;Cat. Res. Vol. 27, No. 5, pp. 829-838, 1993 0043-1354/93$6.00+0.

00
Printed in Great Britain. All fightsreserved Copyright ~ 1993PergamonPress Ltd

BIOFLOCCULATION IN ACTIVATED SLUDGE:

AN ANALYTIC APPROACH
V. URBAn~~, J. C. BLOCK20 and J. MANEM'
'C.I.R.S.E.E., Lyonnaise des Eaux-Dumez, 38 rue du President Wilson, 78230 Le Pecq, France and
2Laboratoire Sant~ et Environement, Facult6 de Pharmacie, 5 rue Albert Lebrun, 54000 Nancy, France

(First received February 1991; accepted in revised form November 1992)

Abatract--A study on the physico-chemical structure of activated sludge flocs was carried out to get a
better insight in its relationship with sludge settleability. For this purpose, 16 sludge samples from different
origins were analyzed in order to provide information with regard to their settleabifity, biomass and
exocellular composition, surface characteristics and internal hydrophobicity. The presence of filamentous
microorganisms was observed in all samples but was not always associated with poor settleability,
supporting to some extent the idea of their role as a backbone in the floes. Relationships between the
measured variables were studied through their linear correlations. A high amount of exocellular polymers
(ECP) was associated with poor settling conditions. The DNA fraction and the C/N ratio of the ECP,
had also a negative influence on the adsorption of a cationic molecule in the sludge samples. Finally, sludge
settleabifity was described with a mathematical model which shows the opposition between ECP and the
internal hydrophobic~tyof the floes. From the model, the positive role of hydrophobic interactions should
provide a new approach in the understanding of flocculation mechanisms in activated sludge.

Key words---activated sludge, settleability, SVI, exocellular polymers, hydrophobicity, model, surface
charge

INTRODUCTION difficult. The internal organization of the floc is

also difficult to modelize and, for example, Li and
Poor settling of activated sludge is an important Gan .caxczyk (1990) did not observe any uniform
ecological as well as technical problem which often structure in stained sections of activated sludge flocs.
leads to a discharge of suspended solids in the The overall floc structure is negatively charged and
environment and operating problems in the waste- is the result of physico-chemical interactions between
water treatment plant (WWTP) itself. A survey on microorganisms (mainly bacteria), inorganic particles
bulking in biological WWTPs (Pujoi and Carder, (sillicates, calcium phosphate and iron oxides),
1989) showed that at least 25% of the plants were exocellular polymers and multivalent cations (Fig. 1).
concerned with settling problems (SVI ~ 150 or Exocellular polymers (ECP) have two different
200 ml.g-I). origins, (1) from metabolism or lysis of microorgan-
The negative influence of filamentous micro- isms (proteins, DNA, polysaccharides and lipids) and
organisms on sludge settling is well-known (Wanner (2) from the wastewater itself (e.g. cellulose, humic
and Grau, 1989). Their control is still very difficult to acids...). Their influence on sludge settling has been
achieve because of the diversity of species and thereby widely studied, but since there is no unified method
of environmental parameters which influence their for their extraction, it is always difficult to compare
(over-)growth. This biological problem hides the results from different studies. Thus, relationships
fact that there are few informations on the physico- between sludge settling and ECP (Magara et ai., 1976;
chemical structure of activated sludge floc and its Chao and Keinath, 1979) and data about the ECP
relationships to the sludge settling capacity. composition are sometimes contradictory (Sato and
Aggregation in activated sludge is an active process Ose, 1980; Horan and Eccles, 1986).
as cells are originally dispersed. This process is Since bacterial surfaces, ECP and eventually inor-
stochastic and heterotypic as fluid motion and differ- ganic particles provide negative adsorption sites,
ent microbial species are involved (Calleja et al., the role of divalent cations in the floc stability
1984). The size of the floes ranges from 20 to 200/tin must be emphasized. Divalent cations such as Ca 2+
according to Mueller et al. (1967) but Parker et al. and Mg2+ are known to be involved in the chemical
(1971) showed a bimodal size distribution from 0.5 structure of bacterial aggregates (Belcourt et al.,
to 5/~m and 25 to 3000 #m. Most of the methods 1974; Eriksson and Aim, 1991) and biofflms
assume one or two principal axes and sometimes a (Turakhia eta/., 1983), because of their ability to bind
sphere-like shape, and because of the three dimen- to negatively charged chemical groups. Steiner et al.
sional structure of the flocs realistic sizing is still (1976) showed that different types of adsorption

829
830 V. Uz~L~q et al.

q-
m

Bacten'a ; PO4n"

OH
N+
I hydr~e m
Exocellular polymers

Inorganic particles
IB
Oivalem cations

CZ+
Fig. 1. Schematic representation of the activated sludge floc on an arbitrary scale of size.

