PCR based fingerprinting techniques to transpire the microbial populations diversity of biofilm based nitrifying packed bed bioreactor in the bioremediation of aquaculture wastewater

To deal with the increasing risk of pollutants from the aquaculture practice, wastewater treatment systems are considered to be an ideal strategy, for reducing the impact on the natural ecosystem. Biological treatment systems per se bioreactors involving microorganisms are efficient in the bioremediation process is determined by the reactor design, operational module i.e., structure of the bacterial community, and their diversity which in turn reveals the essential relationship between its ecosystem and the environmental factors. Microbial diversity and the dynamics of the microbial populations illustrate the principal background of different bacterial communal development performing bioremediation. Progression in genome exploration led to multifaceted technical ease in recent with the emergence of different generation sequencing technologies e.g., Sanger sequencing, 454 sequencing, Illumina/Solexa sequencing, Single-molecule real-time sequencing, and Oxford nanopore sequencing. Though the recent advancements provide a greater profile of information for research, using high throughput sequencing could not be a suitable option in all aspects, especially in consideration of the initial research requirement, the low number of targets, minimal samples, and also the diversity scaling methodologies. In this review, we have discussed the PCR-based molecular fingerprinting techniques which go accessible with sanger sequencing methodologies as well as by combining different statistical and bioinformatic algorithms. The systematic analysis with the elementary molecular techniques combining sequence technologies and bioinformatics tools would enable us to understand the overview and diversity structure of the biofilm and further in-depth research by selecting appropriate sequencing platforms results in the dynamics of the microbial community.


Introduction
Different environmental initiatives have been conducted and taken place during the last two decades in response to the increasing ratios of municipal and industrial wastewater involving pathogens and chemical constituents. The innovation in the treatment technologies is expected to overcome the limitations of the conventional treatment methods like chemical coagulation, adsorption, and activated sludge, that not only advance the operational Page 2 of 21   2:5 modules but also these technologies should answer the sustainability in the wastewater treatment and management strategies [1]. The expeditious development of the aquaculture industry has brought a variety of environmental problems, including water quality deterioration, contaminants, biological invasion, aquatic diseases, and so on [2,3] due to the inorganic, organic, and other pollutants consisting of residual baits, animal wastes, and organic waste from natural sources [4]. Intensification in aquaculture happened to be an inevitable eventuality, led to environmental degradation, threatening its long-term sustainability [5], destruction of coastal mangroves, degradation of land resources, and deterioration of water quality [6] because of the toxicants from aquaculture wastewater e.g. ammonia, nitrite, nitrate, and hydrogen sulfide. The choice of technology, scale, and intensity of its operations in aquaculture vary a great deal with consideration of the environmental and socio-economic impacts [7].
The modern land-based intensive culture through the implementation of recirculating aquaculture systems (RAS) has been developing as a solution to environmental degradation since the effluent wastewater is processed, treated, and reused [8]. Thus, it is expected that the introduction of aquaculture recirculating systems with competitive biofilters and nitrifying bioreactors will take forward aquaculture most environment friendly with sustainability.
Recirculating aquaculture system technology addresses the environmental challenges by decreasing waste output and increasing the recycling of resources up to 90-99% by integrating various components per se sand filters, protein skimmers, and trickling filters [9]. Recycled water and its quality are associated with the animal health and growth rate in a RAS. The water quality parameters such as pH, salinity, alkalinity, hardness, temperature, ammonia, nitrite, carbon-di-oxide, and dissolved oxygen are interrelated and are very much essential to maintain the total viability of the system [10,11]. Over three decades, several researchers worked on RAS, focusing on the process of design, up-gradation, and system integration with different biofilters. The foremost necessity of RAS is an efficient biofilter that prevents toxic metabolites such as ammonia and nitrite [12].
Nitrifying Packed bed bioreactor (PBBR) integrated into RAS is a perfect tertiary waste water treatment system for removal of ammonia as the oxidized products and other free radicals by the involvement of nitrifying bacterial consortia (NBC) [13]. PBBR was activated by immobilizing NBC on polystyrene beads packed in different beds inside the Nitrifying bioreactor (NBR) [14,15]. PBBR is configured to operate under closed recirculating mode with minimal energy and water requirements for continuous operation. NBC is a highly ecofriendly and user-friendly reactor that provides a solution to the aquaculture industry where powerful biological treatment of waste material is required.. Biofilm is a densely packed group of microorganisms composed of different species bound together by a polymeric substance. These polymeric substances excreted by the microorganisms is known as extracellular polymeric substance (EPS) which forms an adhesive matrix attached to the surfaces and holds the biofilm together [16]. Fixed film biofilters showed high efficiency in total ammonia nitrogen (TAN) removal [17]. Under RAS mode, NBC undergoes differential conglomeration of bacterial populations having the diverse function in nitrification including ammonia oxidation, nitrite oxidation, and denitrification based on locales where the system performs [18]. This conglomeration differentiation is the main aspect explaining the dynamism of the NBC indicating the colonized microbial community in the RAS. Dynamics of the microbial population enlightened using molecular techniques helps to explore community diversity, microbial shift, population dynamics, and functional genomics discriminating the nitrogen removal performance of the bioreactor treatment technology. Community and the organizational structure of the biofilm prominently relate the substrate utilization and metabolic interactions. The community structure of any bacterial group is fundamentally based on the function of individual species and their interaction with multiple species in the system [19]. Analysis based on qualitative and quantitative data is crucial and it serves as the basic frame to determine the longterm changes in the community which include diversity, abundance, and population shift. Small subunit rRNA gene-based molecular studies have overcome the traditional analysis of culture-based approaches to identify the microbial composition and also made possible the characterization of microbiomes that cannot be cultured [20]. Development of biomarker-based analysis, specific probes approach to evaluate the functional composition and potency of a habitat provides the understanding on the communal activities of the microbes, their role in the study habitats and the potential impacts in environmental processes [21,22].
16S rRNA gene sequences consist of 1.5 kb molecular size whereas the NGS sequencing platforms Roche-454 Life Sciences , Solexa Genome Analyzer-Illumina , SOLiD System-Applied Biosystems covers 100-700 bp read length of varied accuracy [23]. The distinct difference between NGS the Sanger sequencing is parallel sequencing as well as sequencing during synthesis. Likewise, each sequencing platforms have its inimitable characteristics per se Illumina uses fluorescently labeled nucleotides during DNA synthesis, and during pyrosequencing 454 technology detects chemiluminescent signals of pyrophosphate released, Ion Torrent detects pH changes when hydrogen ions are released. These platforms are unique and useful in the notions and opting the one for the analysis of communities is dependent on the amplicon size, the number of samples, read length, sequence accuracy, and prominently, the budget [23]. Sequences of the 16S rRNA genes are curated and termed as OTUs (Operational Taxonomical Units). Further taxonomic alignment is based on the reference in the database whilst the diversity, and community analysis can be done using diversity-based approaches to explain the community structure. The quality and accuracy of the outputs are highly dependent on the reference database selected for the sequence analysis [23]. This review is to focus on the details of the nitrifying bioreactors in aquaculture wastewater treatment, the usage of molecular techniques, and Sanger sequencing considering the perspective to determine and describe the microbial diversity, community profile, and the metabolic interaction of the nitrifying population with the help of different statistical and bioinformatics tools as equivalent to the high through put methods.

