Culturing the uncultured microbial majority in activated sludge: A critical review

Abstract Activated sludge is a widely applied wastewater treatment process that mainly uses suspended microbial flocs to remove pollutants in wastewater. With the characteristics 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 microbial resources. However, the majority of microorganisms in activated sludge have not been isolated, which substantially limits the improvement 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 cultivation in recent years has spawned the creation of many innovative and high-throughput cultivation techniques. In this review, we summarized the microorganism “wanted list” in activated sludge, reviewed the potential cultivation methods that could extend our understanding of activated sludge microbiota, and discussed the significance and perspectives for activated sludge microbiota cultivation. Graphical abstract


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 environment and human health (Beychok, 1967;Peces et al., 2022). With the complex sources of sewage (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 microorganisms (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 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 treatment 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; and more comprehensive metagenomics (Wang et al., 2021b) to obtain detailed genomes has radically altered our perceptions of microbial diversity in activated sludge. In 2012, researchers investigated 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) . 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 Â 10 23 bacterial cells and has 1.1 ± 0.07 Â 10 9 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)  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, researchers 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(Martiny, , 2020Steen 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 categories 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 techniques (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 cultured, (2) reviewing the potential cultivation and detection methods that can extend our understanding of activated sludge microbiota, and (3) discussing the significance and future perspectives of the large-scale cultivation of activated sludge microbiota.
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 Â 10 9 at the worldwide level based on 16S rRNA sequence data of V4 region while only 96,148 OTUs are defined for bacterial and archaeal phylotypes 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 . 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 cultured 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 directions 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 abundance in 50% of all samples), another research summarized the general core taxa of global activated sludge which have a high fraction of poorly characterized species and once again prove the novelty and insufficient research of microorganisms in activated sludge . Except for many known bacteria associated with nitrification (Nitrosomonas and Nitrospira) and polyphosphate accumulation (Tetrasphaera and Candidatus Accumulibacter), this list also contains glycogen accumulation species (Candidatus Competibacter) and the known filamentous species (Candidatus Villigracilis and Leptothrix). In fact, culturing abundant members that are notyet-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 present 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 analytical tools that could define the priorities and evaluate the potential ecological roles of microorganisms 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 metabolic capacities and ultra-small size (M eheust 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).
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 , 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 nitrification drivers ammonia-oxidizing archaea (AOA) (K€ onneke et al., 2005;Wang et al., 2021a) and the superphylum DPANN archaea 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 waiting to be investigated although they are not the focuses of this review, i.e. viruses, fungi, and  (Castelle & Banfield, 2018) 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 community 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;. Meanwhile, the cultivation of microalgae in activated sludge has emerged since microalgae can assist the functional microorganisms to remove pollutants (Anbalagan et al., 2016;Barros et al., 2015).

The history of cultivation
Microorganisms are the most numerous and diverse life forms on the Earth with an estimated number of 4 Â 10 30 -6 Â 10 30 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  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 highthroughput cultivation but there are no reported studies of large-scale cultivation in activated sludge ecosystem up to now.

Cultivation and metagenomics
Cultivation and metagenomics are completely two different methods to analyze MDMs in microbial ecosystems ( Figure S2). Cultivation could get pure cultures to carry out the physio-biochemical 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 bioinformatic 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 Ju & Zhang, 2015a;Walker et al., 2017). As a culture-independent method, metagenomics can analyze all microorganisms in an ecosystem by an integrative and time-saving workflow 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 heterogeneity 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 cognition about not-yet-cultured microbes, it still cannot get the pure cultures for follow-up biochemical 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 creative and powerful way to explore genome information of unknown microorganisms in communities 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.

Uncultured reasons of microorganisms
Although microbiologists have different opinions on the specific value of the "1% culturability paradigm" (Martiny, 2019(Martiny, , 2020Stewart, 2012), it is undeniable that high proportions of microorganisms across most biomes remain uncultured (Steen et al., 2019). The reasons why microorganisms 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 researchers 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), xanthan 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 community, 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.

