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Article

Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction

by
Jazmin M. Salazar
1,2,*,
Jessica Calle
1,
Steeven Pereira
1,
Paula Cordero
1 and
Carlos Matovelle
1,3
1
Centro de Investigación, Innovación y Transferencia de Tecnología, Universidad Católica de Cuenca, Vía a Bibin, Cuenca 010102, Ecuador
2
Laboratorio de Microbiología, Universidad Católica de Cuenca, Vía a Bibin, Cuenca 010102, Ecuador
3
Laboratorio Hydrolab, Universidad Católica de Cuenca, Vía a Bibin, Cuenca 010102, Ecuador
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(12), 9277; https://doi.org/10.3390/su15129277
Submission received: 7 March 2023 / Revised: 18 May 2023 / Accepted: 24 May 2023 / Published: 8 June 2023
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Abstract

:
Nitrification is part of the nitrogen cycle that occurs naturally in ecosystems. It is related to the presence of microorganisms and their metabolism, especially bacteria, which are involved in oxidizing compounds such as NH4+ and NO2− to NO3−. In this study, we evaluated the nitrification potential in 12 bacteria strains that belong to the genera Aeromonas, Bacillus, Buttiauxella, Mycobacterium, Paenibacillus, Serratia, and Yersenia, which are part of the cultivable microbial community from soil in a native forest and pine forest in The Labrado area within the Machangara micro-watershed in the Andes located in the south of Ecuador. This investigation aims to identify heterotrophic and lithoautotrophic strains using specific culture media for ammonium oxidative (AOL-AOH) and nitrate oxidation bacteria (ONL-ONH). The formation of nitrifying halos in the culture media allowed the identification of 10 strains with nitrifying potential. Five strains were from the pine forest, four were isolated from the native forest, and one strain was shared between both forests. The Serratia and Yersinia genera have a high NO2− oxidation capacity. Their inoculation in synthetic water rich in nitrogenous products allowed us to determine 40% and 94% nitrite reduction percentages and cell retention times of 20 to 40 days. Our results are promising for their possible potential use in environmental bioremediation processes through inoculation in wastewater for the biological removal of nitrogenous compounds.

