Microbial strains were isolated from activated sludge samples collected at two distinct wastewater treatment plants. The first sample originated from domestic sewage treatment and was collected after complete wastewater treatment. The second sample was retrieved directly from the effluent treatment system of a textile industry before the clarification tank. Both samples were collected using sterilized vials and refrigerated until processing. The samples underwent decimal serial dilution with peptone water (0.1%) (Merck, Darmstadt, Germany) to a final concentration of 10^-5 cells for microbial isolation. The samples were then inoculated in duplicate onto R-2A agar plates (Difco, Detroit, MI, USA) and incubated at 30 °C for 5 days. Colonies exhibiting distinct morphologies from the initial plates were selected for further replication, resulting in pure cultures.

The previously isolated microorganisms were cultivated in 10 mL of liquid R2A medium for 48 hours to analyze phosphate granule production. The R2A medium used in this study was composed of (per liter) 0.5 g yeast extract, 0.5 g proteose peptone, 0.5 g casamino acids, 0.5 g glucose, 0.3 g dipotassium phosphate (K₂HPO₄), 0.3 g magnesium sulfate (MgSO₄$\cdot$7H₂O), 0.3 g sodium pyruvate, and 15 g agar. The pH was adjusted to 7.2 before autoclaving. After incubation, the microorganisms were stained using Neisser’s method for polyphosphate detection [4]. Isolates showing positive results were selected to test their inorganic phosphorus removal capacity. The experiment was again conducted in a R2A liquid medium, with total phosphate measured using a spectrophotometer (Model SP-22, Biospectro, São Paulo, Brazil) through colorimetric analysis of vanadomolybdophosphoric acid, following Standard Methods 4500-P [1]. Measurements were taken immediately after inoculation and again after 48 hours. Cultures were kept at 30 °C in a shaker (Model MA420, Marconi, Piracicaba, Brazil). Hydrolysis or digestive oxidation was not required since the targeted phosphate was inorganic (orthophosphate or reagent phosphate). For the analysis, 3 mL of the cultivated cultures were centrifuged in 1.5 mL microtubes at 8000 rpm for 5 min to precipitate the cells (centrifuge, MiniSpin® model, Eppendorf SE, Hamburg, Germany). Following centrifugation, 1.3 mL of the supernatant was diluted with 5.7 mL of distilled water (reducing the phosphate concentration to the method’s detection range) and analyzed using a previously obtained standard calibration curve to determine the inorganic phosphate concentration.

The effluent samples used for phosphorus removal testing were collected from two distinct origins: a local fish-processing industry and domestic wastewater treatment. Samples were refrigerated until filtration through 47 mm glass microfiber filters (Millipore, Burlington, MA, USA) and subsequent sterilization. A pre-culture medium and inoculum of the previously isolated LAMA1607 strain were prepared for the experiments. Pre-culture vials were prepared in 125 mL Erlenmeyer flasks containing 25 mL of nutrient broth and grown at 30 °C for 48 hours. The effluent samples (300 mL each) were divided into two duplicates: one enriched with glucose (0.5%) and another non-enriched. Each replicate was inoculated with 1 mL of the pre-culture medium and incubated at 30 °C with agitation at 150 rpm. A control vial containing 100 mL of sterile effluent sample was also prepared. During growth, pH, microbial growth, and orthophosphate concentration were monitored at 0 h, 24 h, and 48 h intervals.

