Process Biochemistry 42 (2007) 119–133

www.elsevier.com/locate/procbio

Review
Molecular biology techniques used in wastewater
treatment: An overview
José L. Sanz *, Thorsten Köchling
Centro de Biologı́a Molecular, Universidad Autónoma de Madrid, Madrid 28049, Spain
Received 14 June 2006; received in revised form 12 September 2006; accepted 1 October 2006

Abstract
Identification of microorganisms by conventional methods requires the isolation of pure cultures followed by laborious characterization
experiments. These procedures are therefore inadequate for study of the biodiversity of a natural or engineered ecosystem. A new set of molecular
techniques developed during the 1990s revolutionized microbial ecology research. Among these techniques, cloning and the creation of a gene
library, denaturant gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization with DNA probes (FISH) stand out. Cloning provides
very precise taxonomical information, but is time consuming and requires specialized personnel and so its introduction in wastewater treatment has
been slow. DGGE is a rapid and simple method that provides characteristic band patterns for different samples, allowing quick sample profiling,
while retaining the possibility of a more thorough genetic analysis by sequencing of particular bands. FISH makes possible to identify
microorganisms at any desired taxonomical level, depending on the specificity of the probe used. It is the only quantitative molecular biology
technique, although quantification is either complex or tedious and subjective. Combination with a confocal laser-scanning microscope allows the
visualization of three-dimensional microbial structures (granules, biofilms). The methods discussed have deepened our understanding of the
microbiology of biological wastewater treatment. PCR-based methods (cloning and DGGE) have proved suitable for identifying the micro-
organisms that form the sludge. Both DGGE and FISH have been extensively employed. FISH is currently being used for elucidation of the
composition, quantification and distribution of different bacterial groups in granules and biofilms, as well as their structure and architecture.
# 2006 Elsevier Ltd. All rights reserved.

Keywords: Wastewater treatment; Anaerobic digestion; Molecular ecology; Molecular biology techniques; Denaturant gradient gel electrophoresis (DGGE);
Fluorescent in situ hybridization (FISH); Gene library

Contents

1. Molecular microbial ecology: a new approach to study the biodiversity of complex ecosystems . . . . . . . . . . . . . . . . . . . . . . . 119
2. Molecular biology techniques and their applicability to wastewater treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
2.1. Cloning of 16S rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
2.2. Denaturant gradient gel electrophoresis (DGGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
2.3. Fluorescent in situ hybridization (FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
2.3.1. Activated sludge and nutrient removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
2.3.2. Anaerobic digestion, upflow anaerobic sludge blanket (UASB) reactors and granular sludge . . . . . . . . . . . . . . . 125
2.4. Alternatives and new methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

1. Molecular microbial ecology: a new approach to

study the biodiversity of complex ecosystems

* Corresponding author. Tel.: +34 914 978 077; fax: +34 914 978 087. Conventional microbiological techniques, based on isolation
E-mail address: joseluis.sanz@uam.es (J.L. Sanz). of pure cultures and morphological, metabolic, biochemical
1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.10.003
120 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
and genetic assays, have provided extensive information on the
biodiversity of microbial communities in natural and engineer-
ing systems [7]. However, the drawbacks of the existing
conventional methods, such as incomplete knowledge about
their physiological (nutritional and physical–chemical) needs
and the complex syntrophic and symbiotic relations, which are
abundant in nature, make it impossible to obtain pure cultures
of most microorganisms in natural environments. Moreover,
most culture media tend to favor the growth of certain groups of
microorganisms, whereas others that are important in the
original sample do not proliferate. It is therefore generally
accepted nowadays that the number of known prokaryotic
species (including the two domains: Bacteria and Archaea) is
very small compared to the diversity of microorganisms and
illustrates how difficult it is to get a full picture of the bacterial
diversity of an ecosystem by relying only on conventional
methodology. At present, about 7000 bacterial species have
been described (DSMZ, 2005; http://www.dsmz.de), but
according to molecular and ecological estimates, the real
number must be several orders of magnitude higher [3]. This
small known fraction does not reflect the composition and
diversity of a microbial community.
One suitable solution to this problem is to use molecular
biology approaches. The techniques are based on the RNA of
the small ribosomal subunit (16S rRNA for prokaryotes) or
their corresponding genes, considering it as a ‘‘molecular
clock’’ or ‘‘evolutionary chronometer’’. This molecule was
chosen because of its universality and abundance in all living
beings (103 to 105 ribosomes/cell) and the fact that it is a highly
conserved molecule throughout evolution although bears some
highly variable regions. These features allow comparison of
organisms within the same domain, as well as differentiation of
strains of the same species. Moreover, the gene sequence is
sufficiently long to generate statistically relevant data and can
be easily sequenced with current technology. For the first time it
is possible to survey the biodiversity of a natural habitat in a
relatively simple, but complete way.
However, as it will be pointed out throughout the text, the
knowledge and information gained using these techniques may
sometimes be scarce and the usefulness for engineers and
technicians who actually design and operate bioreactors could
be uncertain. For decades a biological reactor has been
considered a ‘‘black box’’. The new insights in microbiology
have helped to improve the design and performance of new
generation reactors [95,42]. Probably it is true that it is not
essential to know the phylogenetic position (or, in other words,
the taxonomic ranking of a microorganism according to its
evolution) of the individual microorganisms that dwell inside a
system for the design of a wastewater facility. Or, does it really
help to know that the predominant microorganism in the
oxidation of nitrite to nitrate is Nitrospira and not Nitrobacter
(as taught in the books) and that this Nitrospira belongs to a
completely different bacterial phylum (hierarchical group of
bacteria at maximum level) than other nitrifying bacteria, all of
which are proteobacteria (other bacterial phylum)? The answer
can be ambiguous. The recent discovery of the new bacterium,
Brocadia anammoxidans, which is capable of oxidizing
ammonium to molecular nitrogen under anoxic conditions,
may serve as an example. This finding has revolutionized
research on nitrogen elimination in wastewater treatment plants
[89]. The focus of this review is to present the main molecular
biology techniques actually used to identify and quantify
microorganisms in wastewater treatment systems, with its main
advantages and drawbacks. These techniques have been
developed very recently and mostly used at lab scale. Its real
potential as tools for design or monitoring full-scale wastewater
treatment plant must be evaluated in a near future.
2. Molecular biology techniques and their applicability
to wastewater treatment
The possibility of identifying specific populations of
microorganisms in their native habitat without the need to
isolate them is revolutionizing microbial ecology and giving
rise to various new applications in numerous research fields.
In wastewater treatment, microbial molecular ecology
techniques have been applied mainly to the study of flocs
(activated sludge) and biofilms that grow in aerobic treatment
systems (trickling filters). This article will briefly outline the
most widely used with a discussion of their potential and
limitations. Due to space restriction we have limited the scope of
the review, placing particular emphasis on anaerobic digestion,
and to the techniques unquestionably most broadly used:
denaturant gradient gel electrophoresis (DGGE), Fluorescent
in situ hybridization (FISH) and cloning of 16S rDNA, although
the generation of genetic libraries is not so popular in this field.
2.1. Cloning of 16S rDNA
Of all the molecular techniques considered in this review,
cloning and sequencing of the gene that codes for 16S rRNA is
still the most widely used in the field of microbial ecology. This
methodology implies the extraction of nucleic acids, amplifica-
tion and cloning of the 16S rRNA genes, followed by
sequencing and finally identification and affiliation of the
isolated clone with the aid of phylogenetic software (Fig. 1).
While amplicons generated from pure cultures of bacteria could
be sequenced directly, in the case of genomic DNA extracts
from microbial communities, the cloning step has to be
included. This is necessary in order to separate the different
copies of 16S rDNA, as a mixed template cannot be sequenced.
Because this approach is so widespread, half of the
approximately 240,000 sequences deposited in the 16S rDNA
NCBI-database (April 2006), belong to non-cultured and
unknown organisms, that is, organisms detected by 16S rDNA
cloning. This illustrates how extensively and successfully the
cloning strategy has been employed since its introduction in the
beginning of the 1990s [99]. However, cloning is time
consuming and so less apt for analyzing larger sets of samples,
for example, when monitoring changes in natural or engineered
microbial communities over time, particularly if several sample
points are required.
Despite their extensive application in the study of microbial
communities in natural habitats, these techniques are less
 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133 121

