Vol. 40, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1980, p. 179-1850099-2240/80/08-0179/07$02.00/0

Comparison of Bacterial Extracellular Polymer ExtractionMethods

MELANIE J. BROWN AND JOHN N. LESTER*Public Health Engineering Laboratory, Imperial College, London SW7, England

Five different bacterial extracellular polymer extraction methods and a com-bination of two of these methods were compared on cultures of activated sludge,synthetic activated sludge, and Klebsiella aerogenes. High-speed centrifugationwas the most effective extraction method for the K. aerogenes culture, based onthe comparatively small amount of cell disruption and the relatively high extra-cellular polymer yield. Steaming treatment was the most effective extractionmethod for the activated sludges, since it released a significant quantity ofextracellular polymers from the flocs and caused less cellular disruption thanethylenediaminetetraacetic acid and sodium hydroxide treatments. Sodium hy-droxide treatment caused extensive disruption in all cultures. Ultrasonicationreleased low concentrations of extracellular polymers from all cultures. However,it caused no significant cell disruption and therefore may be useful as a preliminarytreatment in conjunction with another extraction method.

It has been proposed that bacterial extracel-lular polymers have an important protectivefunction, aiding survival and dispersal of bacte-rial species (27, 31). They have been shown tobe involved in flocculation in bacterial culturesand the adsorption of metal ions from solution(2). The removal of metal ions from the aqueousphase is particularly important in the activatedsludge process of biological wastewater treat-ment (2).The bacterial extracellular polymers extracted

from activated sludges have been mainly of apolysaccharide nature, although protein and nu-cleic acid from autolysis are also constituents ofthe floc polymer matrix (2). Most activatedsludge bacteria are gram negative (22); extracel-lular protein production is very rare in gram-negative organisms due to the complex and im-permeable nature of the cell walls (3). The mostcommon monosaccharides found in bacterial ex-tracellular polymers are the hexoses D-glucose,D-galactose, and D-mannose (27). Steiner et al.(25) found that the monosaccharides present inslime layers extracted from activated sludgesvaried between different sludge samples. Hex-oses, pentoses, and glucuronic acids were presentin all the sludges investigated. The slime andcapsular extracellular polymers of Klebsiellaaerogenes (formerly Aerobacter aerogenes)have been characterized (11, 24). The polysac-charides contained approximately 50% glucose,29% uronic acid, 10% fucose, and 1% galactose(11).Many methods have been used to extract ex-

tracellular polymers from different bacterial cul-

tures. These have included ammonium hydrox-ide extraction (21, 28), sodium hydroxide extrac-tion (29), ethylenediaminetetraacetic acid(EDTA) extraction (19), sulfuric acid extraction(24), trichloroacetic acid extraction (6), boilingbenzene extraction (30), ultrasonication (14),blending (12), high-speed centrifugation (21),and extracting by boiling or autoclaving (11, 13,23).The aim of the present investigation was to

select an extraction method suitable for routine,quantitative analysis of extracellular polymerconcentrations in pure and complex bacterialcultures. This was achieved by comparing theeffectiveness of five extraction methods (and onecombined method) in releasing extracellular pol-ymers from three different bacterial cultures:activated sludge, synthetic activated sludge, anda pure culture of K. aerogenes. The extractionmethods selected for this comparison repre-sented most of the methods which have beenshown to successfully extract polymers frompure cultures or activated sludges.

