Culturing The Uncultured Microbial Majority in Activated Sludge: A Critical Review

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Culturing the uncultured microbial majority in activated sludge: A critical

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Culturing the uncultured microbial majority in

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Yulin Zhang & Tong Zhang
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CRITICAL REVIEWS IN ENVIRONMENTAL SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/10643389.2022.2077063
INVITED REVIEW
Culturing the uncultured microbial majority in activated

sludge: A critical review
Yulin Zhang and Tong Zhang
Environmental Microbiome Engineering and Biotechnology Lab, Department of Civil Engineering, The
University of Hong Kong, Hong Kong, China
ABSTRACT
Activated sludge is a widely applied waste-
water treatment process that mainly uses
suspended microbial flocs to remove pol-
lutants in wastewater. With the characteris-
tics of high biomass content and high
microbial diversity, activated sludge plays
an important role in pollutant removal and
contains various functional microorganisms
as a valuable pool of various useful micro-
bial resources. However, the majority of
microorganisms in activated sludge have not been isolated, which substantially limits the improve-
ment of treatment efficiency and the innovation of process technology in wastewater engineering.
As the basic biological methodology which can extremely expand the downstream studies for
microorganisms, the cultivation of new species in activated sludge is urgently needed to fill the
gaps between the cultured and uncultured microbial communities. The growing emphasis on culti-
vation in recent years has spawned the creation of many innovative and high-throughput cultiva-
tion techniques. In this review, we summarized the microorganism “wanted list” in activated
sludge, reviewed the potential cultivation methods that could extend our understanding of acti-
vated sludge microbiota, and discussed the significance and perspectives for activated sludge
microbiota cultivation.
KEYWORDS 16S rRNA gene sequencing; activated sludge; cultivation; culturomics; microbial dark matters; wastewater
treatment plants
HANDLING EDITORS Eakalak Khan and Lena Ma
1. Introduction
Activated sludge is the most popular biological wastewater treatment application in the world and
has been used for more than a century to treat a large variety of wastewater to protect the envir-
onment and human health (Beychok, 1967; Peces et al., 2022). With the complex sources of sew-
age (including rainfall, residential areas discharge, hospital wastewater, industrial wastewater, and
farm wastewater), activated sludge contains abundant diversity of microorganisms. What’s more,
as a classical wastewater treatment method, activated sludge contains plentiful functional microor-
ganisms (Ioannou-Ttofa et al., 2017) that can remove pollutants in wastewater and transform
them into harmless substances. These functional microorganisms play key roles in the ecological
CONTACT Tong Zhang zhangt@hku.hk Environmental Microbiome Engineering and Biotechnology Lab, Department of
Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Supplemental data for this article is available online at https://doi.org/10.1080/10643389.2022.2077063.
ß 2022 Taylor & Francis Group, LLC
2 Y. ZHANG AND T. ZHANG
biogeochemical cycle and are thought to be important engines for elements energy flow on the
Earth. Therefore, it is critical to decipher the ecology of the activated sludge microbial community
that might bring new insights for future optimization of the biological wastewater treat-
ment process.
The explosion of sequencing technology like 16S ribosomal RNA (rRNA) gene sequencing to
determine phylogenetic relationships (Ju et al., 2014; Saunders et al., 2016; Zhang et al., 2020)
and more comprehensive metagenomics (Wang et al., 2021b) to obtain detailed genomes has rad-
ically altered our perceptions of microbial diversity in activated sludge. In 2012, researchers inves-
tigated the bacterial communities of activated sludge from 14 wastewater treatment plants
(WWTPs) of Asia and North America and defined over 700 genera and thousands of operational
taxonomic units (OTUs) (Zhang et al., 2012). In 2014, the five-year temporal dynamics of the
bacterial communities in a Hong Kong WWTP has been investigated and showed the stability of
the activated sludge system with no significant seasonal succession (Ju et al., 2014). In 2019, a
global water microbiome consortium conducted a worldwide survey of activated sludge covering
269 WWTPs in 23 countries on 6 continents. It was estimated that activated sludge contained
4–6 1023 bacterial cells and has 1.1 ± 0.07 109 bacterial species at the global level (Wu et al.,
2019). The bacteria diversity was three orders of magnitude higher than the human gut and only
one order of magnitude lower than the global ocean microbiome (Wu et al., 2019).
The culture-independent sequencing technologies indeed expand the tree of life due to the
emergence of high-throughput sequencing (HTS) (Reuter et al., 2015) with the platforms like
Illumina, Pacific Biosciences (PacBio), and Nanopore. However, the known microbial genomes
mainly come from a small number of well-studied cultured lineages (such as human microbiome)
(Zhang et al., 2020) and the majority of microorganisms on Earth are still uncharacterized
(Castelle & Banfield, 2018). These unknown microbes are colloquially called “microbial dark
matters” (MDMs), which play necessary ecological roles but do not have representative isolates
yet due to various reasons, such as the low abundance, the lack of unknown nutrients
(Zamkovaya et al., 2021), failure in competition with other microbes (Song et al., 2020), and
undesired cultivation conditions with toxins accumulated (Tanaka et al., 2014). Having abundant
microbial communities, activated sludge is also the residence of MDMs. Cultivation is a basic but
indispensable method that can get both the phenotypes and genotypes of microorganisms
although traditional cultivation methods are painstaking and laborious. In recent years, research-
ers show growing interest in microbial cultivation and related research has a gradual rise over the
past ten years (Figure S1). At the same time, the increased demand for effective cultivation has
enormously promoted the application of many innovative cultivation strategies (Sood et al., 2021)
like high-throughput droplet cultivation (Zhu et al., 2022) and stable isotope labeling for target
cultivation (Lee et al., 2021), and successfully isolated many novel microorganisms. It is indeed
the time to apply all available cultivation methods to overcome the “1% culturability paradigm”
(Martiny, 2019, 2020; Steen et al., 2019) by getting pure cultures of “MDMs” from activated
sludge, and then provide new insights for their ecological roles, such as syntrophy or competition
with the functional groups in activated sludge.
As an important topic, there already have many reviews on cultivation with the following cate-
gories basically: strategies to improve the success rate of cultivation like waking dormant
microbes (Mu et al., 2020) and co-culture (Marmann et al., 2014), innovations in cultivation tech-
niques (Lewis et al., 2020) and microbial identifications (Singhal et al., 2015), and the cultivation
applications such as human (Lagier et al., 2018) and marine (Salam et al., 2020). In this review,
we present a framework for future studies on the cultivation of activated sludge microbiota by (1)
summarizing the “wanted list” of microorganisms in activated sludge that have priority to be cul-
tured, (2) reviewing the potential cultivation and detection methods that can extend our under-
standing of activated sludge microbiota, and (3) discussing the significance and future
perspectives of the large-scale cultivation of activated sludge microbiota.
CRITICAL REVIEWS IN ENVIRONMENTAL SCIENCE AND TECHNOLOGY 3
2. The not-yet-cultured majority and the “wanted list” of activated sludge