isotherms can be obtained with cations such as complex structure of the flocs. Thus in this paper, 14
cobalt, calcium or copper and with a higher attinity analytic methods are utilized to describe activated
for ECP alone than for the whole sludge. A difference sludge from different origins. The results are dis-
of affinity between Ca 2+ and Mg: + ions for the ECP cussed such as to describe the physico-chemical struc-
has also been demonstrated by Forster and Lewin ture of the flocs and its relation with settleability.
(1972) and Forster and Dallas-Newton (1980). In As environmental conditions are known to influence
spite of the importance of divalent cations in floccu- sludge settleability, characteristics of the WWTPs
iation processes, there is a lack of information about and wastewater composition are also related to the
either their accumulation in the exocellular struct~es measured out variables.
of the sludge flocs or their affinity with specific
constituents of the ECP. MA'rKRL,U.S AND NEgTHODS
Biological sludges are highly hydrated structures Aczivazed sludge aamplMg
and little attention has been paid to the role Sixteen activated sludge samples were taken from seven
of liydrophobicity in flocculation. As pointed out different WWTPs (A'G). Four of these plants (A, B, F and
by Eriksson and Axberg (1981), the hydrophobic- (3) were sampled once and the three others (C, D and E)
hydrophilic balance should be considered an import- four times on different days. The sludge samples from plants
C, D and E are really different because large variatioM were
ant factor. Cell surfaces are known to exhibit hydro- observed for the different measured out variables. These
phobic areas (Magnusson, 1980) and hydrophobic large variations show that samples from the same plant but
molecules such as lipids or proteins from the cells can from di~erent times are really different.
be trapped into the flocs. Hydrophobicity has been Charactemties of each plant (mass loading rate Bx)
poorly studied mainly because microorganisms in the and wutewater ¢ompmition (BOD/COD, COD/N and
COD/P) were provided by the laboratory of Metz treatment
sludge are aggregated and most of the methods for plant (E) and from control quality measurements (A, B, C,
testing hydrophobicity of bacteria have been devel- D, F and G) by the LOREAT (technical a ~ ' _ ~ com-
oped for testing dispersed cultures. The contact angle pany, Metz) (Table 1).
technique has been used to relate the flocculation Sludges from plants C, D and E were analysed immedi-
ately after umpling but those from plants A, B, F and G,
performance of activated sludge to its hydropbobic sampled by the LOREAT Company the day before the
characteristic (Valin and Sutherland, 1982). Because analysis, were stored at +4°C overnight. The pmslble effect
hydrophobic surfaces have a natural tendency to of low temperature on the variables was not investigated.
avoid hydrated environments, the hydrophobicity Micrmcopic examination
of activated sludge flocs must be determined from
The relativedensity of filamentous becteria was estimated
disaggregated samples. by means of a micrmeopic obeervation (x 320), aoeording
This short literature review shows that the under- to a qualitiative scale: + / - presence, + + high demity,
standing of the floc structure and its relationships + + + + proliferation.
with activated sludge settling is dependent on Sezth~,~zezz
methods that are able to provide information on its The sludge volume after 30 miss of Settlinl (SV~e) was
chemical composition. Moreover there is still no measured on sludge volumes of 100 ml (King and Forfcer,
single technique allowing a good description of the 1990). The sludge volume index (SVI, nd.g -t) represents
 Bioflocculation in activated sludge 831

Table I. Characteristicsof the wastewater treatment plants (WWTP) (Bx: mass loading rate) and
composition of the influent wastewaters
WWTP Bx(kg-IBOD.kg-IVM.j -') Loading range BODICOD COD/N COD/P
A 0.61 High 0.49 9.3 25.4
B 0.03 Extended aeration 0.31 6.4 17.6
CI 0.03 Extended aeration 0.40 8.6 32.8
C2 0.03 Extended aeration 0.40 8.6 32.8
C3 0.03 Extended aeration 0.40 8.6 32.8
CA 0.03 Extended aeration 0.40 8.6 32.8
DI 0.14 Low 0.41 13.0 68.1
D2 0.40 Intermediate 0.41 13.0 68, |
D3 0.43 High 0.41 13.0 68.1
D4 0.26 Intermediate 0.41 13.0 68.1
El 0.42 High 0.38 5.2 16.5
E2 0.37 Intermediate 0.38 5.8 21.0
E3 0.42 High 0.38 6.5 25.0
FA 0.42 High 0.38 6.2 21.8
F 0.29 Intermediate 0.51 9.4 32.2
G 0.77 High 0.47 8.8 23.4