Development of nitrifying bacterial consortia
After different enrichment series under laboratory-scale ammonia-oxidizing non-penaeid culture (AMONPCU-1) and ammonia-oxidizing penaeid culture (AMOPCU-1), and two nitrite-oxidizing consortia, nitrite-oxidizing non-penaeid culture (NIONPCU-1) and nitrite-oxidizing penaeid culture (NIOPCU-1), were developed to establish nitrification in penaeid and non-penaeid hatchery systems [13]. Seawater enriched with 10 ppm of NH 4 Cl and 2 ppm of KH 2 PO 4 was used for the cultivation of nitrifying bacteria from the marine environment and the enriched cultures were stored at 4 °C [12]. Advantageously, these cultures could be mass-produced and used for the activation of nitrifying bioreactors whilst rapid nitrification would be possible in RAS with a single consortium [12]. Likewise, NBC for the freshwater, brackish, and marine aquaculture systems was mass-produced using different fermenters.
PBBR [15] (Patent No. 241648) was developed to establish sustained aquaculture production, maintaining the health of the animal with high yield and effective technology of waste management. The PBBR facilitates the hatchery operation by minimizing the discharge of spent water and maintaining the water quality [24]. These bioreactors are activated by penaeid nitrifying bacterial consortium (NBC) [13] which includes autotrophic nitrifiers and heterotrophic denitrifiers [25] functioning based on the steady-state biofilm kinetics. One of the advantages of the nitrifying bacterial consortia immobilized on polystyrene beads in PBBR is the minimum start-up time and its integration into any existing hatchery design without any modifications [15].

Immobilization of nitrifying bacterial consortia and Biofilm formation
The ammonia is oxidized to nitrite, and then nitrate which may then be biologically denitrified (reduced to molecular nitrogen). Nitrifiers use ammonia as an electron donor to provide energy for their growth. Biological treatment is relatively cheap and produces no unwanted byproducts. Immobilizing the microorganisms on a support medium (using polystyrene/low-density polyethylene/high-density polyethylene), would ensure that the nitrifiers are retained within the packed beds. Immobilization of nitrifying consortia would increase the bacterial cell concentration to a greater extent as well as the volumetric efficiency tending to better system performance. Immobilization is the best solution to using nitrifiers-based biofilters and the technique is effective in the potential removal of ammonium compounds [26]. The highest ammonia removal rate of 82 g/m 3 per day was observed in the RAS with consortia immobilized beads having a hydraulic retention time of 0.3 h [27]. Nitrification relies on a healthy population of nitrifying bacteria, which can be used for the reactivation of any biofilters. A viable population of AOB and NOB population helps the complete retrieval of the system activity [28]. This can lead to relatively small reactors that may afford protection from toxic shocks and adverse temperatures which would help maintain year-round treatment. Immobilization is an efficient method to retain slow-growing organisms like nitrifiers in continuous flow reactors such as PBBR. Nitrifying biofilters in immobilized and encapsulated forms independent of the specific growth rate [26] in different reactor models revealed vigorous synthetic ammonia removal [29]. Immobilization of bacterial cells can be divided into naturally attached cells (biofilms) with limitations and the artificially attached ones, a well-defined and controllable system by which the NBC was immobilized to the beads in NBR. Mass cultured bacterial consortia with known kinetics and diffusion coefficients are used for the activation and biofilm formation on the polystyrene beads as a mode of biofilm formation. Wood particles from A. altissima (soft wood tree) with a high surface area were identified as immobilization medium for indigenous nitrifying bacterial consortia, and their in-situ application was confirmed as a viable tool for the reduction or removal of TAN in tropical shrimp culture systems. This technology can also be used as a biodegradable carrier [30]. In a biofilm model, the thickness or the density of the biofilm will increase as the system undergoes the continuous operation. Considering the varying amounts of biomass, it is essential to study the dynamics of the system. Competition between different species in a biofilm results in a heterogeneous distribution involving population shift and distribution based on the substrate flux. NBC biofilm enhances the growth of both nitrifying and denitrifying bacterial groups. A biofilm will expand in depth as long as the growth rate is higher than the rate of decay or the rate of attrition [31]. In that context, heterotrophic bacterial groups are relatively fast growers with maximum sloughing, and shear loss when compared to nitrifiers. The immobilized product, a single bioaugmentation product sufficient to bring forth nitrification as well as denitrification can eliminate ammonia with no accumulation of nitrite and nitrate in the system [30].

Packed Bed Bioreactor under recirculation aquaculture system
The PBBR integrated recirculating aquaculture system was identified to be functional under different substrate concentrations and flow rates and exhibited significant nitrification (p < 0.001) potential during operation. The reduction rate of ammonia was gradually increased in the system since the initiation of recirculation mode and considerably the system performance was also improved. Animals reared were found to be a healthy resulting increase in the yield. The presence of nitrifiers was confirmed and their stability in the long-term RAS operation under different flowrate experiments was confirmed using Fluorescent In-situ Hybridization (FISH) analysis of biofilm. PBBR ( Fig. 1) could be integrated into different aquaculture models as closed recirculation systems for maintaining biosecurity and environmental quality [14]. Effluent treatment technologies in aquaculture are distinct, i.e. bio-flocs technology, trickling filters, bead filters, and the rotating biological contactors, fluidized sand biofilters [32] to remove suspended solids, ammonia, nitrate, and nitrite from the aquaculture effluent.
Designing the system and its components used in RAS should be based on the idea to maximize production capacity per unit of capital invested and reduce the cost of the unit even while maintaining reliability. The nitrifying bioreactor is of 6 reactor beds filled with beads made of fiberglass shells with 9 aeration cells each. The surface of the polystyrene beads increases the rate of cell attachment and immobilized NBC. Surface characteristics of the substrata are very important in fixed bed bioreactors which act as perfect support material for both the slowgrowing autotrophic and fast-growing heterotrophic bacteria [33]. The PBBR as a whole consists of a total of 60 kg of beads with a surface area of about 28.26 m 2 and a specific surface area of about 205 m 2 /m 3 directly proportional to the quantity of the biofilm concentration facilitating the removal of harmful nitrogenous compounds from the system (www. nitri fying-biore actor. com). The flow of treated water from the bioreactor back to the tanks would be regulated using the reservoir tank and a collecting tank. The PBBR designed here is flexible and could be interchangeable for prawn (salinity 15 ppt) and shrimp (salinity 30 ppt) larval rearing systems with NBC based on the salinity. Recirculation is one of the prime requirements for shrimp maturation systems from the perspectives of water quality and biosecurity [15]. A PBBR connected to the RAS exhibited high performance by shrimp biomass yield and maintenance of water quality throughout the study period. Operational conditions such as oxygen, water flow, and temperature were optimized as suggested [24] in the study system which was visible by the efficiency of the bioreactors.

Molecular characterization of the microbial population
Marine nitrifying bacterial consortia (NBC) was used as a startup culture for immobilizing the commercialized nitrifying bioreactors for RAS. Nitrifying biofilm operated in the laboratory scale bioreactor was responsible for the high rate of nitrification and nitrogen removal [34]. The addition of these mass-produced nitrifying bacterial products indicated a significant improvement in nitrification efficiency in RAS. Enhanced nitrifying biomass retention in pulsing sequencing batch reactors showed predominant nitrifying bacterial population development, in particular, AOB and NOB in the reactor [35]. The consortia involve autotrophic nitrifiers, and denitrifiers and these denitrifying heterotrophs utilize the metabolites of the nitrifiers, a vital part of the consortia [25]. Both nitrification and denitrification processes can be possible in the bioreactors operated under suspended or attached bacterial growth conditions [25]. A lab-scale Integrated vertical membrane bioreactor (IVMBR) parallelly connected with controlled MBR having both oxic and anoxic zones and phase separators favored both nitrification and denitrification processes in the effluent of synthetic wastewater [36]. The experimental results of the aerated land fill bioreactor revealed nitrification and heterotrophic carbon oxidation as well as bio-stabilization at various ORP levels [37]. Microorganisms will adhere to the internal walls of the reactor and higher cell densities achieved in the retentostat mode with potential microbial interactions eliminating ammonia from the waste water system [38]. In a co-existing community, a high-affinity population can stay active under low ammonium concentrations whilst the secondary population thrives during ammonium peaks [39].

