INTERNATL MICROBIOL (2000) 3:103–106 103

© Springer-Verlag Ibérica 2000 RESEARCH ARTICLE

Frederic B. Gich1, Estefania Amer2,

Jordi B. Figueras1,3, Charles A. Abella1, Assessment of microbial community
M. Dolors Balaguer2, Manel Poch2 structure changes by amplified
1
Laboratory of Microbiology, Institute of Aquatic ribosomal DNA restriction
analysis (ARDRA)
Ecology, University of Girona, Spain
2
Laboratory of Engineering and Environmental
Chemistry, University of Girona, Spain
3
Institute of Biochemistry, Syddasnsk
Universitet-Odense Universitet, Odense, Denmark

Received 25 January 2000 Summary Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method
Accepted 5 April 2000 based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In
this study we have evaluated the suitability of this method to detect differences in
activated sludge bacterial communities fed on domestic or industrial wastewater,
and subject to different operational conditions. The ability of ARDRA to detect these
differences has been tested in modified Ludzack-Ettinger (MLE) configurations.
Samples from three activated sludge wastewater treatment plants (WWTPs) with the
MLE configuration were collected for both oxic and anoxic reactors, and ARDRA
patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice
similarity coefficients was calculated and used to compare these restriction patterns.
Differences in the community structure due to influent characteristics and temperature
could be observed, but not between the oxic and anoxic reactors of each of the three
MLE configurations. Other possible applications of ARDRA for detecting and
Correspondence to:
Frederic B. Gich. Laboratory of Microbiology. monitoring changes in activated sludge systems are also discussed.
Institute of Aquatic Ecology. University of
Girona. Campus Montilivi. 17071 Girona. Spain. Key words Amplified ribosomal DNA restriction analysis (ARDRA) · Wastewater
Tel.: +34-972418396.
Fax: +34-972418150. treatment plants (WWTPs) · 16S ribosomal DNA · Activated sludge · Dice similarity
E-mail: fgich@morgat.udg.es coefficient

oligonucleotides, we can see that up to 90% of the biomass

Introduction present in activated sludge can be metabolically active [17].
Thus, the recent development of molecular biology techniques,
Many problems found in wastewater treatment plants (WWTPs) which do not rely on cultivation methods, allows microbial
that perform biological removal of pollutants are due to ecologists to reveal inhabitants of natural microbial communities
alterations in the microbial communities involved. These which have not yet been cultured [7, 13, 15]. As a result, these
alterations are caused by changes in influent characteristics techniques are now widely applied to characterize microbial
and operating conditions. The study of their influence on the community structures in different environments such as
microbial communities of WWTPs can provide useful biological wastewater systems [6, 12, 14, 15].
information to solve these problems. Plate counting and most Two of these techniques, cloning and sequencing, allow us
probable number (MPN) techniques have been used for the to determine which microorganisms are present in the
study of microbial communities in mixed culture systems. community, but they are time-consuming. Hybridization and
However, less than 1% of microorganisms in the environment probing are faster, but require a sufficient knowledge of the
can be usually cultivated by standard techniques because culture community to choose the appropriate target sequences [2]. In
techniques fail to reproduce in artificial media the niches of this study, another molecular biology technique, the amplified
many microorganisms found in high-diversity environments ribosomal DNA restriction analysis (ARDRA), is applied to
such as activated sludge [13]. Recoveries from activated sludge, activated sludge samples. Even faster than hybridization and
even with optimized media, can be as low as 5% [17]. probing, ARDRA has been used in the analysis of mixed
Traditional thinking, based on viable counts of bacteria, suggests bacterial populations from different environments [1, 8, 10].
that the bulk of bacterial biomass in activated sludge is near Although ARDRA gives little or no information about the type
death. However, by probing with fluorescent-labelled of microorganisms present in the sample, it can be used for a
104 INTERNATL MICROBIOL Vol. 3, 2000 Gich et al.