Over the past decades, the tree of life has been vastly expanded with several archaeal and bacterial
groups of high taxonomic rank to thoroughly facilitate the interpretation of microorganisms in
ecosystems like the human gut. For activated sludge, the total number of bacterial and archaeal
species is estimated to be around 1.1 ± 0.07 109 at the worldwide level based on 16S rRNA
sequence data of V4 region while only 96,148 OTUs are defined for bacterial and archaeal phylo-
types with a 97% similarity threshold (Wu et al., 2019). Up to now, there is no public reference
that can provide the number of microorganisms cultivation ratio in activated sludge, while a
study indicates that averagely 62% of cells and 81% of taxa are not genome-sequenced in different
biomes on Earth (Zhang et al., 2020). The status quo emphasizes the significance of cultivation
and more efforts should be paid to dig out these unknown bacteria and archaea.
Different from the fast-developed molecular techniques, cultivation and isolation remain to be
a time-consuming and laborious task with large uncertainties. Thus, the microorganisms in the
“wanted list” that represents the key targets in the specific systems have the priority to be cul-
tured first. The “wanted list” of activated sludge (Table 1) should be developed from four aspects:
universality, abundance, function, and novelty. According to the universality (occur in >80%
samples) and abundance (relative abundance >0.1%) index, a “most wanted” list of activated
sludge at the global level which contained the core bacterial community with 28 OTUs was for
the first time defined (Wu et al., 2019). These OTUs show high proportions of novel species with
only half of them can be classified to genus level and take important roles in the key functions of
activated sludge, such as nitrification (OTU6_g_Nitrospira) and phosphorus removal
(OTU37_g_Ca. Accumulimonas and OTU25_g_Ca. Accumulibacter). This list indicates the direc-
tions of further research and contributes to the successful isolation of OTU 16 (a new genus of
phylum Proteobacteria that might be a potential phosphorus accumulating (PAO) bacterium)
from a China WWTP in 2020 (Song et al., 2020). With a similar method (>0.1% relative abun-
dance in 50% of all samples), another research summarized the general core taxa of global acti-
vated sludge which have a high fraction of poorly characterized species and once again prove the
novelty and insufficient research of microorganisms in activated sludge (Dueholm et al., 2022).
Except for many known bacteria associated with nitrification (Nitrosomonas and Nitrospira) and
polyphosphate accumulation (Tetrasphaera and Candidatus Accumulibacter), this list also con-
tains glycogen accumulation species (Candidatus Competibacter) and the known filamentous spe-
cies (Candidatus Villigracilis and Leptothrix). In fact, culturing abundant members that are not-
yet-cultured is far from enough since the rare microbial taxa also take significant roles within
their communities such as the conditionally rare or abundant taxa (CRAT), which typically pre-
sent in communities with low abundance but occasionally become prevalent and generally have
profound importance within the communities (Shade et al., 2014). In fact, more microbial analyt-
ical tools that could define the priorities and evaluate the potential ecological roles of microorgan-
isms in the microbial communities should be applied to make the “wanted list” more
comprehensive such as network and correlation analysis (Zamkovaya et al., 2021).
Except for the specific species in the “wanted list,” the resolution improvements of phyla that
are widely presented in activated sludge but have few representatives like the Candidate Phyla
Radiation (CPR) group and the PVC superphylum (named after its three important members,
Planctomycetota, Verrucomicrobiota, and Chlamydiota) (Wagner & Horn, 2006). Patescibacteria
phylum is key member of the CPR group and is abundant in activated sludge with reduced meta-
bolic capacities and ultra-small size (Meheust et al., 2019; Wang et al., 2021b). Belonging to the
PVC superphylum (Wagner & Horn, 2006), Planctomycetota phylum includes many classical
microorganisms with unique metabolic properties and important functions, such as anammox
(Kartal et al., 2010), an interesting bacteria that could use nitrite and ammonium ions to directly
form diatomic nitrogen and water but still no reported representative isolates (Wiegand
et al., 2020).
Table 1. Key targets for microbial cultivation in activated sludge.

Reasons they are of interest for
Targets Phylum cultivation Reference
Global core bacteria in Assorted 28 OTUs distributed in different (Wu et al., 2019)
activated sludge phyla (Betaproteobacteria,
Gammaproteobacteria, and
Bacteroidetes). The majority of
them were abundant in WWTPs
and had close relations with
WWTPs performance. In 2020,
OTU 16 (potential PAO) was
isolated from WWTPs.
Core taxa in activated sludge Assorted The general core taxa contained (Dueholm, 2022)
at global level many known bacteria associated
with key functions such as
nitrification (Nitrosomonas and
Nitrospira), polyphosphate
accumulation (Tetrasphaera, Ca.
Accumulibacter), and glycogen
accumulation (Ca.
Competibacter).
The PVC superphylum The PVC superphylum Planctomycetota contains many (Arrigo, 2005; Wagner &
interesting microorganisms with Horn, 2006)
specific metabolic properties,
such as anammox, a classical
bacteria that could convert
nitrite and ammonium ions
directly into diatomic nitrogen
and water but still no
representative isolates.
The CRP group The CRP group Patescibacteria phylum belongs to (McLean et al., 2020;
the CRP group and widely exists Meheust et al., 2019).
in activated sludge with high
abundance. With reduced
metabolic capacities and ultra-
small size, Patescibacteria
phylum might have various
host-associated sources.
AOA Thaumarchaeota One of the key contributors to (Ashida et al., 2010)
ammonia oxidation in
activated sludge.
DPANN archaea DPANN archaea With small genomes and cell sizes, (Castelle & Banfield, 2018)
DPANN archaea have limited
metabolic repertoires and lack
many core biosynthetic
pathways. The majority of them
are likely to be symbionts or
parasites of other
microorganisms.
At the same time, the minority in the community should not be ignored as they also have
important ecological niches in ecosystems like archaea. The recent achievements in novel archaeal
cultivation, i.e. Asgard, the closest archaeal relative of eukaryotes cultured to date (Liu et al.,
2021), raise increasing interest of researchers in the exploration of archaeal cultivation. It also
hints to us that similar attempts should be paid on activated sludge archaea such as the nitrifica-
tion drivers ammonia-oxidizing archaea (AOA) (K€ onneke et al., 2005; Wang et al., 2021a) and
the superphylum DPANN archaea (Liu et al., 2021; Rinke et al., 2013), the major archaeal group
with small genome size and limited metabolic capabilities.
Except for bacteria and archaea, other important large groups in activated sludge are still wait-
ing to be investigated although they are not the focuses of this review, i.e. viruses, fungi, and
microalgae. AS harbors enormous number of viruses (mainly bacteriophage, a type of virus that
infects bacteria) that are poorly explored but have key roles to play in the shape of AS commu-
nity due to their special physiological behaviors to impact key functional microorganisms (Chen
et al., 2021; Otawa et al., 2007). Fungi are also the important components of activated sludge
which exhibit great diversity and have amounts of biomass comparable to bacteria in activated
sludge (Li et al., 2022; Wei et al., 2018). Meanwhile, the cultivation of microalgae in activated
sludge has emerged since microalgae can assist the functional microorganisms to remove pollu-
tants (Anbalagan et al., 2016; Barros et al., 2015).
3. Microbial cultivation
3.1. The history of cultivation
Microorganisms are the most numerous and diverse life forms on the Earth with an estimated
number of 4 1030 –6 1030 and microbial cultivation is the basis of biological research on
these microorganisms (Locey & Lennon, 2016). Since Robert Koch developed the solid
medium in 1881 and Petri produced the prototype of modern Petri dishes in 1887, scientists
discovered and isolated various microorganisms (such as Anthrax Bacillus (Blevins & Bronze,
2010)), and thus brought the first Golden time (1857–1914) of microbiology (Figure 1). In
the following decades, microbiology entered its second Golden time (1950–1970) mainly
focusing on molecular biology not cultivation. Until 1990, a group of microbiologists began
to use selective or diagnostic medium and isolated lots of bacteria, especially pathogens. And
in 2007, an environmental microbiologist used the diffusion chamber to increase the diversity
of recovered isolates and announced the rebirth of cultivation (Bollmann et al., 2007). In
2012, scientists reported the successful cultivation of various bacteria in the human gut and
for the first time proposed the definition of culturomics (a high-throughput cultivation
method) which opened a new era of cultivation research (Lagier et al., 2012). As researchers
pay more attention to large-scale cultivation, more and more related studies were reported
covering the microbiota of plant roots (Bai et al., 2015) and human (Lagier et al., 2016; Saheb
Kashaf et al., 2022; Zou et al., 2019). We are now in the third Golden time of microbiology
(aiming for microbiome exploration) which has advanced techniques to support high-
throughput cultivation but there are no reported studies of large-scale cultivation in activated
sludge ecosystem up to now.
Figure 1. The history of cultivation.

