Simultaneous heterotrophic nitrification and aerobic denitrification potential of Paenibacillus sp. strain GLM-08 isolated from lignite mine waste and its role ammonia removal from mine waste water

Nitrification and denitrification processes mediated by microorganisms are the most cost-effective nitrogen bioremediation techniques (Padhi & Maiti 2017; Pang & Wang 2021). Concurrent nitrification and denitrification by a aerobic conversion of ammonium (⁠⁠) to N₂ gas occur naturally in habitats such as biomass films, activated sludge, wastewaters, lakes, and benthic habitats (Liu et al. 2010; Palacin-Lizarbe et al. 2019). Simultaneous nitrification and denitrification (SND) is an efficient system to treat wastewater, where nitrification and denitrification occur simultaneously in the same reactor under like operational settings (Schmidt et al. 2003). The conventional biological nitrogen removal is mediated by a two-step process, where aerobic nitrification is carried out by autotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) which are slow growers with strict growth requirements. The efficiency of the nitrification process is limited by the low growth rate of nitrifying bacteria and their sensitivity to pH, oxygen, temperature, and toxic compounds (Hu et al. 2004). The next step is denitrification, where the transformation of nitrate (⁠⁠) to N₂ occurs, which removes fixed nitrogen (⁠⁠) from the environment and returns it to the atmosphere in a biologically inert state (N₂). This process occurs in anoxic conditions under reduced settings, and it is one of the most important metabolic activity involved in the removal of nitrogen from different ecosystems (Devol 2015). Thus, a stringent, and entirely different reaction condition is required for the complete process of nitrification and denitrification, and this makes the function of the nitrogen removal system quite complicated. The discovery of heterotrophic bacteria with the ability to perform simultaneous nitrification and aerobic denitrification has overturned this traditional concept (Robertson et al. 1988).

Earlier, most experts thought that denitrification by heterotrophs could only occur in anoxic environments (Guo et al. 2016). Several studies have characterized the role of heterotrophic bacteria in the nitrification process, and it was also observed that many of them were capable of aerobic denitrification as well (Zhao et al. 2017; Liu et al. 2019; Gupta et al. 2022). The main metabolic pathway of SND nitrogen removal is → NH₂OH → → → → NO → N₂O → N₂. The schematic diagram of the traditional ammonia nitrogen metabolism pathway is presented in Figure 4. Bacterial isolates with the property of SND were derived from municipal activated sludge, saline-alkali lake sludge, marine sediment, piggery wastewater treatment, and lake-wetland sediment (Liu et al. 2010; Ravishankar et al. 2022). The first bacterium Thiosphaera pantotropha having the potential for SND was discovered in the 1980s (Robertson et al. 1988); since then several isolates from diverse sites have been characterized. Bacterial strains reported for SND activity include Comamonas sp. (Patureau et al. 1997), Pseudomonas stutzeri (Su et al. 2001), Alcaligenes faecalis (Joo et al. 2007), Providencia rettgeri (Taylor et al. 2009), Acinetobacter calcoaceticus HNR (Zhao et al. 2010), Anoxybacillus contaminans HA (Chen et al. 2016), Bacillus cereus GS-5 (Rout et al. 2017), Acinetobacter sp. ND7 (Xia et al. 2020), Alcaligenes faecalis WT14 (Chen et al. 2021), and Pseudomonas mendocina X49 (Xie et al. 2021). Several bacteria (Bacillus sp. LY, Rhodococcus sp. CPZ24, Pseudomonas tolaasii Y-11, and Ochrobactrum anthropic LJ81) with SND activity have been efficiently utilized for nitrogen removal from different wastewater systems (Zhao et al. 2010; Chen et al. 2012; He et al. 2015; Lei et al. 2019). Heterotrophic bacteria with SND ability are rapid growers, have potential to utilize different carbon sources for denitrification under aerobic conditions. Due to the simple operation condition and cost-effective nature of the process, growing attention has been given on the application of SND process in nitrogen removal in wastewater treatment systems (Rout et al. 2017; Xia et al. 2020). Heterotrophic denitrifiers carry out the denitrification process by using organic carbon sources as electron donors. This works well in wastewaters with a high C/N ratio (Chung et al. 2014).