1. Introduction

Soil is a complex and dynamic system that provides suitable habitats for various living organisms and produces a significant part of the biogeochemical cycle of elements such as nitrogen. The nitrogen cycle starts in the soil due to the activity of fixing bacteria that introduces N2 from the atmosphere. In addition, N in soils can also come from synthetic fertilizers, manure, and other crop inputs. Some microorganisms are present in the plant roots, and others live free in the soil. Nitrogen is fixated by bacteria and transformed into ammonia which is later oxidized to nitrite in the first place, and oxidized by the bacteria through the nitrification process to nitrate in the second step [1]. These bacteria are lithoorganotrophic organisms responsible for transforming nitrogen into an assimilable form for the plants as nitrates, which will be the main form in the trophic chain [2,3].
It is well known that chemolithoautotrophic and heterotrophic bacteria oxidize nitrite to nitrate, a process present in terrestrial and aquatic ecosystems. In aquatic ecosystems, the accumulation of nitrogen components can cause eutrophication. These nitrogen components can be treated in water through bioremediation with nitrification and denitrification microorganisms [4]. The microbial nitrification and denitrification in the soil contribute to 70% of the global emissions of N2O [5]. In addition to nitrogen fixation and its transformation to ammonia, the waste products of the cell metabolism of microorganisms and organic material (OM) decomposition are the favourite nitrogen source for plants and algae. Nitrate is also the substrate for the bacterial process of denitrification, which reduces nitrate to dinitrogen gas, N2. Most plants cannot use N2 as a nitrogen source; therefore, this gas is lost from the system as soon as it is produced. Furthermore, ammonia is present in the water as a positively charged ion [6].
There are different groups of ammonia-oxidizing microorganisms. Ammonia-oxidizing bacteria (AOB), which are included different bacteria that can generate ATP as reductant from ammonia oxidation and that use this energy to fix carbon dioxide [7]. These are considered obligated autotrophs because they fix their carbon dioxide throughout the Calvin cycle (Figure 1). Some of them, such as the Nitrobacter genera, are chemolithoautotrophies, and others, such as Nitrospina, use the reductive tricarboxylic acid reductive pathway to fix CO2. Many strains are known to have heterotrophic capabilities, and they are considered mixotrophic or facultative autotrophic [8]. Ammonia is their only metabolic and energetic source.
Nitrification is the process of converting ammonia to nitrite, and it begins with the step of nitrosation. Nitrosoma and Nitrosospira carry out this step through the action of the enzyme ammonia monooxygenase (AMO), which uses oxygen to produce hydroxylamine (NH2OH). Hydroxylamine is then oxidized to nitrite by another enzyme called hydroxylamine oxidoreductase (HAO).
Following nitrosation, nitration occurs, which is the oxidation of nitrite to nitrate. Nitrobacter and Nitrospira carry out this process through the action of the enzyme oxidoreductase (NOB), which uses oxygen to produce nitrate [9].
A secondary group that differs from the ammonia-oxidizing microbial communities was recorded and cultivated in 2005. The ammonia-oxidizing archaea (AOA) oxidize ammonia to nitrite, but it is assumed that they are predominantly autotrophs and do not use the Calvin cycle. The third group of bacteria that belong to the phylum Planctomycetes oxidize ammonia using nitrite instead of oxygen and produce N2 instead of nitrite. Their metabolism is anoxic; thus, they are obligate autotrophs and can produce denitrification [7].
Some heterotrophic bacteria and fungi are known to produce nitrification only through ammonia oxidation, but this process does not produce ATP. In general, the biological processes, particularly those from the nitrification metabolism, are affected by temperature, salinity, light, OM concentration, substrate concentration (ammonia or nitrite), pH, and oxygen concentration. In addition, the oxygen concentration in aquatic habitats, between the subsurface water, the sediments, and the soil, is a significant variable for microbial activity regulation and the distribution of nitrogen cycling [10].
Nitrification generally occurs in acid soils. The difficulty of obtaining acidophilic nitrifying agents in culture has been demonstrated, which shows the importance of heterotrophic nitrification in this type of system. It is well known that many nitrification bacteria types can be identified through their gene sequencing in their natural environment, but they still need to be identified in culture collections. Therefore, acidophilic autotrophic nitrifiers may exist but are resistant to cultivation. Salinity and temperature do not appear to place unusual restrictions on the conditions under which nitrification can occur [11].
At a general level, nitrite-oxidizing strains have been studied in some regions, such as the United Kingdom: “the influence of inorganic nitrogen management regime on the diversity nitrite-oxidizing bacteria in agricultural grassland soils” [12]. In one study in the Andes, specifically in the extreme environments of the Chilean highlands [13], which carried out bacterium identification in water and sediments, microorganisms which were extremophile but not directly correlated with the nitrogen cycle were found. Regarding studies of bacteria that intervene in the nitrogen cycle in Andean ecosystems, the following was found: the isolation of nitrogen-fixing bacterial strains [14,15] and the public effect of land use on the density of nitrifying and denitrifying bacteria in the Colombian Coffee Region; it should be considered that these are disturbed ecosystems and at lower altitudes. Molina [16], in their study of the activity of nitrifying microorganisms in a high-altitude Andean wetland, found Nitrosomonas, which oxidize ammonium to nitrite, but no possible oxidants of nitrite were detected. In addition, there have been studies on the isolation of bacteria and their use in the bioremediation of soils contaminated by organophosphates, especially those belonging to the genus Nitrosoma, as well as other studies on their use in contaminated waters [17,18].
None of the previous studies have been carried out in Ecuador, nor have they dealt with the nitrification potential. For this reason, this study aims to determine the nitrification potential of cultivable bacteria isolated from soils in a native forest and a pine forest in the Machangara micro-watershed, Azuay, Ecuador. It is crucial to determine the bacteria strains with nitrification potential and subsequently use these in terrestrial and aquatic ecosystem bioremediation processes [19,20,21,22], contributing in this way to the availability of nitrogen used in plant development.
In the Andean mountains of Ecuador, there are significant pine forest plantations, without them being native vegetation of the area. When the soils of these mountains are abandoned after having been used for cultivation and overgrazing, they are reforested with exotic species in a poorly executed attempt, without technical or scientific criteria, to remedy the damage caused by the loss of native vegetation [23,24,25]. This has led to the existence of specific “patches” of exotic vegetation, such as pine, within areas of ecological interest in the Andean mountains; for this reason, according to the study area, our research proposes comparing the microbiota (bacteria) of this exotic vegetation with that which exists in the native vegetation to identify the potential that these soils may have to harbor bacteria that can later be used for environmental remediation applications. One of the fundamental questions of this study is: whether there are differences in the number of possible strains of bacteria with nitrifying potential between the native forest and the pine forest.