3.2. Cultivation and metagenomics

Cultivation and metagenomics are completely two different methods to analyze MDMs in micro-
bial ecosystems (Figure S2). Cultivation could get pure cultures to carry out the physio-bio-
chemical experiment to validate metabolism and allow the retrieval of complete genomes even
they are minority populations in the original community. At the same time, cultivation is
laborious and time-consuming, and could not provide further function analysis by bioinfor-
matic ways directly. Proposed almost two decades ago, metagenomics is a key technology to
explore the genomic potential from microbes especially the not-yet-cultured ones (He et al.,
2020; Ju & Zhang, 2015a; Walker et al., 2017). As a culture-independent method, metagenom-
ics can analyze all microorganisms in an ecosystem by an integrative and time-saving work-
flow that allows the following functional analysis and has the ability to handle a large number
of samples at one time. However, the results of metagenomics might be affected by the het-
erogeneity of protocols utilized like different DNA extraction methods and bioinformatic
pipelines, and the sequencing depth limits its exploration of microorganisms for the minority
populations. More importantly, though metagenomics dramatically improves people’s cogni-
tion about not-yet-cultured microbes, it still cannot get the pure cultures for follow-up bio-
chemical experiments to further investigate the species. In summary, cultivation and
metagenomics should be integrated to provide insights into communities especially the
“MDMs” in AS. On one hand, cultivation can obtain pure cultures and expand classifications
to reduce unassigned units in metagenomics. On the other hand, metagenomics offers a cre-
ative and powerful way to explore genome information of unknown microorganisms in com-
munities to provide potential metabolism information for cultivation (Ju & Zhang, 2015b). As
a result of the rapid development of metagenomics, it has a great necessity to vigorously
develop cultivation.
3.3. Uncultured reasons of microorganisms

Although microbiologists have different opinions on the specific value of the “1% culturability
paradigm” (Martiny, 2019, 2020; Stewart, 2012), it is undeniable that high proportions of micro-
organisms across most biomes remain uncultured (Steen et al., 2019). The reasons why microor-
ganisms cannot grow as a pure culture are various and complex. First of all, some microbes grow
slowly or are in the dormant period called “viable but nonculturable (VBNC)” state, and research-
ers could try to isolate these “recalcitrant” strains by the enrichment method through not only
abundance accumulation but also resuscitation (Mu et al., 2018). Secondly, some microorganisms
cannot grow on agar causing the “great plate count anomaly” phenomenon and this situation
could be improved by using agar alternatives like gellan gum (Kawasaki & Kamagata, 2017), xan-
than gum, guar gum, carrageenan, isubgol (Das et al., 2015), absorbent pads (Gordon et al., 1952)
which soaked with liquid medium, and so on. Besides, some specific microorganisms need
unknown nutrients, growth factors, or intermedia products that could not be synthesized in
laboratory currently but could be provided by other collaborators or hosts in the original commu-
nity, and co-culture is the first recommended choice to solve this problem (Traore et al., 2019).
Sometimes, the toxins produced or accumulated in medium also hinder microbial growth such as
reactive oxygen species (Tanaka et al., 2014). For example, a simple modification could greatly
improve the culturability of microorganisms by autoclaving phosphate and agar separately to
mitigate oxidative stress (Kato et al., 2018). What’s more, shaped by their native habitats, many
microorganisms are oligotrophic while the medium used to get the pure culture is often eutrophic
(rich in nutrients), and this phenomenon spawns the usage of oligo or diluted medium and gets
gratifying progress (Song et al., 2020). Finally, microorganisms may fail in the competition with
others in medium, and dilution-to-extinction cultivation might be a potential solution.
3.4. The necessity of enrichment