the ratio between SV30(in ml. 1- t) and the dry matter content ate membranes (Millipore GTBP 04700) which were covered
of the sludge (DM, g.l-~). As the standard procedure is with a small volume of filtered MilliQ water. The mem-
based on a 1 1 volume of activated sludge, SVI values from branes were rinsed with 100 ml of MilliQ water and placed
100ml (SVlea) or 1 1 (SVI 0 were compared. From five acti- on a microscope slide with buffered glycerine for immuno-
vated sludge samples (Metz WWTP) (37% < SV30 < 72% fluorescence (Diagnostic Pasteur 74.921) on top, and then
and 2.99g.i -t < D M < 4 . 0 9 g . I -m) deviations of - 1 6 to covered with a glass slip. Fluorescent bacteria were counted
+ 13% were found when comparing SVI0.~ to SVI~, which at immersion ( × 1000) with a microscope under an u.v.-light
is close to the uncertainty on the test itself. source. Ten microscopic fields chosen at random were
observed through a square eye-piece and the results were
Biomass analysis expressed in number of cells per liter of sludge,
The biomass composition was analysed according to four
different analytic methods: the dry (DM) and volatile matter Extraction and analysis of hydropholic ECP
(VM) content of the sludge samples were determined after The ECP were recovered in the aqueous phase of a
centrifugation (50 mi, 10 rain at 2500 g). The pellets placed sonicated sludge sample. The sludge was centrifuged (2500 g
in crucibles were successively dried (24 h at 105°C), calcined 10rain) to a final concentration about 8.5g.I -I (using the
(3 h at 550°C) and weighed at each step. The volatile matter DM = f ( O D ~ ) relationship) in a 30 ml volume of MilliQ
content (VM) was calculated by difference between the dry water. Then 20 ml of this concentrated sludge were soni-
and mineral water values °and the results were expressed in cared (as previously described for the cell counting pro-
grams per liter of sludge. cedure) and mixed with 20 ml of MilliQ water. After a
In order to provide a rapid estimate of the DM concen- contact time of less than 60 rain, since preliminary assays
tration of the sludge, a relation was established between have shown that cell lysis can occur, the hydrophilic ECP
optical density at 600 nm (OD~) of a sonicated sludge were recovered in the aqueous phase by centrifugation
sample and its DM content (DM =5.40 × O D ~ - 0 . 1 2 , (20rain, 14,000g at +4°C) and frozen at -26°C until
• = 0.97 and n -- 42). This relation has been calculated from chemical analysis is performed.
triplicates on three different sludge samples, providing a This aqueous extract was analysed for its polysaccharide,
range of DM concentrations between 0.72 and 4.628.1 -I. protein, DNA, Ca 2+ , Mg2+ composition and its C/N ratio.
The sludge samples (Metz WWTP) were concentrated Polysaccharides in the ECP were measured on diluted
then diluted with 0.22/~m-filtered secondary effluent. Each thawed samples (1/10 in MilliQ water) according to the
concentration (10 ml aliquotes) in a 20 ml plastic test tube, phenol-sulfuric method of Dubois et al. (1956) with glucose
was sonicated (Bioblock sonicator, 50W 20 kHz) in an ice as standard. Proteins were measured immediately after
hath during two 15s periods interrupted by a 10s resting extraction according to the method of Bradford (1976) with
time. Then I ml was mixed with 9 ml of MilllQ water BSA as standard. DNA in the extract was determined
and the ODee0 (Kontron Uvikon 810) was measured. The according to the method of Deriaz et al. (1949) with calf
OD~e-DM relationship gave results from - 9 . 4 to 28.6% of thymus DNA as standard. All these determinations have
the DM content determined in this study with the drying been done in triplicate and the results expressed in rag
and weighing procedure. (equivalents of standard) per liter of sludge.
The C, H and N analysis have been performed on 105°C The elemental C and N composition (in %) of the ECP
dried and ground sludge samples. Determinations were was determined after lyophiliTation of the aqueous extract
made in duplicate using a Carlo Erba C.H.N. analyser and only the C/N ratio was calculated.
(Elemental analyser MOd.II06). The C, H and N values The C,a2+ and Mg2+ contents were measured in the
were expressed as rag. !-~ on the basis of the DM content aqueous extract containing the ECP by atomic absorption
of the sludge. spectrophotometry (Perkin-Ehner 2380) with the standard
The total cell count in the sludge (Xt) was determined addition method to overcome the interference of the organic
after souication, with an epifluorescence counting technique matrix in the samples.
adapted from Malone and Caldwell (1983). In short, 10 ml
of diluted sludge (1/5 in 0.2 #m-filtered MilliQ water) were Sludge surface characteristics
sonicated as described above. The suspension was immedi- The negative sludge surface charges can be estimated
ately diluted from 1/500 to 1/2000, depending on the initial by the adsorption of cationic molecules such as ruthenium
estimated dry matter concentration. In sterile test tubes, red (RR+). The pnx:edure was adapted from Figueroa
I ml of the diluted suspension was mixed with 1 mi of an and Silverstein (1987). Adsorption of RR + ( M W =
acridine orange solution (0.01% final concentration) (Sigma 576.5g.mol -m) was measured at equilibrium with a con-
A 6014). After a 30rain contact time, bacteria were recov- stant sludge concentration and increasing dye concen-
ered by vacuum filtration through 0,2 #m black polycarbon- trations. The adsorptio n data can be approximated by the

Wit 27/~,---G
832 V. U u ~ e t a / .