2. Materials and Methods

2.1. Study Site

The sampling was carried out in the El Labrado sector (2°40′40.23″ S, 79°4′30″ W) (Figure 2) of the upper basin of the Chuico River tributary of the Machángara, located in the austral zone of Ecuador at 3400 m.a.s.l. The area has a daily mean temperature of 8 °C, a min value of 6 °C, and a maximum of 10.6 °C. The area is located between the provinces of Azuay and Cañar. The isolation of the area grabs the attention of many scientists as an important area for biological studies. The area is part of the biosphere reserve “Macizo del Cajas”, which is globally considered an area of high natural, scientific, and cultural value. This national park has a large number of elements that, as a whole, constitute an ecological heritage of great importance [26].
Along the watershed, the land use is distributed in populated areas (6.4%), crops (11.3%), highlands (paramo) (59.1%), native forest (1.2%), grasslands (9.3%), forest plantations (pine and eucalyptus) (4.2%), bushy vegetation (6%), other vegetation (1%), aquatic bodies (1%), and infrastructure (1%) [27].
This study sampled soils under vegetation cover with native forest and pine forest land use. At each site, we established 10 m2 plots with squares delimitated with a grid; we randomly selected ten quadrants per forest. As shown in Figure 3, one can see the “patches” of pine forest within the Andean ecosystem. For this reason, the research plots were built in the pine forest and the native vegetation, allowing us to compare two different ecosystems within the same area.

2.2. Samples Preparation and pH Measurement

Each quadrant collected 1 kg of soil sample at a depth of 10 cm (after removing the organic layer). For the microbiological analysis, we used 10 kg of sample per plot, which was composed of 10 subsamples of soil by Papen and Von Berg [28]. We also measured the pH and temperature in the soils. We took 10 g of soil from each plot and dissolved it in 990 mL of distilled water. The pH was measured using a HACH® model HQ40D pH meter in the native forest; we recorded a pH of 5.6 and a temperature of 15 °C. for the pine forest, a pH of 3.5 and a temperature of 13 °C were recorded.

2.3. Isolation and Identification of Bacterial Strains

Serial dilutions isolated the cultivable bacteria. We weighed 10 g of the composed soil sample, diluted in 90 mL of sterile distilled water. We isolated the strains with the striated technique over a Petri dish with nutrient agar culture medium TM MEDIA® and incubated them at room temperature for 16 to 24 h. The isolates were purified with successive plating. Subsequently, the bacteria strains were conservated in 15% glycerol and stored at 4 °C. Using the MALDI-TOF technique, we initially selected different morphotypes for the bacteria strain identification (gender and species) [29].

2.4. Bacterial Density by Most Probable Number (MPN)

The culture media were selected based on the methodology proposed by Papen [28] (Table 1). The media OAL, ONL, and ONH had a pH of 7 to 7.4, and OAH media had an adjusted pH of 8.6. For the preparation of the sample, the remains of organic material corresponding to the vegetal layer of the soil were removed to obtain a sample that was as homogeneous as possible. Once homogenized, 10 g of the sample was weighed and diluted in 90 mL of sterile water type 1.
Serial dilutions were made in test tubes containing the culture media described above. These were incubated at 30 °C for 3 to 6 weeks. After the incubation period, we added 100 µL of each dilution and 100 µL of methyl red indicator to a microdilution plate to identify the presence of nitrifying bacteria. We verified the presence of nitrifying bacteria by changing the color from yellow to red as positive.
Subsequently, we took 1 mL from all the positive dilutions and inoculated them in 9 mL of 0.85% NaCl saline solution. From this solution, we dispensed 5 µL in Petri dishes with nutrient agar TM MEDIA® and incubated at 25 °C for seven days. We verified the colony growth daily and registered the number of colonies after three days. The resulting data were analyzed with MPN calculator software 3.1 that allowed us to calculate the density of bacteria in one sample.