Once confirm the targets, enrichment is the first step for cultivation with eminent necessity
(Laso-Perez et al., 2018). Generally, enrichment is to use specific growth media and environmen-
tal parameters to make the desired microorganisms grow vigorously over others by taking advan-
tage of specific growth characteristics of different microorganisms. While with the development
of modern instruments and molecular biology techniques, other methods that could boost the
proportions of targeted microorganisms in communities should also be classified as enrichment,
such as cell sorting (the novel nitrite-oxidizing bacteria (NOB) enrichment) (Mueller et al., 2021).
Enrichment has great importance for the subsequent cultivation since it can significantly
increase the success rate of isolation. Firstly, enrichment takes a key role in the transition of the
complicated consortium to simple systems and finally isolates, especially for activated sludge sys-
tems. Compared with human gut microbiota, activated sludge has higher microbial diversity with
the rare circumstance that a single microorganism occupies a large amount of abundance. In this
case, enrichment can simplify activated sludge system and remove many insignificant interfer-
ences by accumulating the abundance of prominent targets to facilitate subsequent isolation pro-
cedures. Secondly, enrichment could activate the VBNC microorganisms from the dormant state
to expand the potential isolate lists (Wei et al., 2018). A cultivation study emphasized that the
preincubation of samples in a blood culture bottle before cultivation will obviously increase the
number of culturable species (Lagier et al., 2016). Besides, the proportions of microorganisms suf-
fering low abundance could be increased via long-term enrichment, which should be the cultiva-
tion goal for most systems as microorganisms with high abundance usually have higher isolates
proportions. What is important but usually ignored is that the naturally enriched consortia could
be used directly as the starting isolation source for the target species. The Earth Microbiome
Project data shows certain microorganisms are already highly enriched in natural samples (Zhang
et al., 2020). For example, several OTUs may account for >50% proportions in communities of
animal gut and corpus indicating the huge potential of animal gut and corpus as the isolation
sources for these not-yet-cultured OTUs (Gilbert et al., 2014). In other words, natural habitats
can be species troves with which we can skip the enrichment process and immediately get the
enriched targets.
However, enrichment process is not always smooth sailing cause the metabolism of microor-
ganisms is complex and sometimes elusive meaning the huge difficulties to make the enrichment
direction exactly perform as expected. Thus enrichment process must be combined with rapid
identification methods to verify the microbial profile in real-time, which will help us to determine
if the community is enriched for the desired microorganisms and allow timely adjustment of
enrichment conditions.
3.5. The current cultivation methods

To maximize productivity, culturing efforts to characterize microbial communities should apply
diverse methods and media. The increasing need for large-scale cultivation emphasizes the
importance of high-throughput methods, and recent innovations in methodology have opened
doors to growing hitherto uncultured species. All currently available cultivation methods can be
divided into four categories with individual characteristics, pros and cons, and applications
(Figure 2 and Table S1), and researchers can select or combine the appropriate methods accord-
ing to specific circumstances for different samples.
3.5.1. Traditional cultivation

Traditional cultivation tries to use various culture conditions for microorganisms to grow with
the keystone to mimic the difference between laboratory and real environment conditions. In this
Figure 2. The summary of current cultivation methods. a Traditional cultivation; b In situ cultivation; c Gene prediction-based
cultivation; d Sorting-based cultivation.
method, the pre-cultivation or enrichment step is necessary as enrichment can promote the
recovery of some VBNC cells (Mu et al., 2018) and thus dig out novel taxa with low abundance
(Zhao et al., 2019). The medium is designed to suppress the majority populations and to promote
fastidious microorganisms at lower abundance, or in summary, to “kill the winner” in the original
community since the dominant species will inhibit the growth of fastidious microorganisms due
to competitive exclusion. Multiple selective pressures can be used to enrich microorganisms,
including but not limited to density, antibiotics, heavy metals, specifical chemical inhibitors, tem-
perature, pH, and cell size (Figure 2a). For instance, Patescibacteria could be enriched with 0.1 or
0.22 mm membrane due to its small size, and b-lactams antibiotics can accumulate
Planctomycetes because of the unique cell-division machinery in this phylum (van Teeseling
et al., 2015). Besides, targeted enrichment can also be achieved by designing the medium for spe-
cific taxa according to their characteristics. As an engineering system, enrichment and isolation of
taxa in activated sludge with particular functions could be achieved by adding special substrate
and nutrients such as the microorganisms involved in nitrogen cycle (ammonia oxidizer, nitrite
oxidizer, and nitrate reducer), phosphorus cycle (glycogen accumulator and polyphosphate accu-
mulator), and sulfur cycle (sulfur oxidizer). Up to now, traditional cultivation is still the most
classic and commonly used method, and most known microorganisms are isolated by it. For
instance, researchers successfully isolated 79 bacterial strains from phylum Planctomycetes that
had previously unknown modes of bacterial cell division: binary fission as well as budding
(Wiegand et al., 2020). Another research got more than 6,000 bacteria isolates and obtained 1,520
high-quality genomes of bacteria in human gut, thus enabling higher-resolution descriptions of
the human gut microbiome from 50% to >70% (Zou et al., 2019).
Based on the experience accumulated from the traditional cultivation, a high-throughput culti-
vation technology that combined with rapid identification by matrix-assisted laser desorption ion-
ization time-of-flight (MALDI-TOF) mass spectrometry (MS) was created, i.e. “culturomics”
(Lagier et al., 2012, 2018). In general, culturomics firstly divides the sample into several parts to
conduct high-throughput enrichment and cultivation with different culture conditions, then
applies MALDI-TOF MS to identify the isolates, and finally adopts 16S rRNA and genome
sequencing to validate and report the new taxa. Culturomics has made advanced progress since
proposed especially after the rapid development and wide application of MALDI-TOF MS
because culturomics cannot stand alone without the fast identification methods. In 2015, a cul-
turomics study summarized 18 powerful media that could cultivate half of the isolated bacteria in
human gut and pointed out the key components of rumen fluid and sheep blood (Lagier et al.,
2015b). And another research introduced different culture strategies of clinical microbiology via
culturomics, especially the specific approaches for those fastidious bacteria such as Mycoplasma
and Spirochetes (Lagier et al., 2015a). In addition to the examples mentioned above, culturomics
also plays a key role in microorganisms resolution of other ecosystems likewise the archaea in
hypersaline environments (Duran-Viseras et al., 2021) and bacteria in plants (Sarhan et al., 2019).
Currently, the major disadvantage of culturomics remains the high workload and inability to test
large samples simultaneously as other methods, for example, metagenomics.
3.5.2. In situ cultivation

In situ cultivation allows microorganisms to grow in their native habitats, and thus gets rid of the
complicated process of medium selection and achieves cultivation without knowing the mechan-
ism. At first, environmental microbiologists created the diffusion chamber with the inoculum
sandwiched between 0.03 lm membranes, and then placed the chamber in the original habitats of
inoculum (Figure 2b) (Kaeberlein et al., 2002). The chamber allowed for the free exchange of
chemicals with the external milieu by diffusion while restricted the movement of cells and was
verified to successfully increase the diversity of recovered isolates from rarely microbial groups
(Bollmann et al., 2007; Kaeberlein et al., 2002; Steinert et al., 2014). Based on the structure of the
diffusion chamber, a device called isolation chip (iChip) consisting of a central plate and two sup-
porting side panels was designed for high-throughput cultivation (Goh et al., 2022; Nichols et al.,
2010). The iChip has multiple matching through-holes that will capture small agar plugs and
house growing microorganisms (Figure 2b). It also promoted the isolation of a previous uncul-
tured bacteria which could synthesize a new antibiotic named teixobactin (Ling et al., 2015).
Recently, a 3D-printed version iChip was built to culture bacteria from a tropical peat swamp in
situ and made the conclusion that iChip was a superior tool for bacteria that might not be able
to grow on media directly in vitro (Goh et al., 2022). Yet, the application of iChip still encounters
many obstacles and is not successful for all members in the communities due to different possible
reasons. First of all, although theoretically appealing, not all the native habitats can be mimicked
due to the complex biological processes. Second, some targets currently fail to be cultured because
of our poorly understood on their lifestyles (such as microbial dormancy). Lastly, even though
the targets are enriched successfully, the following high-throughput cultivation and the mainten-
ance of aseptic environment is still arduous and challenging.
3.5.3. Gene prediction-based cultivation