Denitrification is one of the crucial biogeochemical processes, which serves as the primary sink for fixed nitrogen (Knowles 1982). The nitrogen metabolism and removal pathways have been studied in diverse bacterial strains; however, a clear understanding of the entire heterotrophic nitrification-aerobic denitrification process is still awaiting (Chen et al. 2016; Padhi & Maiti 2017; Qiao et al. 2020). It is important to understand the mechanism of heterotrophic nitrification-aerobic denitrification process for its operation in wastewater treatment. Denitrification involves four enzymes which mediate the conversion of oxides of nitrogen to dinitrogen gas. A membrane-bound reductase (nar) or periplasmic reductase (nap), encoded by the narG or napA gene, respectively, reduces to in the first step. One or both reductase encoding gene, narG and napA are present in the denitrifying bacteria (Smith et al. 2007; Reyna et al. 2010). Cytochrome cd1 and Cu containing nitrite reductases (encoded by the nirS and nirK genes, respectively) reduce to NO, nitric oxide reductase (encoded by the norB) reduces NO to N₂O and nitrous oxide reductase (encoded by nosZ) reduces N₂O to N₂ (Capone et al. 2008). The process can be negatively affected when nitrite accumulation occurs, and the denitrification activity of the bacteria is inhibited. Functional genes encoding enzymes relevant to the denitrification process possess conserved sites and they have been used as molecular markers for the detection and enumeration of relevant bacteria (Bonilla-Rosso et al. 2016). The narG, napA, nirS, nirK, and nosZ genes are used as a biomarker to explore the diversity of denitrifiers (Bonilla-Rosso et al. 2016; Chen et al. 2022). Denitrification is especially significant in wastewater treatment since it removes undesired nitrates and ammonia, lowering the risk of eutrophication and other negative outcomes from the water discharged from the treatment plants. Pilot scale denitrifying bioreactors have been applied for treating nitrogen contaminated mine water. High nitrate removal rates at low temperatures, and without any unwanted ammonium or nitrous oxide production, have shown promising results in these sites (Kiani et al. 2020).

In this study, a new strain capable of heterotrophic nitrification and aerobic denitrification was isolated from lignite mine waste in Barmer Basin, Rajasthan, India. The bacterium was identified as Paenibacillus sp. strain GLM-08 based on its phenotypic and 16SrRNA gene-based phylogeny. The nitrification and denitrification activity of the isolate were investigated in heterotrophic nitrification (HN) and denitrification (DM) medium supplemented with ⁠, and as sole N source. The functional genes relevant to nitrification and denitrification were amplified and the probable pathway of nitrogen conversion is also proposed. The efficiency of the bacterium in total nitrogen removal in real wastewater derived from mine tailings was studied for its application in wastewater systems.

Paenibacillus sp. strain GLM-08 was isolated from a soil sample obtained from Giral lignite mine sites (26.0557°N, 71.2526°E) in Barmer, Rajasthan, India. The enrichment technique was used with a growth medium, containing 0.5 g/L of ammonium sulphate, 5.62 g/L of sodium succinate, 0.5 mg/L of phenol red indicator, and 50 mL of trace element solution, pH 7.5 (Sarathchandra & Chalcroft 1978). The composition of trace elements solution includes (per liter): 5 g of K₂HPO₄, 2.5 g of MgSO₄·7H₂O and NaCl, 0.05 g of MnSO₄·4H₂O and FeSO₄·7H₂O. The soil sample was incubated at 28 °C in a rotary incubator for 21 days. The pure isolate was achieved from the system by streaking onto a peptone-meat extract agar.

Biochemical and physiological characterization of the isolated bacterium was done (Holt 1994). The specific tests are presented in Table 1. The appearance and morphology of the isolates were observed on the nutrient agar plates.

Genomic DNA isolated from bacteria was used as templates to amplify the 16S rRNA gene using universal primers (27F: 5′-AGA GTT TGATCC TGG CTC AG-3′ and 1492R: 5′-GGT TAC CTT GTTACG ACT T-3′). Standard PCR conditions were used (Lane 1991). Amplified PCR products were resolved by agarose gel electrophoresis, purified and nucleotide sequences were obtained.

Sequence similarity was analyzed with the program BLAST and other available nucleotide databases. Similar sequences retrieved from Genbank were aligned using multiple sequence alignment (MSA) programs. Molecular evolutionary genetics analysis software (MEGA 4) was used to construct phylogenetic trees (Tamura et al. 2007).