quick assessment of genotypic changes in the community Data analysis The patterns of each sample were compared by
over time, or to compare communities subject to different identifying, from different samples, fragments of identical size
environmental conditions. in the same digestion. Pairwise comparations of the band
patterns were manually performed, and a presence/absence
matrix was constructed. In this way, the Dice similarity
Materials and methods coefficient [16] was obtained for every pair of samples, enabling
us to generate a similarity dendrogram. The data were computed
Description of the WWTP configurations The activated sludge by using the SPSS program version for Macintosh 4.0.
samples were collected from the oxic and the anoxic reactors of
three WWTPs located in Girona, Spain. The WWTP of Vidreres-
Sils (D1) has an anoxic tank (D1-an) of 350 m3, and an oxic tank Results and Discussion
(D1-ox) of 1500 m3, arranged in a modified Ludzack-Ettinger
(MLE) configuration. It treats about 1500 m3/day of domestic Influent characteristics, operational parameters and
wastewater. The WWTP of Ripoll (D2) was built to operate as removal efficiencies Table 1 summarizes the principal influent
an Orbal but, due to the low influent load, only the two inner characteristics and operational parameters for each of the three
channels are in use. Of these two, the outer one (4500 m3, WWTPs. The nitrite and nitrate concentrations in the influents
designated as D2-an) operates under anoxic conditions, while were always under 1 ppm N. The chemical oxygen demand
the other (3500 m3, designated as D2-ox) is under oxic conditions. (COD) removal efficiencies of all WWTPs were over 85%.
Internal recycling from the inner to the outer channel allows the The nitrification efficiencies were 83% and 94% for reactors
system to work in MLE configuration. The WWTP of Ripoll D1 and IN, respectively, while reactor D2 showed complete
treats approximately 9000 m3/day of domestic wastewater. Finally, nitrification. Reactors D1 and D2 had denitrification efficiencies
the third WWTP studied is a pilot plant that treats industrial of 16% and 64%, respectively. Reactor IN also achieved
wastewater from a food-processing factory. It has an oxic reactor complete denitrification.
of 3.5 l (designated as IN-ox), and an anoxic reactor of 2.2 l
(designated as IN-an) in an MLE configuration. The influent
flow to the pilot plant is 0.83 l/day. Table 1 Principal characteristics and operational parameters of the treatment
plants studied: D1, urban Vidreres-Sils wastewater; D2, urban Ripoll wastewater;
IN, industrial wastewater
Sampling and analytical determinations Samples of the inlet
and the outlet of each reactor were collected in order to determine Reactor
Operational parameter
influent characteristics and reactor efficiencies. The conservation D1 D2 IN
of the samples, their processing and all analytical measurements sCOD (mg/l) 144 157 8539
were carried out according to APHA [3]. Activated sludge pCOD (mg/l) 96 149 1138
samples of 50 ml for DNA extraction were collected in sterile N-NH4+ (mg/l) 11 18 655
sNorg (mg/l N) 2 3 327
Falcon tubes and frozen at –20ºC until processing. pNorg (mg/l N) <1 8 –
MLSSanoxic reactor (mg/l) 1600 3048 7080
16S rDNA amplification DNA extraction was carried out in MLSSoxic reactor (mg/l) 1600 4082 4080
%VSSanoxic reactor 50% 47% 49%
Nalgene sterile tubes by the phenol-chloroform method described %VSSoxic reactor 49% 48% 71%
by Moore [9]. DNA was purified with Bio-Spin chromatography θanoxic reactor (h) 20.3 5.6 63.4
columns (Bio-Rad, Hercules, CA, USA) to eliminate proteins θoxic reactor (h) 15.7 24 100.8
θc (day) >30 22 >30
and nucleotides. PCR amplification of 16S rDNA, using the external recycle (%) 75 290 150
primer set of fD1 and rP1, was performed as described previously internal recycle (%) 200 290 1000
[18]. PCR products were purified using the QIAquick PCR Tmixed liquor (°C) 10 12 38
Purification Kit (Quiagen, Valencia, CA, USA). s: soluble, p: particulate, θ: sludge residence time
COD, chemical oxygen demand
MLSS, mixed liquor suspended solids
rDNA restriction fragments separation by electrophoresis VSS, volatile suspended solids
Double restriction endonuclease digestions were performed
for every sample with AluI+MspI (Promega, Madison, WI, USA)
as described previously [1]. Restriction enzymes were chosen Differences between industrial and domestic WWTP
on the basis of their high average number of restriction sites communities The restriction patterns obtained by electro-
per taxon [11]. Separation of digested products in poly- phoresis are shown in Fig. 1. The differences between restriction
acrilamide gels were performed as described elsewhere [1, 8]. patterns in all the communities subject to domestic wastewater
The gel was digitallized using a scanner AGFA Arcus II and are smaller than those between domestic wastewater WWTPs
the images were contrasted using the NIH Image Program 1.59 and those treating the industrial wastewater, as reflected in the
(National Institutes of Health, Bethesda, MD, USA). dendrogram and in the Dice similarity coefficient matrix shown
ARDRA to study community structure INTERNATL MICROBIOL Vol. 3, 2000 105