Fig. 1. Outline of the cloning procedure for studying a microbial community. The work cycle is as follows: (A) direct nucleic acid extraction, without the need for
previous isolation of microorganisms; (B) amplification of the genes that code for 16S rRNA by polymerase chain reaction (PCR), commonly using universal primers
for bacteria or archaea, resulting in a mixture of rDNA copies corresponding to the microorganisms present in the sample; (C) cloning of the PCR products obtained
into a suitable high copy number plasmid and transformation of competent E. coli cells with this vector; (D) selection of transformed clones with an indicator
contained in the plasmid (the white colonies in the figure); (E) extraction of plasmid DNA; (F) sequencing of the cloned gene, creating a clone library; (G)
determination of the phylogenetic affiliation of the cloned sequence with the help of dedicated computer programs (ARB, Seqlab, PAUP, PHYLIP).

widespread in research of wastewater treatment processes. This
lack of popularity may be due to the need for specialized
personnel and equipment, which are not always readily
available in engineering and chemistry departments. This
technology may be too complex and so its use is limited to that
of a support tool only. Nonetheless, several examples of the
present decade illustrate its potential in the wastewater
treatment area. Cloning was employed to establish with
precision the phylogenetic position of filamentous bacteria in
granular sludge that were previously affiliated, by in situ
hybridization, to the division of green non-sulfur bacteria [86];
or to determine the prevalent sulfate reducing bacteria in a
biofilm [41]. The microbial communities residing in reactors
for treating several types of industrial wastewater have also
been determined by means of 16S rDNA cloning and
sequencing. Egli et al. [27] examined the microbial composi-
tion and structure of a rotating biological contactor biofilm for
the treatment of ammonium-contaminated wastewaters. In their
16S rDNA clone libraries, they found the sequences of several
previously undetected and uncommon microorganisms, as well
as others that were confirmed to be associated with the process
by FISH analysis. The study also confirmed the predicted
functional structure of a mixed aerobic/anaerobic biofilm
developed in the presence of high ammonium concentrations. A
description of the microbial communities responsible for the
anaerobic digestion of manure and manure/lipid mixtures in
continuously stirred tank reactors (CSTR) was published in
2003 by Mladenovska et al. [54]. Phylogenetic analysis of the
sequences obtained showed a narrow range of diversity, with
most of the screened microorganisms belonging to the
Methanosarcina genus. A good example of complementary
application of molecular and traditional culturing methodology
is the research on an anaerobic system for cis-1,2-dichlor-
oethylene degradation, carried out by Hata et al. [37]. The
authors assessed the microbial community structure of the
biomass, and established the phylogenetics of the degrading
bacteria, confirming their activity in batch experiments.
Ferrera et al. [31] studied a microbial biofilm for treatment
of sulfide containing waters employing construction of a clone
library and genetic analysis. The authors highlighted the
suitability of the method for assessing the microbial richness of
an ecosystem, as expressed by the number of different
operational taxonomic units (OTUs). Chen et al. [16]
constructed clone libraries of the microorganisms in a
thermophilic anaerobic hybrid reactor for the degradation of
terephthalate, and compared the community members present
in the sludge before and after operational perturbations (heat
shock, pump failure) had occurred. The article is a good
illustration of the sensitivity of the cloning approach, as
significant changes in community structure and phylotype
abundance were detected by comparing the ‘‘before and after’’
gene libraries. Zhang et al. [104] provided another example of
the cloning approach in systems dedicated to the degradation of
organic compounds. Working with a methanogenic reactor
adapted to phenol degradation, the researchers used cloning in
conjunction with in situ hybridization analysis to give a detailed
picture of the population, as well as to identify the species
responsible for phenol transformation. The work by Sekiguchi
et al. [86] on filamentous bacteria found in UASB reactors
mentioned earlier was continued by the same group [102]. This
work revealed further structural and phylogenetic details of the
observed bacteria, now known to belong to a Chloroflexi
subphylum. Characterization and understanding of these
bacteria is an important field of research as the filamentous
122 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
Chloroflexi pose problems for reactor operation because of
sludge bulking.
In general, cloning and rRNA gene library construction have
been applied in combination with other techniques in waste-
water treatment. Cloning of the whole gene yields far more
exact phylogenetic information than other molecular techni-
ques such as FISH and DGGE. Moreover, the potential of such
an approach as a tool for designing new specific primers and
gene probes for detection and/or quantification of a certain
organism (or group of microorganisms) deserves mention. A
good example of this approach is the work of Crocetti et al.
[18], who extracted genomic microbial DNA from a sequencing
batch reactor, cloned the bacterial 16S rDNA and identified
Rhodocyclus sp. and Propionibacter pelophilus as the
microorganisms responsible for the polyphosphate accumula-
tion taking place in the reactor. These authors then designed
probes for these species that could establish a correlation
between phosphorous removal and the number of hybridized
cells in different sludges. In 2004, with some highly skilled
work Beer et al. were able to design new probes for in situ
hybridization with information provided by 16S rDNA gene
library sequences and DGGE analysis [6]. These new probes
target members of the alpha-proteobacteria, whose role in
anaerobic/aerobic-activated sludge phosphorous removal plants
was analyzed in the study. A similar approach had been taken 3
years earlier by the same authors, but without probe design [47]
to identify the predominant microorganisms in an anaerobic
sequencing batch reactor (SBR) with no phosphorus removal. In
this case, the combination of cloning, DGGE, and FISH provided
a description of the structure and function of the biomass present.
The creation of gene libraries can identify a substantial share
of the microbial species living in a sample, but such a complete
picture of a bacterial habitat is extremely time consuming and
laborious to obtain. The main advantages and disadvantages of
the approach can be summarized as follows:
 Advantages:
 Complete 16S rRNA sequencing allows:
very precise taxonomic studies and phylogenetic trees of
high resolution to be obtained;
design of primers (for PCR) and probes (for FISH).
 If time and effort is not a limiting factor, the approach
covers most microorganisms, including minority groups,
which would be hard to detect with genetic fingerprinting
methods.
 Identification of microorganisms that have not been yet
cultured or identified.
 Disadvantages:
 Very time consuming and laborious, making it unpractical
for high sample throughput.
 Extraction of a DNA pool representative of the microbial
community can be difficult when working with certain
sample types (e.g. soil, sediments).
 Many clones have to be sequenced to ensure most of
individual species in the sample are covered.
 It is not quantitative. The PCR step can favor certain
species due to differences in DNA target site accessibility.
2.2. Denaturant gradient gel electrophoresis (DGGE)
The increased popularity of denaturant gradient gel
electrophoresis (DGGE) is reflected by the increasing number
of studies that use the technique. It is based on the differing
mobility on a gel of denatured DNA-fragments of the same size
but with different nucleic acid sequences, thus generating band
patterns that directly reflect the genetic biodiversity of the
sample. The number of bands corresponds to the number of
dominant species. Coupled with sequencing and phylogenetic
analysis of the bands, this method can give a good overview of
the composition of a given microbial community (Fig. 2).
DGGE is the method of choice when the desired information
does not have to be as phylogenetically exhaustive as that
provided by cloning, but still relatively precise to determine the
dominant members of a microbial community with medium
phylogenetic resolution. Muyzer and Smalla [57] wrote an
excellent review on the potential, problems and applications of
DGGE. Since, its first use in the study of complex microbial
populations [58], this method has been employed in the
characterization of a wide array of habitats, such as soil,
bacterioplankton, hot springs, continental waters, etc. The
technique is less widely used in anaerobic wastewater
treatment, though in recent years DGGE seems to be becoming
increasingly popular. So, for example, DGGE has been used for
the evaluation of the granular sludge’s microbial diversity from
UASB reactors treating brewery [15], alcohol distillery [1], and
unbleached pulp plant wastewaters [11].
In general, this technique is not used alone but rather as
part of a combined approach with other methods, for example
with in situ hybridization in the study of sulfate reducing
bacteria [81] or phosphorous elimination [63]. Both these are
good examples of the advantages of combining fingerprinting
with in situ hybridization. The authors managed to trace the
most probable protagonist in the process by evaluating DGGE
band intensity and then designing a specific probe with the
help of the predominant band sequence, in turn enabling
quantification of the candidate and confirmation of the results
obtained by DGGE. Tang et al. [90] were interested in
developing a micro-aerated thermophilic digester system for
treating municipal solid waste that does not produce the
unwanted and foul smelling H2S gas. The authors made use
of the full rRNA cycle technology available to provide a
detailed description of the microbial populations in both
micro-aerated and strictly anaerobic digesters. The global
proportions of species belonging to bacteria and archaea were
quantitatively surveyed (FISH) and shifts in the archaeal
community structure were detected as a response to aeration
(DGGE, cloning). Rincón et al. [74] combined DGGE with
gene library creation to carry out an exhaustive analysis of a
microbial community in a CSTR for treating solid olive mill
waste. The authors found a wide array of bacteria from
different phyla. A different approach was taken by Zhang
et al. [103], who used statistical methods, such as cluster
analysis of DGGE band patterns to determine the similarity
of bacterial and archaeal populations found in a UASB
reactor for treating municipal wastewaters.
 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133 123

Fig. 2. Schematic representation of DGGE. DNA is extracted from the original sample, in this case as granular sludge from a UASB reactor (A). The 16S rRNA gene
is partially amplified by PCR usually with universal primers to give a mixture of DNA fragments, all of the same length. Each of the different product DNA molecules
resulting from this step represents a different microorganism (B). The DNA mixture is then separated by denaturant gradient electrophoresis on an acrylamide gel with
an increasing urea/formamide gradient. The DNA molecules migrate towards the positive pole and come to a halt on the gel on reaching their corresponding
denaturant force (Tm), which depends on the DNA sequence. Every band on the gel corresponds to a different microorganism in the sample, providing sufficient
information for many requirements (C). The bands can be cut from the gel and the DNA extracted and sequenced (D). Comparison of the sequences with a 16S rDNA
database allows to determine the phylogenetic affiliation of the microorganism (E).

