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Abstract
The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic
sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show
that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1 1.2% of DNA in the
sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For
each gram of volatile solids, formaldehydeNaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic
and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic
substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge
(62% in the EPS extracted by formaldehydeNaOH), whereas protein was predominant in the methanogenic sludge
(41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS
from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated
from confocal laser scanning microscopic observations, formaldehyde NaOH extracted only a limited portion of
EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Extracellular polymeric substances (EPS); Extraction; Formaldehyde; Quantification; Sludge
1. Introduction
Extracellular polymeric substances (EPS) are
metabolic products accumulating on the bacterial
cell surface (Morgan et al., 1990). They form a
protective layer for the cells against the harsh
external environment, and also serve as carbon
* Corresponding author. Tel.: + 852-2859-2660; fax: + 8522559-5337.
E-mail address: hrechef@hkucc.hku.hk (H.H.P. Fang).
0168-1656/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 0 2 5 - 1
250
moved 90% COD, 95% BOD5, and 85 90% nitrogen from wastewater. The acidogenic sludge
was sampled from a fermentor (Biostat B, B.
Braun Biotech) treating a sucrose-rich wastewater. It converted over 95% sucrose with 6 h of
hydraulic retention into butyrate and acetate,
plus a biogas containing 63% hydrogen, 35%
carbon dioxide and 2% nitrogen. The granular
methanogenic sludge was sampled from an
upflow reactor treating phenolic wastewater with
12 h of hydraulic retention. The reactor degraded 97% phenol in wastewater and produced
a biogas comprising 69% methane and 31% carbon dioxide (Fang et al., 1996).
251
The total quantity of extracted EPS was measured by the weight of solids after lyophilization.
The carbohydrate content in EPS was measured
by the anthrone method (Gaudy, 1962) using
glucose as the standard. The contents of protein
and humic substance in EPS were measured by
the modified Lowry method (Frlund et al., 1995)
using bovine serum albumin and humic acid
(Pract., Fluka, USA) as the respective standards.
Uronic acid content was measured by the m-hydroxydiphenyl sulfuric acid method (Blumenkrantz and Asboe-Hansen, 1973) as modified
by Kintner and Van Buren (1982) using glucuronic acid as the standard. The DNA contents
in both EPS and sludge were measured by the
diphenylamine colorimetric method (Sun et al.,
1999) using E. coli DNA as the standard. The
DNA were extracted from the sludge following
the procedures described previously (Zhang and
Fang, 2000).
252
Table 1
EPS extracted from various sludges
Extraction
FormaldehydeNaOH
EDTA
Formaldehyde-ultrasound
Cation exchange resin
Formaldehyde
Control
164.99 3.9
146.89 1.1
77.9 91.1
57.8 91.0
49.7 91.2
25.7 9 0.3
179.09 3.2
104.9 90.7
100.09 0.3
58.4 91.1
56.9 9 1.2
36.9 90.6
102.1 9 1.5
73.2 9 0.5
36.8 9 0.8
30.1 9 0.9
32.5 9 0.4
15.8 9 0.2
Extraction method
Reference
Aerobic
Aerobic
Aerobic
Acidogenic
Methanogenic
Methanogenic
Methanogenic
FormaldehydeNaOH
Cation exchange resin
80 C
FormaldehydeNaOH
FormaldehydeNaOH
Phenol
Formaldehyde at 100 C
165
109
58
179
102
91
74
Present study
Rudd et al., 1984
Liu et al., 2001
Present study
Present study
Veiga et al., 1997
Fang and Jia, 1996
253
Table 3
Constituents of EPS in each gram of aerobic activated sludge
Extraction
Carbohydrate
(mg)
Protein
(mg)
Humic substance
(mg)
Uronic acid
(mg)
DNA
(mg)
Unknown
(mg)
Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control
40.5 91.7
54.6 9 2.0
50.4 9 3.7
4.2 90.4
0.35 90.05
14.8 93.3
12.491.2
28.9 90.9
22.9 9 0.5
20.4 9 1.0
59.29 2.5
18.9 9 1.5
2.1 90.4
1.8 90.1
0.47 90.03
0.13 90.02
49.7 9 3.1
7.8 90.5
12.7 90.4
17.6 9 0.9
16.4 9 0.8
1.2 90.2
0.14 90.02
9.8 91.0
15.9 91.0
7.7 90.1
12.3 9 0.3
7.990.1
10.99 0.6
6.4 90.3
1.1 90.1
0.5 9 0.1
0.07 9 0.01
0.06 9 0.01
9.4 90.3
3.1 9 0.2
Table 4
Constituents of EPS in each gram of acidogenic sludge
Extraction
Carbohydrate
(mg)
Protein
(mg)
Humic substance
(mg)
Uronic acid
(mg)
DNA
(mg)
Unknown
(mg)
Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control
110.99 4.0
25.89 1.0
15.1 9 1.0
5.5 9 0.3
0.15 90.03
21.5 91.1
41.79 1.2
71.6 91.3
6.5 90.4
10.890.7
15.9 9 1.0
5.09 0.4
2.3 9 0.2
2.5 9 0.3
0.22 90.03
0.05 90.01
38.3 91.1
10.0 90.4
38.7 90.8
6.2 90.3
3.090.2
2.2 9 0.1
0.08 90.01
8.2 90.3
39.4 91.0
23.7 90.7
5.990.4
4.49 0.2
2.59 0.3
1.9 9 0.2
1.7 9 0.2
1.0 9 0.1
0.02 90.00
0.02 90.00
7.4 90.3
5.9 90.2
Table 5
Constituents of EPS in each gram of methanogenic sludge
Extraction
Carbohydrate
(mg)
Protein
(mg)
Humic substance
(mg)
Uronic acid
(mg)
DNA
(mg)
Unknown
(mg)
Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control
19.19 0.8
42.19 1.7
23.3 9 2.0
2.1 9 0.2
0.19 90.01
15.3 91.45
6.89 0.5
12.0 9 0.4
12.090.7
13.1 90.7
24.39 0.7
5.690.2
1.2 9 0.1
1.0 9 0.1
0.26 90.03
0.04 90.00
28.3 90.52
5.1 90.78
7.99 0.4
10.69 0.4
5.590.3
0.9 9 0.1
0.05 90.01
5.1 90.95
9.7 90.7
4.1 90.2
11.990.8
5.890.2
4.6 9 0.3
3.1 9 0.1
0.8 9 0.1
0.3 9 0.1
0.03 90.00
0.03 90.00
5.5 90.44
2.5 90.17
254
all sludges were primarily composed of carbohydrate, protein and humic substance, plus small
quantities of uronic acid and DNA. About 34
39% of EPS extracted by EDTA remained unidentified, but only 9 19% of EPS extracted by the
other five processes were unidentified, most of
which might be lipid or phenols (Schmidt and
Ahring, 1996).