2.5. Evaluation of the Nitrifying Potential

Nitrifying potential detection was executed using selective solid media for lithoautotrophic and heterotrophic bacteria with 18 g/L of SIGMA® agar-agar and 0.3% methyl red indicator [30].
We prepared stock solutions from every sterile saline solution isolation and adjusted them according to the McFarland scale to 0.5 standard turbidities. Afterward, the bacteria were inoculated and incubated at 30 °C for 3 to 6 weeks, in 5 μL amounts from each Petri dish solution containing the culture media (two seedings per Petri dish and two replicates per isolate) [1].
Later, we verified the formation of the acidification halo (purple color) generated by the reduction in the pH. We measured the halo diameter (cm) in each incubation. This semiquantitative test shows the nitrifying potential of bacteria [31].

2.6. Detection of Oxidation Products (NO3−)

All strains that showed nitrifying potential and presented an acidifying halo bigger than 0.5 cm in diameter were subjected to the detection of oxidation products, basically the presence of nitrate and based on the selection criteria from Botello [1]. We used culture media ONL, OAL, ONH, and OAH, dispensed 200 mL per strain, and inserted them into bottles. To incorporate the strains into the culture media, we inoculated them in a 2% saline solution, adjusted according to the McFarland scale to 0.5 standard turbidities. Subsequently, we incubated them at 30 °C with a constant agitation of 150 rpm for 3 to 6 weeks.
We used spectrophotometry (spectrophotometer HACH®, model UV-VIS DR6000 Hach Company, 2019) to detect NO3− oxidation products using the cadmium reduction method from the 4500-NO3− NITROGEN (NITRATE) [32]. The resulting data were expressed as the presence or absence of NO3− oxidation products.

2.7. Application of Nitrifying Bacteria in a Synthetic Medium Rich in Nitrogenous Products

We used a synthetic medium rich in nitrogenous compounds, with a concentration of 376 mg/l NO2−, to register the change in the nitrification process [33]. The inoculation of the strains was made with stock solutions, adjusted according to the McFarland scale to 0.5 standard turbidities and poured into reactors containing 250 mL of synthetic medium [34].
All strains and their respective replicas were tested from day 15 with a time interval of 5 days until reaching 40 days. We generated six measurements on NO2− with spectrophotometry under the parameters from method 8153 [32], the “Ferrous Sulphate Method” (Hach Company, 2014), for each strain, respectively.

2.8. Experimental Setup

To evaluate the bacterial efficiency from the identified strains, a batch system was applied, for which we established 10 reactors with a volume of 250 mL of synthetic water rich in nitrogenous products that operated discontinuously, meaning that the reactors were filled during a fixed operating time and later emptied without a constant flow. In these reactors, the strain bacteria were inoculated (Figure 3). The reactors had aerobic conditions to detect the oxidation process. We controlled the dissolved oxygen concentrations of each reactor with a HACH Brand Luminescent/Optical Intellical sensor. We kept the oxygen level at a minimal value of 2 mg/L, similar to the experiment from [35]. This concentration guarantees that the reactors are always aerobic and that the organic matter degradation and nitrification processes are carried out. The reactor’s constant agitation system kept a complete mix and the minimum oxygen supply.
The experimental process makes it possible to know, before application on a larger scale, the efficiency of the microorganisms, minimum cell retention times, process speeds, and optimal operating considerations. Since the bacteria come from the soil, their use in water must be controlled, allowing us to know if they will effectively have a use in environmental remediation.
Using several controlled reactors with the same experimental design allows for evaluating the efficiency of each identified strain individually, since it is unknown how each will respond to this proposed experimentation.
All the systems worked with a cellular retention time (TRH) of 40 days. The aim was to verify and evaluate the proper time of the function of each bacteria strain applied in each treatment. In each reactor, an incubation system maintained and controlled the temperature at 30 ± 1 °C [36].