The most important application of DGGE is monitoring
dynamic changes in microbial communities, especially when
many samples have to be processed. There are multiple
applications of DGGE related to anaerobic digestion processes:
studies on differences between mesophilic and thermophilic
reactors, demonstrating the lower biodiversity in thermophilic
reactors used for the treatment of residual waters generated by
the pharmaceutical industry [48]; analysis of the changes
observed in the bacterial diversity of an anaerobic digester for
treating urban solid waste [87]; studies on the changes in
bacterial communities in a continuous stirred tank reactor
(CSTR) in response to dilution rate [93]. Nakagawa et al. [59]
monitored changes in an ethylbenzene-degrading bacterial
consortium in enrichment cultures under anaerobic, sulfate-
reducing conditions. By monitoring the predominant bacterial
species over a period of 127 days, they identified a dominant
bacterium that was present throughout the whole incubation
period and most likely to be the microorganism responsible for
ethylbenzene degradation. Both spatial and temporal changes
in microbial community profiles were monitored by Pereira
et al. [67], in a study of expanded granular sludge bed (EGSB)
reactors for the treatment of oleic acid. With this approach, the
researchers were able to add another dimension to the analysis
and compare the change in microbial communities in different
layers of the sludge bed, as well as changes over the time. The
start-up phase of garbage composters under fed batch operation
was studied by Narihiro et al. [60]. DGGE fingerprints revealed
a significant shift in the bacterial population from ubiquinone-
containing microorganisms to Actinobacteria during the
observation period. Recently, Xing et al. [101] used DGGE
fingerprinting to monitor changes in the microbial community
of a hydrogen producing bioreactor during the different phases
of the process. The authors detected shifts in the population
during start-up followed by stabilization once the process was
running, and also found that cometabolism and mutual
relationships played an important role in the microbial
community involved in biological H2 production. In an
excellent study, Roest et al. [76] monitored microbial
populations in a UASB reactor for treating paper mill
wastewater over 3 years. With a combination of different
molecular techniques and even conventional microbiological
methodology, the authors were able to accurately describe the
biological component of the process.
Simple analysis of band patterns may be sufficient for
certain purposes. Liu et al. [51] monitored mesophilic and
thermophilic acidogenic reactors for treating dairy wastewater
at start-up by analyzing band patterns generated during the
course of the experiment. By itself, this approach provides
limited information, so Liu et al. completed their research with
the help of in situ hybridization. Recently, Connaughton et al.
[17] used cluster analysis of DGGE band patterns to describe
changes in the microbial community taking place in an
expanded granular sludge bed-anaerobic filter (EGSB-AF)
reactor operating under psychrophilic conditions. Once again,
sequence data were used to complement the cluster analysis.
Park and Lee [65] compared the microbial community
composition of a biofilm with that of the biomass in the liquid
phase of a jet-loop type membrane reactor. As the authors did
124 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133

not analyze individual bands, this study of the composition of a
microbial community composition could only provide relative
abundances.
Although DGGE (or TGGE: temperature gradient gel
electrophoresis) is by far the most widely used genetic
fingerprinting technique in molecular ecology, it is not the only
method based on PCR amplification of nucleic acids and
separation of the product mixture by electrophoresis. An
alternative approach is the generation of SSCP (single strand
conformation polymorphism) patterns, a technique that has been
employed to study anaerobic digesters both on laboratory and
industrial scale [21]. Bouallagui et al. [10] investigated the
microbial community in a two-phase anaerobic bioreactor for the
treatment of vegetable wastes with SSCP to assess changes in the
composition of the bacterial and archaeal biomass. A similar
technique called restriction fragment length polymorphism
(RFLP) was used in Hiraishi’s study of methanobacteria [38].
Both methods require the use of a whole set of molecular
methods and generation of a band or peak pattern, and so their
application in monitoring of wastewater treatment is limited.
The main advantages and disadvantages are summarizing as
follows:
 Advantages:
 Permits rapid and simple monitoring of the spatial-
temporal variability of microbial populations if just band
patterns are considered.
 It is relatively easy to obtain an overview of the dominant
species of an ecosystem.
 It is adequate for analysis of a large number of samples (far
more than cloning).
 Disadvantages:
 Depending on the nature of the sample, extraction and
amplification of representative genomic DNA can be
difficult (as in cloning).
 After the PCR amplification, the DNA copy number –
which depends on abundance of a particular microorganism
and the ease of amplification of the 16S rRNA – can be very
different (as in cloning). The intensity the bands obtained
on a DGGE gel may therefore vary (not quantitative).
 The number of detected bands is usually small, which
implies:
the number of identified species is also small;
the bands correspond, although not necessarily, to the
predominant species in the original sample.
 The sequences of the bands obtained from a gel correspond
to short DNA fragments (200–600 bp), and so phylogenetic
relations are less reliably established than with cloning of
the whole 16S rRNA gene. In addition, short sequences are
less useful for designing new specific primers and probes.
2.3. Fluorescent in situ hybridization (FISH)
An excellent way to overcome some of the problems of
studying microbial populations of a microcosm without
resorting to traditional methodology is to use fluorescent
probes. These are short sequences of DNA (16–20 nucleotides)
labeled with a fluorescent dye. These sequences recognize 16S