Frenndfich model which relates the adsorbed concentration negative charges 0tydrophilic groups) or by a high density
of the molecule (Z) divided by the amount of dry matter of hydrophobic sites inside the flocs.
(m), to the free concentration (Ce) in the form of
X/m •K.Ce tl". The adsorption constant l/n shows the Reproducibility of the methods
non-linearity of the model and K is related to a maximum For practical reasons, analysis on activated sludge
of adsorption when l/n is close to zero. Preliminary exper- samples were not replicated. Preliminary experiments
iments showed that adsorption of RR + can be studied with showed that the reproducibility of the analytic methods
dry matter concentrations near 0.25 g.l -~ and R.R + final described above is quite acceptable. As an example, stan-
concentrations between 0.025 and 0.15 g. l- i. dard deviations of thirteen parameters measured in repficate
The dry matter contents of the sludge sample were on sludge samples from Metz WWTP are shown in Table 2.
estimated (DM = f ( O D ~ ) relationship) and the sludge All coefficients of variation with the exception of IHB and
diluted to 0.25g.1 -I with 0.22/~m-filtered secondary waste- Xt (14%, duplicate tests) are lower than 10%. This 14%
water (250 ml final volume). In 150 mi glass flasks, 18 mi of value may arise from the coarse state of dispersed sludge
this dilution were mixed with 2 ml of RR + solutions from samples from which turbidity (ODeeo) or total cell numbers
0.25 to I g.l -~. In other flasks the diluted sludge was are measured.
replaced by filtered wastewater in order to check for a
possible adsorption onto dissolved material in the liquid. Statistical analysis of data
All the flasks were placed on a rotary table at 250 rpm at A statistic software (Statview II run on Macintosh SI) was
room temperature (~25°C). After three hours of contact used to analyze the data (16 observations, 16 variables)
(Figueroa and Silverstein, 1987) the flasks containing the (Table 3). The linear correlation matrix was computed and
sludge were centrifuged for 10 rain at 2500g and the OD at significant linear relationships between variables were con-
530 nm ()~m RR+) was measured out in the supernatants. sidered when coe~clents of correlation were higher t h a n
The adsorbed (mmol.kg -t DM) and free (mmol.l -l) 0.50 (~ = 5%, n ffi 16) (Table 4). T h e linear relationships
concentrations of RR + were calculated from a calibration were checked graphically in order to prevent from situations
curve. The linear regression analysis on the logarithm of where dispersion around the regression line is high.
the adsorbed concentrations vs free concentrations of RR + Data were also computed by means of multiple regression
gives the value of l/n (slope) and In K (y axis intercept). analysis (stepwise method with 0c and ~ equal to 5%) (Box
et aL, 1978). In order to avoid autocorrelations, care has
Determination of an internal hydrophobicity in the sludge been taken to use absolute units, i.e. rna~ units per liter of
The salt aggregation test developed by Lindahi et al. sludge, except for data from the settling, adsorption and
(1981) for testing hydrophobicity of pure bacterial strains flocculation tests.
was adapted to activated sludge flocs.
Flocculation in a sonicated sludge sample mixed with RESULTS AND DISCUSSION
increamg concentrations of ammonium sulfate in phos-
phate buffer was measured. The ammonium sulfate influ- The chemical structure o f activated sludge floes has
ence was determined from OD~eomeasurements after a 3 h been studied on sixteen sludge samples from seven
contact time with reference to a control without ammonium different wastewater treatm¢nt plants. In this study,
sulfate. Preliminary experiments showed that the difference
22 variables were analyzed. Mass loading rates (Bx)
between the ODeee of the control and the test is a linear
function of the ammonium sulfate concentrations: the in- and wastewater composition ( B O D / C O D , N / C O D ,
ternal hydrophobieity of the flocs (IHB) was expressed by P / C O D ) (see Table 1) were obtained from control
the slope of the linear regression analysis. quafity measurement. The SVIo.~ values, floc compo-
The sludge dry matter concentration was set up to sition (biomass analysis, exocellular hydrophilic
2.5 g.l -j and then sonicated as previously described for the
ECP extraction procedure. In short, I mi samples of soni- constituents) and surface characteristics (cationic
cated sludge were poured in 20 mi plastic test tubes which adsorption and internal hydrophobicity) (see Table 3)
contained 9 ml of ammonium sulfate in phosphate buffer were determined with the methods described herein.
(0.07 M, pH 7 + 0.4) at 0.3, 0.75, 1.5, 2.25 and 2.5 M final
concentrations. After 3 h at room temperature, the tubes General characteristics of the plants and the sludge
were stirred by hand and OD~ e were measured. samples
A high value of IHB is equivalent to a large aggregation
in response to a small increase in the ammonium sulfate According to mass loading rates (Table 2), plants
concentration. It can be explained either by a low density of B and C I - 4 are operated at extended aeration

Table 2. Mean values, standard deviations (SD) and ~ t s of variation (c.v,) for some of
the variables from the analysis of dilfemnt activated sludges (Metz WWTP)
Variables Mean SD c.v. % Replicatm
DM (g-I-i 1.89 0.08 4 3
VM (g'l-') 1.56 0.03 1.9 3
Xt.10I: (I-') 4.11 0.58 14 2
Carbon (g.l-') 0.86 6.24.10-3 <I 3
Hydrogen (g-I-') 0.II 8.13'i0 -3 <1 3
Nitrogen (g.l") 0.18 4.25.10-3 < I 3
Exocellular polysaccharides(ms,l -i) 35.30 1.79 5.1 3
Exocellular proteim (rag-1-I) 212.13 24.94 11.7 3
Exoeeilular DNA (mg.I -I) 16.70 0.31 i.9 3
SVI~I (ml.g-I) 372 17 4.6 3
IHB. 102 2.23 0.28 14 2
l/n 0.24 0.01 4.2 3
K 837 1 0.12 3
DM and VM: dry and volatile matter content of the slmllge;Xt: total number of cells; SVt,:
sludge volume index; IHB: interred hydrophobicity of biologicalfloc~ l/n and £: admrption
contants from the Freundich model.
 Bioflocculation in activated sludge 833

