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Applied and Environmental Microbiology, November 2001, p. 5303-5307, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5303-5307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chlorine-Susceptible and Chlorine-Resistant Type
021N Bacteria Occurring in Bulking Activated Sludges
M. A.
Séka,1
Y.
Kalogo,2
F.
Hammes,1
J.
Kielemoes,1 and
W.
Verstraete1,*
Laboratory for Microbial Ecology and
Technology, Center for Environmental Sanitation, Ghent University,
9000 Ghent,1 and Laboratoire
d'Epuration des Eaux, FUSAGx, 5030 Gembloux,2
Belgium
Received 12 March 2001/Accepted 27 June 2001
 |
ABSTRACT |
Two filamentous bacteria causing bulking in two activated sludges
were examined. Investigations using morphological features, staining
techniques, and fluorescent in situ hybridization identified both
filaments as type 021N. However, an examination of the effect of
chlorine on the sludges revealed a chlorine-susceptible type 021N in
one sludge and a chlorine-resistant type 021N in the other.
| |
TEXT |
The performance of the activated
sludge process is often determined by the gravity separation between
the treated water and the sludge in the final clarifier. The
proliferation of filamentous bacteria in the microbial community of the
sludge, referred to as "filamentous bulking," has often been
reported to hamper this solid-liquid separation (7, 18).
Filamentous bulking results in less dense and less settleable sludge
flocs, and in severe cases the operation of the plant can be totally
compromised. Addition of disinfectants (mostly chlorine based) to the
sludge to selectively kill off the causative filamentous bacteria is
often used in practice as a short-term and cost-effective solution
(7). Jenkins et al. (7) presented sludge
chlorination as a method of choice in the United States to combat
filamentous bulking. They reported several case histories of successful
control of bulking at full-scale treatment plants using chlorine and
described the method as a "universal success" with any filamentous
bacteria. Yet cases of partial success and cases of failure can be
found in the literature. Lakay et al. (11) obtained only a
partial elimination of Microthrix parvicella bacteria at a
high chlorine dose. Hwang and Tanaka (6) found in batch
tests that M. parvicella remained intact at very high
chlorine doses, while the microbial flocs were completely destroyed.
Fontaine (3) could not overcome filamentous bulking in a
pilot plant using chlorination. More recently, Madoni et al.
(12) reported in a survey in Italy that use of
chlorination was successful in only 63% of the cases. Most often, no
relevant explanation was offered for these unsuccessful cases of sludge chlorination. Thoroughly examined unsuccessful cases have been ascribed
to the presence of well-known resistant filamentous bacteria with
hydrophobic cell walls such as M. parvicella and
Nostocoida limicola (6, 18). This paper
presents and discusses the effects of chlorine on two sludges, one
dominated by a chlorine-vulnerable type 021N bacterium and the other
dominated by a chlorine-resistant type 021N bacterium, as a relevant
explanation of the discrepancies noted in the effectiveness of chlorine
in the cure of bulking sludge.
Experimental procedures.
The two types of sludges (sludges A
and B) involved in this study originated from two industrial wastewater
treatment plants (plants A and B) located in the region of Ghent,
Belgium. Plant A employed a two-stage activated sludge system fed
continuously with the wastewater resulting from the manufacture of
mayonnaise and soups. The sludge loading rate (SLR) of sludge A was
0.4 g of COD (g of VSS · day)
1 (where COD is
the chemical oxygen demand and VSS is the volatile suspended solids).