Fig. 3. The in situ hybridization process can be divided in four stages: (A) sampling and immediate fixation in formaldehyde to preserve the integrity of the cells,
especially the ribosomes; (B) hybridization with a specific probe, labelled with a fluorescent dye at its 50 -end and matched with a sequence of the 16S rRNA; (C)
counterstaining with a universal marker (DAPI, which attaches non-specifically to DNA molecules) or another more general probe, labelled with a different
fluorescent dye; (D) visualization via fluorescence microscopy. Direct quantification is possible by manual counting of hybridized cells (epifluorescence and laser
confocal microscope) or by image analysis of digital photos (both microscopes) or automated counting with a flow cytometer. The ratio of cells that have hybridized
with the specific probe to the total cell count with DAPI or a more general probe gives the percentage of bacteria present in the sample, based on the total number of
cells or on the group that was chosen with the general probe (e.g. bacteria or archaea).
 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
rRNA sequences in fixed cells and hybridize with them in situ
(DNA–RNA matching) (Fig. 3). Microorganisms can be
identified, localized and quantified in almost every ecosystem
with hybridization [2]. The specificity of the probe enables
detection/identification on any desired taxonomic level, from
Domain down to a resolution suitable for differentiating
between individual species. The main shortcoming of this
technique lies in the lack of availability of probes targeting the
desired bacterial taxon or group. Although it is possible, in
theory, to design the most apt probe for each application thanks
to the growing rRNA sequence databases (16/18S and 23/28S
rRNA), it may be impossible to develop a probe that specifically
detects certain groups of microorganisms that share metabolic
properties (for example, sulfate-reduction or halo-respiration).
Furthermore, some previous knowledge of the expected
microorganisms in the sample is often required to apply this
method successfully. To target a particular species, a specific
probe must be ready or its 16S rRNA sequence must be
available.
The use of oligonucleotide probes targeting 16S rRNA
presents a revolution in microbial ecology, both for basic
research and practical applications. Within the area of
wastewater treatment, hybridization techniques are by far the
most extensively used ones. Therefore, any review can only be
partial. We present here a few of the most recent or relevant
publications on the application of FISH in aerobic (activated
sludge and N/P removal) and anaerobic (granular sludge)
treatment.
2.3.1. Activated sludge and nutrient removal
The first work in this area involving hybridization was
performed in 1993 [96] and, although the approach was
somewhat superficial (only specific probes for the detection of
alpha-, beta- and gamma-proteobacteria were employed), the
researchers showed that conventional technology relying on
culture and isolation of microorganisms was unsuitable to
describe microbial communities in activated sludges. Since
then, many studies have been carried out and have become
increasingly complete, surveying microbial communities that
grow in activated sludge systems. Some of these studies aimed
to give an overview of the microbiota [44,64], whereas other
researchers addressed very specific questions, such as the role
of protozoa in the size and distribution of sludge flocs [53].
Outstanding studies on the characterization of filamentous
organisms (Haliscomenobacter, Sphaerotilus, Thiothrix, Leu-
cothrix, Gordona, formerly Nocardia) and their significance for
sludge bulking or flotation phenomenon have been also
conducted [22,94]. In this sense, must be pointed out the
studies focused on Microthrix parvicella, the dominant and
most studied filamentous bacterium present in wastewater
treatment plants, and Nostocoida limicola and N. limicola-like
microorganisms for which FISH probes are already available
[45,50,79].
Interestingly, many applications of FISH in the wastewater
treatment field have been directed towards study of the
microorganisms taking part in the biological elimination of
nitrogen and, to a lesser extent, phosphorous. Publications have
125
dealt with the composition of nitrifying populations in
bioreactors [46,55,61], the predominant role of the ammo-
nia-oxidizing Nitrosococcus and the nitrite-oxidizing Nitros-
pira in the nitrification process [19], or practical guidelines for
developing highly efficient nitrifying biofilms [92]. Although
Onda’s work in 2002 [63] on phosphate-accumulating
organisms in activated sludge reactors has already been
discussed, it deserves further mention as an excellent example
of combining different molecular techniques leading, in this
case, to the design and application of a new specific probe for in
situ hybridization.
A recent breakthrough in the nitrogen cycle was the
aforementioned anammox process. FISH successfully identi-
fied anammox bacteria in different reactor types and waste-
waters [26]. Since then, many researchers have applied FISH to
shed some light on this formerly unknown process. With the
help of 16S rRNA-targeting gene probes, Guven et al. [35]
confirmed the dominating presence of the previously described
Brocadia anammoxidans in a CSTR. In another study on the
enrichment of annamox cultures Third et al. [91] reported the
discovery of supposedly new anammox species. Jetten et al.
[43], in a paper dealing with different aspects of the anammox
process, have presented a molecular approach – including FISH
and planctomycetes-specific PCR – for the environmental
analysis of habitats in which the anammox process occurs.
Another industrially important process, enhanced biological
phosphorous removal (EBPR), has also been thoroughly
examined. The results brought into question the role of
Acinetobacter [97,56], thought to be responsible for this
process, and emphasized the participation of members of the
Betaproteobacteria subclass 2 and Actinobacteria [18,97],
Alphaproteobacteria [56] and Pseudomonas [5] in phosphorous
elimination and interrelated removal of phosphorous and
nitrogen [66]. In 2004, Pijuan et al. [70] monitored the presence
of polyphosphate-accumulating organisms (PAOs) and quanti-
fied candidatus Accumulibacter phosphatis and the glycogen
accumulating organism (GAO) candidatus Competibacter
phosphatis from reactor start-up to steady state. In a recent
article on EBPR, Oehmen et al. [62] reported that candidatus
Accumulibacter phosphatis accounted for 63% of the bacterial
population in the test culture.
2.3.2. Anaerobic digestion, upflow anaerobic sludge
blanket (UASB) reactors and granular sludge
The study led by Stahl with pure cultures of methanogenic
archaea [72] formed the basis for all subsequent work in this
area. In this pioneering study, many of the probes currently used
to identify methanogenic microorganisms at different taxo-
nomic levels (order, family, and genus), were described. The
authors themselves used these probes to locate and quantify
methanobacteria living in different anaerobic digesters [73].
Ever since the first studies on UASB reactors in the early
1980s (see also, http://www.uasb.org), granular sludge has been
popular with researchers from different fields of anaerobic
digestion. Despite efforts and multiple articles, including
guidelines for start-up of UASB reactors with granulation
[39,40] and mechanism and models [83,52], the phenomenon
126 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
of granulation is not well-understood. Molecular techniques to
analyze this form of biomass were introduced in the last decade
and they have contributed greatly to the knowledge of its
microbiology. After the pioneering work by Harmsen et al. [36]
and the excellent contribution by Sekiguchi et al. [85] on the
structure, composition and distribution of different microbial
groups, a number of aspects of this remarkable microcosm have
been corroborated, disproved and explained.
Publications that further illustrate the application of FISH in
anaerobic digestion have dealt with the interaction and
distribution of trophic groups, such as sulfate reducing bacteria
and methanogenic archaea in methanogenic/sulfidogenic
reactors [82] or differentiation between hydrogenotrophic
and acetoclastic methanobacteria, and within this group
between Methanosaeta and Methanosarcina [33,75]. The
influence of feeding on granule structure and microbial
composition has been also assessed by FISH [24]. Other work
has emphasized the dependence of the settling behavior of
