composition qna[ysiy The EPS yields in the extracts ation. A HP 8453 UV-Vis diode-array concentr DNA by protein concentrations, and cell lysis was indicated (Gerhardt et al., 1994) method was acid ulfuric phenol-s A used. was r hotomete (190 - 1100 nm) spectrop tion of the Bradford (1976) method, modifica A . used to quantitY Carbohydrate, with glucose as the standard proteins, with Bovine Serum quantify to used was l), Chemica (Pierce e procedur ie called the Coomass was used to quantitY DNA, 1994) al., et t (Gerhard method Albumin (BSA) as the standard. A fluorometric . standard the as sperm with salmon s with protein analysis. Theref~re, a~1 Protein dialysis. Formaldehyde used in the RCF extraction interfere or I Molecularporous DialySIS Spectra/p a through put were n the samples treated after the RCF extractio hyde. The dialysis membranes were formalde the remove to m) (Spectru 8,000 6 MWCO with e Membran An optimum dialysis time of 5-7 placed in a beaker containing 2 liter of continuously stirred MilliQ water. hour time period. 88 an over ations concentr protein ng monitori by ed determin was hours Biofilm samples Washing , recover (A)
Collect pellets Grind in liquid N" freeze-th aw three times Add extracllo n buffer and proteinas e K
Carbohy drate DNA
Figure 2. Procedure for lysing the pellets from biotilms.
during the extractions, the pellets Celll.l'Sis stuc[y In order to evaluate the degree of cell lysis that occurred (Zhou et aJ., 1996). Figure 2 method aw freeze-th a were collected after the RCF extraction and lysed using
Quantifying eXllacellular polymers in biofiIms
215
shows the procedure for lysing the pellets from biofilms. DNA, carbohydrate and proteins from the pellets were quantified. In order to verifY the accuracy of results, the EPS and the lysed pellets were combined and analyzed for DNA, carbohydrate and proteins. RESULTS AND DISCUSSION Contribution of the wasbinK step to the BPS yields It was found that the carbohydrate extraction yield contributed by the washing step varied from 8-50% among the different extraction methods (see Figure 3), which verifies that collecting the wash water from the washing step is essential in biofilm EPS analysis (Gehr and Henry, t 983). Ultracentrifugation (33,000 g) contributed the greatest amount of carbohydrate to the EPS yields from the washing step. Depending on the method used, the more the carbohydrate collected from the washing step, the less the carbohydrate recovered from the stripping step. M
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216
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Extraction method cowpansons Figures 4 and 5 show the extracted carbohydrate, protein and DNA concentrations measured by each extraction method. Among the methods, for both types of biofilms, the RCF extraction provided the greatest amount of carbohydrate, but only relatively small amounts of proteins because formaldehyde cross-links proteins and make them hard to detect. The steammg extraction procedure helps to release the stripped material from the pellets; it gave the second greatest amount of carbohydrate and the greatest amount of proteins. The other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins. In both types of biofilms, the protein yield was lower than the carbohydrate yield In the EPS. The RCF method gave the highest yield ratio of carbohydrate to proteins: 13 7 for the aerobic/sulfate reducing biofilms, and t t.O for the nitrifying!denitrifying biofilms; the rest of the methods gave a range of ratios: 1.54-2.23 for the aerobic/sulfate reducing biofilms, and 2.99-4.64 for the nitrifyingldemtrifying biotilms. A perequlslte for a good extraction method is to remove exopolymer material effectively, with minimal cell lysis (Gehr and Henry, 1983). DNA was used as the cell lysis indicator. The amounts of DNA after extraction may be due either to its release from the exopolymer matrix or from cell disruption. DNA concentrations were very low in both aerobic/sulfate reducing and nitrifymgldenitrifying biofilms, the highestbeing(4.17± 1.53) x 1O"llglllgTS. Cell lySIS study. Although the amounts of DNA in the extracted EPS were very small, it is still difficult to conclude that there was no cell lysis during any of the extractions without knowing the DNA amounts in the pellets. Therefore, an experiment was designed to study the DNA amounts in the pellets, together with a comparison of DNA amounts in the EPS and the blOfilms. Carbohydrate and protein concentrations were measured as well (FIgure 6). A great amount of DNA 18.52 ± 1.55) x 10'3 IJgllJg TS) was detected in the pellets. There was only (0,68 ± 0.15) x 10'3 1Jg!1!8 TS in the EPS. DNA amounts in the pellets were 27 hmes higher than in the EPS on a total solids basis. Generally, greater than 90 % of the cells will be lysed using the freezc-thaw method (Zhou et al., 1996). Apparently, no significant cell lysis occurred during any extractions. The sum of the DNA from the EPS and the pellets almost exactly equaled the DNA amounts in the blOfilms, which verified the accuracy of the measurement.