Phosphorus was quantified using colorimetric analysis of vanadomolybdophosphoric acid, following Standard Methods 4500-P [1]. Solutions A and B were prepared as follows. For solution A, 25 g of ammonium molybdate was dissolved in 300 mL of deionized water. Solution B was prepared by dissolving 1.25 mg of ammonium metavanadate in deionized water by boiling, cooling, and then adding 330 mL of HCl. Solutions A and B were then mixed and diluted to 1 L. The pH was adjusted to 7.0 according to the Standard Methods [1]. A standard phosphate solution was prepared by dissolving 2196 mg of KH₂PO₄ in 1 L of deionized water. A calibration curve was constructed using five different concentrations of the standard phosphate solution (4, 7, 13, 16, and 18 µM). Absorbance readings were measured at a wavelength of 460 nm (see Supplementary Material for details). Sample replicates were prepared for analysis by mixing 35 mL of the sample with 10 mL of the colorimetric reagent and adjusting the final volume to 50 mL using deionized water. Absorbance was measured at 460 nm. Microbial growth was monitored by measuring absorbance at 600 nm. A calibrated pH meter probe (Mettler Toledo, Zurich, Switzerland) was used for all pH measurements. All readings were performed at the same predetermined time intervals. Experimental graphics were generated using the ggplot2 v.3.5.1 package in R (R Project for Statistical Computing, Vienna, Austria; https://ggplot2.tidyverse.org) [2].

Sixty microorganisms exhibiting diverse morphologies were isolated from the two inoculated wastewater samples (domestic and textile industry) onto R2A agar plates. These samples were split equally between the two sources (30 isolates each). Following initial isolation, 17 colonies were excluded due to contamination. The remaining 43 isolates (23 from domestic wastewater and 20 from textile wastewater) were evaluated using Neisser’s staining to assess the presence of polyphosphate inclusions. This analysis revealed that 65% of isolates from domestic wastewater and 85% from textile wastewater stained positive for polyphosphate (Fig. 1A).

Fig. 1.

Preliminary phosphorus removal results. (A) Percentages of total isolates based on polyphosphate accumulation and the presence of polyphosphate granules, as determined by Neisser’s staining method, in domestic (Sample I) and textile industry effluents (Sample II). (B) Isolates can remove phosphorus and their respective phosphorus removal percentages after 48 hours of incubation.

Subsequently, the inorganic phosphorus removal capacity of isolates stained positive for polyphosphate inclusions was assessed. Among the 43 isolates tested, 19 exhibited significant phosphorus removal activity. Notably, isolates from textile industry wastewater displayed a higher removal rate. In total, 10 of the 17 polyphosphate-positive isolates from this source were capable of phosphate removal, whereas only 8 of the 15 positive isolates from domestic wastewater demonstrated the same ability (Fig. 1B). Interestingly, the remaining 13 organisms displayed increased phosphate concentrations after the 48-hour incubation period, suggesting that not all isolates with detectable polyphosphate inclusions possessed phosphate accumulation capabilities. For further characterization, isolate 15 from domestic wastewater was chosen due to its demonstrably high phosphate removal efficiency (Fig. 1B). This isolate was subsequently designated LAMA1607 and served as the target organism for the remainder of this study.

Regarding total phosphate removal, the domestic effluent experiment did not achieve significant removal after 48 hours, with no variations observed between the enriched and unenriched sets (Fig. 2). Conversely, the enriched fish effluent experiment displayed a 70% reduction in initial phosphate concentration by 48 hours, while the non-enriched set exhibited a 30% reduction (Fig. 2A). Microbial growth patterns differed between enriched and unenriched sets across both effluent types (Fig. 2B). In the domestic effluent experiment, the enriched set exhibited a rapid growth spurt in the first 24 hours, followed by a decline (decay phase), which suggests the utilization of readily available carbon. In the fish effluent experiment, the enriched set displayed the most pronounced growth during the first 30 hours (exponential phase), followed by a plateau (stationary phase). Conversely, the non-enriched set in both experiments exhibited a lag phase initially, followed by a slower growth phase (exponential phase). The pH of the culture medium differed between enriched and unenriched sets across both effluent types (Fig. 2C). In the domestic effluent experiment, the enriched set exhibited minimal variations throughout the incubation period. The non-enriched set also displayed minimal variations, with a slight increase in pH after 24 hours. Conversely, the fish effluent experiment revealed a significant decrease in pH in the enriched set, dropping from an initial value of 7.0 to 3.78 by the end of the experiment. The non-enriched fish effluent set exhibited a slight increase in pH (Fig. 2C).

Fig. 2.