UASB granules on their microbial composition and the decisive
role of the positioning of methanogenic archaeas in granules
during sedimentation or flotation [80]. A recent paper by
Yamada [102], mentioned earlier when discussing the use of
16S rDNA-sequencing for the identification of microorgan-
isms, deserves highlighting again, as it dealt with filamentous
bacteria that cause sludge bulking in UASB reactors, and the
involvement of members of the Chloroflexi phylum in this
phenomenon. The authors reported an in situ hybridization-
based survey of the distribution of these microorganisms in
thermophilic, as well as mesophilic UASB sludge granules.
Calli et al. [12] operated UASB reactors for the treatment of
ammonia rich landfill leachates and were able to corroborate
the impact of high ammonia loads on the microbial community
of the sludge granules with FISH. The same authors [13] in a
subsequent work also with UASB reactors operated under high
ammonia concentrations found that the differences in the
efficiencies of reactors were correlated with the proportion of
resistant and sensitive microorganisms dominated in the
reactors.
The most recent approaches combine complementary
techniques. Diaz et al. [25] have studied the microbial
composition and structure of different types of granule in a
UASB reactor that treated wastewater from a brewery. The
authors used FISH, DGGE, cloning, and electron microscopy to
gain insight into the structure, function and physical appearance
of methanogenic granules. The use of multiple techniques was
necessary to elucidate the structure-function relationship of the
different granules. Roest et al. [76] studied in depth the
microbial community of granules from a reactor treating paper
mill wastewater with a similar approach.
In situ hybridization has been also used as a molecular tool
to describe microbial communities in other anaerobic waste-
water treatment systems besides UASB reactors. A few
noteworthy publications include Plumb’s analysis of the
microbial composition of the biomass inside an anaerobic
baffled reactor [71]; various studies of membrane reactor
systems [53,78]; the identification and characterization of
anammox microorganisms in different systems by Jetten et al.
[43]; the observation of anaerobic biofilm development [4,30].
This list is intended only as an overview and should not be
considered as exhaustive.
We will bring this section to a close with two final
comments. First, we should remember that FISH is exclusively
a taxonomic method that is most commonly used to examine
whether members of a specific phylogenetic affiliation are
present in a sample. It cannot, however, reveal information
about the function or metabolic features of the microorganisms,
although these characteristics can sometimes be deduced from
comparison of the microorganisms detected with phylogeneti-
cally-related bacteria. With a view to overcoming this
limitation, Wagner et al. [49] have presented an approach that
combined FISH with microautoradiography, using substrates
labeled with 3H or 14C. This combination allowed them to
determine metabolic activities and simultaneously identify the
microorganisms involved. This strategy can be useful for
analyzing the structure and function of microbial communities,
though it makes the technique much more complex.
The second closing remark refers to the quantitative aspects
of FISH. The possibility of quantitative results represents a big
advantage over the other molecular techniques, but this only
applies to homogeneous and evenly distributed samples. The
bacterial count per region of the microscopic grid should lie
between 30 and 150. Between 10 and 20 regions should be
counted to ensure statistically significant cell counts. Non-ideal
samples and fluorescent background (a common phenomenon
with environmental and sludge samples) can make cell
counting by fluorescence microscopy a tedious and time-
consuming process that can be influenced by the judgment of
the operator and his or her experience. Truth in this case is in the
eye of the beholder, and a standardized and automatic
procedure would be preferred. This is possible with a laser
confocal microscope or an epifluorescence microscope coupled
to a digital camera and a computer workstation to analyze the
pictures. The system usually requires expensive software,
which makes it less accessible, although free simple software
can be found on the Internet (ImageJ, Image Pro Plus).
Confocal microscopy on its own is of course already an
extraordinarily powerful tool for examining the three-dimen-
sional structure and texture of the microbial aggregates
(granules, biofilms) that develop in wastewater treatment
systems.
In short, the main advantages and disadvantages of FISH can
be summarized as follows:
 Advantages:
 easy and fast if required probes are available (a wide array
has already been described);
 allows direct visualization of non-cultured microorgan-
isms;
 generally quantitative;
 quantification of specific microbial groups is also possible,
in contrast to conventional techniques (most probable
number, plate counts) or other molecular techniques;
 differential/preferential detection of active microorgan-
isms;
 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
 apt for routine use, highly trained and specialized personnel
is not necessary, only a basic knowledge of microscopy and
laboratory experience are required.
 Disadvantages:
 a priori knowledge of the ecosystem under study and the
microorganisms most likely to be detected is necessary
(combined use with other techniques may be necessary);
 if a particular microorganism has to be detected and
quantified, its rRNA sequence must be known (if the
corresponding probe has not yet been published);
 the design of a specific and unambiguously restrictive
probe for a certain group of microorganisms is not always
possible, especially if metabolic criteria are applied (e.g.
nitrifying bacteria, halo-respiring bacteria);
 the design and optimization of hybridization conditions for
a new probe is a difficult process that requires experience
and dedication, and the results may not always be
satisfactory;
 quantification can be tedious and subjective (manual
counting) or complex (image analysis);
 structural analysis of aggregates (granular sludge, biofilms)
requires a confocal microscope and an image analysis
environment (expensive, trained personnel necessary).
However, as FISH is a widely applied technique, users have
come up with new ideas and upgrades to overcome some of its
pitfalls. Wagner et al. [98] published a comprehensive survey
on the subject, outlining the FISH technique, its advantages and
drawbacks, as well as the recent developments that add to the
usefulness of the method. Some of the improvements discussed
are especially useful for application in anaerobic digestion, as
researchers in this area frequently have to deal with complex
and dense biofilms, in which in situ hybridization is difficult. To
allow for quantification of the biomass in this sample type,
Daims et al. [20] proposed an extended FISH protocol, which
they called ‘‘Spike FISH’’. Known amounts of E. coli cells are
added to the samples to serve as an internal standard. After in
situ hybridization, microscopical observation, coupled with
automatic image analysis, allows the cells in the sample to be
quantified. Hybridized cells are calculated as the relative
volume of the total biovolume and the spiked bacteria then
allow hybridized cell numbers to be quantified because
biovolume is related to the number of individual cells.
However, for this technique to work, two requirements have
to be met. First, a confocal laser scanning microscope and an
image analysis software has to be available and second, the
target cells of the sample should be of similar morphology and
cell size to those of E. coli. This is only one example of the
many improvements considered in the mentioned review. Other
problems and possible solutions are discussed, such as new
strategies for probe design, FISH variations when working with
cells immune to conventional fixation/preparation protocols,
improvements in analysis of physiological characteristics via
methods that combine FISH with other techniques, such as
microautoradiography, and a whole new FISH extension that
even makes it possible to detect cells with low ribosome
numbers, called CARD-FISH.
127
This latest hybridization technique deserves further men-
tion, as it increases the sensitivity of FISH dramatically and
provides further advances which will be welcomed by
researchers in wastewater treatment. CARD-FISH (catalyzed
reporter deposition-FISH) was first described by Bobrow et al.