MATERIALS AND METHODSBacterial cultures. Return activated sludge was

obtained from Mogden Water Pollution ControlWorks (Thames Water Authority, London) with asludge age of between 5 and 6 days, a suspended solidsconcentration of 8.222 g/liter, and a sludge volumeindex (SVI) of 116 ml/g. Synthetic activated sludgewas obtained from a modified Hussman activatedsludge simulation operated at a sludge age of 6 dayson a synthetic sewage feed (26). The synthetic sewagecontained (grams per liter): Lab Lemco (meat extract)Oxoid L 29, 0.234; bacteriological peptone Oxoid L 34,

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180 BROWN AND LESTER

0.156; NaCI (analytical reagent grade), 0.009; CaCl2(G.P.R.), 0.005; MgSO4. 7H20 (analytical reagentgrade), 0.002; NH4Cl (analytical reagent grade), 0.072;KH2PO4 (analytical reagent grade), 0.006. The syn-thetic activated sludge had a suspended solids concen-tration of 3.105 g/liter and an SVI of 267 ml/g. A purebatch culture of a capsular strain of K. aerogenes(NCTC 8172) was grown in an LKB 1601 Ultrofermfermentation system (LKB Instruments Ltd., SouthCroydon, U.K.) at 25 ± 0.2°C, pH 7.0 + 0.05, and adissolved oxygen saturation of 75 ± 5%. The mediumcontained the following analytical reagent-grade salts(grams per liter): 2 NaO-CO-CH2C(OH)(COONa)-CH2COONa.2H20 (trisodium citrate), 2; NH4Cl, 0.5;MgSO4. 7H20, 0.1; FeSO4. 7H20, 0.01; CaCl2.6H20,0.01; MnSO4.4H20, 0.005; KH2PO4, 0.3; and K2HPO4,0.4. The culture was harvested after 5 days. The totalcell dry weight in the K. aerogenes culture was 0.339g/liter.Suspended solids. Measurements of suspended

solids in the sludges were carried out as recommendedby the Government of Great Britain (15). A measure-ment of suspended solids (cell dry weight) in the K.aerogenes culture was obtained by weighing the resi-due retained on filtration through a cellulose acetatefilter, pore size 0.22,m (Oxoid, London), after dryingfor 2 h at 105°C.

SVI. The SVI was determined by a modification(26) of the standard Government of Great Britainmethod (15).

Viable counts. Samples from the K. aerogenesculture were serially diluted in 0.1 M phosphate buffer;then 0.1 ml of each dilution was inoculated on to threesurface-dried plates of Casitone-glycerol-yeast extractagar (22). Plates were inverted and incubated at 250Cfor 36 h.

Extracellular polymer extraction methods. Allextraction procedures and the separation of sludgeflocs from supematants were carried out on threeseparate 100-ml subsamples from an initial 3-liter sam-ple of each bacterial culture.

(i) Separation of sludge flocs from superna-tants. Samples were centrifuged at low speed (ap-proximately 2,000 x g) in a bench centrifuge (MSE,Crawley, U.K.).

(ii) High-speed centrifugation. The method ofPavoni et al. (21) was used in a modified form. Cen-trifugation was carried out on the samples in a Hi-Spin 21 centrifuge (MSE) at 33,000 x g for 10 min at4°C. The pellet obtained was resuspended to increasethe centrifugal force on the cells or flocs, and centrif-ugation at 33,000 x g was repeated for a further 10min.

(iii) Ultrasonication. Samples were ultrasonicatedfor 10 min in a Kerry 125 ultrasonic bath (KerryUltrasonic Ltd., Hitchin, U.K.) with the voltage re-duced to 120 V by means of a Regulac variable trans-former (Claude Lyons Ltd., Hoddesdon, U.K.); thisgave an output of 18 W. Centrifugation at approxi-mately 2,000 x g was carried out for 10 min to removethe cells.

(iv) Ultrasonication and high-speed centrifu-gation. Samples were ultrasonicated for 10 min andthen centrifuged at 33,000 x g as described above.

(v) Steaming extraction. The steaming extraction

method was a modification of that proposed by theWater Pollution Research Laboratory (23). Sampleswere steamed in an autoclave for 10 min and thencentrifuged while still hot at 8,000 x g for 10 min.During centrifugation the temperature was reduced to15°C. The steaming treatment was used in an attemptto reduce the disruptive effects on the cells of boilingor autoclaving.