3. Results

3.1. Characterization of Strains and Bacterial Density

We characterized 12 cultivable bacterial strains; 5 were isolated from the pine forest soil: Bacillus pumilus, Aeromonas molluscorum, Bacillus muralis, Mycobacterium sp., and Yersinia kristensenii; and six from the native forest soil: Buttiauxella ferragutiae, Buttiauxella gaviniae, Yersinia enterocolítica, Paenibacillus stellifer, Serratia proteamaculans, Serratia plymuthica, and Aeromonas encheleia. The last one was present in both forests (Table 2).
The density of oxidizing nitrate lithoautotrophic bacteria in the native and pine forest soil was insignificant, with a confidence limit of 95%. However, we could identify the bacteria by changing the color after adding methyl red. The results indicated that the analyzed soil samples from both forests did not detect ammonia-nitrifying bacteria (lithoautotrophic and heterotrophic) or nitrite-oxidizing bacteria (heterotrophic).
Several possible explanations exist for the difference between nitrifying bacteria isolated from a pine forest and a native Andean forest. For example, environmental conditions such as temperature, humidity, nutrient availability, and pH can influence the composition of microbial communities and, thus, the presence and abundance of nitrifying bacteria. In this case, when the pH values were recorded, there were differences: the pine forest had a pH of 3 and the native forest a pH of 5.

3.2. Determination of Nitrifying Potential and Detection of Oxidation Products

The presence of an acidification halo allowed us to identify that 10 of the 12 strains (B. ferragutiae A6, B. gaviniae A19, Y. enterocolítica G23, S. plymuthica C2-1 isolated from the native forest; Bacillus pumilus b3R, A. molluscorum b5R, Bacillus muralis b17R, Mycobacterium sp. f2, and Y. kristensenii b2R9 isolated from the pine forest; and A. encheleia A1 found in both forests) showed nitrifying potential (Table 2). S. plymuthica had an acidified halo diameter of 1.6 cm, significantly more prominent than the rest. The strain Y. enterocolítica G23 had an acidity halo diameter of 1.3 cm (Figure 4). In the remaining strains, the acidification halos ranged between 1 and 1.2 cm. The A. encheleia A1 had the smallest halo of acidification, 1 cm. Serratia proteamaculans b1R14 and Paenibacillus stefellifer C2(2) strains in the native forest did not form acidification halos.
There are no standardized data for the values of the diameters. These indicate whether or not the metabolic process under study exists. Since the values were comparable for each bacterium, a descriptive statistical analysis was performed. The mean values for the diameters (standard deviation = 0.17) are presented in Table 2 (p < 0.05).
All culture media with the inoculated strains presented values of NO3− evidencing oxidation products.

3.3. Application of Nitrifying Bacteria in Synthetic Medium Rich in Nitrogenous Products

Figure 5 shows the temporal evolution of the nitrification processes for each species of bacteria evaluated in the synthetic medium. Although the nitrification process presents particular characteristics depending on the specific time of experimentation, this is analyzed to obtain conditions for the operation of biological reactors for wastewater treatment (Figure 6).
Two reactors (R1 and R2) were tested for each bacteria tested, each with the same operating conditions. Even so, the initial tests showed a particular behavior in several processes. For this reason, it was decided to test two reactors for each bacterium, showing that they could have different behavior, even if the established and controlled conditions are maintained.
Later on in the analysis, all nitrate reduction levels were similar to experiments carried out in anoxic reactors for denitrification, achieving in a discontinuous process the elimination of nitrates.
Table 3 shows the analysis of the results obtained in the nitrification process for each bacterium. Some bacteria showed greater efficiency in the oxidation of nitrites, evidenced by the amounts of nitrates recorded, starting from the same initial concentration.
On the other hand, there were strains in which there was no change in the amount of nitrate in their application since no concentrations of NO3− nitrates were found. According to the analysis summarized in the table above, the bacteria that had optimal performance in nitrification were Mycobacterium sp., Aeromonas molluscorum, Yersenia enterocolítica, Buttiavexella gaviniae, and Aeromona encheleia.