[8] and introduced in microbial ecology by Pernthaler et al.
[68]. The technique has proved a potent alternative to
conventional FISH as it includes an enzymatic amplification
step of the fluorescence signal. The gene-probes are not directly
labeled with fluorochromes but covalently bound to horseradish
peroxidase (HRP). In an amplification step, this enzyme
catalyzes the formation of fluorochrome-labeled tyramide
radicals. These highly reactive molecules will bind to tyrosine-
rich regions of the ribosome and other proteins in the vicinity.
With this additional catalytic step, a cell containing only a
single ribosome can theoretically be detected as the fluores-
cence signal is amplified many times by the massive deposition
of labeled tyramide (Fig. 4). CARD-FISH is therefore ideal
when conventional FISH techniques yield low signal and/or the
background signal swamps emission from the hybridized cells.
This is often the case with aquatic sediments, soils, and a
number of biological sludges. In such cases, CARD-FISH may
facilitate their analysis. To the best of our knowledge, CARD-
FISH has so far only been used in the field of anaerobic
wastewater treatment by Sanz’s group [29]. The authors used
CARD-FISH to demonstrate that Thiobacillus denitrificans was
responsible for autotrophic denitrification in UASB reactors.
The technique should soon become widespread for analysis of
samples with high autofluorescent background.
But even this new technique is being improved. The same
authors who introduced CARD-FISH to molecular ecology
have developed another variation in which rRNA, as well as
messenger-RNA (mRNA) is hybridized simultaneously and
works as an extension to FISH and CARD-FISH protocols [69].
The presence of mRNA in living cells is indicative of
metabolism and thus activity. This information is extremely
valuable when studying wastewater treatment processes,
because it allows differential detection of bacteria that are
actively participating in the treatment processes, excluding
those that are only present in their dormant form. This
Fig. 4. Schematic outline of the CARD-FISH procedure, as described in the
text.
128 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
possibility has not been available until now. So far, there are no
publications that describe the use of this new method for
dealing with anaerobic digestion, but it should not be long
before the technique becomes widely accepted and takes its
place in the molecular ecologist’s toolbox.
2.4. Alternatives and new methods
As an alternative to DGGE as a community profiling method,
terminal restriction fragment length polymorphism (tRFLP) can
be applied when treating complex, species-rich samples. This
technique is also PCR based but the further procedure differs
from PCR/DGGE or PCR/cloning. In tRFLP the 16S gene is
amplified with universal primers, one of them being fluorescently
labelled, and the product is digested with frequently cutting
restriction enzymes. Given that each species in the sample has
differences in the amplified gene sequence, the terminal
restriction fragment will differ in size, so can be separated
electrophoretically. Furthermore, it is possible to sequence and
identify the generated fragments via comparison with a sequence
database. The strength of the fluorescent signal yields additional
information on the abundance of the different species, though this
feature should be regarded with caution, just like the band
intensity in patterns of a DGGE gel. This method is fairly
common in the analysis of samples from wastewater treatment
installations and was for example successfully applied by Scully
et al. [84] in a study of the community of an expanded granular
sludge bed reactors treating wastewater. The authors demon-
strated in their paper the suitability of tRFLP as an alternative
high throughput profiling method.
While most of the molecular methods for the identification
of microorganisms are based on the 16S ribosomal RNA gene,
other DNA regions such as those carrying information for
specific and interesting genes or even specific intergenic
regions can be of interest in this type of research. The spacer
region between the 23S and 16S rRNA genes for example is
used as the PCR target in Intergenic Spacer Region Analysis
(RISA). Differences in this sequence’s length and base
composition allow a rapid monitoring method, as well as a
tool to distinguish microorganisms down to subspecies level.
Nonetheless this technique, though available for some years
now, has not been used so far in the profiling of anaerobic
microbial communities in bioreactors.
If a process to be monitored involves a characteristic
biochemical reaction, for example the biological degradation of
a certain organic compound, the gene coding for the
corresponding enzyme can be amplified by PCR and the
products run on a DGGE gel or cloned into a vector and
sequenced. This specific PCR step, involving special primers
which embrace the gene of interest, excludes a priori the
microorganisms which are not of importance for the study, and
allows even to differentiate between different strains of
degrading bacteria in the reactor. An example for the use of
this approach combined with the aforementioned RISA
technique is the publication by Canstein et al. [14]. These
authors monitored the community of a mercury waste treating
bioreactor, they used RISA to describe the system biodiversity,
as well as a specific PCR amplification of the gene coding for a
mercury reducing enzyme in order to assess variations in that
enzyme among the studied bacteria. Similar cases are the work
of Tang et al. [90], which has already been addressed in the
DGGE section of this article, who assessed sulfate reducing
activity in a anaerobic waste digester by amplification of the
dissimilatory sulfite reductase (sfr) enzyme, or the recent paper
by Geets et al. [32], which describes a method that allows
subsequent DGGE profiling of amplified gene fragments
coding for the mentioned enzyme. By exclusively targeting the
population carrying specific enzymes for the breakdown of
substances, the method gives direct information on the presence
and diversity of the biodegradative part of the microbial
population. If certain microbiological groups have to be
analysed in detail which have metabolic features in common,
this strategy helps to focus on what is important for the
research. However, in order to evaluate the biodiversity of an
anaerobic digestion community the 16S approach is more
advisable. Other genes such as recA (coding for a recombinase,
a DNA repairing protein) and groEL (coding for a chaperonine)
also permit the analysis of phylogenetic relations between
different bacteria.
Other methods include phospholipid ester linked fatty acid
(PLFA) analysis, which reveals the presence of certain groups
of bacteria by detection of their characteristic fatty acid
molecules, thus permitting identification without relying on
genes and their amplification [77] and DNA-microarrays for the
simultaneous hybridization with large gene specific probes, in
conjunction with the parallel detection of phylogenetic or
metabolic features [23]. The latter method allows hybridization
with DNA probes targeting enzyme coding DNA regions,
which can give valuable information on degradative character-
istics of the analysed bacterial communities.
Before continuing with this review on the more established
molecular methods we would like to state that the above
mentioned procedures are surely viable options, but their
current state of development or limited availability of
references in the field of wastewater treatment and bioreactors
led us to exclude them from a more thorough analysis. In order
to keep the introduction to the molecular toolbox practicable
our intention is to avoid confusion by simply giving large
amounts of information, not needed for setting up a basic
laboratory devoted to microbial community profiling. The main
features of alternative and complementary methods are
summarised in Table 1.
3. Concluding remarks
Cloning and sequencing of bacterial rRNA genes retrieved
from natural environments have revealed a vast genetic
diversity that was inconceivable when the only tools available
to assess microbial biodiversity were isolation in pure culture
and microscopical observation. The use of molecular probes
has revealed the wholly unexpected existence of active
microorganisms in certain habitats that were thought to be
too hostile for bacteria. Despite some shortcomings, the
techniques discussed in this review are currently the most
 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133 129