conditions, plants D I - 4 and E l - 4 between intermedi-

ate and high loading, plant F at intermediate and
plant G at high loading conditions. The overall
composition of the incoming wastewatcr shows a
. . . ~ . . . ~ g ~ large but typical range of BOD/COD ratio from 0.31
to 0.51. The major types of nutrients (C, N and P) are
assumed to be balanced as the COD/N and COD/P
ratios are about 8 and 27, respectively.
According to our qualitative scale, microscopic
examination of the sludges always revealed the pres-
ence of filamentous structures even with low SVI0.~
values (sludges F, G, DI, D2, D3 and D4) (Table 4)
and ten sludges out of sixteen showed SVIo.~ values
~ N ~ - ~ ~ ~ higher than 150 ml-g-m which is considered to be a
i IC ?1 " ' " ~ ~ ~ 4 ~ limit before bulking (Forster, 1985a). The presence of
filaments in each sample supports to some extent the
role of filamentous structures as a backbone for the
structure of the floes (Sezgin et al., 1978).
Biomass analysis
On the basis of the lowest and highest values
obtained from biomass analysis, the 16 sludge
~ ~ ~ "~ • .~: ..... samples contained 1.8-6.6gDM.I -~ with organic
fractions from 55 to 85% and bacterial cell numbers
from 4 to 12.1012.1-~.
~ '~ " ~ • The cell dry weight calculated on the basis of the
volatile matter concentration and the bacterial cell
number was found to be fairly constant with
an average value of 0.40 (_+0.06).10-ng (n = 16).
This value is quite comparable to those found by
Herbert (1971) with pure cultures of Escherichia coli
~ " ~ ~ ~
(0.12-0.41.10.-12g) between resting phase and log
phase. Herbert's values were calculated on a plate
count determination basis which gives lower results
than the epifiuorescvnce counting method and thus
" 0 ~ 0 ~ 0 ~ 0 0 ~ 0 ~ overestimate the cell dry weight. As our results and
those from Herbert are comparable, microorganisms
in activated sludge could be considered an association
between cells and organic ¢xocellular compounds
~o~o~o--.~...--. =
("cells + ECP").
l=l The bacterial cell number is strongly correlated to
the amount of volatile matter (VM) (r = 0.92) (Fig. 2)
~ ~ .... ~ ..... and C, H and N amount (r > 0.87) of the sludge.
These five variables can be gathered in a homo-
geneous group which describes the organic fraction of
the biomass. These strong linear correlations suggest
that it is not necessary to determine all these vari-
ables. However, correlations between the C/N ratio
or the amount of elemental nitrogen in the sludge and
constituents of the ECP (proteins, Ca 2+ ), settlcability
(SVI0.~) or sludge surface characteristics (adsorption
constant, K) (see Table 4) will provide information
that cannot be obtained from a single method such as
the dry matter determination.
ECP and divalent cation~
Extraction procedure. The amount of ECT rep-
resents a small fraction of the activated sludge mass
since even with a drastic extraction procedure such
as heat treatment (Beccari et al,, 1980; Clarke and
834 V. URBAIN et ol.

Table 4. Linear coeff~ents of correlation statistcally significant at a 0.95 probability level (~ = 5%), i.e. r > 0.5 (abbreviations can be found
in "Materials and Methods")
DM VM C H N C/N Xt EPS Proteins DNA C/N Ca2+ Mg2+ SVI K IHB COD/N
DM 1.00 . . . . . . . . . . .
VM 0.95 1.00 . . . . . . . . . . .
C 0.95 0.99 1 . 0 0 . . . . . . . . . .
H 0,97 0.97 0.97 i.00 . . . . . . . . . . "
N 0.86 0.90 0.91 0.94 1.00 . . . . . . . . . .
C/N -- -- -- I .oo . . . . . . . . .

Xt 0.87 0.92 0.90 0.91 0.88 -- 1.00 . . . . . . . .

EPS -- 0.70 - - - - 1.00 . . . . .

Proteins - - - -0.63 - - 0.85 1 . 0 0 . . . .

DNA -- 0.56 0.57 0.62 0.81 - - 0.67 0.94 0.82 1.00 . . . . .

C/N . . . . . . 0.63 - - 1.00 . . . .