(vi) Sodium hydroxide extraction. Sodium hy-droxide extraction was carried out as described byTezuka (29) with some modifications. Samples werecentrifuged at 2,000 x g for 20 min. The supernatantswere discarded, and 2 volumes of 2 M NaOH wasadded to the pellet. These samples were gently agita-ted at 20°C for 5 h and then diluted with tap water tothe original sample volume (100 ml) to facilitate cen-trifugation and filtration. The samples were centri-fuged at approximately 2,000 x g to remove the cells.

(vii) EDTA extraction. The EDTA extractionmethod was a modification of that described by Ni-shikawa and Kuriyama (19). In this method, 100 ml of2% EDTA (tetrasodium salt) was added to a 100-mlsample and left for 3 h at 4°C. The samples were thencentrifuged at 14,000 x g for 20 min at 4°C.

Filtration. The supernatants obtained on centrif-ugation after all treatments were filtered through 0.22-um cellulose acetate filters in a Pyrex filtering appa-ratus [Millipore (UK) Ltd., London] to ensure thatsamples were free of cells. The pellets were discarded.

Estimation of extracellular polymer concen-trations and cellular disruption by biochemicalanalysis. Hexose sugar and hexuronic acid concentra-tions were used as measures of extracellular capsularor slime polymers; protein and deoxyribonucleic acid(DNA) concentrations were used as measures of thedegree of cellular disruption.

Hexose sugars were measured by the phenol-sul-furic acid method of Dubois et al. (10) and the an-throne method described by Dische (8). Protein con-centration was determined by the method of Lowry etal. (18), and DNA was measured by the Burton di-phenylamine method (4). The Dische carbazolemethod (9) was used to measure hexuronic acids. D-Glucose was used as the standard for hexose sugars,bovine serum albumen was used for protein, the so-dium salt of DNA from calf thymus gland was usedfor DNA, and D-glucuronic acid was used for hexuronicacids. Three sets of standards were prepared for eachcalibration curve. Two standards and appropriateblanks for sodium hydroxide, EDTA, and residualcolor were included with each batch of experimentalsamples. (Activated sludge extracts had varying de-grees of a yellow/brown color, which was pronouncedafter steaming, sodium hydroxide, and EDTA treat-ments.) All tests were carried out in triplicate. Absorb-ance was measured by using an SP8 100 ultravioletspectrophotometer (Pye Unicam Ltd., Cambridge,U.K.). The mean concentrations of hexose sugars,protein, DNA, and hexuronic acids, estimated fromthe calibration graphs in milligrams per liter, wereconverted into milligrams per gram ofsuspended solidsfor each culture.

Bacterial extracellular polymers may contain hex-osamines, uronic acids, and pentose sugars in additionto hexose sugars (2); therefore, concentrations of hex-

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EXTRACELLULAR POLYMER EXTRACTION PROCEDURES 181

oses could be considerably lower than the total extra-cellular polymer concentrations. The use of standardcompounds, for example, D-glucose, to convert opticaldensity readings into weight equivalents provided anestimate of the concentration of hexose sugars, sinceextracellular polymers contain different hexose sugarsin varying proportions and different hexose sugarsshow different absorbances in the biochemical, color-imetric analyses (10). The glucose equivalent concen-tration, however, allowed direct comparison betweensamples of the same origin and is a valuable index ofextracellular polymer concentration. The bovine se-rum albumen, calf thymus gland DNA, and glucuronicacid are likewise indices of protein, DNA, and hexu-ronic acid concentrations.The biochemical analyses carried out on superna-

tants which had been separated from cells or cell flocs(untreated samples) provided a measure of the resid-ual hexose, protein, DNA, and hexuronic acid concen-trations in the culture supernatants. Any increase inthe concentrations of these compounds in extractedsamples over the values in untreated samples repre-sented their extraction or release from the cells orsludge flocs.