4. Discussion

The role of microorganisms in the nitrogen cycle in nature is frequently accepted. In different environments, nitrogen is oxidized mainly by autotrophic nitrifying microorganisms, including chemoautotrophic or chemolithoautotrophic bacteria. Nitrification and denitrification processes are the primary regulators of nitrogen retention in the soil, where bacteria (AOB) intervene in the oxidation from ammonium to nitrite [37], from nitrite to nitrate by BON, and nitrification, which is the passage from nitrate and mineralized N to nitrous oxide and molecular nitrogen. These steps require separate processes and strictly controlled conditions.
The soil samples analyzed do not present a significant variation in bacterial density. The isolated strains belong to a single physiological group detected, lithoautotrophic nitrifying bacteria; these results agreed with analyses carried out in this type of ecosystem, where it is evident that autotrophic nitrification is the main nitrification pathway in acid soils of a pine forest [38]. It has been reported that parameters such as pH, temperature, and altitude have influenced the structure of macro- and micro-communities in Andean ecosystems [13]. Some studies also indicated that altitude is another parameter that can control the activity of AOB and AOA bacteria [16].
However, recent studies have changed the paradigm of aerobic nitrification with the discovery of AOA and their role in this process, which is still under investigation, making it clear that there is no simple relationship between the oxidation rates and relative abundance of AOA and AOB [39].
In the Andean region of Colombia, the authors compared the structure and function of microbial communities in forest soils of different forest types. They found that the presence and abundance of nitrifying bacteria varied according to forest type, with forests with higher plant diversity and more nutrients in the soil having a higher abundance of nitrifying bacteria [40].
On the other hand, the researchers compared the diversity and structure of microbial communities in forest soils of different forest types in Brazil, including pine forests. The results showed that environmental conditions, such as temperature and soil acidity, influenced the composition of microbial communities and that pine forests had a higher abundance of nitrifying bacteria, which are adapted to drier and more acidic environments [41].
These studies support that bacteria with nitrifying potential may vary in abundance and composition depending on environmental conditions and microbial community composition in different forest types.
Research has reported that the genera described in this study have a nitrifying potential, as is the case of S. plymuthica C2-1, which was confirmed to be involved in the nitrification process in the study on the influence of microorganisms on the N cycle, in which the genus Serratia was also identified as a nitrifying bacterium [42]. The genus Bacillus was nitrifying based on the presence of the acidification halo in the two species evaluated. This genus was reported as nitrifying by Sakai [43] in his study on nitrite oxidation and reduction. The author assumes that the bacteria of this genus are facultative as they contribute to the process of nitrification and denitrification, which Mekuto [44] confirmed through a bioremediation process using Bacillus sp. consortia, achieving nitrification and denitrification in a single step. Botello [1] also mentions this genus as nitrifying in his study on detecting the nitrifying potential of bacterial isolates that had the characteristic of being NH4+-oxidizing.
The genus Mycobacterium also showed nitrifying potential in the study on salt-tolerant bacteria for aerobic nitrification and denitrification under cyanogenesis conditions. The nitrifying potential of the genus Yersinia and Serratia was confirmed due to the NO2 oxidation capacity [45], confirming study [42] on Serratia plymuthica.
The genus Aeromonas was tested in [33] in nitrification and denitrification processes applied in wastewater to reduce NH4+, NO2− and NO3− under extreme conditions, which validates the nitrifying potential of this genus and its facultative behavior. The study in [46] highlights the NO2 oxidation capacity of the genus Buttiauxella, as does [45], which tested the NH4+ and NO2 degradation capacity of bacteria of the order Enterobacteriales, to which the genus Buttiauxella belongs, and found them to be facultative.
Regarding nitrifying potential, the experimental setup worked for 40 days using only a compound loaded with nitrite as feed synthetic wastewater, resulting in a stable compound. In the experiment, the bacteria were inoculated and carried out their process suspended in the synthetic medium; according to Sánchez [47], there is no difference in the growth rate compared to those on the solid support. However, the studies conducted by Morita [48] and Dong [49] achieved greater efficiency with encapsulated microorganisms.
In Oliveira’s study [50], a reactor with several phases was operated, with the denitrification phase being operated in an established adequate operating condition and even more when working with a specific bacterial strain.
Other bacteria performed the denitrification process in the anoxic phase of the experiment. Ammonium removal had high efficiency in some strains. However, monitoring was only performed until day 40, unlike the study presented by Cho [51], where they observed effective nitrate reductions from day 61 to 90 because of the time bacteria take to adapt to media with additional organic matter content [52].
Each bacteria had a particular behavior in the medium in which it was inoculated. Therefore, the monitorization was made over 40 days, the optimal cell retention time for this type of bacteria. The nitrification values obtained in the first days of the tests (450 mg/L for the application with Yersenia enterocolitica) are similar to those presented in similar research [53,54]. However, these trials were carried out with biological filtration processes, and the performance of the bacteria and the efficiency of the process yielded comparable results.
Therefore, nitrite was removed efficiently in most aerobic reactors, similar to the information published by Farazaki and Gikas [35], where the highest efficiency was obtained in the aerobic reactor and not in the anoxic one. However, the same reactor was used in two phases in this investigation. Studies attribute this behavior to the presence of facultative characteristics in the genus Aeromonas, Buttiauxella, Yersinia, Bacillus, Serratia, and Mycobacterium [41,42,43,44] that were evaluated in this study.
On the other hand, statistical techniques were applied for the kinetics of nitrification to generate regressive models that allow obtaining growth and nitrification values. The obtained rates ranged between 0.1 and 0.3 d-1; Matovelle obtained similar values [55], although, in this research, a few kinetics applied to water quality models and wastewater discharges were analyzed.
Using the Monod approaches and adjusting the similar Bhattachayra [56] methodology, the growth rates for the selected bacteria were obtained, registering a maximum value of 0.85 d-1 for Yersenia enterocolitica G23, a value slightly higher than that reported by Bhattachayra [57] and to the values (0.62–0.71) used by Mehrani [58] for the application in a model of nitrite oxidation in suspended growth wastewater treatment systems.
On the other hand, an increase in the amounts of NO2 during the evaluation period was observed. This could be explained by the total consumption of energy sources that led to the point of chaos. Here, the amounts of NH4+ rose due to the high mortality rates, contributing to the formation of OM and becoming part of the nitrogen cycle. Graham [59] shows that on instability in biological nitrification, chaos is vital for stabilizing nitrification due to the fragile mutualism between ammonium-oxidizing bacteria and nitrite-oxidizing bacteria.