Ca 2+ -- -- --0.54 -- 0.88 0.93 0.85 -0.62 1.00 - - -- --

Mg2+ -- 0.55 0.57 0.58 0.78 -- 0.60 0.86 0.73 0.92 -0.59 0 . 8 3 1.00 --
SVI - - 0.56 - - - - 0.85 0.64 0.74 0.61 0.63 - - 1.00 - -

K -- -0.50 0.60 . . . . . 0.55 0.52 - - -0.69 -- 1.00 -- --

IHB . . . . . . . . . . . . 1.00 - -

COD/N . . . . . 0.59 . . . . . . 0.67 1 . 0 0

COD/P . . . . . . . . . . . 0.95

Forster, 1982) ECP still accounts for less than 14% on cell structures. Therefore a physical method
of the sludge dry weight. The exocellular organic such as sonication was recommended by Kiff and
matrix in biological floes is really heterogeneous and Thompson 0979).
requires a disaggregation step for recovery. ECP In our experimental conditions, preliminary assays
comes from cell structures, either because of ceil lysis, with ultrasonic treatment have shown that a dead
metabolic excretion, or leakage of exocellular con- level of the total number of cells in sonicated samples
stituents and also from the adsorption of organic was reached. According to our method ECP consists
matter (cellulose, humic acids ...). ECP and poly- of readily extractible macromolecules which are
mers attached to the outer membrane (e.g. lipo- mostly hydrophilic as they are recovered in an
polysaccharides in gram negative species) or to the a q u e o u s extract.
peptidoglycan (e.g. teichoic acids in gram positive T h e results o f t h e analysis o f e x o c e l l u l a r p o l y -
species) are too close to ensure that only non- s a c c h a r i d e s , D N A a n d p r o t e i n s in t h e 16 a c t i v a t e d
covalently bound exocellular structures (ECP) are s l u d g e s a m p l e s a r e r e p o r t e d in T a b l e 6 (lowest a n d
removed by a defined extraction procedure. There- h i g h e s t values) a n d compared w i t h results f r o m o t h e r
fore, the basic requirement of an extraction pro- studies. T h e d a t a f r o m K i f f a n d T h o m p s o n (1979) are
cedure must be the lowest extent of cell lysis, i.e. of in t h e r a n g e o f o u r values f o r D N A , b u t t h e results
intracellular polymers contamination. o f B r o w n a n d L e s t e r (1980) c a n b e c o m p a r e d to o u r
In spite of several comparative studies (Can" and results o n l y f o r p o l y s a c c h a r i d e s a n d p r o t e i n s w i t h t h e
Gancarczyk, 1974; Kiffand Thompson, 1979; Brown s t e a m i n g p r o c e d u r e . T h e r a t i o b e t w e e n s t e a m i n g a n d
and Lester, 1980; Rudd et al., 1982) there is still no s o n i c a t i o n p r o c e d u r e s in B r o w n a n d L e s t e r ' s s t u d y
unified method for the ECP extraction. Shearing r a n g e s b e t w e e n 18 a n d 25 w h i c h s u g g e s t s t h a t their
effect of ultracentrifugation is not an effective method u l t r a s o n i c p r o c e d u r e ( u l t r a s o n i c b a t h : 20W, 2 rain) is
for activated sludge (Novak and Haugan, 1981), t o o w e a k f o r a g o o d e x t r a c t i o n . B r o w n a n d L e s t e r
whereas chemical or heating procedures are too hard (1980) u s e d t h e p r o t e i n / c a r b o h y d r a t e r a t i o f o r t h e i r
c h o i c e o f s t e a m i n g as t h e b e s t m e t h o d f o r t h e e x t r a c -
t i o n o f E C P f r o m a c t i v a t e d sludge b u t for R u d d e t al.

6 0
Table 5. Qualitative scale for the presem~ of filamentous micro-
organisms in the sludge samples (+/--,preuence, + + ,-high
5 density and + + + + = proliferation)

~ 4 oO
Sludge
A
B

e2
SVI0.I (ml.g -~)
315
182
201
223
Density of filaments
+/-
++-F+
++++
++++
c3 201 ++++
c4 204 ++++
D! 139 +/-
D2 76 +/-
D3 69 ++
1 3 6 9 12 134 107 +/-
XI.1012.1-1 El 188 + +
E2 279 ++
Fig. 2. Linear relation between the m o u n t o f volatile matter E3 264 ++
(VM) and the total number o f cells (Xt) in the 16 sludge FA 288 + +
84 +/-
samples. F
 Bioflocculation in activated sludge 835

Table 6. Comparisonof different results on the analysisof cxocellularpolymers(ECP)

ECP at mg. g- t (on dry matter basis)
Reference Extraction procedure P o l ~ DNA Proteins
Our study Sonication 6.50-24.84 9.49-29.66 6.16--163.44
Brown and Letter (1980) Sonication 0.80 + 0.14 0.20 + 0.07 3.20 + 0.68
Steaming 20.30+3.60 3.70+0.31 77.10+12.90
Kiff and Thompson(1979) Sonication ND 17-28 ND
Brown and Lester:9 replicates; Kiffand Thompson: 5 replicates; our study:minimumand maximumvalues;ND:
not determined.