Results in Tables 3 5 show that formaldehyde
NaOH was most effective in extracting carbohydrate, protein and uronic acid for all sludges,
whereas EDTA was most effective in extracting
humic substance and DNA. Ultrasound enhanced
the extraction of all the constituents of EPS by
formaldehyde. Table 3 shows that formaldehyde
NaOH extracted about equal amounts of carbohydrate, protein and humic substance (ranging
2533%) from aerobic activated sludge. Table 4, on
the other hand, shows that carbohydrate accounted
for 62% of the EPS in the acidogenic sludge; this
could be due to the conversion of substrate sucrose
into levans by the levansucrase (Sangiliyandi and
Gunasekaran, 2001).
4. Discussion
255
Acknowledgements
The authors wish to thank the Hong Kong
Research Grants Council (HKU 7004/00E) for
the financial support of this study, and to Tong
Zhang for the CLSM analysis.
References
Alcamo, I.E., 1997. Fundamentals of Microbiology. Benjamin
Cummings, Menlo Park, CA Chapter 11.
Blumenkrantz, N., Asboe-Hansen, G., 1973. A new method
for quantitative determination of uronic acids. Anal. Biol.
54, 484.
Cescutti, P., Toffanin, R., Pollesello, P., Sutherland, I.W.,
1999. Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15. Carbohydrate Res. 315 (1/2), 159 168.
Dignac, M.F., Urbain, V., Rybacki, D., Bruchet, A., Snidaro,
D., Scribe, P., 1998. Chemical description of extracellular
polymers: implication on activated sludge floc structure.
Water Sci. Tech. 38 (8/9), 45 53.
Fang, H.H.P., 2000. Microbial distribution in UASB granules
and its resulting effects. Water Sci. Tech. 42 (12), 201 208.
Fang, H.H.P., Jia, X.S., 1996. Extraction of extracellular
polymer from anaerobic sludges. Biotechnol. Tech. 10 (11),
803 808.
Fang, H.H.P., Chen, T., Li, Y.Y., Chui, H.K., 1996. Degradation of phenol in wastewater in an upflow anaerobic sludge
blanket reactor. Water Res. 30 (6), 1353 1360.
Frlund, B., Griebe, T., Nielsen, P.H., 1995. Enzymatic activity in the activated-sludge floc matrix. Appl. Microbiol.
Biotechnol. 43, 755 761.
Frlund, B., Palmgren, R., Keiding, K., Nielsen, P.H., 1996.
Extraction of extracellular polymers from activated sludge
using a cation exchange resin. Water Res. 30 (8), 1749
1758.
Gaudy, A.F., 1962. Colorimetric determination of protein and
carbohydrate. Ind. Water Wastes 7, 17 22.
Kintner, P.K., Van Buren, J.P., 1982. Carbohydrate interference and its correction in pectin analysis using m-hydroxydiphenyl method. J. Food Sci. 47, 756 760.
256
Sutherland, I.W., 1997. Microbial exopolysaccharides structural subtleties and their consequences. Pure Appl. Chem.
69, 1911 1917.
Sutherland, I.W., Kennedy, L., 1996. Polysaccharide lyases
from gellan-producing Sphingomonas spp. Microbiology
142, 867 872.
Tsuneda, S., Park, S., Hayashi, H., Jung, J., Hirate, A., 2001.
Enhancement of nitrifying biofilm formation using selected
EPS produced by heterotrophic bacteria. Water Sci. Tech.
43 (6), 197 204.
Veiga, M.C., Mahendra, K.J., Wu, W.M., Hollingsworth,
R.I., Zeikus, J.G., 1997. Composition and role of extracellular polymers in methanogenic granules. Appl. Environ.
Microbiol. 63 (2), 403 407.
Wingender, J., Neu, T.R., Flemming, H.C., 1999. Microbial
Extracellular Polymeric Substances: Characterization,
Structures and Function. Springer-Verlag, Berlin Heidelberg Chapter 3.
Zhang, T., Fang, H.H.P., 2000. Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities. Biotechnol. Lett. 22,
399 405.
Zhang, T., Fang, H.H.P., 2001. Quantification of extracellular
polymeric substances in biofilms by confocal laser scanning
microscopy. Biotechnol. Lett. 23, 405 409.
Zhang, X.Q., Bishop, P.L., Kinkle, B.K., 1999. Comparison of
extraction methods for quantifying extracellular polymers
in biofilms. Water Sci. Tech. 39 (7), 211 218.