Phosphorus removal, microbial growth, and pH results for fish and domestic effluent. Measurements were performed at 0 h, 24 h, and 48 h while considering a glucose-enriched substrate and a non-enriched substrate, as well as a control for both variables. Samples were divided between the effluent source (domestic sewage treatment and fish industry wastewater). (A) Phosphorus removal: Absorbance measured at a wavelength of 460 nm. (B) Microbial growth: Absorbance measured at a wavelength of 600 nm. (C) pH measurements.

By analyzing the 16S rRNA BLAST sequences and the phylogenetic tree (Fig. 3), LAMA1607 was identified as Priestia megaterium since this species showed the highest sequence similarity to LAMA1607 in the analysis.

Fig. 3.

Phylogenetic tree of Priestia megaterium LAMA1607. Maximum-likelihood tree showing the phylogenetic relationships of 16S rRNA gene sequences of P. megaterium LAMA1607. Bootstrap 500 replicates.

Genome assembly using CLC Genomics Workbench resulted in 5,234,874 bp, divided into 20 contigs, with an L50 of 3, an N50 of 1,017,811, and a GC content of 38.0%, summarized in Table 1. The RAST software presents the genome annotation divided into subsystems and pathways, which are already known by their role (Fig. 4). In total, 5540 coding sequences (CDs) were identified, forming 325 subsystems and 79 RNAs. A portion of 76% of the genes are not associated with them, while 24% are. The most common subsystems were amino acids and derivatives (360), carbohydrates (313), protein metabolism (195), cofactors, vitamins, prosthetic groups, pigments (160), and nucleosides and nucleotides (109). A total of 19 CDs were associated with the phosphorus metabolism subsystem.

Fig. 4.

Subsystems identified in Priestia megaterium LAMA1607. Overview of the subsystem categories assigned to the P. megaterium LAMA1607 genome. A pie chart was generated using the Rapid Annotation using Subsystem Technology (RAST) server (blue); 76% of genes are related to the identified subsystems features, while 24% are not (green).

Additionally, the antiSMASH server [11] annotation identified eight biosynthetic gene clusters responsible for secondary metabolites, including NI-siderophore, phosphonate, RRE-element containing, type III PKS, and tRNA-dependent cyclodipeptide synthase (Fig. 5B). Table 2 summarizes the 15 enzymes found after manual curation using the Enzyme Commission (EC) number, based on the list of Association of Manufacturers and Formulators of Enzyme Products (AMFEP), which lists the main enzymes with biotechnological applications.

Fig. 5.

Circular view of the genome and biosynthetic gene clusters. (A) A circular view of the genome assembly was generated using PATRIC software. Data from outermost to innermost: contigs (dark blue); forward coding sequences (green); reverse coding sequences (purple); antimicrobial genes (red); virulence factors genes (orange); transporters (blue); drug targets (black); GC content (lilac); GC skew (orange). (B) Gene clusters responsible for synthesizing secondary metabolites were identified using the antiSMASH server. CDS-FWD, Coding Sequences - Forward Strand; CDS-REV, Coding Sequences - Reverse Strand; AMR, Antimicrobial Resistance Genes; VF, Virulence Factors.

The PATRIC server annotation also illustrated 5540 CDs, divided into 3814 proteins with functional assignments, 1726 hypothetical proteins, and 79 RNAs. From these 3814 proteins, 1448 were assigned to subsystems, and 885 were assigned to pathways. The most common subsystems were ribosomal proteins, single-copy (42), ribosome large subunit (LSU), bacterial (36), flagellum (34), cell division initiation-related clusters (28), spore germinant receptors (27), and glycerolipids and glycerophospholipids (27). Furthermore, 95 infectious disease-related genes were identified (Fig. 5A).

In total, 27 phosphorus removal-related genes were found in the P. megaterium LAMA1607 genome; of these, eight encode transport proteins, three encode regulatory proteins, twelve encode enzymes, two encode enzymes related to phosphorus incorporation, and two encode enzymes related to polyphosphate (polyP) granules (Table 3).