Statistical analysis. The results obtained fromthe biochemical analyses on the supernatants fromthe different extracellular polymer extraction methodswere statistically treated. The analysis of variance,using the F-test (1), and Tukey's procedure (1) wereapplied to the results to identify whether the meanconcentrations of the various compounds in the ex-tracts were statistically different for different extrac-tion methods at the 5% level of significance.

RESULTSThe mean concentrations of hexose sugars,

protein, DNA, and hexuronic acids before andafter subtraction of the untreated sample values,the relative standard deviations of the nine rep-licates, and the results of the statistical analysesare presented in Table 1. The relative standarddeviations of samples for which the mean valuewas very close to 0 were much higher than thoseof samples where mean values were above 10.This was due to the nature of the calculation,and therefore a comparison of the error involvedin each of the extraction treatments is not valid.Steaming, sodium hydroxide, and EDTA extrac-tions produced high yields of the various com-pounds in all the tests. The average relativestandard deviations of these methods were verysimilar for all the biochemical tests in the threecultures, ranging from 7.9 to 11.5%. This suggeststhat none of these three methods involved asignificantly greater error than the others.Comparison of extracted extracellular

polymer concentrations. The results of theanthrone and sulfuric acid-phenol hexose sugaranalyses were in very good agreement, althoughthe anthrone test showed slightly lower valuesin the samples from the K. aerogenes culture.

In all three cultures examined, hexose sugars

were identified in the untreated samples. Theseconcentrations were much higher in the K. aer-ogenes culture than in the activated sludges.

High-speed centrifugation and ultrasonicationwere not effective in removing hexose sugarsfrom the flocs of either sludge. The mean super-natant sugar concentration after high-speed cen-trifugation was slightly lower than that of theuntreated sample, although these two valueswere not statistically different. Ultrasonicationproduced a very slight increase in the superna-tant sugar concentration in both sludges.

Steaming, sodium hydroxide, and EDTA ex-tractions all released hexose sugar concentra-tions from the sludge flocs that were at least 15times greater than the concentrations releasedby other extraction methods. These three meth-ods released very similar quantities ofsugar fromthe synthetic activated sludge flocs. Sodium hy-droxide extraction on activated sludge, however,released approximately three times as muchsugar as the steaming and EDTA extractions.The results of both sugar analyses on K. aer-

ogenes showed a different trend from that ob-served for the sludges. Sodium hydroxide treat-ment produced no significant increase in hexosesugar concentration over the untreated sample.Ultrasonication produced a small increase insupernatant sugar concentration. High-speedcentrifugation, steaming, and EDTA extractionall released considerably larger quantities of hex-ose sugars from the cells, at least twice as muchas the ultrasonication method. EDTA extractionyielded the greatest quantity of sugar, andsteaming released a little more sugar from theculture than did high-speed centrifugation.Hexuronic acid concentration was measured

after extraction of the K. aerogenes culture only,since interference occurred in the analysis ofsludge samples. Sodium hydroxide also inter-fered with the hexuronic acid reaction. The re-sults of the hexuronic acid analysis revealed thesame pattern as the hexose sugar tests in sam-ples extracted from the K. aerogenes culture.Hexuronic acid was present in the untreatedsample. Ultrasonication released very little hex-uronic acid into the supernatant. EDTA, high-speed centrifugation, and steaming extractionsincreased the supernatant hexuronic acid con-centrations; the values were not statistically dif-ferent for these methods.The hexuronic acid concentrations measured

in the samples extracted from K. aerogenes werebetween 14 and 22% (average, 18%) of the hexosesugar concentrations in the same samples asmeasured by the sulfuric acid-phenol test andbetween 24 and 35% (average 31%) as measuredby the anthrone test.The combination of ultrasonication and high-