The 5-day biochemical oxygen demand (BOD)5/N/P ratio
for this plant was variable, and addition of extra nutrients (N and P)
was often necessary. Plant B, a small and poorly supervised plant,
operated at an average SLR of 0.3 g of COD (g of VSS · day)1. On the day of sampling, the value measured gave an
SLR of 0.6 g of COD (g of VSS · day)1,
probably due to a recent wastage of overestimated excess sludge. This
plant employed a sequencing batch reactor (SBR) system using approximately 20 h of aeration followed by 2 to 3 h of
sedimentation and 1 h of effluent decantation. Thus, unlike plant
A, plant B was fed discontinuously with the wastewater generated from
the processing of potatoes to make chips. For both systems, sludge samples were collected and transported to the lab within less than
2 h in vessels no more than half filled, thus providing for a
headspace with oxygen. Both sludges reached the laboratory on the same
day and were tested immediately for the appropriateness of chlorine to
cure the filamentous bulking. The rest of the samples were kept at
4°C in vessels no more than half filled. The pHs were 7.3 for sludge
A and 7.1 for sludge B. The concentrations of mixed-liquor suspended
solids (MLSS) and VSS were 3.00 and 2.43 g liter1,
respectively, for sludge A and 2.00 and 1.69 g
liter1, respectively, for sludge B. Filamentous bacteria
were similarly abundant in both sludges and reached level 6 (excessive)
on the scale of 0 (none) to 6 based on the filament scoring system
proposed by Jenkins et al. (7). Sludge A was dominated by
larger flocs (up to 700-µm diameters), while sludge B contained
mostly smaller flocs (60-µm diameters). The sludge volume indexes
(SVIs) were 667 ml g1 for sludge A and 960 ml
g1 for sludge B, compared to 150 ml g1 for
a well-settling sludge.
A stock solution of 1.90 g of Cl2
liter1, prepared by the dilution of a commercially
available solution of sodium hypochlorite (12° Chl 
38.04 g of Cl2 liter1) with distilled water,
was used. For each type of sludge, the following doses were
added to 1-liter volumes of sludge contained in 2-liter Erlenmeyer
flasks and used as activated sludge units: 0 (control), 2, 5, 10, 15, and 40 g of Cl2 (kg of MLSS)1
(designated treatments T0, T1, T2, T3, T4, and T5, respectively). After
20 min on a rotary shaker (90 rpm), 5 ml of mixed liquor was taken from
each Erlenmeyer flask for viability staining and 200 ml was taken for
oxygen uptake rate (OUR) measurements. The remaining sludge (about 800 ml) for each treatment was further fed with 200 ml of a skim milk
solution at an SLR of 0.4 g of COD (g of VSS · day)1 for 24 h using materials and equipment similar to
those described by Séka et al. (16). After this
period of incubation, the SVI and the residual soluble COD were
measured for each treatment. Sludge samples were examined
microscopically with light microscopy (Polyvar; Reichert-Jung, Vienna,
Austria), and digital images were captured with a charge-coupled
device camera (Hamamatsu Photonics GmbH, Herssching, Germany). Filament
scoring (7) was further determined for each treatment.
Floc diameter and filament length and width were determined with
digital image analysis software (Micro Image 4.0; Olympus
Optical Co.,
Hamburg, Germany). COD, MLSS, and VSS testing were
performed as
described by the American Public Health Association
(
5).
The SVI was measured as the diluted SVI (
5). OUR
measurements
were conducted using equipment and protocol similar to
those described
by Gernaey et al. (
4). Viability staining
was performed using
the commercial Live/Dead stain (L-13152) (Live/Dead
BacLight bacterial
viability kit technical information sheet, Molecular
Probes Europe
BV, Leiden, The Netherlands) and equipment and procedures
similar
to those described by Séka et al (
16). For
each sludge, three
preparations per treatment were examined by
fluorescent microscopy.
Bacteria with intact membranes were stained
green and scored as
"alive," and those with damaged membranes were
stained red and
scored as "dead." Digital images were also
captured.
The main filamentous bacteria in both sludges were first identified
using the criteria of Jenkins et al. (
7) and Eikelboom
(
2). These criteria include morphological features and
results
of Gram and Neisser staining. The sulfur oxidation test using
sodium thiosulfate and the sodium hypochlorite test for the
visualization
of sheaths described by Jenkins et al. (
7)
were also conducted.
The presence of a sheath was further investigated
by means of
scanning electron microscopy using a JEOL JSM 840 instrument.
Samples of 10 ml of each sludge type were first centrifuged
and
subjected to double fixation with glutaraldehyde and
OsO
4 (
20)
before being subjected to scanning
electron microscopy. The identification
of the filamentous bacteria
dominating both sludges was further
double-checked by means of
fluorescent in situ hybridization (FISH).
On the basis of the
morphological features, probe 21N (
17),
complementary to
the 16S rRNA sequences of the filamentous bacterium
type 021N, was
used. This probe, obtained from Genset (Paris,
France), was labeled at
its 5' end with Cy3. Probe EUB338 (Eurogentec,
Liège,
Belgium), labeled with fluorescein and hybridizing with
all bacteria,
was used simultaneously with probe 21N to check
the vitality of the
filamentous bacteria that would not react
to probe 21N. The
hybridization was carried out as described by
Wagner et al.