the systems studied, as shown in the dendrogram (Fig. 2A). The

absence of differences between the patterns does not ensure that
the composition of the communities is exactly the same. However,
significative composition changes in the community should be
detected with the restriction enzymes used [11]. Previous works
demonstrated that double restriction endonuclease digestions are
sensitive enough to detect important composition changes in the
community [1, 8, 11]. The absence of differences between the
patterns of the oxic and the anoxic reactors leads to the conclusion
that there were probably no significant changes between the
microbial communities of the two reactors. Similar conclusions
were drawn by Ehlers and Cloete [5] by using protein fingerprints
to evaluate the differences between the microbial community
structures among P-removing, non-P-removing and N-removing
systems. Thus, the similarity of endonuclease restriction patterns
among the samples agrees with the high similarity of protein
fingerprints in bacterial communities of different activated sludge
systems. Given the residence times and the internal recycle values
of the systems studied, the generation times of the microorganisms
are probably too long to observe significant differences in
community composition among the anoxic and oxic reactors.
Therefore, the aerobic and anaerobic populations apparently do
not have enough time to change while inside the oxic or anoxic
reactors, and therefore they merely coexist. Despite the absence

Distance

Fig. 1 Polyacrilamide gel showing AluI+MspI digestions of the whole eubacterial

communities from the six reactors studied: D1-an, Vidreres-Sils wastewater
anoxic reactor; D1-ox, Vidreres-Sils wastewater oxic reactor; D2-an, Ripoll
wastewater anoxic reactor; D2-ox, Ripoll wastewater oxic reactor; IN-an, industrial
wastewater anoxic reactor; IN-ox, industrial wastewater oxic reactor

in Fig. 2. Two main factors can explain these results. First,

industrial WWTP communities are subject to much higher
COD, ammonia and organic nitrogen inputs than the domestic
WWTP communities, as shown in Table 1. Besides, the
temperature was higher in the industrial wastewater reactors
(38ºC) than in all the domestic wastewater reactors, where it
remained between 10 and 12ºC (Table 1).
Fig. 2 Differences in restriction patterns. (A) Dendrogram of eubacterial 16S
Differences between the oxic and anoxic reactors in MLE rDNA-ARDRA similarities obtained by digestion with AluI+MspI. (B) Dice
configurations No significant differences have been observed similarity coefficient matrix. Reactors D1-an, D1-ox, D2-an, D2-ox, IN-an
between the restriction patterns of oxic and anoxic reactors of all and IN-ox, as in Fig. 1
106 INTERNATL MICROBIOL Vol. 3, 2000 Gich et al.

of changes in community composition, there are probably 3. APHA, AWWA and WEF (1992) Standard methods for the examination of
differences in the microbial activity developed in the oxic and water and wastewater. American Public Health Assoc., Washington, DC
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anoxic reactors. In each of these, only the part of the community Pommepuy M (1997) Molecular epidemiological survey of rotaviruses
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bacterial communities in a three-compartment granular activated sludge
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Concluding remarks processes. Appl Environ Microbiol 64:2528–2532
7. Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent
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communities from industrial and domestic wastewater treatment 8. Martínez-Murcia AJ, Acinas SG, Rodríguez-Valera F (1995) Evaluation of
plants, and these differences could be due to influent prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified
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Acknowledgments This work was funded by the CICYT under Project BIO
14. Sellwood J, Litton PA, McDermott J, Clewley JP (1995) Studies on wild
96/1229. F.G. and E.A. acknowledge CIRIT for the FIAP grants received. We
and vaccine strains of poliovirus isolated from water and sewage. Water
gratefully thank the staff at the Vidreres-Sils and Ripoll WWTPs for their help
Sci Tech 31:317–321
and operational data. We also thank Javier Rodríguez for his technical assistance
15. Snaidr J, Amann R, Huber I, Ludwig W, Schleifer KH (1998) Phylogenetic
in SPSS analysis, and Erik T. Buitenhuis for critical comments and revision
analysis and in situ identification of bacteria in activated sludge. Appl
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Environ Microbiol 63:2884–2896
16. Sneath PHA, Sokal RR (1973) The estimation of taxonomic resemblance.
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