Proposed with the maturation of sequencing technology, gene prediction-based cultivation firstly
forecasts the potential metabolic characterizations of microorganisms from genotypes and then
provides corresponding nutrients to promote growth. This method can achieve targeted cultiva-
tion of specific microorganisms and got many exciting achievements in recent years by integra-
tion of multiple omics methods (including but not limited to metagenomics, metatranscriptomics,
metaproteomics, and metabolomics). For instance, scientists mapped the landscape of existing
culture media and build an online Known Media Database (KOMODO) (https://komodo.model-
seed.org/) to predict medium composition of the taxa based on their phylogenetic similarity with
the cultured taxa (Oberhardt et al., 2015) (Figure 2c). Another study coupled metagenomics and
cultivation approaches to isolate novel bifidobacteria from animal feces by adding predicted sub-
strate (Lugli et al., 2019). In 2019, an approach to capture pure cultures of specific microorgan-
isms from complex communities through genome-informed antibody engineering was reported
(Figure 2c). Firstly, the genomes of the target microorganisms belonging to novel or important
clades can be reconstructed from metagenomic data and used to predict and identify the highly
expressed membrane proteins with extracellular domains. Then, the target-protein domain anti-
gen is synthesized and inoculated into an appropriate animal to produce antibodies. And the
purified antibodies will be combined with the fluorescent dye and added to samples to label the
targets. Finally, the targets can be sorted through the fluorescence signal provided by antibodies
and cultivated in growth media. As a result, three Saccharibacteria (TM7) from different species-
level lineages and one SR1 bacterium which was previously considered uncultivable were isolated
successfully (Cross et al., 2019). Although integrating genome information with cultivation
appears to be quite promising as lots of sequencing data is generated every day, there are some
challenges as well. Currently, most of the genes (50%) cannot be annotated with known func-
tions, and therefore only limited metabolic information can be provided for cultivation (Sood
et al., 2021). Besides, the targets should have unique physiological characteristics that allow to dis-
tinguish them from the complex community. In conclusion, this method is a superior tool for
targeted isolates while difficult to realize high-throughput cultivation.
3.5.4. Sorting-based cultivation

Over the past decades, cell sorting is emerging as a new technology in microbiological research
and has obtained excellent achievements in microbial cultivation with the representation of
microfluid and flow cytometry (Liu et al., 2017) (Figure 2d). Droplet microfluid may encapsulate
single cells into nanoliter (even picoliter) droplets and provide the high throughput to isolate
novel microbes from diverse communities (Goh et al., 2022; Kaminski et al., 2016), such as a bac-
terium in the “most wanted” list of Human Microbiome Project (HMP) (Ma et al., 2014), new
species that could degrade polycyclic aromatic hydrocarbons (PAH) from soil communities (Jiang
et al., 2016), and the slow-growing bacteria in marine (Hu et al., 2020). Another research devel-
oped a Raman-based automated sorting platform which used microfluidics and optical tweezers
for targeted single cell sorting from complex microbial communities and targeted cultivation of
novel microbes with specific physiology of interest (Lee et al., 2021).
Unlike the microfluid to manipulate the microbes at the single-cell level, the application of
flow cytometry in cultivation usually happens to collect multiple cells. Combined with other tech-
nologies like fluorescent dyes, fluorescence in situ hybridization (FISH), and isotope labeling, flow
cytometry could realize the rapid and targeted collection of interested microorganisms from envi-
ronments to facilitate subsequent cultivation by escaping the long medium enrichment term. As
early as 2005, researchers began to use flow cytometry to enrich targets in activated sludge with
the help of fluorescent dyes (Park et al., 2005). And similar collections were also achieved for
functional taxa (Ammonia-oxidizing bacteria (AOB) and NOB) in activated sludge with the scat-
tering signatures formed by targets (Abe et al., 2017; Fujitani et al., 2014). FISH is a robust tool
that could provide effective assistance to flow cytometry for targeted sorting while the damage it
caused to bacteria blocks the following cultivation step. To overcome this obstacle, a “live-FISH”
technology that skips cell fixation and permeabilization steps and attempts to chemically trans-
form probes into living cells has been proposed (Batani et al., 2019).
As an important and innovative high-throughput method developed in recent decades, sort-
ing-based cultivation allows for targeted, efficient, and high-throughput screen and enrichment of
cells with interest (especially the functional ones and low-abundance microorganisms). However,
sample conditions should be taken into consideration when applying this method. Likewise,
microorganisms in activated sludge often form flocs and have dense extracellular polymeric sub-
stances (EPS) that could trap the targets and challenge the separation of cell sorting. In these
cases, pretreatment like mechanical dispersion and ultrasonication (Foladori et al., 2010) must be
done to release the microbes free before sorting. Besides, long-term cultivation in droplet micro-
fluid is difficult to achieve for most of current technologies, which is a fatal shortcoming since
certain numbers of not-yet-cultured microbes have slow growth rates. Furthermore, labeling
methods of the targets are key procedures that are worthwhile for updating to ensure the targets
can be separated from other microbes and remain active as well.
4. Identification standards and screening methods

4.1. Identification standards of novel microorganisms: from phenotype to genotype
The creation of the microscope (Murphy & Davidson, 2012) ushers in the era of microbiology by
bringing these tiny but unique organisms into people’s view. Simultaneously, the microscope has
a profound impact on another important field of microbiology—taxonomic classification, which
facilitates worldwide scientists to identify prokaryotes with the same standard. Then the identifi-
cation of microorganisms undergoes the transformation from phenotype (microscope) (Murphy
& Davidson, 2012) to genotype (16S rRNA gene) due to the limited resolution of phenotype
(Woese et al., 1990; Tenaillon & Matic, 2020). Currently, 16S rRNA gene is the gold standard
and first-line tool for evaluating the taxonomic status of a prokaryotic strain and is applied to
define the novel microorganisms in enrichment or single colony via their 16S rRNA gene similar-
ity with cultured species in cultivation-related studies (Yarza et al., 2014). If the similarity of
microbial 16S rRNA gene with cultured species is below 98.65% (Sentausa & Fournier, 2013;
Yarza et al., 2014), it might be the representative of a new species. Similarly, a 16S rRNA identity
of <95% with the phylogenetically closest species with a validated name may indicate that the
isolate is a potential new genus, while <87%, 82%, 79%, and 75% for the potential new family,
order, class, and phylum, respectively (Kim et al., 2014; Yarza et al., 2014).
4.2. Screening methods in cultivation