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EXTRACELLULAR POLYMER EXTRACTION PROCEDURES 183

speed centrifugation did not increase the yieldof hexose sugars and hexuronic acids over theyield from either method alone. In most cases,the supernatant sugar concentrations releasedby the combined method were very similar tothose released by the high-speed centrifugationmethod in any of the cultures.Estimation of cellular disruption caused

by the extraction procedures. (i) Release ofprotein and DNA from the cultures. EDTAinterfered with the protein analysis, intensifyingthe blue color produced by the reaction. For thisreason results are not available for the K. aero-genes culture, and the results reported for theactivated sludges are subject to a greater degreeof error than the protein concentrations pre-sented for the other extraction methods. Theerror in the protein concentration of the sampleextracted by EDTA was increased from 5 to 17%in synthetic activated sludge and from 5 to 22%in activated sludge.Supernatants from untreated samples in all

three cultures contained residual protein andDNA, except in activated sludge, where DNAwas not detectable. Residual protein and DNAconcentrations were particularly high in the K.aerogenes culture. The quantity of protein re-

leased from the activated sludge flocs was ap-

proximately three times greater than the sugarconcentration for all the extraction methods.The reverse was true of the K. aerogenes cul-ture, with the exception of the sodium hydroxidetreatment, which released at least 21 times moreprotein than hexose sugar.Samples from both the sludges extracted by

high-speed centrifugation and ultrasonicationexhibited little or no increase in protein concen-

tration over the untreated samples. Extractionsby these two methods on the K. aerogenes cul-ture revealed small increases in protein concen-trations. Steaming and EDTA extractions on thesludges produced significant increases in super-natant protein concentrations, at least ninetimes as much as high-speed centrifugation andultrasonication, with EDTA extraction releasingmore protein than steaming even when the ad-ditional error in the EDTA values is taken intoaccount. The steaming extraction on the K. aer-

ogenes culture produced a substantial increasein supernatant protein concentration, at leasttwice as much as the ultrasonication and cen-

trifugation methods. Sodium hydroxide extrac-tion resulted in a vast increase in supernatantprotein concentration for all three cultures, atleast three times as much as the steaming andEDTA extractions.The results of the DNA analyses demon-

strated a pattern similar to that for protein in

all three cultiures. High-speed centrifugation andultrasonication produced a very slight increasein DNA concentration in the supernatants of thesludges over the untreated samples. This effectwas slightly magnified in the K. aerogenes sam-ples. Steamring, EDTA, and sodium hydroxideextractions caused an increase in supernatantDNA concentrations over the other methods,with the exception of the synthetic activatedsludge, where sodium hydroxide treatment ap-pears to have failed to increase DNA concentra-tion in extracted samples. In contrast, the quan-tity of DNA extracted by the sodium hydroxidemethod from the activated sludge was muchhigher than that produced by all other methods.

(ii) Effects of ultrasonication on the via-bility of K aerogenes. The K. aerogenes cul-ture contained 168 x 107 viable cells/ml beforetreatment. After ultrasonication, the samplecontained 162 x 107 viable cells/ml. Therefore,no significant reduction in viability occurredduring the ultrasonication treatment.

DISCUSSIONNucleic acids and proteins have been shown

to be bound in the polymer matrix of bacterialflocs (16, 19). An increase in supernatant proteinand DNA concentrations after extraction treat-ment may be due either to cell disruption or tothe release of these compounds from the flocmatrix. If the protein and DNA concentrationsmeasured in supernatants were the products ofautolysis, and had been released from the flocmatrix, a constant relationship would be ex-pected between hexose sugar and protein orDNA concentrations. However, protein concen-trations in samples extracted from the sludgesby any one method varied between 2.5 and 11times the sugar concentrations (this can be cal-culated from Table 1). DNA concentrations alsodid not correlate with sugar concentrations forthe various methods. This suggests that moni-toring supernatant protein and DNA concentra-tions provides an indication of cellular disrup-tion.Untreated samples contained residual

amounts of hexose sugar, protein, and DNA(also hexuronic acid in the K. aerogenes cul-ture). These compounds may have been looseslime extracellular polymers, components of theinfluent medium in the sludges, or the productsof autolysis, since all the cultures were at least5 days old.A difference is apparent in the results from

the two activated sludges and the K. aerogenesculture. Ultrasonication was not effective as anextracellular polymer extraction method in anyof the bacterial cultures examined. However,