(1982) this ratio remains fairly constant (near 3/1) ionic size of cations may influence their binding
irrespective of the severity of the extraction pro- ability to charged (carboxyl) and uncharged groups
cedure. Since in our study this ratio varies from 0.77 (hydroxyl) in the ECP: thus as calcium ions
to 6.25 it seems dependent rather on the composition are "larger" than magnesium ions (Handbook of
of the exoceilular matrix than on the degree of lysis Chemistry and Physics, 1990), their binding may be
caused by the method. favoured. The linear correlations between exocellular
Composition of the ECP aqueous extract. Only Mg 2+ and D N A (r--0.92), bacterial cell number
polysaccharides, DNA and proteins were analyzed in (Xt, r -- 0.60) or volatile matter concentration (VM,
the ECP extract although other macromolecules such r --0.55) or between exocellular Ca 2+ and proteins
as phospholipids, giycolipids.., may be found in the (r = 0.94) or the C/N ratio of the sludge (r ffi -0.54)
exo~llular organic matrix of the floes. It is difficult may indicate a higher "affinity" of Mg 2+ ions for
to classify these polymers for their relative abundance D N A and Ca 2+ ions for proteins than for the other
in the ECP because their molar concentration is polymers. Additional experiments are needed to de-
not known. In addition, they are measured as stan- termine the mechanisms by which these species are
dard equivalents (glucose, BSA ...) which arc surely involved in flocculation.
not representative of their true composition. This There is no correlation between exocellular
is probably the reason for contradictions in the polysaccharides or proteins and organic biomass
literature. For example, Sato and Ose (1980) found variables such as VM and Xt. This means that the floc
more nucleic acids (DNR + RNA) than proteins and structure does not stem from the association of equal
polysaccharides, Horan and Eccles (1986) more poly- amounts of elementary subunits (e.g. [bacteria, ECP,
saccharides than nucleic acids and proteins and in inorganic particles ...] x n) as it is suggested from
our study we found more proteins than DNA and our schematic representation of the floc structure (see
polysaccharides. Fig. 1).
The three chemical types of polymers analyzed in Origin of the ECP constituents. The origin of the
the 16 sludges are strongly correlated with each other exocellular D N A from lysis of dead cells is supported
(r > 0.82) which means that they form an organic by the correlations with organic biomass variables
matrix in a more or less constant ratio: the strongest such as Xt (r = 0.67), VM (r = 0.56) and the C, H, N
positive linear correlation is obtained between composition of the sludges (r >I 0.57).
DNA and polysaceharides (r ffi 0.94) because the
standard deviation on their mean ratio is low
( D N A / E P S = I . 1 9 + 0 . 1 8 , c.v.=15%). The ratio
between proteins and DNA or polysaccharides
O polysacx~harides
shows a greater variability, 2.31 + 1.45 (c.v. = 63%)
and 2.61 + 1.60 (c.v. = 62%) with linear coefficients A DNA
of correlation equal to 0.82 and 0.85 respectively 600
(Fig. 3).
O&
Inside the organic exoceUular matrix of the floes,
divalent cations (Ca 2+ and M g 2+) may act as bridg-
ing agents as they are correlated with the analyzed
polymers (r >I 0.73). This bridging role is supported
by the work of Eriksson and Aim 0 9 9 0 on the
effect of E D T A on sludge characteristics(change of
"-[ OA

the dewatering property of activated sludge and

release of ECP). Ca 2+ ions are measured in higher
amounts 0.62-72 mg.l- l of sludge) than M g 2+ ions ° , ° ,
(1.26-5.63 mg.l-t of sludge) and thisdifferencemay 0 30 60 90 120
be relatedto theiraffinityfor ECP. Forster and Lewin
(1972) have shown a higher binding of Ca 2+ rather poly~ccharldes o¢ DNA (mg.r 1)
than MS# + ions on E C P recovered from activated Fig. 3. Relations between the amount of exo~llular pro-
sludge and Forster (1985b) hypothesized that the teins, polysaccharides or DNA in the 16 sludge rumples.
836 V. Ut~m~ et al.