During the cultivation process, several enrichments or colonies will generate and one must deter-
mine which of these enrichments or colonies contain microorganisms that are of interest with
active growth. Such screening step will help researchers prioritize the cultures for further investi-
gation as maintaining large numbers of cultures is pricey and will decrease the overall efficiency
of research. Currently, the most classic method to screen the potential novel species is to detect
the similarity of 16S rRNA marker gene with cultured species. In recent years, the emerging
MALDI-TOF MS tool is widely used in routine microbial identification to filter out conspecifics
and non-target taxa quickly and cost-effectively by detecting unique protein fingerprints and
metabolites of the targets. This part will introduce the existing screening methods (belonging to
two categories: 16S rRNA marker gene and MALDI-TOF MS tool) with their unique principles,
characteristics, and suitable application conditions (Figure 3). In addition, turnaround time is also
an important index to evaluate the screening methods because high-throughput cultivation needs
the cooperation of rapid identification. Researchers should take many factors into consideration
to adopt the most appropriate detection technique, such as experimental arrangements, costs,
turnaround time, and available equipment (Table S2).
4.2.1. Sanger sequencing for full-length 16S rRNA gene to identify the novel species
Sanger sequencing developed in 1977 is a method to sequence DNA using electrophoresis based
on DNA polymerase’s random incorporation of chain-terminating dideoxynucleotides during
in vitro DNA replication. Sanger sequencing is still a widely used method now although it has
been largely replaced by high-throughput sequencing methods in recent years, particularly for
large-scale automated genome analyses. In cultivation studies, Sanger method is used in the final
identification of a single colony or isolate as it can produce DNA sequence reads of >500 nucleo-
tides and maintains a very low error rate (>99.99%). The new species is identified if its similarity
Figure 3. The illustration of different screening methods. (a) Sanger sequencing for full-length 16S rRNA gene; (b) Illumina NGS
16S rRNA gene fragment sequencing; (c) Pacbio sequencing for full-length 16S rRNA gene; (d) Nanopore sequencing for full-
length 16S rRNA gene; (e) MALDI-TOF MS.
of 16S rRNA gene with cultured species is below 98.65 (Kim et al., 2014; Yarza et al., 2014), and
the current commonly used 16S rRNA databases include NCBI (Sayers et al., 2022), Silva (Quast
et al., 2013), Greengene (DeSantis et al., 2006), and EzBioCloud (Yoon et al., 2017). Once the
new species are confirmed, the corresponding steps should be followed for new species announce-
ment, such as the brief description of sources and growth conditions of isolates, a panel of experi-
ments to evaluate the morphological and biochemical characteristics, genotypic and phenotypic
information for taxonomy classification, and constructions of GenBank 16S rRNA accession num-
bers and an assembly of strain numbers to promote the propagation of novel species.
4.2.2. Short reads sequencing for parts of 16S rRNA gene

At present, the short reads sequencing of amplicons of 16S rRNA gene fragments is the
mainstream technology used to detect microbial species of samples with the characteristics of
high-throughput, inexpensive, and accurate. At this moment, the catastrophic problem of this
amplicon-based short reads sequencing method is the long turnaround time because of the
unavoidable PCR process and cumbersome library preparation step (usually two weeks for the
commercial companies). Besides, this method faces two major challenges. First of all, amplicon
has primer bias which could result in significant chunks of the known microbial diversity being
missing. For example, approximately 10% of environmental microbial sequences such as the
Candidate Phyla Radiation and as-yet-uncharacterized archaea (Eloe-Fadrosh et al., 2016) might
be overlooked by classical PCR-based 16S rRNA gene surveys because of the prevalence of com-
monly used primer sets. Thus selecting suitable primers that can better capture the total breadth
of diversity in a group could be beneficial for the screening of targeted enrichment and cultiva-
tion. For activated sludge, two common sets of primers (V1-V3 and V4 region of 16S rRNA
gene) were evaluated and results indicated the V1-V3 primers were more advisable as they could
provide better taxonomic resolution and least bias in community profiling of abundant process-
important taxa (Dueholm et al., 2022). The second obstacle for short reads sequencing is highly
resolved phylogenetic classification cannot be supported due to limited information provided by
short regions. To overcome these hurdles, some studies tried to leverage the short reads sequenc-
ing to generate accurate 16S rRNA data like synthetic long-read sequencing technology (Callahan
et al., 2021), while other developers are attempting to find solutions from long reads sequenc-
ing directly.
4.2.3. Long reads sequencing for whole 16S rRNA gene

In recent years, long reads sequencing (or third-generation sequencing) has received increased
attention with two currently dominant technologies: single-molecule real-time sequencing from
PacBio and Nanopore sequencing. Compared with short reads sequencing, long reads sequencing
could provide highly resolved phylogenetic classification and detect base modifications thus get-
ting increased appealing in microbiology (Lam et al., 2020; Leggett et al., 2020; Matsuo et al.,
2021). While the widespread application of long reads sequencing is impeded by the high error
rate (1% for Pacbio (Wenger et al., 2019) and 5% for Nanopore (Jain et al., 2018)). To over-
come this issue, manufacturers update the chemicals to improve accuracy, and researchers design
appropriate correction tools (like Racon (Vaser et al., 2017) and Pilon (Walker et al., 2014)) or
combine molecular methods like unique molecular identifiers (UMIs) (Islam et al., 2014; Kivioja
et al., 2012) to get final consensus sequences. For example, the NanoCLUST pipeline was devel-
oped for the correction and classification of full-length 16S rRNA nanopore reads (Rodrıguez-
Perez et al., 2021), and researchers introduced UMIs into Pacbio and Nanopore sequencing to
generate whole rRNA operon with a mean error rate <0.01% (Karst et al., 2021).
Between these two platforms, PacBio firstly receives more attention since its higher accuracy.
But recently, researchers pay more attention to Nanopore due to its convenient portability and
shorter duration time between sampling and data analysis which greatly facilitates the research on
site. Besides, the continuous improvement of accuracy and constantly developed bioinformatic
tools of Nanopore also promote its wider application. As a powerful sequencing tool, Nanopore
has been widely used for 16S rRNA gene sequencing with fast feedback and reliable results, and
studies from diverse areas also show its promising application (Benıtez-Paez et al., 2016).
Nanopore sequencing offered species-level resolution for phytoplankton samples and provided
new insights into the cyanobacteria biodiversity in tropical marine ecosystems (Curren et al.,
2019). Nanopore also has excellent performance in medical research for the rapid identification
of pathogens (Moon et al., 2019; Shin et al., 2016).
Considering the more convenient library preparation, faster detection speed (key for cultiva-
tion which needs timely feedback to monitor and adjust the enrichment process), and more
abundant products (from Flongle to PromethION that provides flexible choices for users),
Nanopore will be more preferred for profiling in enrichment for effective cultivation. Besides, the
manufacturer of Nanopore claims that the community profile can be determined by 16S rRNA
gene sequencing with a turnaround time of only 40 minutes, which is the fastest method among
all the available HTS technologies (including short and long reads sequencing). No matter which
methods you choose, with continuing progress in accuracy, throughput, and cost reduction, long
reads sequencing will definitely make great contributions to cultivation research as a robust
screening tool.
4.2.4. MALDI-TOF MS tool for microorganism identification