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184 BROWN AND LESTER

ultrasonication produced a small increase insugar concentration over the untreated samplesin all three cultures, although a statistical in-crease in hexose sugar concentration released bythe combined method over the high-speed cen-trifugation method is only apparent for syntheticactivated sludge. Since ultrasonication causedno significant cellular disruption, it may be use-ful as a preliminary treatment in conjunctionwith another extraction method.High-speed centrifugation was of no value in

extracting extracellular polymers from activatedsludge flocs; this agrees with the results of otherworkers (5; F. M. Saunders, Ph.D. thesis, Uni-versity of Illinois, Urbana-Champaign, 1975), al-though this treatment was originally reported asan extraction method for activated sludge cul-tures (21). High-speed centrifugation, however,was the most effective method of extractingextracellular polymers from the K. aerogenesculture. Although hexose sugar concentrationsreleased from the culture were slightly lowerthan those released by steaming and EDTAtreatments, the degree of disruption caused dur-ing the extractions was very much lower afterhigh-speed centrifugation. Hence centrifugationat 33,000 x g is recommended as the most reli-able extracellular polymer extraction method forcultures of free bacterial cells in suspension. Theeffectiveness of high-speed centrifugation in ex-tracellular polymer extraction is related to par-ticle size. It has been calculated that the vastmajority of flocs in an activated sludge would besedimented before maximum speed, and hencecentrifugal force, was attained (Saunders, Ph.D.thesis, 1975). This would explain the resultsobtained for the sludges in this experiment. Thefree cells in the K. aerogenes culture would havebeen sedimented more slowly than the sludgeflocs. The cells were, therefore, subjected to agreater shearing force, which acted on individualcells; a large proportion of cells would have beenprotected in the center of the floc.

Steaming, EDTA, and sodium hydroxidetreatments all released significant quantities ofhexose sugar from the activated sludge flocs.The ratios of protein to hexose sugar (as calcu-lated from both methods of hexose sugar meas-urement), however, demonstrate that steamingtreatment released considerably less proteinfrom the activated sludge flocs than did EDTAand sodium hydroxide treatments. This indi-cates that steaming treatment caused less cel-lular disruption than the other two extractionmethods. EDTA extraction also released lessprotein from the flocs than did sodium hydrox-ide treatment. Since the concentrations of DNAmeasured in the samples after extraction werevery low in most cases, the ratios of DNA to

hexose sugar were very similar for the threetreatments and showed no apparent trend. Ad-ditionally, steaming extraction is a physicaltreatment, eliminating the necessity to removechemical extractants before further experimen-tation.Sodium hydroxide treatment caused a vast

amount of cellular disruption in all three cul-tures, greater than any other treatment as meas-ured by protein and hexose sugar concentra-tions. A strong alkali such as sodium hydroxidewould be expected to have severe effects on cells.Farrah and Unz (12) found that bacterial cellsof zoogloeae treated with 0.1 M sodium hydrox-ide appeared largely distorted.

In both the sludges, after steaming extractionthe estimated hexose sugar concentration wasbetween 1.6 and 2.0% of the suspended solids.Since extracellular polymers may also containhexuronic acids, hexosamines, and pentosesugars, the percentage of extracellular polymersextracted from the sludges may be higher thanthis. These values compare well with othersreported for extractions on activated sludges.Carr and Ganczarczyk (5) found that purifiedpolysaccharide extracted from several activatedsludges ranged from 0.15 to 3.8% of the sus-pended solids. The Water Pollution ResearchLaboratory (23) reported that extracellular pol-ymers extracted from activated sludge plantstreating domestic wastes comprised 1.70 ± 1.05%of the suspended solids; polymers extracted fromactivated sludges in conventional sewage treat-ment plants comprised 4.25 ± 0.31% of the sus-pended solids. Kiff (17) extracted between 1.13and 6.47% of suspended solids as extracellularpolymers over a range of retention periods. Theestimated supernatant hexose sugar and hexu-ronic acid concentrations after extraction byhigh-speed centrifugation were between 10 and16% of the total solids in the K. aerogenes cul-ture (depending on the method of hexose sugaranalysis). Parsons and Dugan (20) estimatedthat extracellular polymers extracted from purecultures of Zoogloea ramigera 115, which pro-duces a zoogloeal gel matrix, represented ap-proximately 40% of the total solids. The amountof extracellular polysaccharide, however, may bemany times the cell dry weight under favorableconditions (31).