5. Conclusions

The conditions of the pine forest and the native forest are unique, with factors such as pH, vegetation, temperature, and vegetation cover, so that different types of bacterial species have been isolated, sharing a single genus between the two ecosystems.
The bacterial isolates from both study sites allowed us to first determine that they did not belong to the nitrifying genera, such as Nitrosospira, Nitrosomonas and Nitrosococcus, which belong to the chemoautotrophic bacteria. The results show that the soils of these ecosystems can host new microbial communities involved in biogeochemical cycles. The isolated strains showed nitrifying potential, demonstrating their participation in nitrification and denitrification processes, indicating that they are facultative bacteria, making them potential species for soil and wastewater remediation.
Conversely, the nitrate oxidation processes took place in a shorter retention time, estimated at 15 days; these controlled processes developed in the experimental reactor, based on these data, can be applied on a larger scale for the treatment of wastewater with a high load of nitrogenous compounds.
The bacteria isolated and tested as nitrifiers showed a denitrification capacity when evaluated in discontinuous processes, without oxygen supply, allowing the operation of a reactor for nitrogen elimination. Although the test was carried out over 40 days, the complete nitrification and denitrification results were satisfactory.
This research highlights the uniqueness of the microbiological diversity of Andean soils and provides an interesting perspective on the applications of these microorganisms in environmental remediation.