Key genes (hao, nxrB, narG, narA, nirS, nirK, norA, nosZ, and nif) having a role in N₂ fixation, nitrification and denitrification were studied by PCR based amplification. The genomic DNA of the bacterium was used as a template for PCR amplification using specific primers sets (Supplementary Table 1). The PCR products were resolved by agarose gel electrophoresis (1% w/v) in 1X TAE buffer with 0.5 μg/mL of ethidium bromide.

The nitrifying and denitrifying activity of the isolated bacterium was tested with varying nitrogen sources (50 mg/L of (NH₄)₂SO₄, NaNO₃, and NaNO₂) in the medium. A basal medium with the following composition 3.00 g/L starch, 1.00 g/L Na₃C₆H₅O₇, 1.00 g/L K₂HPO₄·3H₂O, 0.01 g/L FeSO₄·7H₂O, 0.01 g/L MnSO₄·4H₂O, 0.30 g/L KH₂PO₄, 0.05 g/L MgSO₄·7H₂O, and 3.00 g/L NaCl was used (Li et al. 2015). Three sets of 500 mL Erlenmeyer culture flasks containing basal medium supplemented with either (NH₄)₂SO₄, NaNO₃, or NaNO₂ as nitrogen source were inoculated with the Paenibacillus sp. GLM-08. The initial density of the bacteria was adjusted (OD600 ≈ 0.4) in each conical flask for the different nitrogen sources. Basal medium without inoculum was used as a control for each condition. The sample was incubated at 28 °C in a rotary incubator for 24 h under aerobic conditions in a shaker operating at 120 rpm. Periodically, 20 mL aliquot of the sample was taken and used for measuring the cell density, pH, and -N, -N, and -N content. To measure ⁠, and samples were centrifuged, and the resulting supernatant was passed through 0.45 μm pore size membrane. Ammonium (⁠-N) concentration in sample was analyzed by spectroquant ammonium cell test kit (Merck). Nitrate (⁠⁠) and ion concentration was estimated at by IC Plus 883, auto ion analyzer, Metrohm. Each experimental set was performed in triplicate. The removal efficiencies (REs) of -N, -N, and -N were calculated using the following equation: REs (%) = (C₀–C_t)/C₀ × 100%, where C₀ and C_t are the initial and the final concentrations of -N, -N or -N (Li et al. 2015).

The mine water collected from tailing pond in Giral lignite mining site, Rajasthan was used to study the potential role of the isolate in nitrogen removal in real wastewater. The parameters analyzed for the mine water sample is presented in Table 2. The experimental set included, (i) treatment 1 (mine water with 0.05% (w/v) sodium citrate and 1% (v/v) inoculum of Paenibacillus sp. GLM-08), (ii) control (mine water without carbon source and no bacterial inoculum, this set was used as blank control to get rid of the influence of native microorganisms), (iii) treatment 2 (mine water adjusted with NH₄Cl to reach a concentration of 124.6 mg/L ⁠, with 0.05% (w/v) sodium citrate and 1% (v/v) inoculum). All the experimental flasks were incubated at 28 °C with the shaking speed of 120 rpm. The experiments were conducted for 48 h in 500 mL Erlenmeyer flasks containing 250 mL mine wastewater. During treatment, cultures were sampled periodically to determine the optical density (OD600), TN and -N. During the experimental phase, solutions from each flask were cultured on denitrification medium (DM) to enumerate the potential denitrification strains (Lei et al. 2019). The denitrification medium (DM) consists of Na₂HPO₄ 4.2 g, KH₂PO₄ 1.5 g, MgSO₄·7H₂O 0.1 g, Na₃C₆H₅0₇ 4.7 g and trace elements 2 mL (per liter) (Lei et al. 2019), KNO₃ as sole nitrogen source (Lei et al. 2019).

The statistical analysis for ⁠, and removal efficiency Tukey's test was applied (P < 0.05 was taken as the significant difference). Each experiment was done in triplicates, and the results are presented as means ± SD (standard deviation of means). Standard deviations in this experiment were calculated by Microsoft Excel.

Paenibacillus sp. strain GLM-08 was isolated from lignite mine waste in the Barmer basin, Rajasthan, India. The colony has jagged edges and is harsh, opaque, and fuzzy white. The isolate Paenibacillus sp. strain GLM-08 was found to be a short rod-shaped gram-negative bacterium during morphological examinations. The physiological, morphological, and biochemical characteristic of the isolate is presented in Table 1. The isolate showed a positive result for methyl red, indole, citrate utilization, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melibiose, sucrose, L-arabinose, and mannose, however, was negative for Voges-Proskauer test, H₂S production, catalase, and oxidase activity.