Table 1
Brief description of further molecular methods for microbial community analysis
Method Outline Advantages Disadvantages
tRFLP 16S based monitoring method, Relatively simple procedures Heterogenous size of fragments makes
relying on differences in restriction phylogenetic analysis less confident
behaviour (polymorphism)
Making use of fluorescence signal No real advantages in comparison to
strength, abundance of species DGGE, the more established method
can be estimated (roughly though)
RISA Phylogeny with intergenic spacer High sensitivity, down Database for comparative analysis small
region between 23S and 16S to sub-species level in comparison to 16S sequences
rDNA sequences
PCR with genes Microorganisms containing enzymes Direct detection of the presence Global profiling of microbial
coding for enzymes involved in the biodegradation of degradative microorganisms community missing
process are detected (exclusion of bacteria of no interest)
Subtyping on strain level is possible Microorganisms contributing to the
functioning of the process which do
not bear the targeted enzyme gene
are not detected
PLFA Profiling of microorganisms by Molecular characterisation of Not a good choice as a standard
characteristic fatty acid content microorganisms not relying alone method
on genes !
Complementary information Chromatography–mass spectrometry
to 16S based assays system needed for identification
DNA microarray Multisample hybridization method High sample throughput Expensive equipment
Parallel analysis of Difficult handling
different parameters