Author Contributions

Methodology, J.C. and S.P.; Formal analysis, C.M.; Investigation, J.M.S. and P.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors of this work declare that this research has been carried out without any commercial or financial relationship and, therefore, does not present any conflict of interest now or in the future. The funders had no role in the study’s design; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Oxidation phase of nitrite by bacteria (NOB).
Figure 1. Oxidation phase of nitrite by bacteria (NOB).
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Figure 2. Machangara River Sub-watershed.
Figure 2. Machangara River Sub-watershed.
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Figure 3. Location of plots for soil sampling.
Figure 3. Location of plots for soil sampling.
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Figure 4. Acidifying halo measurement of Buttiauxella gaviniae A19.
Figure 4. Acidifying halo measurement of Buttiauxella gaviniae A19.
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Figure 5. Temporal evaluation of nitrification by bacterial strains inoculated in R1-R2 reactors under the same conditions but with different behavior.
Figure 5. Temporal evaluation of nitrification by bacterial strains inoculated in R1-R2 reactors under the same conditions but with different behavior.
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Figure 6. Nitrification system, through identified and inoculated microorganisms.
Figure 6. Nitrification system, through identified and inoculated microorganisms.
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Table
Table 1. Composition of culture media.
Table 1. Composition of culture media.
CompositionCulture Media
OALOAHONLONH
MgSO4·7H2O0.3 0.30.050.05
CaCO37.5 7.50.030.03
KH2PO4110.150.15
FeSO4·7H2O0.030.030.000150.00015
NaCl0.30.30.5-
(NH4)2SO40.5- -
NaNO2 0.20.2
(NH4)2Mo7O24 4H2O 50 (µg)50 (µg)
Sodium pyruvate 0.55
Yeast extract 1.5 1.5
Peptone 1.5 1.5
Table
Table 2. Strains with nitrifying potential and size of the acidification halo.
Table 2. Strains with nitrifying potential and size of the acidification halo.
StrainDiameter (cm)GenusPlot
ONLBP 1BN 2
b3R1.1Bacillus pumilusx
b2R91.2Yersinia kristenseniix
b1R14-Serratia proteamaculans x
G231.3Yersinia enterocolítica x
A11.0Aeromonas encheleiaxx
C2(2)-Paenibacillus stefellifer x
b5R1.1Aeromonas molluscorumx
C2-11.6Serratia plymuthica x
A191.3Buttiauxella gaviniae x
b17R1.1Bacillus muralisx
A61.1Buttiauxella ferraguliae x
f21.2Mycobacterium sp.x
Mean1.15
SD0.17
CV14.7%
1 Pine forest, 2 Native forest.
Table
Table 3. Behavioral dynamics of applied bacteria strains.
Table 3. Behavioral dynamics of applied bacteria strains.
BacteriaOptimal TCR for Nitrification (Days)—Maximum Concentration of NO3−—N (mg/L)Particular Considerations
Mycobacterium sp.20–350After day 20, there was a denitrification process; no oxygen was supplied to consider anoxia
Yersenia kristensenii40–14There was no denitrification process
Buttiavexella ferraguliae40–80There was no denitrification process
Bacillus pumilus-No clear nitrification processes were evident within the times proposed in the experiment
Aeromonas molluscorum20–350After day 20, there was a denitrification process; no oxygen was supplied to consider anoxia
Bacillus muralis-There was no concentration of nitrates, and no nitrification processes were observed
Yersenia enterocolítica15–450There was an increase in nitrates for up to 15 days and, subsequently, an anoxic process of denitrification
Serratia plymuthica35–2On day 35, a slight increase in the concentration of nitrates was seen, indicating a nitrification process
Buttiavexella gaviniae20–220After day 20, there was a denitrification process; no oxygen was supplied to consider anoxia
Aeromona encheleia After day 20, there was a denitrification process; no oxygen was supplied to consider anoxia
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Salazar, J.M.; Calle, J.; Pereira, S.; Cordero, P.; Matovelle, C. Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction. Sustainability 2023, 15, 9277. https://doi.org/10.3390/su15129277

AMA Style

Salazar JM, Calle J, Pereira S, Cordero P, Matovelle C. Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction. Sustainability. 2023; 15(12):9277. https://doi.org/10.3390/su15129277

Chicago/Turabian Style

Salazar, Jazmin M., Jessica Calle, Steeven Pereira, Paula Cordero, and Carlos Matovelle. 2023. "Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction" Sustainability 15, no. 12: 9277. https://doi.org/10.3390/su15129277

APA Style

Salazar, J. M., Calle, J., Pereira, S., Cordero, P., & Matovelle, C. (2023). Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction. Sustainability, 15(12), 9277. https://doi.org/10.3390/su15129277

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