Nitrification
Nitrification is the microbial conversion of ammonia to nitrate associated with the fixation of atmospheric nitrogen to nitrogen removal (denitrification and anaerobic ammonium oxidation (anammox)), which ultimately converts nitrate or ammonium to N 2 gas. Nitrification plays a critical role in reducing the associated risks of elevated dissolved inorganic nitrogen (ammonia, nitrate, and nitrite) found in coastal systems in addition to the loss of biodiversity, toxic algal blooms, and depleted dissolved oxygen. It is estimated that more than 50% of external dissolved inorganic nitrogen inputs to estuaries are removed by these microbial processes [40]. Nitrification is the biological oxidation process by chemoautotrophic organisms: In the first step, ammonia-oxidizing bacteria oxidize ammonia to nitrite Followed by oxidation of nitrite to nitrate by nitriteoxidizing bacteria In wastewater treatment systems, nitrification is predominantly performed by autotrophic Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) that use ammonia and nitrite as their energy source and CO 2 as carbon source. Utilizing denitrification, oxidized inorganic nitrogen compounds, such as nitrite and nitrate are reduced to elemental nitrogen (N 2 ), the process conducted by facultative anaerobic microorganisms with electron donors derived from either organic or inorganic sources from heterotrophic and autotrophic denitrification respectively. Autotrophic denitrifiers prevent the accumulation of toxic sulfide resulting from sulfate reduction in marine recirculating systems. Nitrous oxide is emitted predominantly in the aerated zones, but whether the nitrifying or denitrifying microorganisms are the main source of N 2 O emissions remains unclear [41].

Ammonia oxidation
Ammonia oxidation is thought to be the rate-limiting step in the overall reaction because the free energy of the reaction is significantly greater than that of nitrite oxidation and nitrite rarely accumulates in the environment. In a nitrifying bioreactor, the specific capacity of ammoniaoxidizing organisms determines the design of the nitrifying bioreactor, the upper limits of water treatment, and the environmental characteristics of the receiving water body. From the recent studies, the first stage of ammonia oxidation was described as phylogenetically restricted to the alpha and beta classes of proteobacteria. These chemoautotrophic organisms catalyze the oxidation of ammonia to nitrite via an ammonia monooxygenase encoded by amoA. Two distinct groups of microbes are capable of ammonia oxidation: (1) the recently discovered ammonia-oxidizing archaea (AOA) and (2) the ammonia-oxidizing bacteria (AOB), including beta proteobacteria (β-AOB) from the genera Nitrosomonas and Nitrosospira, as well as gamma proteobacteria (γ-AOB) from the genus Nitrosococcus. There are multiple copies of amo CAB operon present in the beta-AOB whereas it is a single copy in the gamma-AOB. Generally, only a portion of the operon, amoA gene has been used as the molecular marker for the study of AOB which is highly conserved at around 450 bp [42]. A study on amoA gene abundance of AOA and AOB showed a correlation to the ammonium concentrations of influent and effluent water of seven different wastewater treatment plants [43]. It is described that many important processes like nitrification and anammox are solely carried out by the slow-growing microbes with higher doubling time [44]. Ammonia concentrations keep on varying in the completely active RAS based on the systematics i.e., feeding rates, feed utilization ratio, and detritus settled in the tanks which are responsible for ammonia rate and shift of Ammonia Oxidizing Microbes (AOM) population by altering the activity and presence of important AOB. In aquaculture systems, Nitrosomonas marina-like AOB produced at low ammonia conditions and increased rates of Nitrosomonas europaea like and Nitrosospira tenuislike AOB will persist whilst at higher concentrations Nitrosococcus mobilis-like AOB becomes predominant, rather in wastewater treatment systems Nitrosomonas europaea and Nitrosococcus mobilis like AOB prevails the most [45]. The Archaea are prokaryotes (cells without nucleus) that cannot be easily distinguished from bacteria by size or shape. However, some have morphologies that are not found in bacteria, such as polygonal in halophilic archaea or very irregular cocci in particular hyperthermophiles. The metabolic diversity of archaea is also reminiscent of bacteria. Apart from methanogenesis (presently unknown in bacteria) all metabolic pathways discovered in archaea also exist among bacteria. Archaeal genomes encode many informational proteins that have homologs in eukarya but not in bacteria. Crenarchaea is the major among the archaea that possess ammonia monooxygenase gene, and dominant ammonia oxidizers [40]. Based on the comparison of 16S rRNA gene sequences, Nicol and Prosser [46] described that only a specific lineage of archaea was constituted of archaeal amoA due to a distinct but specific phylogenetic association with thermophilic Crenarchaea. A Marine Chemolithoautotrophic strain of Crenarchaeota has been isolated that uses ammonia as a sole energy source and expresses genes related to ammonia monooxygenases [47]. Nitrosopumilus maritimus and Nitrososphaera gargensi are the two reference species of the marine ammonia oxidizers belonging to the novel phylum Thaumarchaeota [48]. Most of the archaeal sequences in the databases are the gene sequences of the environmental samples [49]. In a seawater aquatic system, the abundance and distribution of AOA were found to be larger than those of AOB in the biofilters and it was indicated temperature was the most important influencing factor for the difference in the abundance levels [50]. Aerobic ammonia oxidizers inhabit the different ecological niches even as a cooperated habitant with anaerobic ammonia oxidizers resembling their metabolic complexity and functionality based on different environments they exist and only by multiple complementary techniques it is possible to retrieve those information [42].

Nitrite oxidation
Nitrite oxidation is the subsequent process of ammonia oxidation consisting of different genera per se Nitrobacter, Nitrococcus, Nitrospira, Nitrospina, and Nitrotoga that convert nitrite (NO 2 ) to nitrate (NO 3 ). The growth balance between the AOB and NOB is the vital optimizing characteristic feature of the nitrifying community [51]. The nitrite-oxidizing sequencing batch reactor (NOSBR) sludge is comprised of a complex microbial community of which 89% is the Nitrospira moscoviensis, autotrophic nitrite-oxidizer from the phylum Nitrospira was first reported in a wastewater treatment system [52]. The coexistence of AOB such as Nitrosomonas oligotropha and Nitrosomonas ureae and nitrite-oxidizing populations like Nitrospira and Nitrobacter the nitrification performance in the drinking water distribution systems whereas the presence of NOB indicated the reduction in nitrite associated chloramine decay in the system [53]. NOB exhibits enzymatic deactivation and reactivation during anoxic and oxic conditions in the continuously stirred tank reactor (CSTR) and batch type reactors, although AOB was observed with no performance difference due to periodic anoxic disturbance [54]. Candidatus Nitrotoga is a novel nitrite-oxidizing bacteria with distinct nitrite oxidoreductase isolated from the wastewater treatment systems with high levels of nitrite [55]. Nitrite-oxidizing and ammonia-oxidizing bacteria resemble proximity to previously reported photosynthetic bacteria as they retain the ancestral phylogeny [56]. Nitrobacter winogradskyi belongs to the phylum alpha proteobacterium, a Gram-negative facultative chemolithoautotrophic nitrite-oxidizing bacteria which can also grow anaerobically and initiates denitrification pathway due to the presence of NXR gene, nitrite oxidoreductase complex which comprises of α and β sub units encoded by nxrA and nxrB and this bacterial population was found in co-occurrence with AOB in the nitrifying community [57].