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Quantifymg extracellular polymers in biotilms
217
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Figure 6. Blofibn composition (sulfate/reducmg biofilms, RCF extraction method),
CONCLUSIONS Collecting the wash solutions is essential in analyzing the EPS from biofilm; its contribution varied from 8-50% of total carbohydrate yields, depending on the extraction method. The five extraction methods presented a general trend in their ability to extract the EPS from aerobic/sulfate reducing and nitrit)'ing/denitrit)'ing biofilms. The RCF extraction gave the greatest carbohydrate yield and steaming extraction gave the greatest protein yield for extracted EPS from both types of biofilms. The other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins. DNA concentrations in the EPS were 27 times smaller than in the pellets, indicating no obvious cell lysis occurred during the five different extractions. ACKNOWLEDGMENT We gratefully acknowledge Tong Yu, Jin Li, Qingzhong Wu and Mark Kramer of the University of Cincinnati for their assistance with reactor operation. This study was funded by the Superfund Basic Research Programs of the National Institutes of Environmental Health Sciences (NIEHS), USA. REFERENCES Bradford, M M. (1976). A rapId and sensitive method for the quantification of microgram quantities of protein utilizing the pnnclple of protein-dye binding. Allalyr Bwchem., n, 248-54. Brown. M. J. and Lester, J. N. (1980). Companson of bacterial exlracellular polymer extraction methods. Appl. Ellviroll. M,crobiol.,40(2). 179-185
Forster, C. F. (1982). Sludge surfaces and theIr relation to the rheology of sewage sludge suspensions. J Chem. Techllol. BlOrechllol.• 32, 799-807.
Forster. C. F. and Clarke, A. R. (1983). Theproducbon of polymer from activated sludge byethanohc extraction and its relation to treatment plant operation. Waf. POIIUf. Control. 82, 43()-433. Fr+lund, 8., Palmgren. R.. Keiding, K., and Nielsen, P. H. (1996). Exlraction of eXlracellular polymers from acllvated sludge usmg a calion exchange resm. Waf. Re,·., 30(8). 1749-1758. Gehr, R and Henry. J. G. (1983). Removal of extracellular material- techniques and pitfalls. Waf. Res .• 17(12). 1743-1748. Gerhardt. P.• Murray. R. G. E., Wood. W. A. and Krieg, N. R. (1994) Mefhods for General and Molecular Bacteriology. Amencan Society for MIcrobiology, Washington, D.C. GoodWin. J.A.S. and Forster, C. F. (1985). A further examinatIon inlo the composition of activated sludge surfaces in relation to their selliement characteristics. Waf. Res., 19, 527-533. Harmer. C. (1991). Factors affecling secondary ulllization kinetics of 8%0 dye in wastewater biofilms. M.S. Thesis, UDlversity of Cmcmnatl, Cincinnati, OhIO. U.S.A. Harmer. C. and BIshop. P. L. (1992). Transformation of azo dye AO-7 by waslewater blofibns. Wat. Sci. Tech., 26(3/4). 627-636. Horan, N. J. and Eccles. C. R. (1986). Purification and characterization of extracellular polysaccharide from activaled slUdge. Wat. Res. 20. 1427-1432.
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Jia. X. S., Furumai, H. and Fang, Herbert H. P. (1996). Yields of biomass and extracellular polymers m four anaerobic sludges. Environ. Tech .. 17.283-291. Karapanagiotls, N. K., Rudd, T., Sterritt, R. M. and Lester, 1. N. (1989). Extraction and characterization of extracellular polymers In digested sewage sludge. J. Chem. Technol. Biotechnol., 44,107-120. Morgan, J. W., Forster, C. F. and Evison, L. (1990). A comparative study of the nature of biopolymer extracted from anaerobic and acnvated sludges. Wat. Res., 24(6), 743-750. Sutherland, I. W. (1977). Bacterial exopolysaccharides - their nature and production. In: Surface Carbohydrates of the Procaryotic Cells, I. W. Sutherland (Ed.), Academic Press, Loodon, England, pp. 27-96. Urbam. V.. Block, J. C. and Manem, J. (1993). Bioflocculation m activated sludge: an analytical approach. Wat. Res., 27(5), 829-838. Zhou, J. Bruns, M. A. and Tiejie, J. M. (1996). DNA recovery from soils of diverse composition. Appl. Environ. Microbiol., 62(3),316-322.