The necessity of enrichment
Once confirm the targets, enrichment is the first step for cultivation with eminent necessity (Laso-P erez et al., 2018). Generally, enrichment is to use specific growth media and environmental parameters to make the desired microorganisms grow vigorously over others by taking advantage 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 systems. 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 interferences by accumulating the abundance of prominent targets to facilitate subsequent isolation procedures. Secondly, enrichment could activate the VBNC microorganisms from the dormant state to expand the potential isolate lists . 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 suffering low abundance could be increased via long-term enrichment, which should be the cultivation 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 . 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 . 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 microorganisms 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.

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 according to specific circumstances for different samples.

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 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 . 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, temperature, 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 specific 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 accumulator), 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 cultivation technology that combined with rapid identification by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was created, i.e. "culturomics" (Lagier et al., 2012(Lagier et al., , 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 culturomics 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 (Dur an-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.

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 mechanism. 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 supporting 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 uncultured 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 maintenance of aseptic environment is still arduous and challenging.

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 cultivation of specific microorganisms and got many exciting achievements in recent years by integration 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.modelseed.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 substrate (Lugli et al., 2019). In 2019, an approach to capture pure cultures of specific microorganisms 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 antigen 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 specieslevel 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 functions, 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 distinguish them from the complex community. In conclusion, this method is a superior tool for targeted isolates while difficult to realize high-throughput cultivation.

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 bacterium 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 developed 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 technologies like fluorescent dyes, fluorescence in situ hybridization (FISH), and isotope labeling, flow cytometry could realize the rapid and targeted collection of interested microorganisms from environments 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 scattering 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 transform probes into living cells has been proposed (Batani et al., 2019).
As an important and innovative high-throughput method developed in recent decades, sorting-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 substances (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 microfluid 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.

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 identification 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 similarity 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 Yarza et al., 2014).

Screening methods in cultivation
During the cultivation process, several enrichments or colonies will generate and one must determine 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 investigation 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 nucleotides and maintains a very low error rate (>99.99%). The new species is identified if its similarity of 16S rRNA gene with cultured species is below 98.65 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 announcement, such as the brief description of sources and growth conditions of isolates, a panel of experiments to evaluate the morphological and biochemical characteristics, genotypic and phenotypic information for taxonomy classification, and constructions of GenBank 16S rRNA accession numbers and an assembly of strain numbers to promote the propagation of novel species.

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 commonly 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 cultivation. 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 processimportant taxa . 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 sequencing 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 sequencing 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 getting 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 overcome 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 developed for the correction and classification of full-length 16S rRNA nanopore reads (Rodr ıguez-P erez 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 (Ben ıtez-P aez 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 cultivation 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.

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 cultivation research (Clark et al., 2018). With its fast and high-throughput identification for a single colony, 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 detection greatly improves identification efficiency and reduces the proportion of ineffective cultivation. 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 identification 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).

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 microbiology but also for engineering applications. The identification of new species by various cultivation 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 application 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 taxonomic novelty showed by species and the lacking of isolates, not to mention the majority that is not in the list.

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 generated 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(Callahan et al., , 2017. The microbial community analysis at first happened 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 amplicon 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 provide 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 emphasize our incapacity to make the repertoire of whole microbial genetic diversity available at present. 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 activated 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 resolution (at species even strain level) will be universal and open new doors for microbial ecology of activated sludge.

Correction of microbiota taxonomy
Cultivation can stimulate the evolution of prokaryotic taxonomic methods. Presently, the complete 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 classification 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 singlegene 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 genomic 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, economic, 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 species discovered by cultivation. All of these efforts will refresh our knowledge of the composition of the microbial world.

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 discovery 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 genera) and enormous potential in wastewater treatment . The successful isolation 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 species 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  and microplastics . Besides, new species could be used in clinical treatments such as human microbiota transplantation, a method to treat disease by transferring functional microorganisms into patients' guts to help them restore microbial diversity (van Nood et al., 2013).

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 , 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 specifically 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.

Conclusions
Activated sludge contains high biodiversity of microorganisms that are utilized to remove pollutants. 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 understanding of the ecological roles of MDMs in activated sludge but also blocks the pace of technological 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 activated 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?