Complete Ammonia Oxidation (Comammox)
The discovery of complete ammonia-oxidizing bacteria (CAOB) obtained from the biofilm samples [58,59] revamped the typical aspects of the two-step process of nitrification -Nitrospira, a single organism capable of oxidizing ammonia and nitrite at a stretch as there is a high possibility of horizontal gene transfer between the β-AOB and comammox Nitrospira [60]. The comammox Nitrospira belongs to the lineage-II of two clades A and B and has been categorized based on the ammonia monooxygenase phylogeny [58,59]. The ecological significance, distribution, and diversity of these populations have been explained and reported from different ecosystems including drinking water systems [61,62], recirculating aquaculture systems [63,64], biofilters [65], wastewater treatment plants [66][67][68], agricultural and terrestrial ecosystems [69]. The detection of the spatial overlapping substantiates the co-existence of the AOB, NOB, and CAOB resulting in the diverse population of the microbial communities [70]. The oxygen concentration gradients in the aerated reactor enhance the growth of nitrogen removal bacteria, especially with diverse nitrifying and denitrifying groups including CAOB and their abundance in the biofilms than activated sludge [71] transpired as the operational efficiency of the nitrifiers as attached cells. Fowler et al. [65] designed primers to cover both the clade A and clade B comammox groups simultaneously with primers for AOB and NOB which transpired that the comammox Nitrospira imputed the overabundance of NOB by providing fractions of nitrite produced by them which tends to the nitrification-denitrification loop (NOB utilizes substrate from nitrate reducers other than the nitrite from AOB involving re-oxidation of nitrite in competition with nitrite reducers), typically referred to as ping-pong effect which is referred as NOB takes up acetate as PHB (polyhydroxybutyrate) resulting in mixotrophic growth by mediating acetate dependent nitrate reduction [72].

Denitrification
Denitrification is the stepwise reduction of nitrate to dinitrogen; a respiratory process alternative to the oxygen respiration process under less oxygen and anoxic condition. Heterotrophic denitrification utilizes the external electron, external as well as endogenous organic carbon originating from the waste. Denitrifiers are also involved in other processes in addition to nitrate removal relevant to the water quality control in aquaculture systems i.e. replenishing some of the inorganic carbon lost through nitrification by raising alkalinity whereas, reduced organic carbon discharge occurred when endogenous carbon sources utilized for denitrification [73]. Bacterial population shift was identified in the osmotic membrane bioreactor (OMBR) due to the accumulation of salt from the municipal wastewater treatment [74]. Nitrification performance even at low ammonia substrate levels could be estimated by monitoring the biofilm characteristics in the moving bed biofilm reactor [75]. Partial nitrification in the waste water pipes could reduce the sulfide accumulation that induces toxicity, corrosion, and unpleasant odors [76]. Partial nitrification observed in a reactor connected with a granular sludge anammox reactor attained a high nitrogen removal rate with consistent efficiency of 15.0 kg-N m −3 [77].
Nitrite reductase catalyzes the reduction of nitrite to nitric oxide, which further reduces to nitrous oxide is catalyzed by nitric oxide reductase, an integral membrane protein. Finally, nitrous oxide reductase catalyzes the reduction of nitrous oxide to dinitrogen. Unlike Gramnegative bacteria, these four enzymes are found membrane-bound in Gram-positive bacteria and archaea [78]. Denitrification is carried out by bacteria predominantly the phylum proteobacteria and also archaea isolated frequently from soils, sediments, and aquatic zones. Acidobacteria, Chlorobi, Bacteroidetes, and Planctomycetes are the other groups of microorganisms found in common in denitrification systems in addition to Rhodospirillales, Burkholderiales, Pseudomonadales, Rhizobiales, Dechloromonas, and Bradyrhizobium [79]. Nitrate reductase is a membrane-bound enzyme that catalyzes the reduction of nitrate to nitrite which is then transported into the periplasm via NarK2. Based on the location there are two types of dissimilatory nitrate reductase: a membrane-bound (Nar) with three subunits (i) catalytic α subunit encoded by narG containing a molybdopterin cofactor, (ii) soluble β subunit encoded by narH, and (iii) γ subunit, encoded by narI containing two b-type haems and a periplasmic-bound (Nap) nitrate reductase [80]. Two-component sensor-regulator pair NarXL identified in the chromosomal region of Pseudomonas stutzeri, denitrifying bacteria found linked with the narG operon for respiratory nitrate reductase which also encodes the nitrate or nitrite translocases (NarK and NarC) [80]. Nap is a hetero dimer that includes NapA, a catalytic subunit containing a molybdopterin cofactor, and the NapB containing two c-type haems whereas NapC contains four c-type haems is membrane-bound which donates an electron to the NapAB complex in the periplasmic region [81]. Nap gene-based nitrate reduction system has been identified in Desulfovibriodesulfuricans, Alcaligenes eutrophus, T.pantotropha, E. coli, P. denitrificans, Ralstoniaeutropha and Rhodobacter species [82].
The reduction of nitrite to nitric oxide is the key step catalyzed by cd1 and copper nitrite reductase (nirS and nirK gene), a soluble dimer of two identical subunits, one with heme c and the other with heme d1. The copper nitrite reductase was identified from the Archaeon like Haloferax denitrificans, Haloarculamarismortui, and bacteria per se Achromobacter cycloclastes, Nitrosomonas europaea, R. sphaeroides, and Bacillus halodenitrificans [80]. The functional markers of nitrogen metabolism like copper-containing dissimilatory nitrite reductase gene (nirK), nitric oxide reductase (norB), and putative nitrite reductase (nirS) have been found in aerobic and anaerobic AOB [42]. Different Alcaligenes sp. was the most common denitrifying group identified in the environmental samples of aquatic systems using PCR amplification of different combinations of nir gene primers [83]. Likewise, samples from forest upland and wetland soils explored the heterogeneity of nir gene denitrifying populations such as Pseudomonas stutzeri, Alcaligenes faecalis, Paracoccus denitrificans, Achromobacter xylosoxidansand Alcaligenes xylosoxidans identified using PCR-RFLP analysis [84] and having both subunits found predominant in the group proteobacteria whilst the nirK gene detected in the Firmicutes and Bacteroidetes respectively [85].
Nitric oxide reductase (nor) consists of two subunits norB, norC which is the protein responsible for the reduction of toxic NO to N 2 O, an inert form intermediate reported in Ps. Stutzeri [86], Paracoccus halodenitrificans [87], Alcaligenes eutrophus [88], Rhodobacter sphaeroides [88], and recently identified in Ralstonia eutropha [89]. In a denitrifying system, nitric oxide accumulated at very low concentrations until the system underwent different oxygen inhibitions where then the high emission of NO was observed [73]. Enzyme synthesis regulation and activity of nitric oxide and nitrite reducing systems are interconnected [90]. Elimination of nitrous oxide from the environmental systems is very important that the reduction of such green-house gas is possible by the nitrous oxide reductase (nos) multicopper enzyme consists of two identical subunits including nosZ, nosR, and nos DFYL genes [80], the terminal enzyme of the energy preserving denitrification pathway in different denitrifying bacteria [91]. The diversity of the abundance of the denitrifying population is based on the dissolved organic carbon and dissolved oxygen rate prevailed in the environment that has been revealed in the community composition and the abundance of the nos Z gene [92] of the denitrifiers in the biofilms of alluvial wetland. The gene-based quantification of the different denitrifying functional genes compared to the total bacterial population with 16S rRNA gene abundance explore the proportion of bacterial functional groups operating in the system [93].

Nitrogen fixation
Other than denitrification, biological nitrogen fixation is a process of biological utilization of dinitrogen, vital for nitrogen availability in different systems such as freshwater, marine, and terrestrial ecosystems [94]. The reaction is catalyzed by the nitrogenase enzyme consisting of proteins, dinitrogenase, and dinitrogenase reductase encoded by nifHDK genes. Under oxic conditions of the system, NifL protein prevents the synthesis of the nif gene which is regulated by NifA protein, and whenever the system sensed limited ammonia the NtrC protein (usually inactive in the presence of ammonia) gets activated to promote NifA transcription [95]. This process of shutting down nitrogenase activity by ammonia is called the ammonia switch-off effect [96]. Such regulation is mediated in different autotrophic and heterotrophic bacteria per se the species of Thiobacillus [97], Rhodobacter [95], Alcaligenes [98], and Rhodospirillum [99], which are common members identified in the aquatic ecosystems also. Therefore, the buildup of ammonia in the system by enzymatic conversion of nitrogenase and other indirect means stimulates the process of nitrification in the presence of oxygen, oxidized to NO 3 ̅ by specific bacteria (AOB, NOB) and archaea (AOA) respectively [100,101].