(
17) and Manz et al. (
13). FISH preparations
were viewed by means of an Eclipse E600 microscope (Nikon Europe
BV,
Badhoevedorp, The
Netherlands).
Characteristics and identification of the main filamentous bacteria
in both sludges.
The filaments dominating both sludges were very
long (0.3 to 1.2 mm in sludge A and 0.5 to 1.1 mm in sludge B),
unbranched, coiled or smoothly curved, Gram and Neisser stain negative,
sheathless, and multicellular. Individual cells within the filaments
were not uniform in shape (discoid or barrel) or size (1.0 to 1.8 µm in width and 0.9 to 1.3 µm in length in sludge A and 1.5 to 1.8 µm
in width and 0.8 to 1.5 µm in length in sludge B). Septa between the
cells were clearly visible. Before the sulfur oxidation test, no sulfur
granules were present in sludge B while very few could be seen in
sludge A. After the sulfur oxidation test, few granules were present in
sludge A and very few were present in sludge B. Overall, the filaments
dominating both sludges showed quite similar morphological traits and
were identified as type 021N. When FISH was used, probe 21N hybridized
with the filamentous type dominating both sludges, confirming the
identification according to morphology. The fluorescence obtained with
both probes was in general of low intensity, and it could be seen that
not all cells in a given filament produced fluorescence. Probe EUB338
also hybridized with filamentous bacteria, which did not hybridize with
probe 21N (data not shown). The weak fluorescence generally observed on
the filaments could be explained by the fact that the bacteria were
less active. Indeed, prior to hybridization, the sludge samples were
kept at 4°C for 2 weeks, corresponding to the delay in receiving the
molecular probes. The viability staining performed at the end of the 2 weeks revealed a high number of dead cells on most of the filaments.
Effect of chlorine on the sludges.
The effect of chlorine on
the sludges was studied using a variety of methods and parameters:
viability staining, OUR inhibition, inhibition of COD removal, decrease
of filament abundance, and decrease of SVI. Overall, the viability
staining evidenced increasing numbers of damaged type 021N bacteria and
largely intact microbial flocs in sludge A with chlorine doses
increasing from treatment T0 to T4. Microbial flocs were significantly
affected only with treatment T5, at which dose all filaments were
killed. In contrast, type 021N bacteria were largely intact and
microbial flocs were increasingly damaged in sludge B with chlorine
doses increasing from treatment T0 to T4. Type 021N filaments were
significantly damaged at treatment T5, at which dose no microbial floc
survived. This contrasting effect of chlorine on the sludges was best
illustrated by the results from treatments with 15 g of
Cl2 (kg of MLSS)1 (Fig.
1). It suggests the existence of a
chlorine-susceptible type 021N in sludge A and a chlorine-resistant
type 021N in sludge B. The results obtained for the OUR, the residual
soluble COD, the SVI, and the filament abundance are shown in Fig.
2. Standard deviations were derived from
the coefficients of variation determined according to OUR (sludge A,
10%; sludge B, 12%), COD (A, 12%; B, 13%), SVI (A, 6%; B, 4%),
and filament scoring (0% for A and B) from four replicates of
treatment T0. According to the OUR and COD measurements, the microbial
activity decreased with increasing chlorine doses for both sludges. For
the same chlorine dose, this decrease appeared more rapidly in the case
of sludge B than of sludge A. SVI and filament abundance decreased with
increases in the chlorine dose in the case of sludge A, while in the
case of sludge B these parameters hardly varied. The micrographs
corresponding to the control and to treatment T4 (Fig.