MALDI-TOF MS is an emerging technology for the identification, characterization, and typing of
microorganism pure cultures by their unique protein fingerprints and metabolites in the cultiva-
tion research (Clark et al., 2018). With its fast and high-throughput identification for a single col-
ony, the rapid development of MALDI-TOF MS greatly promotes the research progress of
cultivation by permitting a dramatic reduction in not only identification costs (1.5 dollars per
strain) but also turnaround time (1 min per strain) (Condina et al., 2019), and such rapid detec-
tion greatly improves identification efficiency and reduces the proportion of ineffective cultiva-
tion. However, MALDI-TOF MS has a fatal flaw: only microorganisms that are closely related to
those already in the database can be identified with high confidence. Presently, MALDI-TOF MS
is mostly used in the identification of clinical microorganisms, rendering their databases unsuited
to identify the taxa from other environments like activated sludge and the novel isolates (Santos
et al., 2016). Besides, there are still discrepancies in the identification of some species by MALDI-
TOF MS. For example, MALDI-TOF MS could not distinguish Escherichia coli from Shigella spp.
(Tan et al., 2012) and did not allow differentiation between different tested species of
Enterobacter (Pavlovic et al., 2012). What’s more, this system currently only be used to identify
the single colony and cannot distinguish samples in which several species are present cause no
clear profile will be produced under this circumstance (Dumolin et al., 2019). Therefore, efforts
should be paid to build and broaden the microbial database of activated sludge to mine the
potentials of MALDI-TOF MS in the environmental field thus making it a useful taxonomic iden-
tification platform for large-scale cultivation. At the same time, MALDI-TOF MS should be used
as a preliminary screening method to exclude the cultured microorganisms and the potential
novel isolates should be verified with confirmed identity (for example, full-length 16S rRNA gene
and whole-genome sequencing).
5. The future of cultivation in activated sludge microbiota

5.1. Expansion of the cultured repertoire in activated sludge
Pure cultures from important environmental samples like activated sludge should be preserved to
construct specific strain libraries as they are valuable resources not only for environmental micro-
biology but also for engineering applications. The identification of new species by various cultiva-
tion strategies will enrich the repertoire of microorganisms and decrease the proportions of
MDMs in activated sludge. Additionally, these novel taxa with critical ecological niches will not
only expand the tree of life but also deepen our knowledge of the complex microbe-microbe
(Burns et al., 2016) and chemical-microbe (Gao, 2021) interactions in activated sludge, which
might influence the wastewater treatment efficiency by producing chemicals or metabolites to
communicate or kill other microbes. According to reports, the application of cultivation to
human-related microorganisms has significantly expanded the cultured species repertoire (Lagier
et al., 2018; Poyet et al., 2019; Renwick et al., 2021). This result makes us confident in the appli-
cation of activated sludge cultivation and greatly inspires us about the future because activated
sludge has a much higher biodiversity than humans gut microbiota (three orders of magnitude
higher than human gut) (Wu et al., 2019). Besides, the “wanted list” of this review indicates the
necessity and eagerness for the cultivation application of activated sludge since the high taxo-
nomic novelty showed by species and the lacking of isolates, not to mention the majority that is
not in the list.
5.2. Methodology shift of microbial community research

Apart from increasing the number of known microorganisms, cultivation will also result in a shift
in methodologies used to describe the unknown ones. OTUs are clusters of sequencing reads gen-
erated in high-throughput analysis that differ by less than a fixed dissimilarity threshold (97%
usually) (Rognes et al., 2016). Amplicon sequence variants (ASVs) are generated by a different
algorithm which could analyze the sequences at the single-nucleotide level to keep the real
sequence variants (Callahan et al., 2016, 2017). The microbial community analysis at first hap-
pened at the OTUs level while the clustering method may cover up some sequencing errors and
neglect the real variation of the sequences due to the loose similarity. With the continuous
improvement of sequencing quality, the increase in sequencing data size, and more reference
genomes of isolates, researchers begin to purchase the algorithms with higher precision for ampli-
con research, i.e. ASVs at present (Amir et al., 2017; Callahan et al., 2016). This methodology
shift has already happened in the research of activated sludge and is necessary since it will pro-
vide us a deeper and clearer knowledge of the community (Peces et al., 2022).
Each OTUs or ASVs represents a biological interpretation in communities, and the unassigned
OTUs or ASVs highlight our knowledge limitations on activated sludge communities and empha-
size our incapacity to make the repertoire of whole microbial genetic diversity available at pre-
sent. For activated sludge in Hong Kong, >50% ASVs cannot be classified to any known genera
with the current databases. This phenomenon limits our further exploration of MDMs in acti-
vated sludge and cultivation can ameliorate this situation by increasing the number of cultured
species. With the continuous expansion of databases by cultivation and endless improvement of
sequencing technologies (Callahan et al., 2019), discussion of scientific questions with higher reso-
lution (at species even strain level) will be universal and open new doors for microbial ecology of
activated sludge.
5.3. Correction of microbiota taxonomy

Cultivation can stimulate the evolution of prokaryotic taxonomic methods. Presently, the com-
plete 16S rRNA gene is applied to provide taxonomic information and define novel species
(Yarza et al., 2014). However, taxonomic studies show that 16S rRNA gene is not sufficient to
estimate the full range of microbial diversity for genera that have a higher level of 16S rRNA
gene variability (Sentausa & Fournier, 2013). In the era of large-scale sequencing, many new clas-
sification methods are proposed like whole operon, DNA–DNA hybridization (Goris et al., 2007),
average nucleotide identity (Parks et al., 2020), Amino Acid Identity (Thompson et al., 2013),
and multilocus sequence typing (Sentausa & Fournier, 2013). Among them, the Genome
Taxonomy Database (GTDB) based on ANI sets an imperative milestone in the development of
microbial taxonomic research and greatly revises the existing tree of life by extending the single-
gene classification system (16S rRNA gene) to a set of single-copy marker proteins (Parks et al.,
2020). These advances also spawn a new discipline called taxonogenomics, which is to use genetic
and phenotypic criteria established by the scientific community as well as the most recent gen-
omic and proteomic data to define new microbial species. At present, we already have more than
380,000 prokaryotic genomes (almost 28,000 complete genomes) in the public database. More
and more isolates by cultivation can provide diverse complete genome information to further
expand the database and improve microbial resolution. Besides, the breakthrough of fast, eco-
nomic, and long reads sequencing techniques will enable the establishment of powerful tools for
genome capture and thus give birth to advanced taxonomy tools to describe new microbial spe-
cies discovered by cultivation. All of these efforts will refresh our knowledge of the composition
of the microbial world.
5.4. Discovery of novel functional microorganisms in activated sludge