powerful instruments available. These tools have healthy
prospects for future application in research into the microbial
diversity of biological reactors. Table 2 summarizes these
techniques and their possible fields of application within the
area of wastewater treatment.
The genetic fingerprinting techniques produce characteristic
band patterns that reflect the complexity of a microbial
community and the changes in its composition over time.
However, techniques based on nucleic acid extraction and
amplification by PCR are not quantitative. As Lapara et al. [48]

Table 2
Comparison of the main molecular biology techniques and their application in bioreactors for wastewater treatment
Gene library DGGE FISH
Required skill level in High Medium Low
molecular biology
Specific facilities PCR PCR, DGGE Fluorescence microscope,
confocal laser scanning
microscope (not mandatory)
Requires sequencing Yes Yes (conditional, No
band pattern could
be sufficient)
Quantitative No No Yes
Time demands High Medium (low if Low
sequences are not required)
Phylogenetic precision High Medium Low/medium
Structural analysis No No Yes (requires confocal laser
(3D-architecture) microscopy)
Sample throughput Low High High
Observation of spatial- No Yes Yes (only for a few key probes)
temporal changes
Detection of a particular No No Yes
taxonomic group or species
Apt for monitoring bioreactors No No/yes (only if band pattern Yes, easy to standardize process
shows obvious differences) for indicator species
Applicability in anaerobic digestion Low Medium High
PCR: polymerase chain reaction; DGGE: denaturant gradient gel electrophoresis; FISH: fluorescent in situ hybridization. Estimates are relative to one another and
have been kept as general as possible, avoiding exceptions to the rule.
130 J.L. Sanz, T. Köchling / Process Biochemistry 42 (2007) 119–133
point out, both PCR-derived methods (DGGE and cloning) can
result in the detection of similar diversity patterns of bacterial
populations in an ecosystem, but they do not give a valid
estimate of the relative species distribution. This suggests one
or both methods are not suitable for determining total
biodiversity, a parameter that depends on both the number of
different species and their relative abundance and distribution.
In situ hybridization with fluorescently labeled probes can
overcome the problems associated with PCR-based methods:
localization of the microorganisms to be examined in situ
(topology) and its quantification.
The techniques here described are not mutually exclusive
and should be considered complementary. In an ideal situation,
the methods required for a given task are used simultaneously.
Thus, PCR-based techniques and sequencing allow the
construction of a gene rDNA library (identification but not
quantification) and the design of new specific probes for in situ
hybridization. These, in combination with general probes or
specific ones already described, can be used to break down the
biodiversity of the system into absolute cell numbers and
relative proportions, as well as to reveal structural character-
istics and possible functional relations between microorgan-
isms. This brings us back to the idea of the full-cycle rRNA
approach mentioned earlier [3,100].
Returning to the question posed at the beginning: is exact
knowledge of the identity of a microorganisms in a bioreactor
or wastewater treatment stage necessary for the engineer? The
answer depends on the particular research objective. It might
not be essential for the functioning of the process to know that
Acinetobacter is indeed the microorganism responsible for
phosphorous accumulation and elimination. In contrast, the
operator would very much like to know whether an excessive
growth of Microthrix, Nostocoida or Sphaerotilus is occurring.
It is feasible to think that the ‘‘colorful’’ visualization (by FISH)
of the specific bacteria responsible for bulking can replace
routine visual inspection of activated sludge. Once optimized,
this method is easy and will be more sensitive and quantitative.
In addition, the discovery of the anammox bacteria and their
detection in denitrifying reactors certainly will help in the
development of more efficient reactors, facilitate the completion
of nitrogen balances and help researchers to understand the
interrelation between nitrogen and sulfur cycles resulting in the
simultaneous elimination of both elements [28,88,91]. The
recent advances in determining the composition of the microbial
community and structure in granular sludge will deepen our
understanding of the underlying mechanisms, such as granule
formation and metabolic functionality. This extended knowledge
in turn has practical benefits, allowing the design of more
efficient systems and bioaugmentation strategies, optimized for
the characteristics of the different types of wastewater [34,95].
A final illustrative example of the potential of these new
tools is the work of Bouchez et al. [9]. They added a pure
culture of Microvirgula aerodenitrificans to two SBR reactors
to increase the denitrification rate. Contrary to what they
expected, the process became less efficient. FISH was able to
determine the cause of this change in the behavior of the
reactor: the scientists observed the complete disappearance of
the bacterium within 2 days of augmentation, whereas the
digestive vacuoles of the protozoa present showed a strong
hybridization signal with the M. aerodenitrificans-specific
probe. This led to the design of a new form of bioaugmentation
in which the bacteria were embedded in alginate beads to
protect them from predation. M. aerodenitrificans was
incorporated into the sludge flocs and the denitrifying capacity
of the reactor was improved.
As a concluding remark, we would like to quote literally a
paragraph of Peter Wilderer et al. at the T.U. Munich: ‘‘Does a
colorful photograph obtained with a confocal laser scanning
microscope and analyzed by digital image analysis provide any
information of practical value? Is it important to the engineer to
know the phylogenetic position of functionally important
bacterial species in wastewater treatment plants’’ [100]. The
engineers are right—the names of the bacteria are irrelevant
unless they can be associated with the properties or the
functionality of the reactor. It becomes evident that the
combined effort of microbiologists (fundamental research) and
chemists and engineers (applied sciences) is necessary to
ensure that the contributions from microbiology and molecular
biology can be incorporated into the design, operation and
control of bioreactors and wastewater treatment systems. This
is the biggest challenge facing environmental microbiology.
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