Anaerobic ammonium oxidation (Anammox)
Anaerobic ammonium oxidation (anammox) with nitrite as an electron acceptor is a recently discovered pathway of the nitrogen cycle. Anaerobic ammonia-oxidizing bacteria (AOB) catalyze ammonium directly to nitrogen gas with nitrite as the electron acceptor under anoxic conditions, is considered to be the significant contributor to the removal of inorganic nitrogen other than denitrifiers [79]. Hydrazine, a unique intermediate of anammox, is formed from nitric oxide and ammonium by a hydrazine-forming enzyme and is subsequently oxidized to dinitrogen gas by a hydrazine oxidizing enzyme (hzo), a member of the octaheme cytochrome c hydroxylamine oxidoreductase protein family. Hzo gene is the potential functional marker for the anaerobic ammonia oxidation which explored the abundance and distribution of anammox groups in different marine ecosystems with high niche-specific community structures [102]. Anammox process is mediated by a group of bacteria within the phylum Planctomycetes. All five described anammox genera CandidatusBrocadia, Kuenenia, Scalindua, Anammoxoglobus, Jettenia have been detected in different wastewater treatment systems. The abundance and diversity of anaerobic ammonia-oxidizing bacteria in eight different nitrogen removal reactors had been studied with molecular techniques such as 16S rRNA gene analysis, FISH, and real-time PCR [103]. Planctomycetes specific 16S rRNA gene primers were used to detect the anammox bacteria and it was only Candidatus Kuenenia stuttgartiensis was the organism identified alongside the ammonia oxidizers in oxygen-limited environments in the reactor operated for more than 500 days [104]. The abundance and distribution of the hzo gene unraveled high taxonomic diversity than the 16S rRNA gene-related diversity shown in a single group, whereas the correlation of gene abundance determined by real-time PCR with the environmental factor suggested that the nitrite concentration is the prominent factor regulating the anammox population [105]. One of the other functional genes that determine the anammox group in the community is the Scalindua-nirS gene of Candidatus scalindua which represents the putative cytochrome cd1-containing nitrite reductase gene (nirS) and it is completely discrete from the nirS gene of denitrifiers [106]. Anammox community utilizes electron donors for energy conservation including acetate, H 2 , formate, and simple organic compounds yielded during the breakdown or fermentation of polysaccharides present in the EPS [107]. Using the rRNA and non rRNA based approaches together would comprehensively provide complete information on the anammox bacterial population activity, abundance, and dynamics in a particular ecosystem [21].

Physiochemical parameters influencing nitrogen metabolism
Physiochemical parameters are correlated to bacterial diversity and distribution [108]. The efficacy of the bacterial consortia controlling the ammonia level was evaluated by measuring water quality parameters such as total ammonia, nitrate, and pH of the water whereas, the bacterial community structure was studied using denaturing gradient gel electrophoresis revealed the presence of both ammonia-oxidizing and nitrite-oxidizing bacteria [109]. Environmental conditions are one of the factors strictly related to the presence of AOA. The possible niches of AOA are based on pH, sulfide, and phosphate levels as it was reported that AOA might be active within the nitrogen cycle in low-nutrient, low-pH, and sulfidecontaining environments [110]. Nitrification in the bacterial film of the biofilter involves biological processes of waste treatment governed by different parameters such as substrate and dissolved oxygen rate, organic matters, temperature, pH, alkalinity, and salinity. The impacts of physio-chemical parameters upon nitrification kinetics can give a better understanding of the performance of biofilters [11]. The retained dissolved organic solids were reduced by aerobic and anaerobic digestion processes within the biofilm of a membrane aerated bioreactor [111]. Drying of soil with sunlight reduced soil pH while tilling tended to increase pH. Pond bottom soils containing black and glutinous organic sludge are required to be treated before the shrimp culture crop follows because of the toxic materials and high numbers of shrimp pathogens get accumulated to form the previous culture [112]. A decrease in the optimum temperature of about 7-9 °C showed a combined reduction of 10% nitrification in the immobilized biomass reactors (Laboratory-scale continuous stirring tanks) [29]. The value of water quality depends strongly on the species, size, and culture objectives. In water reuse systems, fine solids, refractory organics, surface-active compounds, metals, and nitrate may become important since they directly correlated with the nitrification of the system [10].

Nitrifiers and their application in bioreactors
A detailed study on the abundance and transcription levels of specific gene markers of total bacteria, ammoniaoxidizing beta proteobacteria, nitrite-oxidizing bacteria (Nitrospira), and denitrifiers were analyzed using quantitative PCR (qPCR) and reverse transcription qPCR showed different variant response for each parameter by each target group [113]. Ammonia oxidizing bacteria and the nitrate oxidizing bacteria functions in the removal of nitrogen from the municipal wastewater system with partial nitrification under the anoxic condition in the continuous plug flow processes [114]. Coupling aerobic consortiums like enriched autotrophic nitrifying and heterotrophic denitrifying consortiums implied a huge potential in the removal of heavy nitrogen from lowstrength domestic wastewater [115,116]. Performance of the nitrifying bacteria revealed the increase in nitrification potential concerning granulation of the nitrifying sludge in a continuous flow airlift reactor (ALR) [117]. The hydrodynamic parameter in continuous flow airlift bioreactor is the main aspect in the improvement of nitrification potential for the granulation of the nitrifying bacterial groups in ALR [118]. Data on the denitrification process in a bioreactor under RAS are very few. Proper design of a denitrification reactor based on a comprehensive understanding of the dynamics of nitrogen, carbon, and other inorganic nutrients is highly recommended for RAS [73]. NOB composition in the sequencing batch reactors (SBR) sustains with distinct NOB composition at low F: M-C: N conditions [119]. Ammonia oxidation by Candidatus Nitrospira and the coexistence with Nitrosomonas determine the dominance based on the copper levels in the full-scale groundwater treatment bioreactor [120]. Particle image velocimetry (PIV) analysis attained with a larger velocity gradient and stronger shear stress in rotating flat sheet membrane bioreactor (RFMBR) employed to start up anammox process, in comparison with a conventional membrane bioreactor (CMBR) [121]. Nitrifying bacterial communities studied in the bench-scale submerged bed bioreactor revealed that the changes in the hydraulic retention time will significantly affect the community structure of the nitrifying bacteria [122]. Microbial density and population structure of the nitrifying bacterial community in the membrane bioreactors (MBR) determine the efficiency of the bioreactor [123][124][125]. Anaerobic ammonia oxidation rate in a fluidized bed bioreactor or the effective removal of nitrogen was found to be a successful method with a high nitrogen loading rate and short hydraulic retention time with good sludge settling properties [126].