When comparing the two sets of replicates across effluent samples (enriched and non-enriched), the enriched sample obtained better phosphorus removal rates in both cases. However, this can be easily explained by the metabolic phosphate demand during substrate utilization. These phosphorus removal results also correlate with the genetic findings of P. megaterium LAMA1607.

Further investigation also showed that phosphate utilization was higher in the effluent from the fish processing industry. This evidence is supported by the presence of volatile fatty acids (VFAs), an essential nutrient for phosphate utilization in EBPR systems [12, 13]. Considering the amino acid-rich substrate provided by the fish effluent, the drastic drop in pH also suggests a possible metabolic utilization alongside glucose. There was a minor increase in pH in the non-enriched set, indicating a potential uptake of carbonic acids, such as acetate, in the absence of glucose. This is supported by earlier evidence showing the importance of VFAs in EBPR systems [14, 15]. Furthermore, the low phosphate removal and low content of VFAs in the domestic effluent might have affected these results. However, despite low phosphate removal, significant growth can still be observed in the enriched and domestic effluent samples. This can be explained by the presence of genes responsible for adaptability to low phosphorus availability in the substrate, detected through gene annotation. The non-enriched set registered higher pH readings after 24 hours than the fish effluent samples, which is further evidence of the utilization of carbonic acids, as mentioned before.

Gene annotation has elucidated that P. megaterium LAMA1607 can interact with phosphorus in different ways and displayed other biotechnologically relevant enzymes for effluent treatment and industrial use, including hydrolytic enzymes, lipases, proteases and amylases (Table 3), alongside biosynthetic gene clusters of interest, such as NI-siderophore (Fig. 5B). These findings demonstrate a genetic repertory of products of interest for biotechnological applications, available for cloning and further use in other works [10, 16] as they are not only significant for the existence of the organism itself but also useful for effluent treatment. Moreover, the genome size and characteristics of LAMA1607 resonate with previous bacterial genomes that possessed biotechnological potential.

Among the regulatory genes detected (Table 3), three were connected to the phoPR system: a two-component signal transduction system that consists of a histidine kinase, which is a sensory protein (phoR) located in the cytoplasmic membrane, and a cognate response regulator (phoP). The third component is a membrane-associated metal binding protein (phoU) that does not control phoR activity but helps to regulate intracellular phosphate metabolism. Furthermore, a previous study proposed that phoU can inhibit phoR when the pst complex actively transports phosphate, operating as a mediator between the systems [17]. This system controls the Pho regulon and mediates the adaptation under phosphate limitation [18]. The most common members controlled by the phoPR are extracellular enzymes, Pi-specific transporters (such as pstSCAB and ugpBAEC), and enzymes involved in storing and saving the nutrient [19].

Among the transport proteins, four were related to the pstSCAB ABC-type transporter. These consist of a periplasmic protein that binds the phosphate and leads it to the membrane channel (pstS), two transmembrane proteins that establish the transport channel (pstA and pstC), and a dimer whose function is to provide adenosine diphosphate (ADP) for the transport (pstB) [18]. Similar to the pstSCAB operon, the ugpABCE genes encode an ABC transport system for glycerol-3-phosphate and glycerophosphodiester, consisting of two ABC permease transporters (ugpA and ugpE), a periplasmic binding protein (ugpB) and an ATPase (ugpC). The regulation of ugp is mainly phoBR-dependent [20]. Both pstSCAB and ugpBAEC are high-affinity transporters. Thus, in effluents, P. megaterium LAMA1607 can continue to obtain phosphorus even at low exogenous concentrations, growing increasingly compared to other microorganisms without these transporters.

Regarding the enzymes, nine genes were related to alkaline phosphatases, monomeric enzymes that catalyze the hydrolysis of both phosphomonoesters and phosphodiesters [21]. Specifically, two were identified as alkaline phosphatases A and D (PhoA and PhoD). These enzymes are regulated by P availability via the two-component regulatory system phoBR. Phosphatase is considered a key enzyme for utilizing dissolved organic phosphate [22]. In addition to the alkaline phosphatases, an inorganic pyrophosphatase (Ppase) was discovered, which catalyzes pyrophosphate into two orthophosphate molecules [23]. Due to a lack of available orthophosphate in waters, the production of these two groups of phosphatases plays a crucial role in the effluent treatment by breaking complex phosphorus molecules and making Pi ready for the bacteria to assimilate.