In the condition where NaNO₃ is used as the sole nitrogen source, initially concentration was recorded as 49.7 mg/L, a gradual decline in the ions in the medium was observed as 89.2% of ion was removed in 12 h. In presence of as the sole N source, the bacterium was able to grow exponentially, and the highest cell density was recorded in 12 h. In the denitrifying medium with NaNO₂ as the sole N source, the initial condition was 49.35 mg/L, and a gradual decline in the concentration was observed and nearly 93% of was removed at the end of 16 h. Some amount of was accumulated during the initial 12 h of incubation which declined thereafter and was not detected after 24 h. This indicated potential nitrification by the isolate. The simultaneous denitrification ability of the isolate led to the complete removal of nitrogen when ⁠, or was used as the only nitrogen source. Similar phenomena of SND are also reported in several heterotrophic bacteria derived from diverse habitats (Zhang et al. 2017; Lei et al. 2019; Xia et al. 2020). If we compare the growth pattern of the isolate, we observed a correlation between an increase in the cell density and the subsequent removal of nitrogen. In the presence of either of the nitrogen sources, the initial OD was kept between 0.4 and 0.425, the isolate showed nearly no lag phase perhaps due to high inoculum density, and displayed exponential growth which stabilized by 8–12 h. It was during this period maximum nitrogen removal was recorded thus confirming the rate of removal was related to bacterial growth. The removal efficiency of the bacterium was recorded 100% for ⁠, ⁠, and after 24 h of incubation. The average nitrogen removal rate of the strain was found to be 4.775 mg/L/H, 5.66 mg/L/H, and 5.01 mg/L/H for ⁠, ⁠, ⁠, respectively. The nitrogen a removal rate was highest when -N was used in the medium followed by -N and -N. The average nitrogen removal rate of the isolate Paenibacillus sp. strain GLM-08 was comparable to or greater than that reported for several bacteria studied for SND ability. A recent study reported 89.7% removal of -N in 12 h with an average removal rate of 3.81 mg -N /L/h by Acinetobacter sp. ND7 (Xia et al. 2020). Similar studies have been done for Bacillus sp. LY with removal efficiency of 0.43 mg -N /L/h (Zhao et al. 2010), Rhodococcus sp. CPZ24 with removal rate of 3.4 mg -N /L/h (Chen et al. 2012), Pseudomonas tolaasii Y-11 with removal efficiency of 2.04 mg -N /L/h (He et al. 2015), and Ochrobactrum anthropic LJ81 with removal rate of 3.846 mg -N /L/h (Lei et al. 2019).

During the SND process, we observed a nearly negligible amount of accumulation, which was completely removed from the system by 24 h. However, any inhibition to the total and removal was not recorded, and 100% removal efficiency was achieved in 24 h. There are reports on some of the strains (Proteus mirabilis strain V7 (Zhang et al. 2014) and Chryseobacterium sp. R31 (Kundu et al. 2014) capable of SND where accumulation during the process occurred. This led to a reduction in the total nitrogen removal efficiency of the bacterial strains perhaps due to the inhibition of denitrification ability. Based on the results, it could be concluded that the strain Paenibacillus sp. strain GLM-08 exhibited tremendous capability for heterotrophic nitrification and aerobic denitrification, and nitrate or ammonia might be the suitable nitrogen source for the strain.

It has been observed that nitrogen concentration varies significantly in wastewater obtained from different sources, from low strength ammonium to even very high strengths reaching to more than 100 mg/L (Wan et al. 2009; Ashok & Hait 2015; Chen et al. 2018). Low temperatures limit the course of numerous biochemical processes, the most sensitive of which is the nitrification process. One such treatment facility is the municipal and industrial wastewater treatment plant in Vistula River Oswiecim (Poland), where inoculation with cultures of nitrifying bacteria is the sole effective method for restoring stable nitrification following disturbances (Paśmionka et al. 2021). Considering the complexity of real waste mine water which contained various inorganic and organic ions and indigenous microbes, bioaugmentation with Paenibacillus sp. strain GLM-08 provide clear evidence for the application of this bacterium in terms of wastewater treatment, even in situations where high nitrogen load exists.