3) illustrate the different evolutions of
filament content between sludge A and sludge B after the 24-h
incubations on a shaker. These observations are in agreement with the
preliminary effects of chlorine on the filamentous bacteria as revealed
by the viability staining. Indeed, the rationale for curing filamentous
bulking by means of chlorination is that the causative filamentous
bacteria protruding from flocs are more exposed to toxic compounds than
the bacteria inside the flocs (18). Therefore, a reduction
in the filamentous bacteria in the microbial population of a bulking
sludge, accompanied by an improvement of the sedimentation of the
sludge, is a sign of the effectiveness of chlorine. In the present
study, this was observed with sludge A but not with sludge B. Thus,
chlorination was effective against type 021N in sludge A but not in
sludge B, although the filaments were protruding out of flocs in both
sludges. The decrease in microbial activity with increasing chlorine
dose shown by OUR and residual COD measurements confirms the adverse
effects of chlorine on the microbial populations of the sludges. This
excludes the hypothesis of an important influence of the chlorine
demand from the water content of the sludges. Nevertheless, OUR and
residual COD measurements did not reflect the effects of chlorine on
the filamentous bacteria alone. The decrease in microbial
activity was even more pronounced with sludge B than sludge A. This
result confirms the inappropriateness of the activity parameters for monitoring the effect of chlorine on filamentous bulking sludges mentioned by Bitton and Koopman (1). It should be
mentioned that all the observations and trends reported above were
confirmed by repeating the experiments on the effects of chlorine on
the sludges (data not shown).
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FIG. 1.
Comparative micrographs of sludges A (A1 and A2) and B
(B1 and B2) after viability staining. (A1 and B1) Not treated
(control);(A2 and B2) treated with 15 g of Cl2 (kg of
MLSS)1. The size bars on A1 and B1 apply to all four
panels.
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FIG. 2.
OUR, effluent COD, SVI, and filament scoring of sludges
A ( ) and B ( ) after treatment with similar chlorine doses.
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FIG. 3.
Comparative micrographs of sludges A (a and b) and B (c
and d) 24 h after similar treatments. (a and c) Not treated
(control); (b and d) treated with 15 g of Cl2 (kg of
MLSS)1. The size bar on panel b applies to all four
panels.
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Thus, viability staining, filament abundance, and SVI, used to examine
the effects of chlorine on the filaments, led to the
identification of
a chlorine-vulnerable type 021N bacterium in
sludge A and a
chlorine-resistant type 021N bacterium in sludge
B. Bacteria within the
same strain or species that exhibit variations
in resistance to
chlorine have already been mentioned by other
authors (
10,
14). Type 021N is traditionally known as a chlorine-susceptible
filamentous bacterium (
7). The exact causes for the
development
of the chlorine-resistant variant that was observed in this
study
have not been investigated. However, the growth resulting from
the starvation conditions imposed by the endogenous periods of
the
sequencing batch reactor is suspected to be the most probable
cause.
Indeed, according to Roszak and Colwell (
15), factors
such
as starvation may alter cell membrane composition and affect
its
permeability. The fact that the disinfecting efficiency of
chlorine
depends on penetration rate through the cell wall (
19)
suggests that type 021N in sludge B developed a less permeable
cell
wall. Moreover, the slow or difficult staining of certain
bacteria by
viability stains mentioned in other studies (
9,
16) and
attributed to poor cell membrane permeability was also
noticed with
type 021N in sludge B (data not
shown).
The two different reactions of type 021N to chlorine may also imply the
existence of different species of type 021N bacteria,
as suggested by
Kanagawa et al. (
8). This could not be confirmed
by our
relatively limited FISH study. Unfortunately, additional
FISH assays of
freshly collected sludge samples were unsuccessful
because, due to the
dynamic feature of the activated sludge, the
microbial populations had
changed.
So far, unsuccessful bulking correction using chlorination is poorly
understood and is often attributed to inappropriate application.
The
results of this study, particularly concerning sludge B, showed
that
chlorination will not always be efficient in curing bulking,
even if
the rules established by Jenkins et al. (
7) are followed.
These results offer striking evidence that may help to reconcile
the
successful and unsuccessful cases of the application of chlorine
for
controlling filamentous bulking in activated sludge that have
been
reported so far. Therefore, verification of the effectiveness
of
chlorine on filamentous bulking sludge in adequate preliminary
trials
such as OUR determinations and Live/Dead staining prior
to full-scale
application appears to be of prime
importance.
| |
ACKNOWLEDGMENTS |
We are thankful to N. Boon for his guidance in the implementation
of the FISH and to P. De Boever for his critical comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory for
Microbial Ecology and Technology, Center for Environmental Sanitation, Ghent University, Coupure L 653, 9000 Ghent, Belgium. Phone: 32 9 264 59 76. Fax: 32 9 264 62 48. E-mail:
Willy.Verstraete{at}rug.ac.be.
| |
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Applied and Environmental Microbiology, November 2001, p. 5303-5307, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5303-5307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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