Molecular approaches in the study of microbial diversity
Nucleic acid-based methods such as genetic fingerprinting techniques provide the genetic structure of the bacterial population in the community and are being developed for the assessment of the physiological activity of specific groups and their contribution to ecosystem processes. The composition of microbial communities is typically determined by analysis of the 16S rRNA gene, and functional gene cloning comprehends the information on phylogeny including genetic diversity of different taxon, their richness, and relative abundance indicating the hegemony of functional groups. The advantages of molecular methods enable the targeting of specific functional groups, providing finer scale analysis of the activity of microbial groups using association, diversity of operational taxonomic units (OTUs) [127], microbial shift in the total population, and their genetic distinctness in the communities.
The cultivation-based approaches of nitrifying communities are aggravated due to isolation, enumeration, and identification difficulties of these organisms per se of ammonia oxidizers. The 16S rDNA-based procedures have been successful in ecosystem studies to monitor the ecological population shifts and quantify microbial populations. Diversity assays based on the 16S rRNA gene sequences are referred to as versatile due to the availability of the multi-dimensional data in combination with high throughput analyses such as metagenomics, proteomics, transcriptomics, and lipidomics [23]. Microbial diversity has been defined as the range of the significantly different populations of microorganisms and their relative abundance in an assemblage or community. Microbial ecology in the natural environment can be described using PCR based profiling methods including RFLP (Restriction Fragment Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism), ARDRA (Amplified Ribosomal DNA Restriction Analysis), DGGE (Denaturing Gradient Gel Electrophoresis), TGGE (Temperature Gradient Gel Electrophoresis), ARISA (Automated ribosomal intergenic spacer analysis), Ribosomal Intergenic Spacer Analysis (RISA) and RAPD (Random Amplified Polymorphic DNA) [128], FISH (Fluorescent In situ Hybridization) [129]. Molecular techniques like ARDRA and RFLP are too sensitive to characterize community diversity with reliable and robust information. Low-resolution techniques such as DGGE, and hybridization techniques using high definition probes [25] helps to monitor the changes at the community level. Techniques based on 16S/18S rRNA/rDNA were found to be more classic, and accurate and offer evidence on molecular characterization, microbial population, and classification scheme than traditional methods [130]. The coexistence of AOB and NOB in the wastewater treatment system was revealed using the terminal restriction fragment length polymorphism (TRFLP) targeting the 16S rRNA gene and ammonia monooxygenase (amoA) functional gene study [131]. Taxonomical identification of the eubacterial population based on the 16S rRNA genes with a small number of primers is an effective alternative to the conventional phenotypic and biochemical identification [132]. Archaeal and bacterial community diversity at two different decomposition phases (initial methanogenic phase (IMP) and stable methanogenic phase (SMP) were examined using 16S rRNA PCR based 454 pyrosequencing [133]. Likewise, 16S rRNA PCR amplification explored the widespread archaeal population in the water and soils [134].

Denaturing Gradient Gel Electrophoresis (DGGE)
DGGE is a molecular fingerprinting method that separates PCR-generated DNA amplicons to determine microbial composition. During DGGE, PCR products encounter increasingly higher concentrations of chemical denaturant as they migrate through a polyacrylamide gel. The patterns of DGGE bands are based on the different sequences of DNA (from different microbes) denature at different denaturant concentrations. Each band theoretically represents a different microbial population present in the community. Muyzer [135] explored that DGGE is a reliable, reproducible, rapid molecular tool that allows the study of the complexity and behavior of microbial communities. DGGE allows the simultaneous analysis of multiple samples to understand the community changes over time. PCR analyte molecule electrophoresed in a DGGE gel remains double-stranded until it reaches the denaturing gradient, and when it reaches the gradient that melts (not the GC clamps) [129]. Multivariate DGGE band patterns of 16S rRNA gene amplified DNA from the replicate samples of the different soils of grasslands were analyzed statistically using ordination of canonical variates to explain the community variation in the soils [136]. PCR-DGGE technique is highly useful in 16S rDNA clone screening and microbial diversity [137]. PCR-DGGE band profiles of species-specific amplicon were investigated for the identification of microbial groups in commercial probiotic yogurts and lyophilized products, was turned out to be an appropriate cultureindependent approach for rapid detection of the predominant species in mixed probiotic cultures [138]. Phylogenetic analysis of DNA sequences retrieved from 16S rDNA PCR-DGGE analysis of river biofilm samples showed that plastidial DNA of phototroph eukaryotes accounted for the diversity of microbial assemblage [139]. Reduction in the microbial composition and the species abundance due to the heavy metal contamination was analyzed using the different band profiles obtained in 16S rDNA PCR DGGE [140]. Two strong cadmium resistant bacterial strains were found to be dominant in the DGGE band profile among the microbial diverse cadmiumresistant bacterial strains, and these groups were selected based on morphological, physiological, and biochemical characteristics, also 16S rDNA sequencing [141]. Microbial dynamics and the community shift of the photosynthetic bacteria were highly revealed in the PCR-DGGE bands of the photohydrogen producing reactor scrutinized using PCR-DGGE [142]. The combination of both culture-dependent and the PCR DGGE approaches reveals the microbial diversity and dynamics during sorghum fermentation, an essential component of starter cultures in the commercial production of ting [143].
The community profiling and characterization of amoA genes, determining abundance and growth of Thaumarchae, and the total population of mesophilic Crenarchae (ammonia oxidizers) was carried out using the DNA finger printing technique [46]. Community analysis of the ammonia-oxidizing bacteria has been done using inosine containing newly designed amoA gene primers for the PCR-DGGE to resolve the different band patterns than universal primer bases [144]. At different denaturant gradient concentrations of the DGGE gel, Nitrosospira and Nitrosomonas sequences were migrated revealing the microbial diversity of the ammonia-oxidizing bacteria [145]. DGGE was a suitable technique for the identification of important members of the microbial community in the artificial consortia [146]. The sequence retrieved from the excised DGGE bands revealed the dominant species of the oxidizing populations which provides insight idea into the diversity and distribution of the ecologically important group in membrane bioreactors [147].
DGGE band profiles of Ammonia oxidizers were identified and characterized using specific hybridization with oligonucleotide probes for the comparison of the community composition of multiple samples over time [148]. PCR-DGGE is a robust and cost-effective approach that helped the identification of Alphaproteobacteria and Betaproteobacteria taxa that remained in the roots of the transplants [149].

Statistical Analysis using DGGE band profiles
DGGE band patterns were analyzed and clustered using Quantity-One 4.5.2 software package (Bio-Rad, USA) using the unweighted pair-group method and Canonical correspondence analysis (CCA) using CANOCO 4.5 (Biometrics, Wageningen, Netherlands). This work suggested the relationship between environmental variables and bacterial community structure in the eutrophic lake sediments [150]. DGGE analysis supported with nonmetric multidimensional scaling (NMDS) analysis (considering the relative intensity of each band) proved to be a powerful tool to monitor community structure changes, allowing the throughput which is required to study ecological variations [151]. Nei and Li [152] developed the theory of mathematical expressions that is dependent on the assumption that all nucleotides are distributed at random over the DNA sequences with given G + C content. DGGE profiles were analyzed in three ways [153] Page 12 of 21 Rangaswamy and Singh Green Technology, Resilience, and Sustainability (2022) 2:5 as i). Bacterial diversity was compared between samples by using the Shannon-Wiener index (also known as the Shannon-Weaver index). ii). A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. iii). Individual DGGE bands and their intensities for different samples were compared using logistic regression analysis. Changes in the microbial community structure of the matured biofilm formed in the urban river were determined by non-metric multidimensional scaling analysis from DGGE patterns [154]. The stress value in an NMDS plot representing the similarity of the community structure between the different soil samples was calculated using the Kruskal stress formula [155]. Significant changes in the total bacteria and ammonia-oxidizing bacterial communities were estimated using Bray-Curtis similarity index and < 0.1 stress values [156] using Primer 6 software (Primer-E Ltd., Ivybridge, UK; http:// www. primer-e. com/). [157] performed principal component analysis (PCA) with Pirouette 2.6 software package (Infometrix Inc., USA) to determine the change in the community structure over time, by calculating the amount of variance accounted by each principal component represented by eigenvalue [158] and recommended that the study would be helpful to understand the dynamics of the microbial population with additional molecular approaches. Functional principal component analysis (FPCA), a multivariate analysis method based on DGGE indicates the neighborhood structure of the microbial community [159].