To understand the mechanism of heterotrophic nitrification and aerobic denitrification, the role of potential enzymes in the process was deciphered by PCR amplification of key genes (hao, nxrB, narG, narA, nirS, nirK, norA, nosZ, and nif) having a role in N₂ fixation, nitrification, and denitrification. In Paenibacillus sp. strain GLM-08, three potential genes, nxrB, nosZ, and nirS, were amplified. Amplicon specific to nxrB (450 bp), nosZ (500 bp), and nirS (400 bp) genes was obtained with Paenibacillus sp. strain GLM-08 genomic DNA as the template (Supplementary Figure 1). While hao, napA, narG, nirK, norB, norC, and nifH genes could not be amplified. The presence of nosZ and nirS genes effectively point to denitrification ability in Paenibacillus sp. strain GLM-08. In this study, three functional genes (amoA, amoB, and hao) have a role in the conversion of to was also targeted, however, any amplification was not obtained. Further, the genes encoding specific nitrate reductase (napA and narG) and nitrite oxide reductases (norA) were also not amplified using the specific primer sets. It is quite possible that this bacterium may contain these genes with sequences sufficiently divergent from the primer sequences used in this study. However, the confirmation of existence of the three essential genes nxrB, nirS, and nosZ further suggests that Paenibacillus sp. strain GLM-08 was capable of heterotrophic nitrification and aerobic denitrification. Based on our results, a probable pathway for the process is depicted in Figure 4. In this pathway (Figure 4), the main function of the ammonia monooxygenase enzyme encoded by amoA is to catalyze the conversion of ammonium to hydroxylamine (NH₂OH) (Soler-Jofra et al. 2021). The amoA has been widely regarded as a functional and phylogenetic marker gene for ammonia oxidizers (Zhao et al. 2019). Hydroxylamine oxidoreductase encoded by hao gene is responsible for the dehydrogenation of hydroxylamine to nitrite (the second step of ammonia oxidation) (Bergmann et al. 2005). The next major enzyme involved in one-step oxidation is nitrite oxidoreductase (NXR), a membrane bound NXR enzyme which catalyzes nitrite to nitrate conversion (Daims et al. 2016). The amplification of the nxrB gene in Paenibacillus sp. strain GLM-08 points towards the role of NXR enzyme in nitrite oxidation (nitrification). The conversion of nitrate to nitrite (⁠-N → -N) during denitrification is catalyzed by nitrate reductase. Two kinds of functional genes encoding nitrate reductase, namely, napA and narG are known (Sparacino-Watkins et al. 2014). The presence of nirS gene in this strain confirms the transformation of to nitric oxide. This gene has been extensively used for analyzing the diversity of denitrifying bacteria (Capone et al. 2008). The norB gene encoding the membrane-bound nitric oxide reductase subunit (NORB) catalyzes the denitrification step of NO → N₂O (Braker & Tiedje 2003). The last denitrification step involves nosZ gene, which aids in the conversion of N₂O into free nitrogen. Based on overall results, nitrification-denitrification metabolic pathway of -N → -N → -N → NO → N₂O → N₂ could be proposed for Paenibacillus sp. strain GLM-08 under aerobic conditions.

Paenibacillus sp. strain GLM-08 isolated from a lignite mining site demonstrated simultaneous nitrification and denitrification potential in presence of either ⁠, or as the sole nitrogen source. The nitrogen removal efficiency of the bacterium was recorded nearly 100% for ⁠, ⁠, and after 24 h of incubation. The strain showed the highest nitrogen removal during the exponential phase of growth in the presence of / / as the sole N source. Among the three N sources tested, this bacterium had the highest N removal rate when was used in the medium. This bacterium was also found to be highly effective in nitrogen removal from mine wastewater containing complex chemical constituents. Bioaugmentation of mine wastewater with Paenibacillus sp. strain GLM-08 demonstrated N removal of 86.6% even under high (<100 mg/L) concentration of ⁠. The efficiency of this bacterium in nitrogen removal from mine wastewater provides clear evidence for the application of this bacterium in treatment of wastewater containing high nitrogen load along with other inorganic and organic substances which may be toxic. The PCR amplification results confirm the presence of essential genes nxrB, nirS, and nosZ relevant to the nitrification and denitrification process. The confirmation of these genetic determinants in Paenibacillus sp. strain GLM-08 indicate the metabolic and enzymatic pathway involved in SND process, however, further studies are required to characterize the detailed molecular mechanism involved.

Authors are grateful to Banasthali Vidyapith, Rajasthan for providing necessary support and research facilities.

Animal models were not used in the experiments conducted for this manuscript.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.