Phosphonoacetate hydrolase (PhnA) was also found, homologous to alkaline phosphatases with the exception that it hydrolyzes a carbon–phosphorus bond, converting phosphonoacetate to acetate and inorganic phosphate [24]. Hence, It can be considered an extra source of inorganic phosphorus acquisition for incorporation by the microorganism.

Still among the enzymes, PhnP, a phosphodiesterase with specificity towards ribonucleoside 2^′,3^′-cyclic phosphates, was found. The phn operon encodes a multienzyme system that enables bacteria to metabolize organophosphonates when the inorganic phosphate is scarce [25]. However, the other operon components were not found in this research, although these genes may not have been present in the part analyzed during the sequencing/procedure. Another explanation could be that it is just an enzyme with a very similar structure to the one of interest (PhnP); therefore, experimental evidence is required to confirm the presence of the desired enzyme in the genome. Nevertheless, this protein can be part of a system that allows the bacteria to assimilate phosphate from organic molecules.

Two enzymes associated with polyphosphate granules were found. One is a polyphosphate kinase (PPK), related to the formation of the polyP granules by catalyzing the conversion from ATP into polyP and ADP [26], and the other one is an exopolyphosphatase (PpX), responsible for the polyP hydrolysis and Pi release [27]. These two enzymes are important for effluent treatment as they are considered crucial enzymes for EBPR from wastewater; they are also closely related to anaerobic phosphorus release and oxic phosphorus uptake [28]. For the storage of Pi, most bacteria induce the expression of PpK, which can accumulate polyphosphate as a Pi reservoir and enable its reuse when necessary [29].

Concerning the proteins related to the incorporation of phosphorus, the enzyme glyceraldehyde 3-phosphate dehydrogenase converts glyceraldehyde 3-phosphate and Pi to 1,3-bisphosphoglycerate, in the first step of glycolysis that leads to ATP formation. The last enzyme targeted was succinyl-coA ligase, which catalyzes succinyl-CoA conversion to succinate. In this reaction, a phosphoryl group is transferred to ADP (or Guanosine diphosphate (GDP)) to form ATP (or Guanosine triphosphate (GTP)) in the citric acid cycle [30]. In these reactions, phosphorus is incorporated by P. megaterium LAMA1607 through the formation and utilization of ATP, providing a function to the inorganic phosphate that has been removed from the effluent. From this analysis of the genomic data, a model has been constructed that contains all the identified mechanisms that may be important for effluent phosphorus removal (Fig. 6).

Fig. 6.

Proposed model for transport, accumulation, and incorporation of phosphorus in Priestia megaterium LAMA1607. The entire phosphorus metabolism is controlled by the regulatory system PhoPR, which recognizes the presence of phosphorus through interaction with PhoU and activates the transcription of genes that encode proteins responsible for the phosphorus removal mechanisms. Phosphorus is transported from the extracellular medium to the cytoplasm through the PstABCS and UgpABCE transporters. Alkaline phosphatases (PhoA and PhoD) and other enzymes (PhnA, PhnP, and PPase) break down different phosphorus-containing molecules, in turn contributing to the increase in inorganic phosphorus (Pi) in the intracellular environment. The organism incorporates the free Pi in the cytosol with the assistance of the enzymes glyceraldehyde 3-phosphate dehydrogenase and succinyl-CoA ligase. Two-component regulatory system PhoPR (PhoPR), phosphate transport system regulatory protein PhoU (PhoU), alkaline phosphatase A (PhoA), alkaline phosphatase D (PhoD), phosphate ABC transporter PstABCS (PstABCS), glycerol-3-phosphate ABC transporter UgpABCE (UgpABCE), phosphonoacetate hydrolase PhnA (PhnA), phosphodiesterase PhnP (PhnP), inorganic pyrophosphatase Ppase (Ppase), nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH) and coenzyme A (CoASH).