Amplified ribosomal DNA restriction analysis (ARDRA)
Amplified ribosomal DNA restriction analysis (ARDRA) is a simple restriction procedure based on restriction endonuclease digestion of the amplified bacterial 16S rDNA and it is a highly accepted method for taxonomical identification, also phylogenetic relationships within and between populations. The selected restriction enzymes based on theoretical digestions cleave the known 16S rDNA (rRNA) sequences and would obtain restriction patterns that are normalized and can be combined utilizing the pattern recognition and analysis software [56]. Finally, numerical analysis allows the individual representatives to be grouped according to the similarities in their combined ARDRA patterns. Restriction typing and mapping of DNA from different isolates provide a convenient method for fingerprinting species, varieties, and even individual strains [160]. ARDRA is an efficient technique for the identification of closely related DNA groups A. calcoaceticus, and A. baumannii belonging to the Acinetobacter group [161], also a highly discriminatory method for the identification and elucidation of the ecology [162]. It is a promising approach to evaluate the evaluation of the changes in the communities of wastewater treatment plants caused by modifications in influent composition, temperature, and other operational conditions [163]. Archaea and bacterial population distribution and their dynamics were determined using ARDRA and it has been characterized phylogenetically that revealing the metabolic variations in the community [160]. Double enzyme digestion patterns were compared to study the difference between activated sludge bacterial communities fed on industrial and domestic waster collected from oxic and anoxic reactors under different operational conditions and were evaluated using the dice coefficient method [163]. The bacterial community of the grassland was characterized using amplified rDNA restriction analysis correspondingly denaturing gradient gel electrophoresis (DGGE) to compare the dynamics and greater replication in the community of different soils in grasslands [136,164]. Similarly, the Bacterial diversity of the anaerobic ammonia-oxidizing bacteria has been investigated using 16S rRNA gene clone libraries, and the functional genes quantified using quantitative real-time PCR [133].
Screening of virtual patterns of restriction and simulation of the sites before the experimentation optimizes the ARDRA technique being the most simple method to determine phylogenetic relationships between organisms concerning the taxonomic delineation level [165]. Certainly, such phylogenetic analyses supported the taxonomic profiling and evolutionary distance with the different microbial genomic patterns i.e. division of Archaea into Crenarchaeota and Euryarchaeota and existence by their functional adaptations [134], identification of the majority of lactic acid bacteria present in the wine making process [166], comparative differentiation of the Ralstonia and Pandoraea species [165], the relationship between species of the lactic acid bacteria developed during fermentation of grape [166], identification, characterization, and differentiation of Azotobacteraceae species belonging to the free-living nitrogen-fixing bacterial group found in soil samples [167]. Rapid assessment of many individuals at once is one of the major advantages of PCR fingerprinting, thus making it particularly useful for preliminary analysis of variation in different regions of DNA before sequence analysis [168].

Dendrogram and Cluster Analysis
The ARDRA restriction patterns are defined as the different haplotypes for each enzyme and the samples are characterized by the same combination of haplotypes obtained with various enzymes will be grouped, defining a phylotype which is often used to term the 16S rRNA clones differentiated by restriction enzymes [169]. Haplotype diversity (H) was estimated using the Shannon -Weaver index [170] which was calculated as: whereas, p i is the frequency of the i th haplotype. Genetic distance between phylotype is calculated using the Nei and Li coefficient [152] and Dice coefficient [171] using the equation: in which n xy is the number of restriction fragments shared between individuals, whereas n x and n y are the total numbers of all restriction fragments in the individuals x and y, resulting distance matrix used for the UPGMA (Unweighted Pair Group Method with Arithmetic mean) cluster analysis [171] and the Neighborjoining mid-point analysis using NTSYS pc Ver.2.2 [172]. The consistency of each node was estimated by the bootstrap method of at least 1000 replications [173]. Similarity matrices based on the presence or absence of comigrating restriction fragments were analyzed with UPGMA using NTSYS, Fitch Margoliash, and KITSCH analysis using PHYLIP [168].

Software and online tools for the 16S rDNA Sequence Data Analysis
16S sequence artifacts or the sequence errors, chimeric sequences can be detected and filtered to reduce the noise during diversity calculation using different software such as DECIPHER [174], UCHIME [175], Black Box Chimera Check (B2C2) [176], Chimera slayer [177]. 16S rRNA gene sequences of the bacterial and archaeal population in complex microbial communities exhibit statistical, bio-informatics, and computational challenges [178]. Sequences obtained by different molecular fingerprinting techniques applied using PCR amplified rRNA genes of environmental DNA samples provide the population composition of the complete communities [179]. Further processing of the sequences can be achieved with different sequence processing software packages such as MOTHUR [180] with command-based language in C + + , QIIME [181] written in Python, and also analysis platforms altogether like Galaxy project [182] which includes i) quality control i.e. data cleaning and optimization, ii) sequence alignment, iii) taxonomic classification using different relational databases for the easy positioning to the pre-existing alignments i.e. RDP [183], SILVA [184], Greengenes [185], and iv) OTU clustering and diversity analysis. Different data visualization framework (HTML5) tools support the visualization of outputs of these sequence processing platforms i.e. Phinch-http:// phinch. org [186], Biological Observation Matrix (Biom) format meta-data files from the computational pipelines were used for the visualization of the exploratory data, (5) H = − pilnpi (6) D = 1 − 2 n xy / n x + n y and secondly, Krona [187] chart resembling a pie chart sub divided into different segments embedded with hierarchy of taxon, sectional areas representing the magnitude of relatedness to the particular taxa.
Microbial community analysis based on the rDNA sequences was also carried out using the SILVAngs online pipeline (https:// www. arb-silva. de/ ngs/) where the multiple fast format files representing the samples to be loaded as a single project. It is a data analysis service for the rRNA gene-based sequences which uses SILVA rRNA database projects, alignments, and taxonomies [188,189]. VITOCOMIC2 [190] is another online tool available for the combined mapping of the taxonomic visualization and determination of evolutionary distance. VITCOMIC compares the 16S rRNA sequences (conducting gene copy normalization) of the user especially the uncultured taxa with the reference sequences in the database by CLASTV, a graphics processing unit-based sequence identity search tool. VITCOMIC2 comparison with the statistical coefficients Jaccard similarity, Pearson correlation, and Yue and Clayton theta similarity coefficients for pairwise community comparison helps out to identify the difference between the different communities. The functional profiles of the microbial communities can be predicted using 16S rRNA gene dataset with Tax4Fun [191], an open-source R software package freely available for download at (http:// tax4f un. gobics. de/). Tax4fun is a powerful tool for the characterization of phylogenetic and functional diversity, assessing and comparing the metabolic structure of the microbial communities.
As a matter of interest, several other tools also would be operable for the processing of sequences and data visualization; Gene Tool (Bio Tools Incorporated) a platform-independent software, and graphical user interface (GUI) using a special language called Smalltalk prominently used for DNA sequence analysis, chromatogram, sequence editing, to assemble and alignment of sequences, also primer designing. EzTaxon (http:// www. eztax on. org/), is a web-based tool used for the sequence similarity search, pairwise similarity calculation, multiple sequence alignment, and the construction of phylogenetic tree [192]. The set of sequences after the similarity search could be transferred for further processing with the following programs PHYLIP (http:// evolu tion. genet ics. washi ngton. edu/ phylip. html), MrBayes (http:// mrbay es. csit. fsu. edu/), and PAUP (http:// paup. csit. fsu. edu/). Molecular Evolutionary Genetics Analysis (MEGA (http:// www. megas oftwa re. net/)), the program works on both GUI and command-line interfaces and comprehends the evolutionary patterns of genes and genomes based on the sequence comparative analysis of protein and DNA [193]. The software helps in the calculation of mean distance, and evolutionary distance of different populations using various distance models such as nucleotide substitution models, gamma distances and rates, heterogenous patterns, and the construction of the phylogenetic tree. The Interactive Tree Of Life-iTOL (https:// itol. embl. de) is another tool for the visualization, and manipulation of phylogenetic trees, especially QIIME2 trees and their associated annotations [194]. The online software promotes the visualization of phylogenetic trees in the file format like a plain text file, Newick, Nexus, PhyloXML,.jplace files generated by RaxML, pplacer, and.qza trees generated by QIIME2.