The linear correlation between proteins and the The exocelhilar organic matrix in the floes plays an
C/N ratio of the ECP (r = -0.63) is not surprising as important role in their surface characteristics. Both
proteins represent one of the nitrogenous constituent the nitrogenous content of the sludge, exocellular
of the exocelhilar organic matrix in activated sludge. D N A and Mg2 + ions are able to reduce the negative
The origin of proteins and polysaccharides from cell charge of the sludge surface, as they are negatively
metabolism is supported by the negative relationship correlated with the adsorption capacity of the sludge
between exopolysaccharides (EPS) and the COD/N for a cationic dye (expressed as K). Owing to the fact
ratio. Inspite of the r value of -0.59, there is a high that low values of these variables are related to low
dispersion around the regression line and the linear SVI0a values, a high surface charge (high values of K)
relationship must be considered only as a trend. This may not be inconsistent with good settling con-
relation can not be explained by an inducing effect of ditions. This statement agrees with the results of
a nitrogen limitation on EPS synthesis (Sutherland, Pavoni et al. (1972) who showed that the surface
1977) as the COD/N ratio is not growth limiting. charge reduction is not the prime mechanism in
bioflocculation because polymers are able to bridge
Influence of ECP and divalent cations on sludge surface the cells either electrostatically or physically. Like-
and settling wise, Ries and Meyers (1968) have shown with a
The linear coefficients of correlation between K synthetic system, that charge neutralization and
and the N composition of the sludges (r = 0.60) bridging could not act simultaneously. Neither did
or the C/N ratio of the ECP (r = 0.52) show that Barber and Veenstra (1986) find any relationship
nitrogenous constituents which are accessible at between electrophoretic mobility of sludge particles
the surface of the flocs do not provide adsorption and SVI of sludge samples from full-scale plants.
sites for a cationic dye (ruthenium red). Exocellular However, the electrophoretic mobility of activated
DNA too (r = -0.55), seems to hinder adsorption of sludge panicles has been shown to be directly
ruthenium red as it is negatively correlated with K. (Forster, 1968), as well as inversely (Magara et al.,
Divalent cations may be able to reduce the number 1976) related to SVI.
of negative charges at the surface of the flocs as there
Internal hydrophobicity and ECP related to settling
is a negative linear correlation between the amount of
Mg 2+ ions measured out in the ECP extract and K capacity
(r = -0.70), but this hypothesis may proceed from Multiple regression analysis has been performed
the relation of this divalent cation with DNA. on data with SVIoa (ml. g - ~) as a dependent variable
All the analyzed constituents of the ECP are and five independent variables, namely the sludge
positively correlated with SVI0a (r > 0.61), high con- C/N ratio (g. g-~) and volatile matter content (VM,
centrations of polysaccharides, proteins . . . resulting g. 1- ~), K (dimensionless), IHB (I02OD~ unit. mol- '
in a worsening of sludge settleability. This negative of ammonium sulfate) and exocellular polysaccha-
influence has already been described in the literature rides (EPS, mg.l-t).
in the case of EPS (Table 7). Results from different In the range of SVIoa values between 69 and
studies are not easily comparable, but irrespective of 315m1.g -~ and with two variables out of five, a
the experimental conditions, there is in most cases a model in the form of SVIoa = 3.3 x [EPS] - 35.1 x
positive linear relationship between SVI and ECP. IHB + 178 with a coefficient of correlation equal to
The exception comes from Goodwin and Forster's 0.89 can be computed. The sign associated to these
study who found a negative linear relationship be- coefficients shows that at low concentration of EPS
tween SVI0a and ECP. The authors assumed that and when the internal hydrophobicity is high, settle-
when the sludge settled poorly, EPS became less ability is improved. If EPS are kept in the model, they
amenable to extraction. A more likely explanation are in fact only the "leader" of a larger group of other
refers to the units for ECP in terms of their total variables which characterize the hydrophilic ECP.
sugars content on the basis of total organic carbon Except for proteins, when DNA, Mg 2+ or Ca 2+ ions
from ECP. The lack of linear correlation between SVI are introduced instead of EPS, similar models are
and ECP in the study of Chao and Keinath (1979) is obtained. However, it must be pointed out that this
quite surprising in reference to the five other relation- model is relevant to a small number of values and
ships, but the extraction procedure and the range of should be verified by the analysis of a larger group of
SVI values are really different from the other studies. sludges.

Table 7. Relationshipsbetween the amount of exocellular polymers(ECP) and sludgesettlcability from the literature and fromour results
Reference SVI range (ml.g-I) ECP units Linear rgeressionanalysis n •
Our study 69-315 mg ge" i - ' SVI - 3.46ECP + 14.90 16 0.89
Forster (1971) 46-190 gge.100g DM-' SV1- 3.04ECP+ 48.99 II 0.84
Mapra et at. (1976) 37-152 g TOC.100g DM-' SVI ,- 4.2$ECP- 6.28 3 0.99
Kiff (1978) 65-400 ms ECP-100S DM - ' SVI m 57.60ECP- 27.34 4 0.90
Chao and K©inath (1979) 100-725 mgge. 100 g D M - ' No ~,lation -- - -

Goodwin and Fortser (1985) 17~-345 mgge.100 S TOC-' SVI - - 19.03ECP + 6.47 8 0.87
ge: glucoseequivalents; DM: dry matter content of the sludge;TOC: total organiccarbon; n: number of data.
 Bioflocculation in activated sludge 837

In a highly hydrated structure such as a biological their overgrowth is always associated with settling
sludge, little attention has been paid to the role of problems.
hydrophobicity in flocculation. Valin and Sutherland Exocellular polymers from metabolism, cell lysis or
(1982) correlated flocculation in activated sludge wastewater are involved in the formation of a three-
with hydrophobicity on the basis of contact angle dimensional matrix or gel where divalent cations,
measurements, but this method describes rather at least Ca 2+ and Mg 2+, act as bridging agents with
an external surface characteristic, as the sludge is probably specific affinities for each kind of exoceilular
not dispersed before analysis, than the hydrophobic polymer.
interaction inside the floe. Biological sludges are In such a highly hydrated system as biological
composed of many different species of micro- sludges, internal hydrophobic bondings are involved
organisms and Singh and Vincent (1987) have iso- in flocculation mechanisms and their balance with
lated one Pseudomonas sp, strain which has proved hydrophilic bondings determines the sludge settling
hydrophobic only when grown in a diluted medium, properties. From the model for SVIo,, one may
this hydrophobic character being associated with the assume that if the famous exopolysaccharides are
capacity to form aggregates. really needed in the floc structure, hydrophohic areas
This opposition between hydrophilic and hydro- in between the cells act as essential adhesives.
phobic interactions has been shown by Wrangstadh
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