LITERATURE CITED

1. Bowker, A. H., and G. J. Lieberman. 1972. Engineeringstatistics, 2nd ed. Prentice-Hall, Inc., Englewood Cliffs,N.J.

2. Brown, M. J., and J. N. Lester. 1979. Metal removal inactivated sludge: the role of bacterial extracellular pol-ymers. Water Res. 13:817-837.

3. Bull, A. T. 1972. Environmental factors influencing thesynthesis and excretion of exocellular macromolecules.J. Appl. Chem. Biotechnol. 22:261-292.

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4. Burton, K. 1956. A study of the conditions and mecha-nisms of the diphenylamine reaction for the colorimetricestimation of deoxyribonucleic acid. Biochem. J. 62:315-323.

5. Carr, D. F., and J. Ganczarczyk. 1974. Activated sludgeexocellular material extraction methods and problems,p. 250-261. In J. E. Zajic and N. Kosaric (ed.), Proceed-ings of the 9th Canadian Symposium on Water Pollu-tion Research in Canada. Institute for EnviromnentalStudies, University of Toronto, Toronto, Ont.

6. Davies, D. A. L 1955. The specific polysaccharides ofsome gram negative bacteria. Biochem. J. 59:696-704.

7. Deguid, J. P. 1951. The demonstration of bacterial cap-sules and slime. J. Pathol. Bacteriol. 63:673-685.

8. Dische, Z. 1962. Colour reactions of hexoses. MethodsCarbohydr. Chem. 1:488-494.

9. Dische, Z. 1962. Colour reactions of hexuronic acids.Methods Carbohydr. Chem. 1:492-501.

10. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers,and F. Smith. 1956. Colorimetric method for determi-nation of sugars and related substances. Anal. Chem.28:350-356.

11. Dudman, W. F., and Wilkinson, J. F. 1956. The com-position of the extracellular polysaccharides of Aero-bacter-Klebsiella strains. Biochem. J. 62:289-295.

12. Farrah, S. R., and R. F. Unz. 1976. Isolation of exocel-lular polymer from Zoogloea strains MP6 and 106 andfrom activated sludge. Appl. Environ. Microbiol. 32:33-37.

13. Forster, C. F. 1971. Activated sludge surfaces in relationto the sludge volume index. Water Res. 5:861-870.

14. Friedman, B. A., P. R. Dugan, R. M. Pfister, and C.A. Remsen. 1968. Fine structure and composition ofthe zoogloeal matrix surrounding Zoogloea ramigera.J. Bacteriol. 96:2144-2153.

15. Government of Great Britain, Department of theEnvironment. 1972. Analysis of raw, potable and wastewaters. Her Majesty's Stationery Office, London.

16. Kato, A., K. Isaka, and H. Takahashi. 1971. Floc-forming bacteria isolated from activated sludge. J. Gen.Appl. Microbiol. 17:439-456.

17. Kiff, R. J. 1978. A study of the factors affecting biofloc-

culation in the activated-sludge process. Water Pollut.Control 77:464-470.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with Folin phenolreagent. J. Biol. Chem. 193:265-275.

19. Nishikawa, S., and M. Kuriyama. 1968. Nucleic acid asa component of mucilage in activated sludge. WaterRes. 2:811-812.

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