T-RFLP
T-RFLP is one of the molecular techniques used in comparative analyses to sketch out the genetic differences between the species in the population of a community. It is the method of measuring the polymorphism in the terminal restriction fragments of the PCR amplicon. TRFLP fingerprint analysis was used to track the bacterial communities functioning in the denitrifying bioreactors using 16S rRNA and nosZ genes, which showed that the microbial groups represented were responding in a consistent manner resulting that the wastewater treatment systems need not be chaotic [195]. Based on the cloning analyses T-RFLP fragment sizes were not predicted from the database, not identified in the clone library, and it could be achieved with more specific primers [196]. Additive Main Effects and Multiplicative Interaction (AMMI) model, principal component analysis (PCA), and detrended correspondence analysis (DCA) were the robust statistical methods that provide perfect ordinations for T-RFLP fingerprinting analysis by comparing different multivariate ordination methods to explore diverse microbial communities. Samples of higher heterogeneity were well described using nonmetric multidimensional scaling (NMDS) with Sorensen and Jaccard distance, exploring the complex gradients in the community [197]. There are online tools like MiCA (http:// mica. ibest. uidaho. edu/) with (APLAUS +) and (PAT +) available for the insilico restriction digestion of 16S rDNA fragments from database using different enzymes to predict the restriction patterns [198]. T-RFLP analysis Expedited (T-REX), a web-based tool that facilitates the analysis of T-RFLP data by producing data matrix complexity with interaction effects and sample heterogeneity. AMMI is the analysis model used [199].

RISA
RISA is based on the PCR amplification of the intergenic region between the 16S and 23S rRNA genes in the rRNA operon. The 16S/23S intergenic region targeted with oligonucleotides resembles the significant heterogeneity of the bacterial community. The resultant band patterns correspondingly represent the profile and composition of the bacterial population in the particular community of the dataset [200]. In ARISA, PCR is performed with a fluorescence tagged oligonucleotide primer and subsequently, the electrophoresis is carried out with an automated system facilitating the laser detection of fluorescent DNA fragments. The technique of using ARISA with the combination of a genetic analyzer (GA-ARISA) due to its higher resolution and reproducibility, would be the better option to determine the changes in the population [201]. Electropherograms of ARISA was precisely quantified by using the GeneScan analysis software and the similarity levels among communities were assessed using indices like Sorenson's and Jaccard's [202]. Screening of specific microbes of large-scale processing multiple samples with fixed gel concentration I possible by RISA method by resolving higher and sensitive gradients of finger printing patterns [203].

Real-time Polymerase reaction
The real-time PCR technique was used to quantify the multiple copies of the gene in a DNA and to compare the different samples of DNA to determine the gene abundance and estimate the functional interaction between the microbial populations in the community. Amplifying different samples at equal concentrations will enable us to calculate the sample which has the highest copy number of the target of interest. Replicates of DNA or RNA extracted have to be analyzed for each sediment or water sample to minimize biases associated with sample heterogeneity and extraction efficiency on working quantitative PCR [40]. The efficiency of the reaction can be precisely calculated using real-time PCR which is one of the benefits of other approaches. A real-time PCR study for quantification of nitrifying populations showed a high correlation to nitrification performance, a good indicator of stable nitrification [204]. Combined analysis of DGGE with real-time PCR determines the abundance of AOB and AOA congruently characterizes the microbial community, their diversity, composition, and population shifts [205]. The Nitrifier population forms a role model for the total microbial community. Increased nitrite concentrations not only signal a disruption of nitrifiers but possibly also of the total configuration of the microbial community [206]. In a wastewater treatment system, the magnitude of the AOB population in a bioreactor under a stable nitrification state was found to be more dominant than the AOA community, estimated using real-time PCR [207]. Quantification of functional genes such as NirK [83,93,208], NirS [83], NosZ [93], NarG [93,209], revealed the community shift, abundance and the metabolic interaction of the operational microbial flora particularly ammonia-oxidizing bacteria [210], denitrifiers [211], in different ecological niche [212]. Hydrazine-oxidizing enzyme (hzo) gene quantification suggested that Anaerobic AOB consists of 16% of the total bacterial population in the anammox reactor [104]. The significant quantification of the AmoA gene of AOA and AOB showed a correlation with the influent and effluent water of the highest ammonium concentrations at seven different wastewater treatment plants [43]. The C-methylation of C2 and C7 is possibly catalyzed by NirE, NirDL, NirGH, and NirJ (dissimilatory nitrite reductase genes) involved in symmetrical reactions [213]. The current knowledge on denitrifying genes and their compared genetic organizations emerges by way of using new sequences resulting from the analysis of microbial genomes with special attention to the clustering of genes encoding different classes of reductases [80]. Nitrifying and denitrifying communities could be analyzed by parallel DNA extractions and clone library construction of functional genes i.e. amoA, norB, nirS and nosZ which is useful in the study of bioremediation strategy and understanding of nitrogenous metabolites through functional genes [213]. Quantification of nitrifying and denitrifying genes (nirK, nirS, nosZ) using real time PCR gives a detailed in-sight on the community structure of the activated sludge in wastewater treatment plants [214].

Conclusion
Molecular ecological approaches have revealed some of the conundrums and it is still a way ahead for a precise understanding of the functional interaction mediated changes in the community diversity and dynamics of microbes within bioreactors. The microbial communities functioning inside the reactors are dynamic and complex, and likely impact on process efficiency and stability of the bioreactor [215,216]. NGS techniques are newfangled and intricate, going on advancing by day which is very essential in the current scenario. However, it is as expensive as the expanse of information obtained from the technique which even increases with the increase of samples. PCR-based molecular techniques are equally factual and comprehensive with the appropriate computational and analytical methods, and software [23]. These techniques are a bit time-consuming but very efficient in the study of multiple sample sets to be analyzed e.g., in the case of microbial ecological change in different time frames. Using NGS in the relevant scenarios and hypotheses would improve the quality of research to a greater extent. Molecular characterization of the microbial communities in the PPBR sampled at different stages of the system operation starting from the designing, development, and installation. The fate of the microbial population in the reactor from the activation period till the completion of the treatment process and the trend in the microbial community structure during each stage of the duration were analyzed using DGGE in combination with statistical interpretation of the shift in the bacterial population and ARDRA based phylotypes analysis and identification of the community profile of the bacterial consortium. At the same time, the count of samples likewise in NGS which requires a huge investment.
The utility of both conventional and contemporary technology is possible by patterning the methodology/ research plan. Furthermore, the combination of fingerprinting techniques and NGS would allow exploring potential data amplifying the microbial ecology than preferring one [217]. Fingerprinting techniques provide necessary information on the trends in the community structure, changes, and dynamics of different samples and it is sensible of opting NGS techniques to go into detail on the specific sample from that point to get in-depth data [198] of the community mapping and functional interactions. Microbial ecology studies are too crucial and it is mandatory to have a better strategy on molecular approaches to be used. Whichever the technical approach may represent, scrupulous understanding and skill are equally important to confront the technical bias using artifacts, especially in the computational analyses. The outcome from these molecular/technical aspects thus presents the foresight technology of the bioremediation leading to a safe and effective environment ahead.