As an engineering ecosystem, the most important role of activated sludge is to remove pollutants,
which highlights the significance of new microbial species excavation with key functions. Because
they not only can provide valuable information for technology improvement but also may have
the possibility to subvert traditional theories with unique metabolisms. The most classic example
is the discovery and isolation of comammox (Daims et al., 2015), a bacterium that has the ability
to complete the nitrification process in one step (oxidizing ammonia to nitrate directly). Its dis-
covery completely changes the two-step nitrification dogma and brings the nitrogen cycle research
into a new era. Additionally, two new PAOs in the genus Dechloromonas were verified in 2021.
These two PAOs were the most abundant species in worldwide enhanced biological phosphorus
removal systems but still no isolates yet (Petriglieri et al., 2021). In the “wanted list” of this
review, around half of the species show taxonomic novelty (cannot be classified to existing gen-
era) and enormous potential in wastewater treatment (Dueholm et al., 2022). The successful isola-
tion of OTU 16 (a new genus of phylum Proteobacteria and PAO bacterium) (Song et al., 2020)
also proves this opinion. We now are looking forward to the next surprise that cultivation will
bring to us in the near future.
Other than the possibility to improve pollution removal efficiency, the cultivation of new taxa
has unlimited potential in the applications associated with human health. For example, new spe-
cies may lead to the discovery of novel antibiotics as microorganisms are the major source of
antimicrobial agents and we need to keep discovering innovative antibiotics to counteract the
emergence of antibiotic resistance (Folgori et al., 2017). What’s more, the new taxa in activated
sludge could be the potential degraders of the emerging pollutants such as antibiotics (Liu et al.,
2021) and microplastics (Liu et al., 2019). Besides, new species could be used in clinical treat-
ments such as human microbiota transplantation, a method to treat disease by transferring func-
tional microorganisms into patients’ guts to help them restore microbial diversity (van Nood
et al., 2013).
5.5. Optimization of cultivation flow for microorganisms in activated sludge

As more and more species in activated sludge are cultured, researchers will gain deeper insights
into their metabolisms. Based on this information, we should improve and establish cultivation
flow for activated sludge specifically. For example, a pretreatment step should be established for
the characteristics of activated sludge flocs to facilitate the subsequent operation since the targets
might be blocked in the dense EPS and the pretreatment like mechanical dispersion, enzymatic
digestion (Liu et al., 2019), and ultrasonication (Foladori et al., 2010) must be done to release the
microbes. At the same time, the boom of sequencing and bioinformatic technologies will bring
faster, more accurate, and cheaper screening methods and researchers should take advantage of
them to build the rapid and accurate identification methods for the potential new species. Finally,
like human microbiota cultivation studies (Lagier et al., 2015b), the culture media that are specif-
ically designed for activated sludge community should be proposed to allow the growth of the
majority of microorganisms in activated sludge. Adopting the same methodology of medium
design for human gut, some key components like sterile filtrate or homogenates of activated
sludge can be added to the medium to provide the unknown nutrients or growth factors for
microorganisms. With, but not limited to, the above suggestions, the optimization of cultivation
flow for activated sludge microbiota will facilitate us with the experimental procedures, improve
the cultivation efficiency, and finally get more novel taxa from activated sludge.
6. Conclusions
Activated sludge contains high biodiversity of microorganisms that are utilized to remove pollu-
tants. Many surveys even long-term observations of worldwide activated sludge communities have
been done via bioinformatic analysis, and results show the majority of the key taxa with crucial
functions in activated sludge are not-yet-cultured. This phenomenon not only hinders our under-
standing of the ecological roles of MDMs in activated sludge but also blocks the pace of techno-
logical innovation of WWTPs. The creation of pure cultures that represent an ecosystem and
novel taxa is no doubt a useful way to reveal the roles of MDMs in activated sludge but also one
of the biggest challenges in microbiology. However, we are currently witnessing a paradigm shift
in which the rapid growth of high-throughput and innovative cultivation methods have opened
doors to culturing previously uncultured taxa thanks to the strong development of instruments
and technologies in recent years. These methods are theoretically feasible and have been validated
on some real samples, but further excavation is needed to investigate their applications in acti-
vated sludge, a community with high complexity. In addition, continued effort must be paid to
develop advanced cultivation technologies for the cultivation of new taxa. At present, there are
already many reports that applied cultivation to other ecosystems, and several studies that pointed
out the targets for cultivation in activated sludge. With the strong support of technologies and
knowledge, large-scale cultivation of activated sludge microbiota should be carried out as soon as
possible, especially the functional ones that are still in the dark state. If not now, when?
Acknowledgments
YZ would like to thank the University of Hong Kong for the Postgraduate Studentship. The authors sincerely
thank Miss Vicky Fung for her technical support.
Authors’ contributions
YZ and TZ designed and wrote the manuscript. The authors read and approved the final manuscript.
Disclosure of interest
The authors declare no conflict of interest.
Funding
This work was supported by Hong Kong General Research Fund (GRF 17206120).
ORCID
Yulin Zhang http://orcid.org/0000-0003-3476-8811
Tong Zhang http://orcid.org/0000-0003-1148-4322
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Culturing the uncultured microbial majority in activated

HANDLING EDITORS Eakalak Khan and Lena Ma

2. The not-yet-cultured majority and the “wanted list” of activated sludge

Table 1. Key targets for microbial cultivation in activated sludge.

Figure 1. The history of cultivation.

3.2. Cultivation and metagenomics

3.3. Uncultured reasons of microorganisms

3.4. The necessity of enrichment

3.5. The current cultivation methods

3.5.1. Traditional cultivation

3.5.2. In situ cultivation

3.5.3. Gene prediction-based cultivation

3.5.4. Sorting-based cultivation

4. Identification standards and screening methods

4.2. Screening methods in cultivation

4.2.2. Short reads sequencing for parts of 16S rRNA gene

4.2.3. Long reads sequencing for whole 16S rRNA gene

4.2.4. MALDI-TOF MS tool for microorganism identification

5. The future of cultivation in activated sludge microbiota

5.2. Methodology shift of microbial community research

5.3. Correction of microbiota taxonomy

5.4. Discovery of novel functional microorganisms in activated sludge

5.5. Optimization of cultivation flow for microorganisms in activated sludge

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